EFFECT OF PARTIAL INCORPORATION OF MALTED MILLET IN

JAND BASED ON SENSORY AND PHYSICO-CHEMICAL ATTRIBUTE

AND ITS QUALITY EVALUATION

by

Ananta Ghimire

Department of Food Technology

Nagarik College, Nawalparasi

Institute of Science and Technology

Tribhuvan University

2023

i
EFFECT OF PARTIAL INCORPORATION OF MALTED MILLET IN
JAND BASED ON SENSORY AND PHYSICO-CHEMICAL ATTRIBUTE
AND ITS QUALITY EVALUATION

A dissertation submitted to the Department of Food Technology, Nagarik College, Tribhuvan

University, in partial fulfillment of the requirement for the degree of B. Tech in Food
Technology.

by

Ananta Ghimire

Department of Food Technology

Nagarik College, Gaindakot, Nawalparasi

Institute of Science and Technology

Tribhuvan University, Nepal

2023

ii
 Tribuvan University

Institute of Science and Technology

Department of Food Technology

Nagarik College, Gaindakot-2, Nawalparasi

Approval letter

The dissertation entitled Effect of partial incorporation of malted millet in Jand based on
sensory and physico-chemical attribute and its quality evaluation presented by Ananta
Ghimire has been accepted as the partial fulfillment of the requirement for the degree of B.
Tech. degree in Food Technology.

Dissertation committee
1. Chairperson ______________________________________
(Mr. Madhab P. Tiwari, Asst. Lecture)

2. Principal ______________________________________
(Mr. Chabbi Lal Kandel)

3. External Examiner ______________________________________

(Prof. Dhan Bahadur karki)

4. Internal Examiner _____________________________________

(Mr. Krishna Chalise, Asst. Lecture)

5. Supervisor ______________________________________
(Mr. Madhab P. Tiwari, Asst. Lecture)

January 21, 2023

iii
 Acknowledgment

I would like to express deep gratitude to my supervisor, respected Madhab PD. Tiwari (Co-
ordinator, Nagarik College of Food Technology) for his effective guidance, encouragement,
inspiration and his valuable time throughout this dissertation work.

I am greatly thankful to principal of Nagarik College, Mr. Chhabilal Kandel for providing the
necessary amenities, support, and encouragement for the work on time. I would also like to express
my humble gratitude respected sir Mr. Hukum chalise, for sharing his extra ideas needed for my
work and all the staff of Nagarik College for creating a friendly environment throughout the
dissertation. I greatly appreciate Ms. Sofi poudel, and Mr. Shiva paudel for their cooperation in
the laboratory work, and Mr. Anil Chaudhary, Mr. Suman sapkota and Mr. Bishal bhandari for
their generous support during my work. Also, I am thankful to all my friends for their
encouragement and precious advice during the work period.

Last but not the least, I express sincere thanks to all those names which have not mentioned
individually but helped me directly and indirectly in this work.

Date of submission: February 3, 2023

Ananta Ghimire

iv
 Abstract
Finger millet (Eluesine coracana) was collected from Bullingtar (VDC in Nawalpur district,
Gandaki province). A study was conducted to optimize partial incorporation of malted millet in
jand. Malted millet was prepared by soaking in water for 24h, germination 4 days at about 24℃
followed by cabinet drying which was done in three steps: 1st step at about 50°C up to moisture
content 23%, 2nd step at about 65°C up to moisture content 12%, and 3rd step at about 85°C up to
moisture content 4-6% then acrospires are removed and sound malt is taken for grinding. Malted
millet incorporated jand was prepared in lab with the incorporation of different proportion 0%,
5%, 10%, 15%, 20%, and 25% respectively with normal millet. The sensory evaluation of malted
millet incorporated jand of different concentration was carried out for consumer acceptability. The
obtained data were statistically analyzed by using one way ANOVA at 5% level of significance.

The proximate analysis for moisture (db), crude protein (db), crude fat (db), crude fiber (db), total
ash (db) and carbohydrate (db) of malted millet was done and the values were found to be 6.10±
0.08, 7.20±0.20, 2.43±0.10, 5.15±0.09, 2.04±0.11 and 83.18±0.12 respectively. Similarly, the malt
extract analysis for pH of wort, extract% (db.), color of wort, saccharification time (min) and
degree plato (0Bx) was done and the values were found to be 5.94±0.10, 5.83±0.08, 75.27±0.69,
5.62±0.09, 12.67±0.58 and 8.31±0.09.The stastical analysis showed that 10% malted millet
incorporated Jand was superior to all jand formulation in terms of sensory characteristics.
.

v
 Contents
List of tables.................................................................................................................................. ix

List of Figures ................................................................................................................................ x

List of Appendices ........................................................................................................................ xi

List of Abbreviations ................................................................................................................. xiii

Introduction ................................................................................................................................... 1

1.1 General introduction .......................................................................................................... 1

1.2 Statement of problems ....................................................................................................... 2
1.3 Objectives .......................................................................................................................... 3
1.3.1 General objective ........................................................................................................ 3
1.3.2 Specific objective ....................................................................................................... 3
1.4 significance of study.......................................................................................................... 3
1.5 Limitation of work ............................................................................................................ 3
Literature review .......................................................................................................................... 4

2.1 Historical background of alcoholic beverage .................................................................... 4

2.2 Raw material for fermentation .......................................................................................... 5
2.3 Some alcoholic beverages prepared from cereals ............................................................. 5
2.3.1 Rice ............................................................................................................................. 5
2.3.2 Maize .......................................................................................................................... 6
2.3.3 Wheat .......................................................................................................................... 6
2.4 Finger millet ...................................................................................................................... 6
2.4.1 Structure of finger millet ............................................................................................ 7
2.4.2 Nutritional composition of finger millet ..................................................................... 8
2.5 Malting .............................................................................................................................. 9
2.5.1 Steeping .................................................................................................................... 10
2.5.2 Germination .............................................................................................................. 10
2.5.3 Kilning ...................................................................................................................... 11
2.6 Changes during malting .................................................................................................. 12
2.6.1 Physical changes during malting .............................................................................. 12
2.6.2 Enzyme activity during germination ........................................................................ 13

vi
 2.7 Benefit of malting............................................................................................................ 14
2.8 Traditional alcoholic beverage of Nepal ......................................................................... 15
2.8.1 Jand ........................................................................................................................... 15
2.8.2 Nigar ......................................................................................................................... 16
2.8.3 Raksi ......................................................................................................................... 17
2.9 Murcha: Indigenous starter culture of jand ..................................................................... 17
2.10 Flavoring compounds produced in alcoholic beverages ............................................... 18
2.9.1 Esters ........................................................................................................................ 18
2.10.2 Aldehydes ............................................................................................................... 18
2.10.3 Organic acids .......................................................................................................... 19
Material and methods ................................................................................................................. 21

3.1 Materials .......................................................................................................................... 21

3.1.1 Raw Materials ........................................................................................................... 21
3.1.2 Chemicals, equipment, and utensils ......................................................................... 21
3.2 Methods ........................................................................................................................... 21
3.2.1 Preparation of malt ................................................................................................... 21
3.2.2 Preparation before fermentation ............................................................................... 23
3.2.3 Proximate analysis of raw materials ......................................................................... 25
3.2.4 Analytical methods ................................................................................................... 25
3.2.5 Physical analysis of Finger millet ............................................................................. 28
3.2.6 Experimental design for laboratory analysis ............................................................ 29
Result and discussion .................................................................................................................. 30

4.1 Proximate analysis of raw materials................................................................................ 30

4.2 Malt extract analysis of finger millet malts ..................................................................... 32
4.3 Physical properties of normal millet and malted millet .................................................. 33
4.4 Germination capacity ...................................................................................................... 34
4.5 Sensory evaluation of jand from normal and malted millet ............................................ 34
4.5.1 Color and appearance ............................................................................................... 34
4.4.2 Flavor ........................................................................................................................ 35
4.4.3 Aroma ....................................................................................................................... 37
4.4.4 Mouthfeel.................................................................................................................. 38
4.4.5 Overall acceptance .................................................................................................... 39

vii
 4.5 Selection of best jand ...................................................................................................... 40
4.6 Chemical analysis of jand of normal and best optimized malted jand ............................ 40
4.6.1 pH of jand ................................................................................................................. 40
4.6.2 Protein of jand .......................................................................................................... 41
4.6.3 Alcohol content of jand ............................................................................................ 42
4.6.4 TSS of jand ............................................................................................................... 43
4.6.5 Acidity of jand .......................................................................................................... 44
4.6.6 Reducing sugar of jand ............................................................................................. 45
4.6.7 Aldehyde content of jand .......................................................................................... 46
4.6.8 Ester content of jand ................................................................................................. 47
Conclusion and recommendations ............................................................................................ 48

5.1 Conclusions ..................................................................................................................... 48

5.2 Recommendation ............................................................................................................. 48
Summary ...................................................................................................................................... 49

Refrences ...................................................................................................................................... 50

Appendices ................................................................................................................................... 61

Photo plate ................................................................................................................................... 72

viii
 List of tables
Table No. Title Page No.

2.1 Proximate composition of finger millet 9

2.2 physiochemical parameters of millet jand 16

3.1 Experimental design for laboratory analysis 29

4.1 proximate analysis of raw and malted millet 31

4.2 Malt extract analysis of finger millet 33

4.3 Physical properties of normal and malted millet 33

ix
 List of Figures
Fig No Title page No.

2.1 Structure of finger millet 8

3.1 Flowchart for malting process 22

3.2 Process flowchart for preparation of Jand 24

4.1 Mean sensory scores for color of jand 34

4.2 Mean sensory score for flavor of jand 35

4.3 Mean sensory scores for Aroma of jand 37

4.4 Mean sensory score for mouthfeel of jand 38

4.5 Mean sensory score of overall acceptance 39

4.6 Mean sensory scores for pH of jand 40

4.7 Mean sensory scores for protein of jand 41

4.8 Mean sensory scores for alcohol of jand 42

4.9 Mean sensory scores for tss of jand 43

4.10 Mean sensory scores for acidity of jand 44

4.11 Mean sensory scores for reducing sugar of jand 45

4.12 Mean sensory scores for aldehyde content of jand 46

4.13 Mean sensory scores for ester content of jand 47

x
 List of Appendices
Annex No. Title Page No.

A.1 Equipment and glassware used 61

A.2 List of chemical used 62

B Hedonic Rating Test 63

C.1 One way ANOVA for moisture content of both millet 64

C.2 One way ANOVA for crude protein of both millet 64

C.3 One way ANOVA for crude fat of both millet 64

C.4 One way ANOVA for crude fiber of both millet 65

C.5 One way ANOVA for ash content of both millet 65

C.6 One way ANOVA for carbohydrate of both millet 65

D.1 One way ANOVA for color of both millet 66

D.2 One way ANOVA for flavor of both millet 66

D.3 One way ANOVA for aroma of both millet 66

D.4 One way ANOVA for taste of both millet 67

D.5 One way ANOVA for overall acceptance of both millet 67

E.1 T-test for pH of Jand 68

E.2 T-test for protein of Jand 68

E.3 T-test for alcohol content of Jand 69

E.4 T-test for tss of Jand 69

E.5 T-test for acidity of Jand 70

E.6 T-test for reducing sugar of Jand 70

xi
E.7 T-test for aldehyde content of Jand 71

E.8 T-test for ester content of Jand 71

xii
 List of Abbreviations
S.No. Abbreviation Full Form

1 abv Alcohol by volume

2 ANOVA Analysis of Variance

3 AOAC Association of Official Analytical Chemists

4 db/wb Dry Basis/Wet Basis

o
5 BX Degree Brix

6 H Hours

7 LAB Lactic Acid Bacteria

8 LSD Least Significant Difference

xiii
 PART I
Introduction
1.1 General introduction
In Nepal, the primary occupation of many people is agriculture. As cereal production is considered
as major crops which is used both as cash crop as well as food crops. Varieties of the cereal-based
fermented beverage are consume worldwide (Blandino et al., 2003) which are used for nutritional
and medicinal purpose (as antiseptic and analgesic). The millet based alcoholic beverages are also
known to have provided several health-promoting benefits (Amadou, 2011 refrence). Chyang,
Raksi, Sake, Bouza, Pito, and Burukutu are some of the cereal-based alcoholic beverages
consumed around the world (Bamforth, 2004; Blandino et al., 2003; Karki,1986) Among them
jand is one which is also considered as an indigenous alcoholic beverage on Nepal.

Fermentation is one of the oldest biotechnologies for the production of food products with
desirable properties such as extended shelf-life and good organoleptic properties (Smid &
Hugenholtz, 2010). Finished fermented foods usually have an improved microbial stability and
safety and some can be stored even at ambient temperatures. Furthermore, there are several
examples of fermentation processes which lead to an increase in nutritional value or digestibility
(Jägerstad et al., 2005)of food raw materials. Finally, food fermentation processes also deliver
products with increased palatability for consumers. All these arguments have boosted the interest
to explore natural food fermentation processes and more precisely to link the diversity of the
community of fermenting microbes and their properties to the energetics of the process and to
product quality.

In Nepal, the term jand is generic name for all cereal-based alcoholic beveragess; Tibetan
call it ‘minchha chhyaang’ and the Lepcha call it ‘mong chee’(Thapa & Tamang, 2004). It is
prepared by solid-state fermentation of starchy raw material like rice, finger millet, Wheat, barley,
sorghum etc. The basic step followed in the traditional cereals fermentation are: cooking of the
prepared cereals, cooling of cooked mass to room temperature, mixing with murcha powder,
leaving for 1 to 2 days for biomass build up and alcoholic fermentation. The common cereal of
choice for jand preparation is finger millet but other cereals like maize, wheat and rice are also
used (S. R. Rai, 2006). Finger millet is the most preferred substrate for jand preparation and is

1
considered to yield an unmatched quality product. The basic reason behind it is the uniqueness in
flavor and taste of the product.

Millet is the group of small seeded cereal crops. Major millets grown in Nepal are finger
millet [Eleusine coracana (L.) Gaertn.], proso millet [Panicum miliaceum L.,] and foxtail millet
[Setaria italica (L.) P.Beauv.].Besides, barn yard millet [Echinochloa frumentacea Link],
sorghum [Sorghum bicolor (L.) Moench], pearl millet [Pennisetum typhoides (Burm.f.) Stapf &
C.E.Hubb.] etc are also grown. Eleusine indica (L.) Gaertn. and Eleusine africanaa Kenn.-
O'Byrne are the two wild forms that have been reported their existence in Nepal. Millets are
under-utilized but important food crops for rural poor farming communities living under
subsistence and marginal environments in the Hills of Nepal. They are also known as Himalayan
super-foods due to their nutrient dense nature (Bhandari et al., 2017)

Malting, a combined process of controlled germination and drying, is done to obtain a

desirable physical and biochemical change within the grain by the subsequent development of
hydrolytic enzymes .The main processes carried out to assure the required biochemical changes
include moistening of grain from 12% to 40% This is followed by germination to synthesize
enzymes and endosperm hydrolysis. Finally, a kilning process is carried out to stop the previous
enzymatic activity (Ojha et al., 2020).

Murcha is used as starter culture in preparation of alcoholic beverage like jand. The term
Murcha is a Nepali word and the different ethnic communities of the region call it by their own
dialect such as Khesung by Limbu, Bharama by Tamang, Bopkha by Rai and Buth/Thanbum by
Lepcha (Karki, D. B., & Kharel, 2007).It contains saccharifying molds, Lactic acid bacteria and
fermenting yeast and is therefore the result of concerted action of these microorganism on the
cooked cereals (KC et. al.,2004)

1.2 Statement of problems

Regarding various research on jand production and quality assessment, the risk of faulty
optimization is still high. The basis of manufacturing of alcoholic beverage is alcoholic
fermentation. To determine the quality of final product, control of fermentation is the pre-
requisite. In case of jand, significant researches have been made on finger millet (kodo) but
researches regarding best proportion of incorporation with malted millet is still insufficient.

2
1.3 Objectives
1.3.1 General objective
The general objective of this dissertation work is to incorporate malted millet in jand in various
proportion and its quality evaluation.

1.3.2 Specific objective

The specific objective of my research are as follow:

 To prepare jand from partial incorporation of malted millet replacing the normal millet in
formulation process.
 To find best proportion of malted millet in incorporated jand.
 To perform physicochemical analysis of prepare jand.
 To perform sensory analysis.

1.4 significance of study

Despite being highly nutritious and economic feasible, traditional alcoholic beverages are not
getting enough improvement and their production has been confined to household level only. So,
researches must be carried out for their improvement in production methodology and raw material
composition. Moreover, finger millet is usually considered as poor man‟s cereal which can be
utilized for alcoholic fermentation. It usually grows under harsh conditions in most parts of the
rural hills and lower Himalayan belt of Nepal .My research is directed toward showing significance
of malted millet in jand. Introduction of partial malt in jand may bring improvement in its quality
and may further support for commercialization of indigenous food to worldwide market.
Furthermore, it might also serve as the foundation for marketing indigenous foods to worldwide
markets, allowing us to bring our ethnic communities, tribes, and nation’s identities to every corner
of the globe.

1.5 Limitation of work

 Market murcha sample will be used as inoculum rather than isolated one.
 Fermentation is carried out using traditional method.
 Activity of enzyme is not studied..
 Shelf life of jand will not be studied.

3
 Part II

Literature review
2.1 Historical background of alcoholic beverage
Humans have been controlling the fermentation process for thousands of years, primarily in the
form of fermented beverages in the earliest days. Evidence of a fermented alcoholic beverage made
from fruit, honey, and rice found in Neolithic China dates back to 7000-6600 BCE. Wine-making
dates to around 6000 BCE in Georgia, in the Caucasus region of Eurasia. There is also strong
evidence that people were fermenting beverages in Babylon around 3000 BCE. The use of wheat,
rye, millet, rice, oats, barley, potatoes or grapes in early fermentation processes paved the way to
the technologies that are in existence currently (Jones, 1985)

Alcoholic drinks have long been incorporated into ceremonies and celebration throughout
Nepal also. Chyaang, raksi, tongba are some alcoholic beverage which are considered as ethnic
food in Nepal. Ethnic foods are defined as foods originating from a heritage and culture of an
ethnic group who use their knowledge of local ingredients of plants and/or animal sources.
Moreover, Jand and raksi are common drink traditionally prepared by almost all Mangarantis
(Mangarantis being one of the ethnic groups of the Nepalese that include Magar, Gurung, Rai,
Limbu, Sherpa, Bhote, and Lepcha Castes).The word Jand, derived from the Mangaranti language,
is known by many synonyms (Chiang by Tibetans; Ou by Thai, which is especially drunk with a
straw(Teramoto et al., 2011); Chii by the Rong and Toongba by the Nepalese who take jand in
bamboo container) (Misihairabgwi & Cheikhyoussef, 2017).

Kodo ko jand is one of the most nutritious and traditional drinks in the Himalayan region
of India, Nepal, Bhutan and Tibet in China(Targais et al., 2012). Jand is said to be the best remedy
to ward off the severe cold of the mountains. It reputedly has many healing properties for
conditions such as common cold, fevers, allergic rhinitis, and alcoholism among others.
Toxicological data suggests it as non-toxic and safe for human consumption. Among the ethnic
beverages of the region, the mild-alcoholic sweet flavored Chyang because of high calories,
vitamin content, beneficial lactic acid bacteria and yeast is considered more as food than an
alcoholic beverage. It quenches thirst, gives energy and provides nutrition. Jand forms part of
socio cultural life of the people in the region. Drinking and making offerings of Jand are part of
many pan-Tibetan social and religious occasions, including settling disputes, welcoming guests,
4
and wooing. The traditional method of preparation of Jand may vary from one region to other
using barley chiefly as the substrate; the alternate substrate is ragi (Thapa & Tamang, 2004). In
the face of increasing population and stagnant wheat and rice productions, millets can be a
promising alternative in solving the problem of food insecurity and malnutrition.

2.2 Raw material for fermentation

Usually starchy raw materials are used as substrate for production of jand. Starch, which has been
gelatinized by heating, can be readily hydrolyzed to fermentable sugars by enzymes. Such starch
occurs in cereal grains (rice, wheat, barley, millet etc.), root crops (cassava), or tubers (potatoes).
All of these materials have been used for the production of whiskey, and the uses of potatoes for
the production of vodka are well known(Prescott, S. C., Dunn, C. G. and Reed, 1987). In Nepal,
cereal grains (rice, wheat, barley, millet etc.) are used for the production of traditional alcoholic
beverages viz. jand (undistilled) and rakshi (distilled) using murcha as a starter.

Although the term jand is commonly used in the finger millet beer, beers from maize, rice
etc. are also called jand the name of the beer is deriving from the raw material used for fermentation
e.g. makai jand (maize beer),wheat jand (ghau jand), bhate jand (rice beer) (Tamang et al., 1988).

2.3 Some alcoholic beverages prepared from cereals

2.3.1 Rice
Rice is one of the world’s most important commercial crop plants, ranking second only to wheat.
Before it is suitable for brewing, rice has to be processed. Japanese "Sake" is a clear, pale yellow,
rice wine, with a characteristic aroma, little acid and slight sweetness (Murakami, 1972). The
alcohol content may vary between 14-20 %( v/v) (Humphreys, T. W. and Stewart, 1978)

It is generally believed that the technique originated in China, but comparison of the
production processes for sake and Chinese alcoholic beverages shows marked differences,
especially in respect to the microorganisms concerned. According to earliest records, sake was
originally brewed from rice that has been chewed to achieve saccharification, followed by natural
fermentation. Sake brewed in this way was used as a sacred wine in the worship of the Shinto gods
(Humphreys and Stewart, 1978).

5
2.3.2 Maize
Maize is the largest of all grain varieties with respect to plant height and seed size. Maize (corn)
starch is used as a substitute for malt. However, the starch must be purified beforehand (dry and
wet milling ).Moreover, it is used as the primary source of carbohydrates for some traditional beer-
like alcoholic beverages in Latin America and Africa and as an adjunct for mass-market beer
production throughout most of the world. Today, maize is most commonly germinated (malted) to
produce amylase for starch conversion in chicha production. Maize malt provides ample alpha-
amylase but is low in beta-amylase and has limited quantities of other diastatic enzymes present
in barley (Haggblade & Holzapfel, 2004).

2.3.3 Wheat
Wheat (Triticum aestivum L) is the most extensively grown cereal crop in the world. Wheat malt
is the raw material for the production of wheat beer in Southern Germany, in particular Bavaria.
Belgian wheat beers as well as the spontaneous fermented beers of the lambic type contain raw
wheat. Up to 50% unmalted wheat is added to the throw of these beers. Since wheat has a relatively
low gelatinization temperature, barley malt hydrolases can degrade wheat biopolymers without
prior boiling (Little, 1994). In the case of lambic beers it is even desired that dextrins and starch
are still available for the overlapping distinct fermentations of a wild microflora (e.g.
Brettanomyces bruxellensis, Brettanomyces lambicus, Saccharomyces spp., Kloeckera spp.,
Pedioccocus spp., Lactobacillus spp.)(Hanke et al., 2005)

2.4 Finger millet

Finger millet is the first important crop among the millets in Nepal in terms of area and production
followed by proso millet and foxtail millet. Sorghum, barn yard millet, pearl millet, little millet
and kodo millet are the other crops that have been reported to be grown in parts and parcels of the
country. It is also known as ragi (Takhellambam et al., 2016) which is consumed without dehulling
(Gull et al., 2015). The grains are staple cereal food in some parts of Africa and India (Saleh et al.,
2013). Although a gluten-free grain with low-glycemic index with nutritional and nutraceutical
advantages, FM is neglected and underutilized (Amadou et al., 2013, Jideani and Jideani, 2011).
Finger millet belongs to the family Poaceae and originated in Ethiopia (Shiihii, Musa, Bhati, &
Martins, 2011) before reaching India (Siwela et al., 2010). In terms of production in semi-arid

6
regions, FM ranks fourth after sorghum, PM and foxtail millet (Shiihii et al., 2011, Upadhyaya et
al., 2011).

In context of Nepal, finger millet occupies an average of 7.9% (268,050 ha) of the total
area covered by cereal crops and accounts for 3.3% (308,488 mt) of total cereal production
(MoAD, 2014) . It has been cultivated from Tarai; Kachorwa (85 masl) of Bara district(Tiwari
et al., 2005) to High Hill: Burounse (3150 masl) of Humla district (Baniya et al., 1992) in
Nepal with cultivation records in all 75 districts. The major production districts of Nepal for this
crop are Khotang, Sindhupalchowk, Baglung, Syangja, Kaski, Gorkha and Sindhuli. It is
considered very important in terms of food and nutrition security in both mid hills and mountains.
A total of 237,862 tons (77%) of finger millet produced was from hill districts followed by
61,417 tons (20%) from mountain districts (Sherchan, 1989)

2.4.1 Structure of finger millet

Finger millet grains are globular in shape and its diameter varies from 1.0 to 1.5 mm (Siwela,
2009; Gull et al., 2014). The predominant cultivar of FM grains is the brown (purna) cultivar, with
few varieties occurring as white (hamsa) (Ramashia, 2018) and red cultivars (Naushad
Emmambux & Taylor, 2013). Sood et al.( 2017) reported that FM grains consist of a unique grain
characteristic of a utricle instead of a true caryopsis, thus making the pericarp not to be completely
fused with the testa. The term caryopses refers to a single-seeded fruit in which the fruit coat or
pericarp surrounds the grain, adheres tightly to the grain coat (Wrigley, 2010) and has a brick red-
colored seed coat (Patel et al., 2014).

The uniqueness of FM grain imparts a characteristic of allowing the pericarp to be easily

removed upon rubbing the grains with mortar and pestle. Another unique structural characteristic
of FM grain is its five (5) layered-testa which has been implicated as one of the likely reasons for
the presence of a high dietary fibre content in the grain (Shobana et al., 2013). The principal
anatomical parts of the FM grains are pericarp, germ and the endosperm. The pericarp is an outer
thin layer which covers the grain and it is known as the glume. The grain pericarp consist of three
(3) layers with varying thickness: the epicarp (outermost layer), mesocarp (middle layer) and
endocarp (inner layer) (Ramashia, 2018)

7
 Fig. 2.1 Structure of finger millet
Source: Ramashia (2018)

2.4.2 Nutritional composition of finger millet

Finger millet grains are gluten-free, non-acid-forming (Muthamilarasan et al., 2016), easy to digest
with low glycemic index foods (Manjula & Visvanathan, 2014).

The protein content of finger millet is ranged from 6-8% on dry basis. As in other cereal
protein, finger millet protein is also deficit in lysine. However finger millet protein contains
significantly high amount of methionine which is deficit in legume protein. Finger millet diet
combined with legume gives good result. The fat content of finger millet is hardly 1.5%. The
energy or calorie content of 100 g is about 350 kcal. Finger millet contains about 56-65% of starch
of which 17-29% is amylase. The special feature of finger millet carbohydrate is the high
proportion of non-starchy polysaccharides (NSP). These NSP serves as dietary fiber that helps in
prevention of constipation, lowering cholesterol and releasing glucose, after each meal, so that the
blood glucose level does not increase rapidly similar to rice diet. Because of this reason, finger
millet diet is recommended to diabetics. Finger millet contains fairly high level of vitamins also.
Thiamine, riboflavin, choline are important vitamins of finger millet. Finger millet is the excellent
source of calcium (340 mg/100 g) and it also contain good amount of phosphorus (Devi et al.,
2014).

The grains contain a high amount of calcium which is an essential macro-nutrients

necessary for growing children, pregnant women and the elderly. This is due to calcium's
importance for normal growth of body tissue such as strengthening bone and teeth. FM has also
been reported to be rich in essential amino acids, such as methionine, tryptophan and lysine

8
(Jideani, 2012). FM contains low amounts fat which contributes to reducing risks of diabetes
mellitus and gastro-intestinal tract disorders (Muthamilarasan et al., 2016). The grains are also rich
in vitamin B complex such as thiamine, riboflavin, folic acid and niacin (Saleh et al., 2013).
Utilization of the plant involves its use as a folk medicine for treatment of liver disease, measles,
pleurisy, pneumonia and small pox. Starch extracted from FM grains are used in the
pharmaceutical industries in the preparation of granules for tablets and capsule dosages (Shiihii et
al., 2011). Application of grains also involves its use in the preparation of baked products,
composite flour, weaning foods, beverage and non-beverage products (Verma & Patel, 2013).

Table2.1 Proximate composition of finger millet

Proximate Composition (Ramashia, 2018) (Hulse et al., 1980)
g/100g %(db)
Moisture 11.2
Protein 11.50-12.3 7.7
Fat/lipids 2.38-4.3 1.5
Minerals 0.47-3.3
Dietary fiber 2.5-8.5
Carbohydrate 60.9-75.2 72.6
Ash 2.6
Crude fiber 3.6

2.5 Malting
Malting is a biotechnological technique which involves the controlled germination of a cereal grain
which aims at activating enzyme systems that catalyse the hydrolysis of polymerised reserved food
materials, notably, proteins, starches and cell-wall substances, thus, extracting fermentable
materials (Rose, 1977; Ogbonna, 2011). It aims to convert or modify the physical structure of the
barley grain and allow synthesis or activation of a series of enzymes such that the final product,
malt, is more readily used in the subsequent stages of brewing, distilling, or food manufacture.

During the malting process, hydrolytic enzyme production and/or release is maximized leading to
cell-wall degradation and protein solubilization with minimal starch breakdown. In order for this
to occur, malting aims to both accelerate germination and retard embryo growth, essentially

9
conflicting activities. Any shoot or root growth produced during the malting process is physically
removed from the final product prior to storage and delivery, and therefore, minimizing embryo
growth reduces losses incurred in the process (MacLeod & Evans, 2015).

The malting process consist of 3 stages which are:

2.5.1 Steeping:

The malting process begins with the steeping process, in which dried grains are imbibed in water
in order to elevate the moisture level to around 42-47% (Schwarz & Li, 2011). Steeping comprises
a series of imbibition in water (2- 4 days) each followed by an air-rest period, during which the
water is drained and carbon dioxide (co2) is evacuated using fans. Sufficient aeration of the steep
vessel with water under controlled temperature (12℃ for partly dormant and 16-18℃ for less
dormant grains) with efficient co2 removal is necessary during steeping process as lack of oxygen
brings about microbial development, anaerobiosis, and souring while excessive aeration results in
unwanted growth and starch loss (D. E. Briggs et al., 2004) .

2.5.2 Germination
The process of germination is marked by embryo development, manifested by the growth of the
rootlets and increment in shoot length, with the concomitant modification of endosperm. In this,
steeped grains are spread on the floor and the grain bed is manually turned over to keep the grains
loose, allow adequate airflow, avoid matting and prevent overheating from respiratory heat of
grains. Currently, this process is conducted using a pneumatic system in the vessels of different
sizes and shapes such as drums with circular germination vessels or rectangular Saladin boxes.

Germination occurs under aerobic and humid conditions at 16-200C for 3-4 days depending
on the process conditions and raw material (Poutanen, 2020). Grain moisture must be about 45-
46% on day 2 and 42-43% on day 5(Palmer, 2018). During the germination stage, GA (Gibberellic
acid) diffuses to the aleurone layer and promotes the biosynthesis of hydrolytic enzymes, which
are released into the inner endosperm for depolymerization of endosperm cell wall polysaccharides
and proteins; the process is referred to as grain modification. This process transforms the hard
endosperm of the grain into the soft (friable). Hydrolyzed products are then translocated to the
embryo via scutellum to provide energy and building blocks for its respiration and growth. It was
recently indicated that GA1 is the most abundant bioactive form synthesized as a glycosyl
conjugate in the scutellum which moves to the aleurone and activates de novo synthesis of GA3

10
conjugate and GA4 (Betts et al., 2020). Ideal modification is considered when the first shoot
(acrospire) has developed to around three of fourth of the grain length (Schwarz & Li, 2011). The
germinated grain is known as green malt after which it is moved toward the kilning process.

2.5.3 Kilning
The green malt is shifted to the kilning chamber where hot air is blown to kill the embryo and
terminate the germination (Oser, 2015). The kilning process aims to discontinue the internal
modifications, reduce the moisture content of malt below 5%, and to ensure the product stability
for storage, transport, and to prevent enzyme denaturation. Kilning also promotes the formation of
melanoidins via the non-enzymatic Millard reaction between amino acids and sugars (Howe,
2020). During curing phase of kilning, this browning reaction can be utilized to add color to the
malt. Usually, kilning is performed in a double-deck kiln. It is conducted in a stepwise manner
with a gradually rising temperature from 50 to 85℃ for around 21 hr. (Poutanen, 2020).

After kilning process, the dried rootlets or culms are removed owing to their extremely
hygroscopic nature. B. E. Briggs et al.( 1981) observed that about 3 to 5% of the original barley is
retrieved as culms in the traditional malting processes. Before being placed in storage bin, malt is
aged up to at least 3 weeks to allow uniform distribution of residual moisture throughout the grain.

11
 Raw material (Finger millet)

Sorting and washing

Steeping (18h)

Draining and washing

Germination (25-30℃)

Kilning (60-65℃)

Removal of rootlet

Malted cereal

Fig. 2.2 General flowchart of Malting

2.6 Changes during malting

2.6.1 Physical changes during malting
The grain swells and increases in its volume by one third during the steeping process. Space is
allowed in the steep tanks for the accumulation of swollen grain. During germination, the grain is
characterized by white coleorhizae or root that breakthrough pericarp and testa; produced from the
base of the corn. In time seminal roots also called rootlets, culms also appear. The shape and size
of the finger millet seem to be changed during the germination process. There is an increase in the
length of the shoot (acrospire). There is a loss in dry weight of barley grain due to excessive growth

12
and oxidation of substances during germination. The leakage of material occurs during the steeping
and germination process which results in malting loss.

2.6.2 Enzyme activity during germination

The aim of germination during malting is the hydrolytic enzyme production in the grain, which
may be classified into three major groups: cell wall hydrolases (arabinoxylanase and β-glucanase);
starch hydrolases {α-amylase, β-amylase, limit dextrinase [LD] [debranching enzymes], and α-
glucosidase} and proteolytic enzymes.

2.6.2.1 Cell wall hydrolases

Better malting performance is associated with lower level of β-glucan and higher level of β-
glucanase in grain (Narzis,2012). The high concentration of β-glucan leads to incomplete
hydrolysis of cell walls, which in turn impede the diffusion of enzymes produced during the
mobilization of kernel reserves and disrupts various quality parameters of finished malt
(Habschied et al., 2020). Therefore, degradation of β-glucan is essential.

2.6.2.2 Starch hydrolases

Amylases

During malting, α-and β-amylases are the principal enzymes responsible for hydrolyzing starch
into fermentable sugars. These enzymes are released from the starch granules after the outer layers
have been already hydrolyzed by cell wall hydrolases. The two amylases have different optimum
temperatures, α-amylase has higher optimum temperature at around 73.89℃ and lower pH of 5.2
and while, β-amylase pH of 5.5 and optimum temperature at 62.78℃ (Sammartino, 2015). Alpha
amylases are the most abundant proteins synthesized in response to GA during germination.

2.6.2.3 Proteolytic enzymes

A large complex of proteolytic enzymes carries out the breakdown of storage proteins during the
grain germination and subsequently malting. Ungerminated barley contains little proteolytic
activity, but it rises rapidly during the early stages of the germination step (Harris, 1962). During
mashing, proteolytic enzymes hydrolyze cell wall protein resulting in the exposure of the starch to
the hydrolytic enzymes. Unlike other hydrolytic enzymes, proteases are not specific to a particular
substrate (protein) but they are specific to certain structural features of the peptide chain. They are

13
categorized into two groups depending upon cleavage site in the proteins: exopeptidases and
endopeptidases. During the germination process, proteins are firstly digested by endoproteases and
subsequently by exopeptidases (Benešová et al., 2018).

Among proteolytic enzymes, most thermostable is carboxypeptidases. The optimal temperature of

their activity is 40-50℃, gets inactivated above 70°C. Their optimal pH is close to that of mashing,
therefore, 80% of the released amino acids are subsequently cleaved during mashing by these
enzymes(Benešová et al., 2017). At germination, about 35-40% of the proteins are broken down
into low molecular compounds, such as oligopeptides and amino acids, which are essential for
yeast nutrition.

2.7 Benefit of malting

 Malting increases vitamin and enzymes: Malting process aims to change grains into malt
with high enzyme and vitamin content. Malting induces important beneficial biochemical
changes in grains. Soaking generates grain softening and increases water availability. The
enzymes produced during germination lead to the hydrolysis of starch and proteins with
release of sugar and amino acids directly available. Proteolytic enzymes improve amino
acid availability, particularly lysine, methionine and tryptophan that are lacking in
cereals. Malt enzymatic power is highly variable with the variety of grain and according to
soaking, germination and drying conditions. Many studies have shown that, by improving
phytase activity, malting process can contribute to the reduction of phytate level of
grain and improve iron and zinc availability. Other studies reveal that during the
germination, there is a decrease in tannins and total phenolic contents. Vitamin (B and C)
content increases significantly during germination (El-Adawy et al., 2003). Kaushik et al.
(2010) found that germination improves calcium, copper, manganese, zinc, riboflavin,
niacin and ascorbic acid content.
 Malting enhances nutrients and flavor: In cereal grains, germination increases
oligosaccharides and amino acids concentration as observed in barley (Rimsten et al.,
2002) , and rice(Manna et al., 1995). Decomposition of high molecular weight polymers
cause generation of bio-functional substances and improvement of organoleptic qualities
due to softening of texture and increase of flavor in grains which leads to particular flavour

14
 given to the derived products . In Japan, germination was used to enhance flavor and
nutrients in brown rice apart from softening the texture (Ohtsubo et al., 2005). Relative
nutritive value of sprouted sorghum increases from 54.6 to 63% and protein efficiency
index increases from 1.5 to 1.7(Amadou et al., 2011)
 Increase the fiber content of grain: Dietary fiber plays an important role in bringing
health advantages n chickpea and helps in lowering plasma cholesterol. (Vasishtha &
Srivastava, 2013) reported that three days germination increased the contents of cellulose,
hemicelluloses, lignin of grain.
 Increases the bioavailability of nutrients: Malted grains are extensively used in weaning
and geriatric foods. A study examined the influence of malting of finger millet, wheat, and
barley on the bio accessibility of iron, zinc, calcium, copper, and manganese. Malting
increased the bio accessibility of iron by >3-folds from the two varieties of finger millet
and by >2-folds from wheat, whereas such beneficial influence was not seen in barley.
The bio accessibility of zinc from wheat and barley increased to an extent of 234 and 100%,
respectively, as a result of malting. However, malting reduced the bio accessibility of zinc
from finger millet. The process marginally increased the bio accessibility of calcium from
white finger millet and wheat. Where as it did not exert any influence on bio accessibility
of copper from finger millet and wheat, and significantly decreased (75%) the same from
barley. For manganese the bio accessibility from brown finger millet(17%) and wheat
(42%) increased. It was reported that malting could be an appropriate food-based strategy
to derive iron and other minerals maximally from food grains (Platel et al., 2010).

2.8 Traditional alcoholic beverage of Nepal

2.8.1 Jand
Jand is a generic term that refers to Nepalese traditional sour-sweet cereal beer made from grains
like millet, rice, wheat, etc. by using murcha as the starter culture (C. Subba, 1985) and bears
similarity with many traditional beers of the world. Jand is very popular among the rural mass of
Nepal (B. K. Rai, 1991). The annual production of jand is higher than that of any other indigenous
fermented products and that trade is probably the single-most important economic activity among
most ethinic groups of low income category (Subba et al., 2005). Kodo ko Jand is the most

15
commonly fermented alcoholic beverages prepared from dry seeds of finger millet locally called
as Kodo in the Eastern Himalayan regions of Darjeeling hills and Sikkim in India, Nepal and
Bhutan (Thapa & Tamang, 2004) . Some aspects of jand have been reviewed by Aidoo et al.
(2005). They have described the role of mucaraceousfungi 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 served in
different forms and modes. Strained jand is prepared by leaching out the readily extractable
contents from the mash with luke warm water. The beverage is cloudy in appearance and has a
very short shelf-life, of the order of few hours. The shelf life of strained jand can be extended to a
few months by in-bottle heat treatment (pasteurization) but the time-temperature regime has to be
worked out carefully to take into account the compounded influence of alcohol content, pH,
acidity, total soluble solids, and packed volume of jand (Mongar & Rai, 2005). Table 2.2 show
some physiochemical property of jand made from different cereals.

Table 2.2 physiochemical parameters of millet jand

Parameter Values
Tss (0Bx) 3.27
pH 4.17
Total acidity, as lactic acid (% m/v) 0.33
Alcohol (% v/v) 6.2
Reducing sugar as dextrose, % m/m 1.58
Esters, as ethyl acetate (mg/100ml) 10.6
Aldehyde (g of acetaldehyde/100 lit absolute alcohol) 24.15
Source: Karki et al.,(2007) & Rai (2006)

2.8.2 Nigar
Nigar is the clear liquid that spontaneously accumulates during prolonged anaerobic fermentation
of jand. The product likens sake and is highly praised by the drinkers. Nigar can therefore be
classified as a cereal wine rather than beer (B. K. Rai, 2006)
16
2.8.3 Raksi
Raksi (also spelt rakshi, rukshi) is an unaged congeneric spirit obtained by pot distillation of the
slurry of jand. The product is somewhat similar with whiskey and has highly varying alcohol
contents (KC et al., 2004), generally between of 15 and 40% (C. Subba, 1985). Several basic
researches have been done on raksi production from different cereals using murcha as well as
isolated pure cultures but there seems to be general lack of attention towards process development
such as preparation of good starter culture, increasing efficiency of traditional distillation apparatus
and separation of feints and foreshots for improving quality of rakshi (B. K. Rai, 2006).

2.9 Murcha: Indigenous starter culture of jand

Murcha or Marcha are the indigenous starters that are used to ferment the locally produced
beverages in Nepal. These are known by different names according to ethnic groups and mostly
prepared by Rai, Limbu, Tamang, Gurung, Newar, and Tharu communities in Nepal. With the
long history of the existence of these starters, there is no evidence of the time it gets started. The
ingredients and process of making starters is kept secret that not daughters, only daughters-in-law
are taught.

This starter culture is a mixed type which comprises various saccharifying molds,
fermentative yeast, and acidifying lactic acid bacteria where the loads were found to be 106 CFU/g,
108 CFU/g, and 107 CFU/g respectively (Tamang & Sarkar, 1995).

The general method of preparation involves soaking of rice (Oryza sativa) in water for 8-
10 hours and crushing into fine powder. Mixing of different ingredients, like roots, leaves, and
flowers from various murcha plants, ginger, dry red chili and previously prepared murcha is
performed. Water is added to the mixture and kneaded into cakes of different shape and size. Then,
they are kept for fermentation for 1-3 days. The distinctive aroma and swollen structure of cakes
specify the end of fermentation which is sun-dried for next few days. Among the 42 plants known
to be used in the preparation of these starters, Plumbago zeylanica, Buddleja asiatica, Vernonia
cinerea, Polygala arillata, etc. are major murcha plants. The use of plants in marcha is based on
the specific ethnic groups and distribution of plants (KC et al., 2001). Although various groups of
microorganisms are present, selective growth of desirable microflora is crucial that includes
S.cerevisiae, S. fibuligera, C. versatilis, Rhizopus sps and P. pentosaceus (Shrestha et al., 2002).
The two main types of marcha are Mana and Manapu.

17
2.10 Flavoring compounds produced in alcoholic beverages
The volatile component profiles of alcoholic beverage products consist of a wide range of
compounds, including acids, alcohols, aldehydes, and other trace level flavor compounds (Egan et
al., 1984). Flavour compounds spirits originate from the raw materials used for fermentation and
from alcoholic fermentation by yeasts (Saccharomyces cerevisiae) and other microorganisms
which metabolize carbohydrate, amino acids, fatty acids and other organic compounds.

2.9.1 Esters
An ester is a compound formed from the reaction between a carboxylic acid and an alcohol. Esters
are numerically the largest group of organoleptic compounds in alcoholic beverages. Lower esters
have pleasant odors that are usually described as fruity (Barnett et al., 1990). Although some ester
formation may occur during the distillation of spirits, the most common esters are produced by the
yeast during fermentation stage. Nout(1992) proposed that esters were produced in the yeast cell
by an enzymatic reaction between acetyl CoA derivatives of fatty acids and free alcohols rather
than by an extra cellular chemical reaction. Experiments involving the addition of individual acids
and alcohols have indicated that the there is competition between different alcohols and acids in
ester formation such that the most abundant esters are derived from the most abundant acids and
alcohols. Since ethanol is the most abundant alcohol the ethyl esters are the most abundant,
followed by isoamyl and propyl esters. Acetate is the most abundant acid formed by yeast during
fermentation, so acetate esters of ethanol and higher alcohols are the most abundant. Ethyl acetate
at up to 50mg/l in beer and 175 mg/l in certain whiskies is the most abundant ester in alcoholic
beverages(Batra & Millner, 1974). It has a characteristic fruity odor (Barnett et al., 1990).

2.10.2 Aldehydes
Aldehydes are synthesized by yeast as intermediates in the formation of alcohols through the
decarboxylation of keto acids. The majorities are further reduced by alcohol dehydrogenase, but a
small amount may be oxidized to acids. During the active phase of fermentation, excess quantities
can be excreted into the fermentation broth. The corresponding aldehydes to most of the alcohols
formed by yeast have been detected in alcoholic fermentation. Acetaldehyde is thus quantitatively
the most significant compound of this group as ethanol is the dominant alcohol formed during
alcoholic fermentation. Generally, aldehydes have flavor threshold two to three orders of
magnitude below the alcohols. The aroma of the lower aldehyde is generally perceived as fruity.
Acetaldehyde has a characteristic pungent odor, but its solution in water, have an agreeable fruity

18
odor (Batra & Millner, 1974). However, as the chain length increases they become more
unpleasant, being cardboard-like and bitter (Messens & De Vuyst, 2002).Parameters which
increases the initial fermentation rate, such as aeration, readily utilizable sugars and other nutrients,
higher temperature, fast fermenting yeast strains and higher pitching rates result in increased
accumulation of aldehydes. The final concentrations of aldehyde in yeast fermentation are a
balance between those which are formed in the initial stages of fermentation, and those which are
utilized in the later stages. In addition, the presence of antioxidants which form complexes with
aldehydes, such as sulphite ions and sulphur dioxide, can enhance the final concentrations
(MacDonald et al., 1984).

2.10.3 Organic acids

Some 100 organic acids have been reported in alcoholic beverages. These arrive from three areas
of yeast metabolism. Those such as acetate, succinate, α-ketoglutarate, malate and citrate are
derived from pyruvate via limited tricarboxalate acid cycle. Pyruvate itself constitutes a
qualitatively important group of acids. They may have direct effect on flavor (e.g. the mouth feel
flavor of pyruvate), but they also contribute to the pH of the beer. Some such as isobutyric and
isovaleric acids are probably derived from the amino acid biosynthetic pathways, but the major
groups are produced form malonyl CoA by the fatty acid synthetase pathway (Lynen, 1967).
Shorter chain fatty acids such as hexanoic (caproic) acid, octanoic (caprylic) acid and decanoic
(capric) acid are produced. They have been considered to have been leaked form the main
biosynthetic pathway. These fatty acids are important flavor compounds in their own right and
have been reported to give a caprylic, gouty, soapy or fatty flavor to beer and when released by
autolysis during the maturation of beer they have been associated with a yeasty flavor (MacDonald
et al., 1984).

2.11 Commercialization in Jand

Traditional alcoholic beverages consumed by the local tribes in the Himalayan region not only
are related to rituals and occasions but also are known to provide increased nutrition such as
proteins, vitamins, added minerals, phytochemicals, phytosterols, and dietary fibers to the
consumer (Das et al., 2016). Tribal people used to drink these alcoholic beverages mostly in the
morning before having breakfast, for health benefits. Alcoholic beverages have also discussed
“Ayurveda” for their medicinal importance. Several workers have reported the health benefits of
traditional alcoholic beverages such as rice beer, which has been found to be effective in diarrhea
and urinary problems, headache, body ache, inflammation, worms treatment, etc.(Samati &
Begum, 2007).

19
The commercialization of jand is quite weaker in market due to lack of proper modernization in
its packaging and proper research on its quality improvement. It is estimated that 250, 225, and
325MT of cereal grains are used annually for Jand making in Dharan, Dhankuta & Terhathum
respectively. Considering that half this amount is sold in the market places, the total annual income
comes to about US$870.000(as of 2006). With the advent of alcoholic beverages based on new
technology, Jand has earned itself social stigma among the well-offs, simply because it happens to
be a poor man’s beer (D. K. Subba et al., 2005)

20
 PART III

Material and methods

3.1 Materials
Materials used in jand preparation were malted and non- malted finger millet, Murcha, water, and
air tight containers.

3.1.1 Raw Materials

Finger millet (Eleusine coracana) was collected from local market of daldale (i.e. Tulsi Kirana
pasal) lied in Nawalpur district and these millets were claimed to bought from bulingtar by shop
owner. 6kg of millets was brought and it was 11 months old (time after harvesting time). Murcha
was also bought from same shop. This shop has been selling good quality murcha since long time.
Moreover, the shop owner claimed that they have been sold raw material of jand from long time.

3.1.2 Chemicals, equipment, and utensils

All the chemicals, equipment, and utensils were used from the laboratory of Nagarik College,
plastic packages and air-tight plastic containers with proper lids were bought from local shop of
Naryanghat.

3.2 Methods
3.2.1 Preparation of malt
Finger millet grains were cleaned by winnowing and washed with tap water to remove extraneous
matters such as dust & stones, foreign grains etc. The clean grains were then subjected to steeping
in tap water for 18h in ambient condition and drained. The grains were then kept in muslin cloth
and swirled to remove excess water. They were then spread over aluminum tray, covered with
moisten muslin cloth and allowed to germinate. During germination period the grains and muslin
cloth were frequently moistened with help of water sprayer at interval of 12h to prevent them from
drying out. The grains were allowed to germinate at about 240c for 96 hours.

In order to stop germination and further enzymatic activities, drying was carried out in cabinet
drier in 3 different stages as follows;

 The first stage, at about 500C up to moisture content 25% for 16h
 The second stage, at about 650C up to moisture content 12% for 4h and
 The third stage, at about 85°C up to moisture content 4-6% for 2h

21
After drying, rootlets were removed and the prepared malt was ground in a grinder and the size of
malt was reduced.

Finger Millet

Steeping (18 h at 220 c)

Draining and washing

Spreading in a tray

Covered with muslin cloth

Mixing and sprinkling water (twice a day)

Germination (96 h at 220c)

Drying/Kilning

Removal of rootlets

Finger Millet malt

Fig. 3.1 Flowchart for malting process

Source: Adelekan et al., (2013)

22
3.2.2 Preparation before fermentation
Required physical and chemical analysis of millet was done then cleaned, washed, and cooked in
steel pot for 25 minutes. Moreover, 1.75 parts (by vol.) of previously boiled water is added to each
part (by wt.) of millet then after cooked for 25 minutes. The gelatinized millet product was then
spreaded in tray for about 30 minutes so that temperature dropped down to below 400 c (to protect
organism present in murcha from high temperature).

Simultaneously, Murcha was ground slightly to get in powder form, which was then sprinkled in
the cooked cooled millet and kneaded through to obtain uniform mixing. Furthermore, the overall
experimental detail is given below:

23
 Cleaning, De-husking and Winnowing of finger millet (6kg each batch)

Washing with tap water

Addition of 1.75 part of volume of boiled water and cooked for 25 min

Addition of 5%, 10%, 15%, 20%, 25% of malted millet in powder form

Air drying of grain for few minute about 5 minute

Murcha about 2% by substrate weight is added to cooked substrate and mixed thoroughly

Mixture is sprayed in tray for 4 hours then transferred to sterile plastic jar of 2 liter capacity

Kept for 2 days for saccharification at about 280c in aerobic condition

After 2 day, the mouth of jar was closed by tight lid and sealed to maintain the anaerobic
environment.

Kept for about 15 days at about 280 c for fermentation at anaerobic condition

After completion of 15 days, water extraction of beverage is done by adding lukewarm water
at the rate of 2 ml/g of fermented substrate (each extraction is kept for about 15 min)
Source: (Ray et al., 2016)

Fig 3.2 Process flowchart for preparation of Jand

24
3.2.3 Proximate analysis of raw materials
3.2.3.1 Moisture content

The moisture content of sample was determined by heating in hot air oven at 105±20c till constant
weight is achieved as per Ranganna (1986).

3.2.3.2 Crude fat content

Crude fat content was determined by the Soxhlet method as describe in Ranganna (1986).

3.2.3.3 Crude fiber content

The crude fiber was determined gravimetrically by acid-alkali treatment as described in Ranganna
(1986).

3.2.3.4 Crude protein content

Crude protein content in the sample was determined by the kjeldahl method (by estimating
nitrogen content) according to Ranganna (1986).

3.2.3.5 Total ash content

The total ash content of the samples was determined by ashing in Muffle Furnance at 5500c
according to Ranganna (1986).

3.2.3.6 Carbohydrate content

The carbohydrate content of the sample was determined by the difference method as per Ranganna
(1986). Carbohydrate (%db.) = 100- (protein +fat+ ash+ crude fiber)

3.2.4 Analytical methods

3.2.4.1 Determination of pH and TSS

PH was determined by pH meter (Hanna Instrument) and TSS by portable refractometer (Hanna
Instrument). The instrument was standardized by using a buffer solution of pH 7 and 4 at the
temperature required Ranganna (1986).

3.2.4.2 Determination of acidity

25
The determination of acidity in jand was done by the preparation of aqueous or solution of the
whey and titration with standard NaOH using phenolphthalein indicator. The result is expressed
as a percentage of dominant acid. 10 ml of jand pipette out 10 ml in conical flask. Add about 10
ml distilled water and mix well. The pipette was rinsed by drawing in and releasing a small amount
of diluted jand. Titrate with 0.1N NaOH using 2 drops of phenolphthalein indicator to a persistent
pink end point. Note the initial and final reading Ranganna (1986).

Acid% = (Titer× N of NaOH × 90 ×100) ÷ (Aliquot × 1000)

3.2.4.3 Determination of Alcohol content

Alcohol content was determined by pycnometric method(S. R. Rai, 2006) taking 200 gm 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. Then weight of dry pycnometer, distillate in pyconmeter and water
in pycnometer was taken; and room temperature was noted. The specific gravity of water was
calculated, and alcohol % (v/v) was found by chart.

3.2.4.4 Determination of Ester content

Method described by Kirk & Sawyer (1991) is used to calculate total ester content, with slight
changes. In a reflux flask, 50 ml of the distillate is neutralized with 0.1 M NaOH and 1 percent
phenolphthalein indicator. The condenser is then attached after 10 ml of 0.1M NaOH was poured.
It is computed using the formula below. 0.0088g of ethyl acetate is comparable to 1 ml of standard
alkali.

Ester content (g ethyl acetate/ L alcohol) = (880×titer)/ %alcohol by volume

3.2.4.5 Determination of Aldehyde content

As indicated in Table below, total aldehyde is calculated as g acetaldehyde/100L alcohol using

Kirk & Sawyer (1991) with minor changes. The following are used to make solutions A, B, C, and
D:

26
 Reagents Preparation method
A Potassium meta bisulphite (15 g) ismixed with 70 mL conc. HCI and
diluted to 1 L with distilled water.
B Na 2 HPO 4 .12H,0 (188 g), 21 g NaoH and 4.5 g EDTA is dissolved in distilled
water and diluted to 1 L with distilled water.
C Conc. HCL (250 ml) is diluted to 1:1 with distilled water.
D Boric acid (100 g) and 170 g NaOH is dissolved in distilled water and diluted to
1 L with distilled water.

300 mL boiling and cooled water and 10 mL solution A are combined in a 1000 mL conical flask.
40 mL distillate is added to this combination, the flask is twisted, and the stopper is put. After then,
it is allowed to stand for 15 minutes. After that, 10 mL of solution B was added, swirled together,
and set aside for another 5 minutes. Then 10 ml of solution C and 10 ml of fresh 0.2 percent starch
solution are added, swirled together, and iodine (0.1 M) is added to eliminate the excess bisulphate
and turn the solution a faint blue hue. Finally, 10 ml of solution D is added, and the released
bisulphate is titrated to the same faint blue end point using 0.05 M iodine solution. Total aldehyde
as g acetaldehyde per 100 L alcohol is calculated using following expression:

Total aldehyde (g acetaldehyde/100 L alcohol) = (Titer × 2.2)/ S

Where, S= % alcohol by volume in the sample.

3.2.4.6 Determination of Reducing Sugar

Total reducing sugar is determined by Lane and Eynon method as described by Ranganna (1986)
.25 g of neutralized sample is clarified and maintained with distilled water in 250 ml volumetric
flask. 10 ml of such sample is titrated with standard Fehling mix solution, and calculation of total
reducing sugar is carried out as % dextrose.

27
3.2.4.7 Sensory evaluation of jand

The coded sample of the jand was evaluated for appearance, color, body, taste, aroma and overall
acceptance on 9 points hedonic scale by 8 semi- trained panelist of Nagarik College. They were
instructed to give 9 points to extremely like and 1 point to the extremely disliked sample. The
coded sample were randomly presented before the panelist. The specimen card for sensory
evaluation is shown in appendix. A. Differences in the quality were determined by statistical
analysis according to Ranganna (1986).

3.2.5 Physical analysis of Finger millet

3.2.5.1 Determination of 1000 kernel weight

The 1000 kernel weight of raw material and final products were determined by measuring the
weight of 1000 kernels of millet grains after selecting appropriate sample size by quartering
method. The grain samples were weighed using digital electronic balance with 0.01 g accuracy
(Sangamithra et al., 2016).

3.2.5.1 Determination of bulk density

The bulk density was measured as mentioned in (Clementson et al., 2010) by pouring the grains
into the funnel shaped hopper, the hopper was centered over the measuring bushel, the hopper
valve was opened quickly, and the grains were allowed to flow freely into the measuring bushel.
After the bushel was filled, the excess material was leveled off with gentle zigzag strokes using
the standard Seedburo striking stick. The filled measuring bushel was then weighed, and the mass
of grains in the bushel was determined by subtracting the mass of the measuring bushel itself. The
bulk density (ρ) of grain was then calculated using the following expression:

Bulk density= mass of grain ÷ volume of bushel

3.2.5.2 Determination of Arithimetric mean diameter (AMD)

The arithmetic mean diameter (mm) of the sample was obtained using the methods of (Ramashia
et al., 2018). Arithmetic mean diameter was calculated from the dimensional values using below
equation:

AMD= (L+B+T)/3

28
L = length (Longest dimension)

B = breadth (Second longest dimension)

T = thickness (Third longest dimension)

3.2.6 Experimental design for laboratory analysis

Manual research have been done with control sample, 5 % malted millet with 95% normal millet,
10% malted millet with 90% normal millet, 15% malted millet with 85% normal millet, 20%
malted millet with 80% normal millet, 25% malted millet with 75% normal millet.

Table 3.1 Experimental design for laboratory analysis

No of sample Parts of malted millet Parts of normal millet
A 0 100
B 5 95
C 10 90
D 15 85
E 20 80
F 25 75

3.2.7 Statistical analysis

The analysis was conducted in triplicates. For significance analysis, data were analyzed by one-
way ANOVA and means were compare using Lsd and Tukey at 5% level of significance.

29
 PART IV
Result and discussion
Finger millet was taken from Bulingtar which was planted in early June in warm climates and
harvested after 11 months of plantation. Proximate and physical analysis of raw material was
performed. Millet was then cleaned and were cooked for about 25 min until they were thoroughly
cooked and were soft in texture. Millet jand was prepared in the laboratory following the traditional
methods used in the common household of various jand consuming ethnic communities of Nepal.
Murcha is used as starter culture which was inoculated in jand and allowed to ferment in small
plastic jars. Every jar was subjected to same environmental conditions in order to ensure the
uniformity in fermentation process. One jar at a time was withdrawn as a sample which prevented
the contamination and thus spoilage of the product. This part presents the results and discussions
regarding proximate and ultimate analysis, sensorial analysis of raw materials and final products.
The following sub- heading deal in detail about these all.

4.1 Proximate analysis of raw materials

Proximate analysis gives inexpensive yet very important information, particularly from the
nutritional and biochemical points of views. The results normally expressed in percentage and
because of the fairly general nature of test employed for the determination, the term crude is
usually used as a modifier; for instant, crude protein, crude fat and crude fiber. Analysis of
particular element or compound, such as vitamins, reducing sugars, etc., is termed ultimate
analysis. In general, ultimate analysis is a more detailed analysis of proximate constituent or the
analysis of components found in very small amounts, e.g., vitamins.

The proximate composition of malted and non- malted finger millet were carried out and the results
are tabulated in table 4.1

30
Table 4.1 proximate analysis of raw and malted millet

Parameters (% in db) Normal millet Malted millet

Moisture 11.26±0.12 6.10±0.08
Crude protein 7.96±0.14 7.20±0.20
Crude fat 3.30±0.13 2.43±0.10
Crude fiber 4.14±0.07 5.15±0.09
Ash content 2.29±0.13 2.04±0.11
Carbohydrate 82.31±0.11 83.18±0.14
The values are the mean ± standard deviation of triplicate analysis, different letter in a rows
indicate a significant differences (p<0.05). Results are ranked in descending order, a>b. %db
indicate percentage dry basis.

The average mean scores of proximate analysis are shown in table 4.1. Statistical analysis
showed normal and malted rice are significant effected (p<0.05) to each other. The mean scores
of normal millet along with standard deviations for moisture content, crude protein, crude fat,
crude fiber, ash content and carbohydrate are 11.26±0.12, 7.96±0.14, 3.30±0.13, 4.14±0.07,
2.29±0.13 respectively and also the mean score of malted rice with standard deviation for moisture
content, crude protein, crude fat, crude fiber, ash content and carbohydrate are 6.10±0.08, 7.20±20,
2.43±0.10, 6.30±0.07, 5.15±0.09, 2.04±0.11, respectively as shown in table **.The proximate
composition of raw finger millet obtained in the laboratory is somehow comparable to the data
provided by (Devi et al., 2014). Slight differences might have occurrence in various parameter due
to differences in analytical methods or due to difference in variety of millet used.

The moisture content of malted millet is decrease significantly at p (≤0.05) which is due to
enzyme inactivation process during malting i.e. kilning. The hydration process during germination
activated a wide array of enzyme which hydrolyzed and solubilized food reserves. There crude
protein content of the malted millet sample decreased significantly at p (≤0.05). The slight change
in protein content may attributed to the fact that water soluble nitrogen was lost during soaking of
seeds and also, during seed germination, part of the protein was utilized for the growth and
development of the embryo. During germination, starch and protein were degraded to soluble
sugars and amino acids, respectively. Their degradations indicated the metabolic system
interference to reserve starch and protein by amylases and proteases(Elbaloula et al., 2014) . The

31
crude fat content of the malted rice slightly increased which may be due to its proportional increase
as a result of decrease in other food reserves like non-reducing carbohydrate.

The crude fiber content of the malted sample increased significantly p (≤0.05). This increased
could be attributed to increased bran matter and the building of dry matter during the growth and
development (germination) of plant. Narsih reported the increase in ash content of rice malt which
is similar to the results of our study. Germination would increase the mineral content due to an
increase in fitase enzyme activity during germination. The enzyme will hydrolyze the bond
between the protein-enzyme minerals become free, therefore increasing the availability of minerals
(Narsih, 2012). The carbohydrate content was also found to be decreased in malted rice this
significant decrease may be due to the activity of enzymes. The carbohydrate may have been
digested to simple sugar by amylolytic enzymes as a result there is significant increase of reducing
sugar on the malted sample at p (≤0.05).

4.2 Malt extract analysis of finger millet malts

Quality malt is essential for the uniform production of beer (Kent, 1982). The malting process is
the formation of hydrolyzing enzymes in the kernels for further decomposition of the starch during
the brewing process to this operation and about 4.5% of the resulting fragments are completely
metabolized. The fermented beverage is then normally clarified and dispensed in an effervescent
condition. Malt extracts analysis of finger millet was carried out in Cg Brewery lab which is given
in table below:

32
Table 4.2 Malt extract analysis of finger millet

Parameter Millet malt extract

Moisture 5.94±0.10
pH of wort 5.83±0.08
Extract% (db.) 75.27±0.69
Color of wort 5.62±0.09
Saccharification time (min) 12.67±0.58
Degree Plato(0Bx) 8.31±0.09
Aroma of wort Malty
Biological matter No
Rate of filtration Normal
Clarity of wort Slightly hazy
*The values in the table are the mean of the triplicates ± standard deviation.

4.3 Physical properties of normal millet and malted millet

The physical properties of millet grain and malt was determined. The result obtained are presented
below:

Table 4.3 physical properties of normal and malted millet

Physical property Normal millet Malted millet

1000 kernel weight (g.) 2.54±0.01 2.39±0.04
Bulk density( kg/HL) 74.62±0.05 68.06±0.07
Arithmetic mean diameter 1.68±0.04 1.72±0.02
*Values are the means of triplicate determination ± S.D*

(Khatoniar & Das, 2020) reported the value of 1000 kernel weight, bulk density and diameter as
3.12±0.06 g, 71±0.02 kg/HL and 1.76±0.02mm of millet grain which is a bit similar to the mean
value of unmalted finger millet of our study.

The thousand kernel wt. and bulk density got decreased while diameter was increased on
malting. The decrease may be due to synthesis of amylases, proteases and other endogenous
hydrolytic enzymes during germination. During the process, the hydrolytic enzymes migrate from
the germ into the endosperm where starch and protein are hydrolyzed to sugars and amino acids,

33
respectively. These are then transported into the germ where they are further metabolized by the
growing seedling(Baranwal, 2017).Thus, decrease in weight may result due to dry matter loss
during malting and utilization of nutrient by growing roots. The removal of this rootlet and
decrease in moisture content after kilning process is also responsible for it.

4.4 Germination capacity

We found 90% of whole millet grain were germinated based on counting method 0f 1000 kernel.
Germination capacity was conducted for millet grain to ensure favorable germination of grain
during malting.

4.5 Sensory evaluation of jand from normal and malted millet

The sensory evaluation of jand was done by 10 semi- trained panelists of Nagarik College. It was
conducted under six parameters color, flavor, aroma, mouthfeel and overall acceptance among the
sensory evaluation was performed under 9-point hedonic scale which is described in the following
sub- headings.

4.5.1 Color and appearance

10
9 b b b ab
8 aa a a
7 ba
Sensory score

A
6 b
B
5
C
4
3 D
2 E
1 F
0
A B C D E F
Sample

Fig. 4.1 Mean sensory scores for color and appearance of jand

*Each bar in the plot is the average and vertical error bars represent ± standard deviations of scores
given by 10 panelists. Different letters indicate significant differences among sample means
(P<0.05) of different formulations (where A is control, B, C, D, E and F contain 5%, 10%, 15%,
20% and 25% malted millet respectively).

34
 The mean sensory scores of colors are shown in Fig 4.1. Statistical analysis showed that
partial substitution of malted jand with normal jand had significant effected (p<0.05) on the color
and appearance. The mean scores along with standard deviations for color of samples A, B, C, D,
E and F were found to be 7.90±0.57, 7.70±0.48, 7.80±0.63, 7.40±0.52, 7±0.47 and 6.80±0.42
respectively as shown in Fig 4.1. The maximum sensory perception was found to be highest for
sample A.

The higher sensory perception for sample A was possibly due to light yellow color then
after sample C got higher perception due to pale yellow color developed by the addition of 10%
malted millet however sample with more than 10% malt simultaneously got lower perception
because of highly yellowish color and haziness due to higher proportion of malt. The yellowish
color may be because of the greater amount of Millard reaction between reducing sugars and
proteins(Roumelioti et al., 2018) .

4.4.2 Flavor

10
9 c bc
b bc
8
a a
7
A
Sensory score

6
B
5
C
4
D
3
E
2
F
1
0
A B C D E F
Sample

Fig 4.2 Mean sensory score for flavor of jand

35
*Each bar in the plot is the average and vertical error bars represent ± standard deviations of scores
given by 10 panelists. Different letters indicate significant differences among sample means
(P<0.05) of different formulations (where A is control, B, C, D, E and F contain 5%, 10%, 15%,
20% and 25% malted millet respectively).

The mean sensory scores of flavor are shown in Fig 4.2. Statistical analysis showed that
partial substitution of malted jand with normal jand had significant effected (p<0.05) on flavor of
jand. The mean scores along with standard deviations for flavor of samples A, B, C, D, E and F
were found to be 7.40±0.52, 7.50±0.53, 8.20±0.57, 7.50±0.85, 6.20±0.63 and 5.90±0.57
respectively as shown in figure 4.1.The maximum sensory perception was found to be highest for
sample C.

Sample C has got highest score as compared to other because of acceptable malt flavor as well as
sweetness. Flavoring compound such aldehyde and ketones may be increased during malting. A
similar increase in malt flavor with the supplement of malted rice was noticed by Briggs (1998). .
Kilning also promotes the formation of melanoidins via the non-enzymatic Millard reaction
between amino acids and sugars which is also responsible factor for flavor in malt (Smart, 2019).
Similarly, decomposition of high molecular weight polymers cause generation of bio-functional
substances and improvement of organoleptic qualities due to softening of texture and increase of
flavor in grains which leads to particular flavour given to the derived products (Holopainen et al.,
2005).

36
4.4.3 Aroma
10
9 b b b
b
8 a a
7
Sensory score

A
6 B
5
4 C
3 D
2 E
1 F
0
A B C D E F
Sample

Fig. 4.3 Mean sensory scores for Aroma

*Each bar in the plot is the average and vertical error bars represent ± standard deviations of scores
given by 10 panelists. Different letters indicate significant differences among sample means
(P<0.05) of different formulations (where A is control, B, C, D, E and F contain 5%, 10%, 15%,
20% and 25% malted millet respectively).

The mean sensory scores for flavor of jand sample of different formulations are shown in
Fig 4.3. Statistical analysis showed that partial substitution of malted jand with normal jand had
significant effected (p<0.05) on flavor of jand. The mean scores along with standard deviations for
aroma of samples A, B, C, D, E and F were found to be 7.40±0.70, 7.60±0.52, 8±0.47, 7.30±0.48,
6.40±0.70 and 6.30±0.67 respectively as shown in Fig 4.3. The maximum sensory perception was
found to be highest for sample C.

The aroma of samples C was found to be aromatic with a malty flavor. Kilning and roasting
is the main reason for generating flavor and aroma in malts. Due to kilning and mashing, the
enzymatic activity occurs and the starches and proteins are hydrolyzed into fermentable sugars and
amino acids. The free sugars and amino acids are the sources for Millard reactions. The aroma and
flavors in malts are due to the production of Millard products during kilning and mashing (Beal &
Mottram, 1993). The different aroma and flavors such as burnt, smoky, caramel, chocolaty, burnt
coffee, and malty are associated with specific malt types. Malty flavor in malts is due to the
37
formation of caramel-like compounds 4hydroxy-2,5dimethyl-3(2H)-furanone (HDMF) and 4-
hydroxy-2(or 5)-ethyl-5(or 2)-methyl3(2H) furananone (HEMF) (Blank & Fay, 1996).

4.4.4 Mouthfeel
10
c
9 bc
b b
8 a a
7
Sensory score

A
6 B
5
4 C
3 D
2 E
1
F
0
A B C D E F
Sample

Fig 4.4 Mean sensory score for mouthfeel of jand

*Each bar in the plot is the average and vertical error bars represent ± standard deviations of scores
given by 10 panelists. Different letters indicate significant differences among sample means
(P<0.05) of different formulations (where A is control, B, C, D, E and F contain 5%, 10%, 15%,
20% and 25% malted millet respectively).

The mean sensory scores for mouthfeel of jand sample of different formulations are shown
in Fig 4.4. Statistical analysis showed that partial substitution of malted jand with normal jand had
significant effected (p<0.05) on flavor of jand. The mean scores along with standard deviations for
mouthfeel of samples A, B, C, D, E and F were found to be 7.30±0.48, 7.40±0.52, 8.10±0.57,
7.20±0.63, 6.30±0.67 and 6±0.67 respectively as shown in Fig 4.3.

The maximum sensory perception was found to be highest for sample C. In alcoholic
beverages, mouth feel has to do mostly with the body (gravity), which depends on the degree of
attenuation.(Berry & Chamberlain, 1986) observed that alcohol and esters content may improve
the mouthfeel of alcoholic beverages mostly. But, too much lower and higher level of esters will
also impair sensory quality of product.

38
4.4.5 Overall acceptance
10
bc c
b b
8 a a
Sensory score

A
6 B
4 C
D
2
E
0 F
A B C D E F
Sample

Fig 4.5 Mean sensory score of overall acceptance

*Each bar in the plot is the average and vertical error bars represent ± standard deviations of scores
given by 10 panelists. Different letters indicate significant differences among sample means
(P<0.05) of different formulations (where A is control, B, C, D, E and F contain 5%, 10%, 15%,
20% and 25% malted millet respectively).

The average means sensory scores of overall acceptances are shown in Fig 4.5. Statistical
analysis (one-way ANOVA) showed that overall acceptability scores were significantly (p<0.05)
higher for sample C as compared to others. The mean scores along with standard deviations for
overall acceptance of samples A, B, C, D, E and F were found to be 7.30±0.48, 7.40±0.52, 8±0.47,
7.20±0.63, 6.30±0.48 and 6.10±0.57 respectively as shown in Fig 4.6.

Sample C scored highest in overall acceptability of the sensory conducted among the
panelists while in term of color, most of panelist prefer a bit more control sample than sample C
due to light yellow color in sample A. Statistical analysis from the experimental data showed that
the partial substitution of malted millet in samples showed significant difference (p<0.05) in
overall acceptability of samples. Sample F showed lowest score in overall acceptability which
could be as a result of higher amount of malted millet incorporated in it. On the other hand, sample
C scored highest in overall acceptability which maybe as a result of optimum malted millet
incorporated in it. The overall experimental analysis showed that optimum malt is essential while

39
with increase of malt than optimum amount, the quality of jand simultaneously degrade in various
parameter as shown in figure above.

4.5 Selection of best jand

By sensory evaluation and obtained data interpretation, 10% malt incorporated jand was the best
product as it shows significantly higher values in almost all attributes. The conclusion derived in
the present study is based on sensory analysis of a limited number of panelists. The result may be
different when subjected to other populations. So, the experimental finding needs to be taken with
some reservation.

4.6 Chemical analysis of jand of normal and best optimized malted jand
The chemical composition of jand includes pH, protein, acidity, TSS, reducing sugar, alcohol%,
ester and aldehyde. These parameter help in assessing the quality of jand. The chemical analysis
of jand was done between control sample and best sample (i.e. 10%malted sample) using
Independent-Samples T-test analysis.

4.6.1 pH of jand

4.5
4.0
3.5
3.0
2.5
pH

2.0 A
1.5 B

1.0
0.5
0.0
A B
Sample

Fig 4.6 pH of jand of different sample

40
* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample. Different letters indicate significant differences among sample means
(p<0.05) of different formulations (where A is control sample and B is 10% malted millet sample)

The pH of the jand of different sample was shown in Fig. 4.6. The pH of jand of sample A
and B was found to be 3.94±0.09 & 4.11±0.03 respectively. Sample B has maximum mean score,
than sample A due to incorporation of malted millet on it. The pH of the malt affects the activity
of enzymes and is critical for the amylases responsible for saccharification (conversion of malt
starch into fermentable sugars, particularly maltose) and liquefaction (Fox & Henry, 1993). In
solid state fermentation of finger millet jand, pH was founded about 4.17(Bahadur Karki & Prasad
Kharel, 2007).The too high and too low pH may create unacceptable i.e. astringent off-flavor.

4.6.2 Protein of jand

9
8
7
6
Protein

5
4 A
3 B
2
1
0
A B
Sample

Fig 4.7 Protein of jand of different samples

* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample Different letters indicate different formulations (where A is control
sample and B is 10% malted millet sample).

The protein content of the jand of different samples was shown in Fig 4.7. The protein
content of jand of sample A & B was found to be 8.58±0.11 & 8.47±0.04 respectively.

Control sample has greater protein content in compare to partially malted one. Protein get
simultaneously decrease with increase in amount of malt incorporation. This may be due to the

41
fact that storage nitrogen reserves may have been mobilized during sprouting after hydrolysis by
proteolytic enzymes (which digest the macromolecular proteins into the more easily assimilable
peptides and amino acids) to play a role in the synthesis of its cellular materials for the rapidly
growing roots and shoots during germination (Ogbonna et al., 2012)

4.6.3 Alcohol content of jand

10
9
8
7
6
Alcohol

5
A
4
B
3
2
1
0
A B
Sample

Fig 4.8 Alcohol of jand of different samples

* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample. Different letters indicate different formulations (where A is control
sample and B is 10% malted millet sample).

The alcohol content of the jand of different samples was shown in Fig 4.8. The alcohol
content of jand of sample A & B was found to be 8.94±0.12 & 9.09±0.07. Alcohol content was
found greater in 10% malted millet jand than control one. As malt provides the fermentable sugar.
Beta and alpha amylase activity acts on starch and the conversion of a dark color to yellowish
occurs i.e., non-fermentable sugar converted into fermentable sugar which represents amylase
activity of malt that directly related to the extraction of fermentable sugar (Muoria, Linden, &
Bechtel, 1998). In brewing, the majority of enzymes such as a-amylase, B-amylase, and

42
limitdextrinase act on starch and cleave into fermentable sugar. The increase in enzyme activity
results increases in alcohol content. (Evans et al., 2005).

4.6.4 TSS of jand

7
6
5
4
TSS

3 A
B
2
1
0
A B
Sample

Fig 4.9 TSS of jand of different samples

* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample. Different letters indicate significant differences among sample means
(p<0.05) of different formulations (where A is control sample and B is 10% malted millet sample).

TSS mean amount of total soluble solid present in the unit volume of solution. The TSS
content of the jand of different samples was shown in Fig 4.9. The TSS content of jand of sample
A & B was found to be 4.50±0.10 & 6.70±0.17 respectively .TSS is found significantly increased
with increase in malt proportion. This may be due to enzymes produced during germination that
lead to the hydrolysis of starch and proteins with release of sugar and amino acids directly available
(Baranwal, 2017).

43
4.6.5 Acidity of jand

1.4

1.2

1.0
Acidity

0.8
A
0.6
B
0.4

0.2

0.0
A B
Sample

Fig 4.10 Acidity of jand of different samples

* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample. Different letters indicate different formulations (where A is control
sample and B is 10% malted millet sample).

The acidity content of the jand of different samples was shown in Fig 4.10. The acidity content of
jand of sample A & B was found to be 1.06±0.06 & 0.93±0.04 respectively. Higher acidity on
control sample may be due to the oxidation of aldehyde to form acetic acid. So, it can be said that
aldehyde present in the samples were converted into acetic acid. Oxidation of ethyl alcohol to
acetic acid may be reason for increase in acidity as well.

44
4.6.6 Reducing sugar of jand

1.4
1.2
Reducing sugar

1
0.8
0.6
0.4
0.2
0
A B
Sample

Fig 4.11 Reducing sugar of jand of different samples

* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample. Different letters indicate different formulations (where A is control
sample and B is 10% malted millet sample).

The reducing content of the jand of different samples was shown in Fig 4.11. The reducing
content of jand of sample A & B was found to be 0.98±0.06 & 1.08±0.06 respectively. It would
be due to the hydrolysis of starch into reducing sugars by activity of amylase enzyme produced
during malting. Similar increase in reducing sugar content with increase in germination time was
observed in millet(Latha & Muralikrishna, 2009).

45
4.6.7 Aldehyde content of jand

0.40
0.35
0.30
Aldehyde(g/l)

0.25
0.20
A
0.15 B

0.10
0.05
0.00
A B
sample

Fig 4.12 Aldehyde content of jand of different samples

* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample. Different letters indicate different formulations (where A is control
sample and B is 10% malted millet sample).

The aldehyde content of the jand of different samples was shown in Fig 4.12. The aldehyde content
of jand of sample A & B was found to be 0.21±0.06 and 0.26±0.08 respectively. Aldehyde is
formed during glycolysis which is synthesized by yeast as intermediates in the formation of
alcohols through the decarboxylation of ketoacids and is released under two conditions; when
ethanol formation is blocked due to absence of alcohol dehydrogenase, or when NADH is being
used for some other purpose and does not need to be recycled duringend product production.
Thus, the excess quantities of acetaldehydes are produced when the reduction, catalyzed by alcohol
dehydrogenase is rate limiting(Swiegers & Pretorius, 2005).

46
4.6.8 Ester content of jand

0.9
0.8
0.7
0.6
Ester(g\l)

0.5
0.4 A

0.3 B

0.2
0.1
0
A B
Sample

Fig 4.13 Ester content of jand of different samples

* Each bar in the plot is the average and vertical error bars represent ± standard deviations of the
value of different sample. Different letters indicate different formulations (where A is control
sample and B is 10% malted millet sample).

The ester content of the jand of different samples was shown in Fig 4.13. The ester content of jand
of sample A & B was found to be 0.61±0.07 & 0.69±0.03 respectively. The compound like fatty
acid, co- enzymes (CoASH) and higher alcohol usually involve in ester production. Moreover, It
has been shown that presence of high level of unsaturated fatty acids namely oleic, linoleic and
linolenic acids into wort results in a decrease in ester synthesis(Peddie, 1990).

47
 Part V

Conclusion and recommendations

5.1 Conclusions
In this study finger millet jand was prepared following the traditional method of fermentation using
murcha as starter culture and various parameters were analyzed in the lab. On the basis of results
following conclusions were drawn.

1. Finger millet malt was prepared from local variety. Proximate composition of raw and
malted millet were analyzed. The proximate composition of malted millet is significantly
different from the unmalted millet at 5% level of significance. The malted millet is superior
in term of crude fiber as compared to the unmalted millet. Diameter is also increased in
malted millet.
2. Preparation of malted millet incorporated jand can be carried out successfully. The
statistical analysis showed that formulation with 10% malted millet was significantly
superior in terms of flavor, texture, taste and overall acceptability among formulations.
3. Chemical analysis (pH, acidity, protein, alcohol, tss, reducing sugar, aldehyde and ester)
were carried out in lab in order to further assessments of quality of product.

5.2 Recommendation
On the basis of study following recommendations are presented which may guide or show the
direction to the other researchers planning to work in Jand.

1. Further analysis can be made to determine micronutrients like vitamins and minerals in
jand. Especially B vitamins can be determined in Jand.
2. Microbial analysis could be done in various media.
3. Further research on antioxidant activity can be done.

48
 Summary
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. It is very popular among the rural mass of Nepal. It is regarded as poor man’s wine. It
contain high calories, vitamin content, beneficial lactic acid bacteria and yeast which is considered
more as food than an alcoholic beverage. Further value of jand can be added by partial
incorporation of malted millet. Incorporation of normal millet with malted millet to make jand
provides a good opportunity to improve the nutritional quality of the fiber, TSS and minerals
consumed by jand consumer. A study was carried out to know about the effects of incorporation
of malted millet on jand quality. Finger millet collected from Nawalpur district was used for
malting. Grains were steeped for 18 h at 24℃. After steeping the grains were germinated for 4
days at normal room temperature and 85%RH. Then after that kilning was done in three steps: 1st
step at about 50℃ up to moisture content 23%, 2nd step at about 65℃ up to moisture content 12%,
and 3rd step at about 85℃ up to moisture content 4-6%.The dried malt samples were then taken
for analysis. Analysis of physical, chemical and functional properties of both grain and malt
samples were performed.

The diameter of millet grain increased from 1.68 mm to 1.72 mm while 1000 kernel weight
and bulk density were found to decrease after malting from 2.54 g to 2.39 g and 74.62 kg/HL to
68.06 kg/HL respectively. Six different jand formulations were prepared by traditional process
with the incorporation of murcha. The proximate analysis for moisture (db.), crude protein (db.),
crude fat (db.), crude fiber (db.), total ash (db.), carbohydrate (db.) and reducing sugar (db.) of
normal millet and malted millet was done and the values were found to be

All the prepared products were subjected to sensory evaluation in terms of appearance and clarity,
color, aroma, flavor, body, and overall acceptance as their sensory qualities and all the
experimental jand were evaluated on a nine-point hedonic rating (1=dislike extremely, 9=like
extremely) by different semi-trained panelists. The obtained data was analyzed statistically by
Analysis of Variance (ANOVA) at 5% level of significance. The Statistical analysis showed that
10% malted millet incorporated jand was superior to all jand formulations. Mean sensory score of
formulation of sample C regarding color, aroma, flavor, body and overall acceptance was
significantly better from other formulations. So product C was selected as the best product.

49
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 Appendices
Appendix A

Annex A.1 Equipment’s and glassware used

Equipment and glassware used Manufacturer

Measuring cylinder, Beaker, pipettes Borosilicate glass, India
Muffle furnance Sonar Company Limited
What man filter paper no 4 (125mm) GE Healthcare UK Limited, made in china
pH Meter Sonar Company Limited
Volumetric flask, test tube, conical flask Borosilicate, India
Weighing balance Ohauscorp, USA
Bunsen filter Ohauscorp, USA
Grinder Asmeet IS: 4520, India
Soxhlet Apparatus Dolphin Pharmacy Instruments Private Ltd
Hot air oven H.L Scientific Industries, India
Kjeldhal distillation and digestion set H.L Scientific Industries, India
Refrigerator LG Company, India
Air Conditioner
Cabinet Dryer Bionics Instruments India
Refractometer Jiangsu Victor Instrument Co., Ltd.

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Annex A.2 List of chemicals used

Chemicals Manafacturer
Boric Acid Qualike 741m Fine Chemical Pvt. Ltd
Sulphuric Acid Merck Specialties Pvt. Ltd, India
Hydrochloric acid Thermo Fisher Scientific India Pvt. Ltd
Potassium metabisulphite Qualikem Fine Chemical Pvt. Ltd
Potassium Chloride Qualikem Fine Chemical Pvt. Ltd
Sodium Hydroxide Merck Specialities Pvt. Ltd, India
Methylene Red Solution S d fine chem. Limited
Phenolphthalein Qualikem Fine Chemical Pvt. Ltd
Acetic acid S d fine chem. Limited
Selenium Dioxide Hi Media laboratories Pvt. Ltd
Nutrient Agar Hi Media laboratories Pvt. Ltd
Sodium Benzoate Thermo Fisher Scientific India Pvt. Ltd
Dextrose
Carrez solution ( I & II)
Fehling’s Solution (A & B)

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 Appendix B

Hedonic Rating

Specimen card for sensory evaluation

Name of panelist: Date:

Product: normal and malt incorporate jand

Dear panelist, you are provided with 6 samples of Jand. Please taste samples and score the
products according to the characteristics given below in the table. Panelists are requested to give
ranks on their Individual’s choice. An honest expression of your personal’s feelings will help me.

Sample Color Flavor Aroma Mouth feel Overall

Code acceptance
A
B
C
D
E
F

Comments if any:

Signature:

Description of scale
9=like extremely 4=Dislike slightly
8=like very much 3= dislike moderately
7=like moderately 2=Dislike very much
6=like slightly 1= dislike extremely
5=neither like nor dislike

63
 Appendix C

Annex C.1 One way ANOVA for moisture content

Sample Sum of Squares DF Mean Square F Sig.

Between Groups 39.835 1 39.835 4200.555 .000

Within Groups .038 4 .009

Total 39.873 5

Annex C.2 One way ANOVA for Crude Protein

Sample Sum of Squares DF Mean Square F Sig.

Between Groups .866 1 .866 28.191 .006

Within Groups .123 4 .031

Total .989 5

Annex C.3 One way ANOVA for Crude Fat

Sample Sum of Squares DF Mean Square F Sig.

Between Groups 1.144 1 1.144 88.917 .001

Within Groups .051 4 .013

Total 1.196 5

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Annex C.4 One way ANOVA for Crude Fiber

Sample Sum of Squares DF Mean Square F Sig.

Between Groups 1.510 1 1.510 229.370 .000

Within Groups .026 4 .007

Total 1.536 5

Annex C.5 One way ANOVA for Ash content

Sample Sum of Squares DF Mean Square F Sig.

Between Groups .099 1 .099 6.776 .060

Within Groups .058 4 .015

Total .157 5

65
 Appendix D
Sensory of Jand
Annex D.1 One way ANOVA for Color and appearance of sample
sample Sum of Squares DF Mean Square F Sig.

Between Groups 10.133 5 2.027 7.496 .000

Within Groups 14.600 54 .270

Total 24.733 59

Annex D.2 One way ANOVA for Flavor of sample

sample Sum of Squares DF Mean Square F Sig.

Between Groups 38.683 5 7.737 21.425 .000

Within Groups 19.500 54 .361

Total 58.183 59

Annex D.3 One way ANOVA for Aroma of sample

sample Sum of Squares DF Mean Square F Sig.

Between Groups 22.933 5 4.587 12.767 .000

Within Groups 19.400 54 .359

Total 42.333 59

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Annex D.4 One way ANOVA for Mouthfeel of sample
sample Sum of Squares DF Mean Square F Sig.

Between Groups 29.750 5 5.950 16.822 .000

Within Groups 19.100 54 .354

Total 48.850 59

Annex D.4 One way ANOVA for Overall acceptance of sample

sample Sum of Squares DF Mean Square F Sig.

Between Groups 25.750 5 5.150 18.417 .000

Within Groups 15.100 54 .280

Total 40.850 59

67
 Appendix E
Chemical analysis of Jand

Annex E.1 Independent Samples T-test for pH of jand

Levene's Test for
Equality of
Variances T-test for Equality of Means
95% Confidence
Interval of the
Difference
Sig. (2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 5.068 0.088 -3.021 4 0.039 -0.16667 0.05518 -0.31986 -0.01347
variances
assumed
Equal -3.021 2.432 0.074 -0.16667 0.05518 -0.36790 0.03457
variances
not
assumed

Annex E.2 Independent Samples T-test for protein of jand

Levene's Test for
Equality of
Variances T-test for Equality of Means
95% Confidence
Interval of the
Difference
Sig. (2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 3.539 0.133 1.431 4 0.226 0.09000 0.06289 -0.08462 0.26462
variances
assumed
Equal 1.431 2.225 0.277 0.09000 0.06289 -0.15602 0.33602
variances
not
assumed

68
 Annex E.3 Independent Samples T-test for alcohol of jand
Levene's Test for
Equality of
Variances T-test for Equality of Means
95% Confidence
Interval of the
Difference
Sig. (2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 1.566 0.279 -1.866 4 0.135 -0.14667 0.07860 -0.36489 0.07156
variances
assumed
Equal -1.866 3.107 0.156 -0.14667 0.07860 -0.39198 0.09865
variances
not
assumed

Annex E.4 Independent Samples T-test for Tss of jand

Levene's Test
for Equality of
Variances T-test for Equality of Means
95% Confidence
Interval of the
Sig.
Difference
(2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 2.000 0.230 -19.053 4 0.000 -2.20000 0.11547 -2.52060 -1.87940
variances
assumed

Equal -19.053 3.200 0.000 -2.20000 0.11547 -2.55482 -1.84518

variances
not
assumed

69
 Annex E.5 Independent Samples T-test for acidity of jand
Levene's Test for
Equality of
Variances t-test for Equality of Means
95% Confidence
Interval of the
Difference
Sig. (2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 1.058 0.362 3.542 4 0.024 0.13667 0.03859 0.02953 0.24380
variances
assumed
Equal 3.542 3.332 0.032 0.13667 0.03859 0.02051 0.25283
variances
not
assumed

Annex E.6 Independent samples T-test for reducing sugar of jand

Levene's Test for
Equality of
Variances T-test for Equality of Means
95% Confidence
Interval of the
Difference
Sig. (2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 0.012 0.917 -1.942 4 0.124 -0.09667 0.04978 -0.23487 0.04154
variances
assumed
Equal -1.942 4.000 0.124 -0.09667 0.04978 -0.23487 0.04154
variances
not
assumed

70
 Annex E.7 Independent Samples T-test for aldehyde of jand

Levene's Test for

Equality of
Variances t-test for Equality of Means
95% Confidence
Interval of the
Difference
Sig. (2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 1.192 0.336 -0.918 4 0.411 -0.05333 0.05812 -0.21470 0.10803
variances
assumed
Equal -0.918 3.476 0.418 -0.05333 0.05812 -0.22475 0.11809
variances
not
assumed

Annex E.8 Independent Samples T-test for ester of jand

Levene's Test for

Equality of
Variances T-test for Equality of Means
95% Confidence
Interval of the
Difference
Sig. (2- Mean Std. Error
F Sig. t df tailed) Difference Difference Lower Upper
Equal 1.915 0.239 -1.644 4 0.175 -0.07333 0.04460 -0.19715 0.05049
variances
assumed
Equal -1.644 2.717 0.208 -0.07333 0.04460 -0.22400 0.07733
variances
not
assumed

71
 Photo plate

Malted paddy Murcha mixed millet

Removing ascospire control and best sample

72
73