Bikram_Subedi_2010-2014 (2)

PREPARATION OF MIXED FRUIT WINE FROM BAEL,
PINEAPPLE AND GRAPES AND ITS QUALITY EVALUATION
by
Bikram Subedi
Department of Food Technology
Central Campus of Technology
Institute of Science and Technology
Tribhuvan University, Nepal
2015

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Preparation of Mixed Fruit Wine from Bael, Pineapple, Grapes and its
Quality Evaluation
A dissertation submitted to the Department of Food Technology, Central Campus of
Technology, Tribhuvan University, in partial fulfilment of the requirements for the
degree of B. Tech in Food Technology
by
Bikram Subedi
Central Campus of Technology, Dharan
Tribhuvan University, Nepal
July, 2015

iii
Tribhuvan University
Central Campus of Technology, Dharan
Approval Letter
This dissertation entitled Preparation of Mixed Fruit Wine from Bael, Pineapple and
Grapes and its Quality Evaluation presented by Bikram Subedi has been accepted as
the partial fulfilment of the requirement for the B. Tech. degree in Food Technology.
Dissertation Committee
1. Head of the Department
(Mr. Basanta Kumar Rai, Assoc. Prof.)
2. External Examiner
(Mr. Pramod Koirala, Senior Food Research Officer,
RFTQC, Biratnagar)
3. Supervisor
(Mr. Navin Gautam, Teaching Assist.)
4. Internal Examiner
(Dr. Surendra B. Katawal, Prof.)
Date: July 6, 2015

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Acknowledgements
I would like to express my deepest gratitude to my advisor, Teaching Asst. Navin Gautam,
who gave me this rare opportunity to work on wine research project. I am grateful for his
sincere commitment in offering invaluable support, advice and motivation during the course
of the study. Deepest gratitude is also due to the Mr. Nirat Katwal, Mr. Roshan Dangol, Mr.
Prakshit Raj Shakya, Mr. Bibek Khatiwada, Mr. Bijay Prakash Timalasina, Mr. Bikram
Poudel, Mr. Arjun K.C., Ms. Priyanka Chudal and Ms. Swaeksha Basnet, without whose
helpful technical assistance, this study would not have been complete.
Special thanks go to the Campus chief of the Central Campus of Technology, Prof. Dr.
Dhan Bahadur Karki, faculty members for always being available for consultation, Chairman
of Department of Food Technology, Assoc. Prof. Basanta Kumar Rai, Teaching Asst. Dev
Raj Acharya, Teaching Asst. Arjun Ghimire and Teaching Asst. Kabindra Bhattarai. I would
also like to convey thanks to the staffs of laboratory Mr. Laxmi Shrestha, Mr. Mahesh
Koirala, Mr. Som Dangol, Mr. Prajwal Bhandari and Ms. Kamana Bantawa Rai.
Finally, I wish to express my love and gratitude to my beloved family, my dad, Lekhnath
Subedi, my mom, Sakuntala Subedi and my elder borther, Arjun Subedi, for their simple acts
of charity as they constantly prayed for the successful completion of this dissertation.
Date: July 6, 2015 Mr. Bikram Subedi

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Abstract
Mixed fruit wines were prepared from three musts’ M1, M2 and M3 containing bael (Aegle
marmelos), pineapple (Ananas comosus) and grapes (Vitis venifera) in ratios 17:2:1, 16:3:1,
15:4:1 maintaining constant TSS 24 ºbrix and 3.9, 4.1, 4.3 pH value, 0.40, 0.41, 0.43 acidity
as citric acid respectively. Fermentation was carried out at ambient temperature around 27
°C using baker yeast. Sensory attributes (such as smell, taste, mouth feel, flavour, color and
overall acceptance) of the wines were evaluated using 9 points Hedonic scale rating test
ranging from dislike extremely (1) to like extremely (9) to identify the best product. The
assessed data were analyzed for one-way and two-way ANOVA using Genstat (Genstat
Discovery Edition 12, 2014) at 5% significance level.
The fermentation kinetics were significantly different (p<0.05) to each other but the
patterns of change on pH, TSS and acidity in all fermentations were similar (p<0.05). The
average pH, TSS, alcohol content, total sugar and acidity of the most acceptable product
were found to be 3.58, 8ºBx, 13.33 % m/v, 0.43 % dextrose anhydrous and 0.43 % citric
acid, respectively. The tannin, polyphenol, ester, vitamin-C and methanol were 594 mg
tannic acid/L, 1062 mg gallic aicd/L, 35.5 g/100 ml alc., 591 % mg and 9.39, mg/100L
respectively. Sensory analysis showed that there was significant difference (p<0.05) among
all the products with respect to flavor, mouth feel, taste and overall acceptance but there is
no significant difference (p>0.05) in color and smell. The cost of the best product was
calculated and found to be NRs 116.4 for 650 ml.

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Contents
Approval Letter ..................................................................................................................iii
Acknowledgements .............................................................................................................iv
Abstract ................................................................................................................................ v
List of tables ........................................................................................................................xi
List of figures .....................................................................................................................xii
List of abbreviation ..........................................................................................................xiii
1 Introduction ...............................................................................................................1-5
1.1 General introduction............................................................................................. 1
1.2 Statements of the problem .................................................................................... 2
1.3 Objectives ............................................................................................................. 4
1.3.1 General objective .................................................................................... 4
1.3.2 Specific objectives .................................................................................. 4
1.4 Significance of the study ...................................................................................... 4
1.5 Limitations of the study........................................................................................ 5
2 Literature review .....................................................................................................6-32
2.1 Historical background of alcoholic beverage ....................................................... 6
2.1.1 History of wine making...........................................................................7
2.1.2 Major wine producing region of the world ............................................. 8
2.1.3 Winery.....................................................................................................8
2.1.4 Scope of beverage industries of Nepal.................................................... 8
2.2 Raw materials used............................................................................................... 9
2.2.1 Bael .........................................................................................................9
2.2.2 Pineapple.................................................................................................9

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2.2.3 Grapes ...................................................................................................10
2.3 Classification of wine ......................................................................................... 11
2.4 Health benefits of red wine................................................................................. 12
2.5 General cultural condition for fermentation ....................................................... 13
2.5.1 pH..........................................................................................................13
2.5.2 Temperature .......................................................................................... 13
2.5.3 Sugar concentration............................................................................... 14
2.5.4 Wine yeast............................................................................................. 14
2.6 Alcohol ............................................................................................................... 15
2.6.1 Alcoholic fermentation .........................................................................15
2.7 General method of wine preparation ..................................................................17
2.7.1 Selection of raw material ......................................................................17
2.7.2 Blending/Crushing ................................................................................ 18
2.7.3 Sulphiting/Preservatives........................................................................19
2.7.4 Yeast......................................................................................................20
2.7.5 Fermentation ......................................................................................... 21
2.7.6 Racking .................................................................................................23
2.7.7 Fining and filtration .............................................................................. 24
2.7.8 Stabilization of wine ............................................................................. 24
2.7.9 Maturing and aging of wine..................................................................24
2.7.10 Bottling................................................................................................ 25
2.7.11 Pasteurization...................................................................................... 25
2.7.12 Finishing.............................................................................................. 25
2.7.13 Storage of wine ................................................................................... 26
2.7.14 Yield....................................................................................................26
2.8 Wine analysis......................................................................................................26

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2.8.1 Physical and chemical analysis............................................................. 26
2.8.2 Sensory evaluation ................................................................................ 28
2.9 Colour of wine....................................................................................................29
2.10 Nutritional aspects of wine............................................................................... 30
2.11 Wine from grapes with other ingredients ......................................................... 31
2.12 Wine defects and spoilage ................................................................................ 31
2.12.1 Wine defects caused by yeasts.......................................................... 31
2.12.2 Wine defects caused by bacteria ....................................................... 32
2.12.3 Prevention of wine spoilage.............................................................. 32
3 Material and methods ..............................................................................................33-39
3.1 Raw materials .....................................................................................................33
3.1.1 Bael .......................................................................................................33
3.1.2 Pineapple............................................................................................... 33
3.1.3 Grape.....................................................................................................33
3.1.4 Table sugar............................................................................................ 33
3.1.5 Wine yeast............................................................................................. 33
3.2 Methods .............................................................................................................. 33
3.3 Experimental procedure...................................................................................... 33
3.3.1 Preparation of bael juice .......................................................................33
3.3.2 Preparation of pineapple .......................................................................34
3.3.3 Preparation of grapes ............................................................................ 34
3.3.4 Preparation of must ............................................................................... 34
3.3.5 Pitching .................................................................................................34
3.3.6 Fermentation ......................................................................................... 35
3.3.7 Racking, pasteurization and bottling..................................................... 36
3.4 Analytical procedure........................................................................................... 36

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3.4.1 Determination of total soluble solids (TSS)..........................................36
3.4.2 pH determination................................................................................... 37
3.4.3 Total acidity determination ...................................................................37
3.5 Analysis of prepared wine .................................................................................. 37
3.5.1 Sensory evaluation ................................................................................ 37
3.5.2 Quality analysis..................................................................................... 37
3.6 Statistical analysis............................................................................................... 39
4 Results and discussion...........................................................................................40-51
4.1 Chemical composition of must........................................................................... 40
4.1.1 TSS........................................................................................................41
4.1.2 pH..........................................................................................................41
4.1.3 Acidity...................................................................................................41
4.2 Fermentation kinetics ......................................................................................... 41
4.2.1 TSS........................................................................................................41
4.2.2 pH..........................................................................................................42
4.2.3 Acidity...................................................................................................44
4.3 Sensory evaluation.............................................................................................. 45
4.3.1 Colour....................................................................................................46
4.3.2 Smell .....................................................................................................46
4.3.3 Taste......................................................................................................46
4.3.4 Flavour ..................................................................................................47
4.3.5 Mouth-feel............................................................................................. 47
4.3.6 Overall acceptance ................................................................................ 47
4.3.7 Conclusion of sensory results ............................................................... 47
4.4 Chemical properties of the products...................................................................47
4.4.1 pH..........................................................................................................48

x
4.4.2 TSS........................................................................................................49
4.4.3 Acidity...................................................................................................49
4.4.4 Total sugar............................................................................................. 49
4.4.5 Tannin ...................................................................................................49
4.4.6 Total phenolics...................................................................................... 50
4.4.7 Alcohol content..................................................................................... 50
4.4.8 Ester ......................................................................................................50
4.4.9 Vitamin-C.............................................................................................. 50
4.4.10 Methanol ............................................................................................. 51
4.5 Best product identification.................................................................................. 51
4.6 Cost evaluation ...................................................................................................51
5 Conclusions and recommendation ............................................................................ 52
5.1 Conclusions ........................................................................................................52
5.2 Recommendations .............................................................................................. 52
6 Summary ..................................................................................................................... 53
References...............................................................................................................54-60
Appendixes .............................................................................................................61-74

xi
List of Tables
Table No Title Page No.
2.1 Composition of various wines ……………………………………….. 12
2.2 Elemental requirement and source for yeast nutrition ……….………. 21
2.3 Components of wine ……………….………………………………… 27
4.1 Chemical composition of musts ……….…………………………….. 40
4.2 Chemical composition of A, B and C………………………………… 48

xii
List of Figures
Figure No. Title Page No.
2.1 Simplified pathway of alcohol synthesis by yeast ……...………… 16
2.2 Flow chart of red table wine preparation ….……………………… 19
3.1 Flow-sheet for mixed fruit wine preparation ………..…………..… 35
4.1 Patterns of TSS change during fermentation ….……………........... 42
4.2 Patterns of pH change during fermentation ….……………............. 43
4.3 Patterns of acidity change during fermentation...………………….. 44
4.4 Mean scores of the sensory analysis of produced wines ..………… 46

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List of Abbreviation
Abbreviation Full Form
ANOVA Analysis of Variance
TSS Total Soluble Solid
ITDG Intermediate Technology Development Group
FAO Food and Agricultural Organization of United Nations
LAB Lactic Acid Bacteria
HDL High Density Lippoprotein
OA Overall Acceptance
°brix Degree Brix
o
C Degree Celcius
Abv Alcohol percentage by volume

Part I
Introduction
1.1 General introduction
From an enological point of view, the term “wine” is defined as the drink resulting from the
fermentation by the yeast cells and also in certain cases by the cells of lactic bacteria, of the
juice from the crushing or maceration of grape cells (Peynaud, 1984). However, fermented
products of others berries, fruits and honey are also called wines. These are designated by
the substrate from which they were made (Banwart, 1987). Wine is one of the God’s choicest
gift to man and history is almost a romance. The oldest testament is the Bible, gives evidence
of wine existing but there is a definite evidence of its use in China and Egypt in 2000 and
3000 B.C. respectively (Andrew, 1980).
Different types of raw materials have been used for the preparation of wine, either for
flavour or for enrichment of wine with chief chemical constituents (Gubhaju, 2006).
Different herb incorporated wines are also on practices throughout the world. For e.g.,
Ginger wine, is an alcoholic beverage made from a fermented blend of ground ginger
(Zingiber officinale) and raisins fermenting by the yeast, Saccharomyces cerevisiae. It is a
popular beverage in Europe (Rai, 2012) Cereals like rice can also be used to make some
forms of wine. For examples, the Japanese sake (Steinkraus, 1987).
Bael (Aegle marmelos) is a tropical seasonal fruit harvested during the months of May–
June in Nepal, India and other Asian countries. The bael plant is a slow growing, medium
sized tree up to 12 to 15m tall with short trunk and spreading sometimes spiny branches
(Srivastava and Singh, 2004). The bael pulp is highly mucilaginous with 10 to 15 seeds and
is rich in β-carotene. The bael fruit pulp contains many functional and bioactive compounds
such as carotenoids, phenolics, alkaloids, coumarins, flavonoids, terpenoids and other
antioxidants which may protect us against chronic diseases. The total dietary fiber found in
this fruit can be divided into insoluble dietary and soluble dietary fiber (mucilage and pectin).
In addition, it also contains many vitamins and minerals including vitamin-C, vitamin-A,
thiamine, riboflavin, niacin, calcium and phosphorus (Dikshit and Dutt, 1930; Parmar and
Kaushal, 1982; Roy and Khurdiya, 1995).

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Hence bael fruit may indicate that it is one of the important plants used for indigenous
traditional medicine. There are innumerable references of its uses in traditional medicine
(Arseculeratne et al., 1981; Karunanayake et al., 1984; Nagaraju and Rao, 1990; Singh,
1986). Bael contains an excellent flavor, nutritive and therapeutic values. There is a great
commercial potentiality for processing it for herbal medicines and food products. A large
number of food products have been developed from bael pulp such as slab, toffee etc. (Roy
and Singh, 1979). Recently a ready-to-serve drink was developed from bael pulp (Patanjali
Ayurved Ltd., Haridwar, India) and has been marketed as a component in mixed fruit juice
by Tropicana and other companies. However, there is no report yet on processing of bael
fruit pulp into fermented beverages such as wine (Panda et al., 2014). Pineapple (Ananas
comosus) belonging to the Bromeliaceae family. Pineapples are sliced and eaten fresh in the
homes or processed into fruit juices for consumption or concentrates for future use.
Pineapple as a fruit crop has a lot of economic, nutritional, medicinal and industrial
importance (Sarah et al., 1997). Pineapple (Ananas Comosus), a leading member of the
family Bromeliaceae comprises about 2,000 species mostly epiphytic and many strikingly
ornamental and varies from nearly white to yellow in color (Morton, 1987). It is an
herbaceous perennial plant which grows to 1.0 to 1.5 m tall with 30 or more trough-shaped
and pointed leaves, 30 cm long, surrounding a thick stem. It is a multiple fruit, forming what
appears to be a single fleshy fruit. Pineapples contain good sugar proportion which makes it
suitable for wine making (Thapkaew and Chomsri, 2013).
1.2 Statements of the problem
Over the last three decades, the production and consumption of United States (US) wines
have demonstrated a consistent growth. According to Estreicher (2004), the total economic
impact of the wine industry reached estimated 4.7 billion dollars with Washington State
contributing more than 3 billion dollars. Due to the fast changing global wine trends and
highly competitive market, there is a pressing need to invest even more on focused research
to meet this challenge (Villamor, 2012). For the competition and survival of wine industry,
mixed fruit wine have been introduced in the world to give the diverse taste and
characteristics in single bottle of wine (Estreicher, 2004).
Bael (Aegle marmelos) occurs in abundance in wild, scattered all along the tropical belt
and mid-hills of Nepal. There are ample scope for its exploitation. Few years ago, Green

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Energy Mission (Nepal) and Intermediate Technology development Group (ITDG) jointly
took the initiative in promoting large-scale use of bael, ranging anything from herbal
concoctions to squashes, jam, jellies and marmalades. At present, they are actively involved
in promoting the value-addition and marketing activities in five districts, viz., Dhankuta,
Bardiya, Nawalparasi, Bara and Mahotari. A cottage level squash/jam industry was
established at Dhankuta, for instance, is an example of the success-story of the initiative
taken by ITDG (Nepal) and Green Energy Mission Nepal (Shakya, 2002). The bioactive
components such as tannin, polyphenols and esters are present in high amount in bael. It
contains an excellent flavor, nutritive and therapeutic values. There is a great commercial
potentiality for processing it for herbal medicines and food products due to its high
therapeutic value (Panda et al., 2014).
Pineapple (Ananas comosus) are sliced and eaten fresh in the homes, or processed into
fruit juices for consumption or concentrates for future use. Pineapple as a fruit crop has a lot
of economic, nutritional, medicinal and industrial importance (Sarah et al., 1997). Pineapples
contain good sugar proportion which makes it suitable for wine making (Thapkaew and
Chomsri, 2013).
Grapes are the most important raw material for making wine. A good understanding of
grape composition is essential to understanding the process of wine making and making
better quality wine. The important group of compounds from the winemaking point of view,
include sugars, organic acids, phenolic compounds, nitrogenous compounds, aroma
compounds, minerals and pectic substances (Adhikari, 2012).
In Nepal, the history of commercial wine making is not very long (Bhandari, 1992).
Although the practice of making some forms of traditional wines can be traced to times
immemorial (Rai, 2012). There is drastic change in wine drinking culture in Nepal within
few years. It was estimated that over 450,000 liters of wine is consumed in Nepal in 2009.
There are several popular local wine brands in the market like Hinwa, Grapple, Big Master,
Nettlange, Himali Dadhagahre etc. Most of the wine products of these brands are not made
from grape only. Wines are mostly produce from grapes but the domestic production of
which is not sufficient for satisfying the needs of growing winery sector. Hence the need for
alternative material such as bael which is abundantly available as wild-crops to be used along
with grapes for wine production (Bhandari, 1992).

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This study provides lots of opportunity of bael, pineapple and grapes for wine production
in Nepal. Also it provides an idea for the production of mixed fruit wine as mixed product
are highly demanded by consumer to get more taste from single product. These fruits have
lots of useful component for wine production and contains aromatic components in high
amount along with high therapeutic value. As grapes is the ideal berry for wine production,
the blend of this two fruit along with grapes gives typically new wine with high tannin,
polyphenol and esters. This study may provide enough frame work for new product
development.
1.3 Objectives
1.3.1 General objective
The general objective of this dissertation was to prepare the mixed fruit wine from bael,
pineapple and grapes and study the fermentation kinetics along with its quality evaluation.
1.3.2 Specific objectives
The specific objectives are as follows:
1. To prepare mixed fruit wine from bael, pineapple and grapes using different
formulation.
2. To carry out the sensory and chemical analysis of prepared wines and select the best
among them.
3. To study the potential utilization of bael in commercial production of wine.
1.4 Significance of the study
This work will be aimed mainly on fermentation of mixed juice made from bael, pineapple
and grapes in different proportion. This study will also focus on the preparation of the best
mixed flavored wine and also be an initial step towards promoting the utility of bael and
pineapple converting them to wine. The pure bael wine was prepared by Shakya (2002) but
I have focused on the preparation of mixed fruit wine to get more flavor from single bottle.
Furthermore, the quality parameter of all produced mixed fruit wine will also be analyzed.
It is also hoped that data generated from this study will be useful and beneficial to scientists

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and scholars carrying researches and also to the emerging commercial wineries of Nepal and
to domestic level of wine productions sectors.
This study emphasized on the production of mixed flavor wine containing high amount
of health beneficial components like vitamin-C, tannin and phenols. This study can be a
pioneer step in the winery industries of Nepal for making a complete new blended wine from
bael, pineapple and grapes with superior quality in terms of flavor, aroma and taste and also
in medicinal sector by imparting essential components (tannins, polyphenols, anti-oxidants,
etc) of bael to wine.
Hence, this study focused mainly on the bael, pineapple and grapes optimization in wine
making and study of fermentation kinetics. It helped to promoting the utility of bael and
pineapple in wine in Nepal. This study also found the therapeutic component present in
prepared wine.
1.5 Limitations of the study
1. The fermentation could not be done in properly controlled environment because of
the unavailability of temperature control instrument in laboratory.
2. Pectinase enzyme was not used during fermentation.
3. Limited analysis of wine was carried out due to technological and time constraints.

Part II
Literature review
2.1 Historical background of alcoholic beverage
Wine has always been indicated as safe and harmless beverage. Red wine polyphenolic
compounds have been found to act as powerful vasodilators and help preserve the integrity
of endothelial tissue (Stoclet et al., 2004). Polyphones, such as resveratrol and quercetin,
have been shown to increase serum antioxidant capacity, which may protect against damage
caused by free radical production in the body and prevent the increase of inflammatory
agents and the oxidation of low density lipoprotein (Berliner and Heinecke, 1996; Kaindl et
al., 2007). It also provides calories and vitamins. At a time when foods are not the best, wine
was the most important food adjunct. During periods when life was often strenuous, it
offered relaxation and very real surcease from pain (Amerine et al., 1980).Wine is one of the
god’s choicest gift to man and its history is almost a romance (Andrew, 1980). The French
have a saying that good wine results from the happy marriage of art and science. Wine
making is by nature, a credit of skill and experience but the changes taking place in the vat
and cask are chemical reaction and the interpretation of these is a science. An important
aspect of wine is its intimate association with man’s artistic, cultural and religious activities
throughout history (Andrew, 1980). Alcoholic beverages are believed to have originated in
Egypt and Mesopotamia some 6000 years ago (Jones, 1995).
Despite this early application of microbiology, the ability of microorganisms to stimulate
the biochemical changes was demonstrated several years later. Alcoholic fermentation was
first identified by Gay Lussac in 1810 but at that time yeast was not recognized as causative
organism. Schwan in 1835 demonstrated that yeast could produce alcohol and carbon
dioxide when introduced in sugar containing solution. He termed yeast Zuckerpilz meaning
sugar-fungus, from which the name Saccharomyces originated. Saccharomyces group
possesses almost all the credit of producing alcoholic beverages (Prescott and Dunn, 1987).
The production and consumption of alcoholic beverage is one of the man’s oldest activities.
Today brewing, wine making and distilling are of major commercial importance in 21th
centuries of many non-Islamic countries and through taxation, can be an important source of
government revenue (Prescott and Dunn, 1987).

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In Nepal, the history of alcoholic beverage dates back to ancient times. At that time, these
technologies were developed by ethnic groups while celebrating various festivals and
settlement of marriage. The knowledge of home brewing has been passed on to generations
but they are quite ignorant about the broad dimensions of microbial biochemistry or their
complex mechanisms. In fact the exact nature of fermentation is still not fully known to them
(Gubhaju, 2006).
2.1.1 History of wine making
Alcoholic beverages are believed to have originated in Egypt and Mesopotamia some 6000
years ago (Jones, 1995). Probably at first time by the accident from honey, later cereals were
used then grapes and then various fruits (Pederson, 1971). Wine is one of the god’s choicest
gift to man and its history is almost a romance (Andrew, 1980). Wine is one of the
foundations of Western Civilization. The story of wine is that of medicine, religion, war,
discovery, science and dream. The importance of wine to the ancients can be seen in many
places. Vine cutting sin silver sleeves were sometimes buried with the dead, probably in the
hope that vineyards could be planted in the afterlife. Wine related bas-reliefs were carved on
Assyrian buildings. Vines were prominent on many ancient coins (Estreicher, 2004).
As stated by Sir John Malcon in his first account of Persian that during the reign of King
Jamshed viniculture flourished and it is he who is credit with the discovery of fermentation
(Andrew, 1980). According to Finish Foundation of Alcohol Studies (1977), alcohol
consumption increased rapidly after the Second World War in most of the European
Countries. Alcoholic beverages are among most popular and most appreciated food products
all over the world. According to Roy and Khurdiya (1995), Alcohol was discovered in 8327
B.C on a warm afternoon by "Grog" who returned to his cave and drank the fermented milk
of a coconut that had been cracked and left out in the sun. Beer and berry wines were made
for the first time in 6400 B.C while grape wines were made in 300-400 BC.
During the year 1991- 1998, Italy was the top wine producing country in the world
whereas France was second in number. The ten wine producing countries are given in
Appendix-V.

8
2.1.2 Major wine producing region of the world
Grape wine found widely distributed throughout the world. The most important species Vitis
vinifera is believed to have been brought by man from Southern Russia to Asia Minor.
Europe is obviously the most important wine-producing area with more than 75% on average
and over 68% of wine production comes from European countries, with France and Italy
capturing nearly 45% of total production (Amerine et al., 1980). Major wine producing
region of the world are shown in appendix V.
2.1.3 Winery
Today France and United States have the maximum number of wineries, including
household type. Most of the household wineries are based on traditional technology having
limited capacity (Cistern, 1998). Modern wineries are automatic, computerized and are
capable of producing 3-4 million liters of wine with only handful of people (Birch and
Lindley, 1985). More recently the use of tower fermentation (Berry and Watson, 1987) with
timer and programmer for the production of both wine and cider have been demonstrated
(Shakya, 2002).
2.1.4 Scope of beverage industries of Nepal
Beverage covers a wide range of liquor and drinks. Today’s consumers are more or less
health conscious and equally needs the refreshment in their daily life. Beverage products are
basically made for the refreshments and enjoyments from the daily drinking habit. People
also want their health upgrade or in proper condition while they enjoy or refresh themselves
by drinks. Many of the beverages have fulfilled this demand though some alcoholic drinks
are not good for health. Beverage industries and their products has been an inevitable essence
of modern society. Traditional drinks like tea, coffee and traditional wines are taken the
symbols of human civilization and they are being used in almost every society around the
globe from the very ancient periods. These drinks are the part of our daily drinking habit.
Non- alcoholic soft drinks and alcoholic hard drinks are the modern concept of drinking habit
which is also used all over the world in very wide range. These all type of drinks has been a
civilization symbol today. This signifies the scope of beverages industries and their product
in Nepal (Panday, 2010).

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2.2 Raw materials used
2.2.1 Bael
Bael fruit (Aegle marmelos) is a tropical fruit native to Southeast Asia and belongs to the
Rutaceae family. It is grown throughout Nepal as well as in India, Sri Lanka, Pakistan,
Bangladesh, Burma, Thailand, and most of the Southeast Asian countries (Roy and Singh,
1979; Suvimol and Pranee, 2008). The bael pulp is highly mucilaginous with 10 to 15 seeds
and is rich in β-carotene. The bael fruit pulp contains many functional and bioactive
compounds such as carotenoids, phenolics, alkaloids, coumarins, flavonoids, terpenoids and
other antioxidants which may protect us against chronic diseases (Dikshit and Dutt, 1930;
Parmar and Kaushal, 1982; Roy and Khurdiya, 1995). Bael contains an excellent flavor,
nutritive and therapeutic values. Nowadays, the world market for functional foods and
nutraceuticals is large and growing (Roy and Singh, 1979).
According to Suvimol and Pranee (2008), the bael pulp contains 39.50 °Brix TSS, about
twice as high as in most other fruits. pH, reducing sugar, total acidity and moisture content
of 5.37, 39.60 mg glucose/g fresh weight (fw), 0.94%, and 67.74%, respectively. The
antioxidant activity of pulp is 6.21 μg (dw/μg) DPPH. The major antioxidants in bael fruit
are phenolics, flavonoids, carotenoids and vitamin-C (Morton, 1987; Roy and Singh, 1979).
Bael fruit pulps had total phenolic content of 87.34 mg GAE/ g dw. Total phenolics in bael
fruits were in the range of traditional Chinese medicinal plants associated with anticancer
(2.2-503 mg GAE/g dw), as well as higher than for common fruits and vegetables including
kiwifruit, orange, pear, garlic, carrot and spinach (1.2-10.8 mg GAE/ g dw) reported by Cai
et al. (2004). It contents several flavoring compounds among them monoterpenes and
sesquiterpenes seem to be the main volatile constituents’ of bael fruit. Limonine was one of
the major constituents that produces the characteristic bael fruit flavor (Suvimol and Pranee,
2008).
2.2.2 Pineapple
Pineapple (Ananas comosus) belonging to the Bromeliaceae family is grown in different
parts of Nepal either for export or for the local market. Pineapples are sliced and eaten fresh
in the homes or processed into fruit juices for consumption or concentrates for future use.
Pineapple as a fruit crop has a lot of economic, nutritional, medicinal and industrial

10
importance (Sarah et al., 1997). Pineapple is the second harvest of importance after bananas,
contributing to over 20 % of the world production of tropical fruits. Nearly 70% of the
pineapple is consumed as fresh fruit in producing countries. Its origin has been traced to
Brazil and Paraguay in the Amazonic basin where the fruit was domesticated. It has been
defined as the most probable area of origin. The zone comprised from upper Panama and
Brazil, Paraguay and Argentina, including the northern Amazonian forest and the semi-arid
regions of Brazil, Venezuela and Guyanas. Worldwide production started by 1500 when
pineapple was propagated in Europe and the tropical regions of the world. The most spread
variety is Cayena lisa (Smooth Cayenne) which was first introduced in Europe from French
Guyana. It was until late 19th
century when canned pineapple was produced commercially in
Hawaii (FAO, 2004).
Pineapple as a fruit crop has a lot of economic, nutritional, medicinal, and industrial
importance (Sarah et al., 1997). Thailand, Philippines, Brazil and China are the main
pineapple producers in the world supplying nearly 50 % of the total output (FAO, 2004).
Other important producers include India, Nigeria, Kenya, Indonesia, México and Costa Rica
and these countries provide most of the remaining fruit available (50%) (Thapkaew and
Chomsri, 2013).
Pineapple as food for human consumption contains about 81.2-86.2% moisture, 13-19%
total solids of which sucrose, glucose and fructose are the main components, 2-3% fibre and
rich source of vitamin-C. Lipids and nitrogenous compounds constitute 0.1% of which 25-
30% of the nitrogenous compounds are true proteins. The fruit is also rich in calcium which
has proteolytic activity due to the enzyme bromelin. This makes pineapple suitable raw
material for wine production (Dull, 1971).
2.2.3 Grapes
Grapes are the most important raw material for making wine. A good understanding of grape
composition is essential to understanding the process of winemaking and making better
quality wine. The fruit of the grape is a berry. Berries are attached to the stem. Many berries
make up the cluster or bunch of grapes. The essential parts of the berry include the skin,
pulp, and seeds. The main components in the skin are coloring matter (red and yellow
pigments), tannins, aromatic substances and potassium and other minerals. Below the skin
layer lies flesh or pulp which makes up most of the berry volume. When the berry is gently

11
crushed, the fragile cells in the pulp are broken and the juice is released. This juice is
commonly referred to as the free run. The seeds are localized in the center of the flesh. The
berry contains two to four seeds. They are rich in tannin which is extracted during
fermentation in red wines. The important group of compounds, from the winemaking point
of view, include sugars, organic acids, phenolic compounds, nitrogenous compounds, aroma
compounds, minerals and pectic substances (Adhikari, 2012).
2.3 Classification of wine
According to Prescott and Dunn (1987), there are several classification scheme of wine
but the main classification of wine categorized into five classes as follows:
 Appetizer Wine: It contains 20 % alcohol by volume. For example Sherry and
Vermouth.
 Red Table Wine: In the production of red wine, the red anthocyanin pigments are
extracted from the skin of red grapes by allowing the fermentation to take place in
contact with the skin. For example, Burgundy, Claret, Barbera, Gamay, Rose etc.
 White Table Wine: White wines are made from white grapes, the skin is separated
before fermentation. White wine is low in pH (below 3.21), high in acidity (0.65 %
minimum), very dry (below 0.2 % sugar). It is very light in color, fresh and fruity with
not over 11 % alcohol.
 Dessert Wine: It is also known as fortified or appetizer wine, to which distilled spirit
is added during or after fermentation. This increases the alcohol content from 15- 20%.
For example Sherry, Malaga, Tokay, etc.
 Sparkling Wine: Sparkling wines are those which are made effervescent by secondary
fermentation in the bottle or in the bulk or by carbonation. For example, Champagne,
Sparkling Burgundy etc.
The composition of various wines is given in Table 2.1

12
Table 2.1 Composition of some wines
Parameters Port Sherry Claret Burgundy Champagne
Specific
gravity
0.995-1.050 0.992-1.015 0.990-1.001 0.995-1.001 1.040-1.055
Alcohol
(gm/100ml)
13.5-20.0 13.5-20.5 7.5-12.5 7.5-12.5 10.0-14.0
% Total solid 3.3-13.0 2.0-9.6 2.0-3.5 2.0-3.5 9.5-18.0
% Free volatile
acid (as acetic
acid)
0.05-0.10 0.15-0.23 0.09-0.15 0.2-0.35 0.03-0.20
% Fixed acid
(as acetic acid)
0.35-0.55 0.25-0.50 0.30-0.50 0.3-0.60 0.30-0.45
% Ash 0.25-0.35 0.35-0.55 0.20-0.30 0.2-0.4 0.25-0.45
% Sugars 2.5-12.0 2.0-7.0 0.0-0.7 0.03-0.55 8.5-16
Source: Pearson (1981)
2.4 Health benefits of red wine
It is well known that France has lower rates of obesity and reduced incidence of mortality
due to cardiovascular disease despite having a diet high in saturated fat. This observation is
known as the “French Paradox” (Renaud and Delorgeril, 1992). A possible explanation for
this phenomenon may be associated with the consumption of red wine by the French people.
Studies show that moderate red wine consumption may have potential health benefits due to
its alcohol content and the substantial amounts of phenolic compounds present in it (Serafini
et al., 1998; Simonetti et al., 2001; Whitehead et al., 1995). According to Friedman and
Kimball (1986), individuals with moderate alcohol consumption, including red wine reduce
the risk of dying from heart disease by 40 %, increase high density lipoprotein (HDL)
cholesterol and promote other cardio-protective effects. Polyphenols are a class of

13
compounds found in plants that exhibit antioxidant characteristics. Red wine polyphenolic
compounds have been found to act as powerful vasodilators and help to preserve the integrity
of endothelial tissue (Cai et al., 2004). Polyphonols such as resveratrol and quercetin, have
been shown to increase serum antioxidant capacity which may protect against damage
caused by free radical production in the body and prevent the increase of inflammatory
agents and the oxidation of low density lipoprotein (Berliner and Heinecke, 1996; Kaindl et
al., 2007).
2.5 General cultural condition for fermentation
Cultural condition refers to the environment of yeast i.e. fermentative media on which the
propagation of yeast as well as final quality of wine is largely depended (Prescott and Dunn,
1987). Following are the few parameters which determine cultural condition of the
fermentative media.
2.5.1 pH
The optimum pH for wine production varies from types of the selected fruit but generally
3.8-4.5 is supposed to be optimum. At higher pH, the concentration of glycerine is increased
during fermentation and at lower pH, there is a noticeable effect of log phase (Prescott and
Dunn, 1987).
2.5.2 Temperature
The optimum temperature for the fermentation is dependent upon the types of wines
produced. For white wine, the temperature is 10-15º C and that for the red wine is 20-30ºC.
There is possibility of ‘stuck’ fermentation if it is carried out at higher temperature. On the
other hand, low temperature may delay onset of fermentation. At high temperature, the loss
of alcohol and aroma substance takes place. Also, a large amount of by-product like glycerol,
acetaldehyde may be formed. An imbalance of these constituents can be very detrimental to
wine quality. It has been reported that at higher temperature the formation of higher alcohol
decreases. The advantage of lower fermentation temperature are the fresher and fruitier
character of wine, smaller losses of ethanol and less danger of producing volatile acidity
(Prescott and Dunn, 1987).

14
2.5.3 Sugar concentration
The ‘must’ having very high sugar concentration imparts high osmotic pressure which in
turn has a negative effect on yeast cells, since both growth of yeast and fermentation activity
are lowered. The optimum sugar concentration in terms of total soluble solid is 20- 24 °Brix.
The tolerance of higher sugar concentration varies according to the yeast species (Prescott
and Dunn, 1987).
2.5.4 Wine yeast
Wine yeast is the members of the Saccharomyces cerevisiae group. The main originates from
the Greek words sakchar means sugar and mykes means fungus, referring to the strong sugar
fermenting properties of the genus in general. Although, Hansen regarded them as a separate
species, they are more ellipsoid in shape than the round or ovate cells of brewery and bakery
yeasts. Hansen restricted the name S. ellipsoideus to them. In the nomenclature of Dutch
school, these yeasts are classified as a variety of S. cerevisiae and consequently named S.
cerevisiae var. S. ellipsoideus. In general articles, however, one will see them briefly
described as ‘ellipsoidal yeasts’ or ‘true wine yeasts’ (Austin, 1968).
Good wine yeast is one which will impart a vinous or fruit like flavor. They ferment sugar
to a low content producing 14-18 % alcohol and is characterized by remaining in suspension
during fermentation and then agglomerating to yield a coarse granular sediment that settles
quickly and is not easily disturbed in racking (Pederson, 1971).
In general, good wine yeast should have the following four properties (Austin, 1968).
 High alcohol tolerance, i.e. the yeast should continue to ferment despite the increasing
concentration of the alcohol, giving stronger, drier wines with up to 16 % alcohol (v/v)
or even up to 18 % (v/v) where the yeast is fed by periodic additions of sugar in small
amounts.
 Good degree of agglutination, i.e. the tendency of the yeast to flocculate into small
lumps that give a cohesive sediment as fermentation ceases, so that racking is simple
and the wine clears easily.
 Steady, persistent fermentation capacity, this leads to wines of better quality than when
the fermentation falls away after a tempestuous start.

15
 Absence of unpleasant flavors generated by dead and dying cells.
2.6 Alcohol
There are many different kinds of alcohol but when the term is used loosely as by
winemakers, it invariably applies to the potable alcohol called ethyl alcohol or ethanol. It
mixes easily with water in any proportion and where quantities are mixed there is a
contraction in volume. It has a low boiling point i.e. 78.4º C, compared with water. It burns
easily in air so that oxidation is possible and then gives a blue, smokeless flame, producing
water and CO2 (Austin, 1968).
There are three main classes of alcoholic beverages i.e. wines, malted beverages and
distilled liquors. The essential step in all the fermentation processes is the conversion of
glucose into alcohol by yeast (Manay and Shadashaswaswany, 1987). The intermediate
products are methyl glyoxal (CH3C:OCH:O), acetaldehyde (CH3CHO) and pyruvic acid
(CH3: OCOOH). Ethyl alcohol is produced by the zymase complex of enzymes in yeast (Lal
et al., 1986).
2.6.1 Alcoholic fermentation
Alcoholic fermentation is simply the production of alcohol by using carbon and nitrogen
substrate. Sugar and nitrogen compounds are the principal substrates for alcohol
fermentation (Prescott and Dunn, 1987).
2.6.1.1 Biochemistry of alcohol fermentation by yeast
The organism uses EMP pathway, generating 2 ATP per mole of glucose converted to
ethanol plus CO2. Ethanol, which is the end product, is primary metabolite. In an industrial
fermentation, the basic strategy is to maintain Crabtree effect during the fermentation (Rai,
2012). A truncated form of the metabolic pathway for ethanol synthesis is given in Fig. 2.1
2C2H5OH + 2CO2
Ethanol Carbon Dioxide
C6H12O6
Hexoses

16
Fig. 2.1 Simplified pathway of alcohol synthesis by yeast
2.6.1.2 Stoichiometry
Ethyl alcohol is the product obtained from alcoholic fermentation of sugar by the action of
enzyme zymase in yeast. In alcoholic fermentation, one molecule of glucose produce two
molecules of ethyl alcohol and carbon dioxide.
However, alcoholic fermentation is fortunately a much more complex process. At the
same time as this overall reaction proceeds, a lot of other biochemical, chemical and
physiochemical processes take place, making it possible to turn the juice into wine. Besides
ethanol, several other compounds are produced throughout alcoholic fermentation such as
higher alcohols, esters, glycerol, succinic acid, diacetyl, acetoin and 2,3-butanediol.
Simultaneously, some compounds of juice are also transformed by yeast metabolism
(Zoecklein et al., 1997).
2.6.1.3 Malo-lactic fermentation
It refers to secondary fermentation in which lactic acid bacteria are allowed to metabolize
malic acid to lactic acid and carbon dioxide. This fermentation is particularly useful if the
titrable acidity of wine is to be reduced. Wines with low levels of acidity should be protected
from malo-lactic fermentation. Wine quality decreases if the acid level falls too low. Malo-
lactic fermentation can be easily prevented by early racking, cool storage and maintaining
100 ppm or more of SO2. On the other hand, if such fermentation is desired, it can be
facilitated by leaving the wine on the lees (yeast sediments) for prolonged periods at higher
temperatures. This storage causes lysis of yeast cells and releases amino acids and other
nutrients needed for the growth of the ‘contaminant’ lactic acid bacteria. Malo-lactic
fermentation has an important bearing in the quality of wine. It is a natural way of reducing
2 Acetaldehyde
Alcohol dehydrogenase
2 Ethanol
4ADP2 ATP 2ADP
2[1, 3-di P glycerate]Glucose
2 Pyruvate
2[NAD+
] 2[NADH+H+
]
2CO2

17
acidity in wine. Besides this, the fermentation also results with greater softness and
mellowness in wines. The bacteria implicated for malo-lactic fermentation are
Leuconostocoenos, Lactobacillus and Pediococcus. The first one being the most important
(Rai, 2012).
2.7 General method of wine preparation
Wild yeast and other microorganisms are present on the skin of the grapes and these pass
into the juicy pulp (known as must) when the fruit is crushed. These are destroyed by adding
sulphurdioxide (or KMS) in the required quantity. If the sugar content is low, sucrose is
added to the desired strength. Next, the must is inoculated with a pure culture of actively
growing yeast (S. ellipsoideus). The temperature and duration of fermentation depend upon
whether dry or sweet wine is required. Fermentation usually lasts 4 to 10 days. When
fermentation is complete, the clear wine is siphoned from the yeast sediment into barrels
(racking) and the wine allowed to age. During this period, secondary fermentation takes
place and wine also losses its raw and harsh flavor and mellows down. During this period of
maturation, clarification takes place in natural way. It can also be achieved by fining and
filtration. Next, the wine is bottled and allowed to mature. The time of this maturation
extends to a number of years depending upon the quality desired (Manay and
Shadashaswaswany, 1987).
2.7.1 Selection of raw material
A suitable raw material is chosen to function as a substrate. Compared to cereals, fruit juices
are more readily utilizable substrate by yeasts for the alcoholic fermentation. The later is also
a suitable media for the yeast to grow (Vernam and Sutherland, 1994). Following criteria
should be fulfilled when selecting for proper raw material for fermentation.
 It should be readily available.
 It should be good source of carbon and nitrogen.
 It should have sufficient amount of fermentable sugar.
 It should not contain any toxic compound nor should impart any undesirable odor or taste.
 It should be clean, sound and mature.

18
2.7.2 Blending/Crushing
This step is carried out to extract the juice from the fruit. It has been suggested that the
process should be very gentle. If the blending and crushing machine is constructed of mild
steel or cast iron then the iron causes “ferric casse-cloudiness” of wine. Actually iron will
react with the tannin of the juice to form ferric-tannin complex. Bronze equipment is also
used but may cause dissolution of copper and tin from bronze equipment and it will affect
the colour. Usually, stainless steel is used for the crushing machine. Water may be added
during blending/crushing for smoothness of operation (Vernam and Sutherland, 1994).
At initial step, purple grapes are collected and destemming is done. After that grapes are
crushed by hand or crushing machine. 75 ppm to 125 ppm SO2 is added in must to prevent
the growth of undesirable microorganisms. Primary fermentation is started at aerobic
condition by adding yeast for about 1-3 days depending upon environmental temperature.
As the primary fermentation completed, it is subjected to secondary fermentation through
drawing off and pressing. Secondary fermentation is main part for alcohol production and is
done until the production of CO2 is ceased. After secondary fermentation, racking, blending,
fining, malo-lacic fermentation and ageing is done. Then it is subjected to filtration. From
this, free run wine is subjected to pasteurization at 72°C for 1 min. Pasteurization is the
process of destruction of microorganisms. After pasteurization, it is cooled and then bottling,
labeling and casing is done. Flow chat of red wine preparation is given in Fig. 2.2.

19
`
Press wine
Pomace
Purple Grapes
Destemming
Crushing
Must
Must Treatment
Primary Fermentation
Drawing off and Pressing
Free- Run Wine
Secondary Fermentation and Filling
Racking, Blending, Fining,
Malo-Latic Fermentation
Aging
Filtration and Tartrate Stabilization
Polishing
Pasteurization
Bottling, Labeling, Casing
RED TABLE WINE
SO2, to make
75 ppm
Yeast
Propagation
SO2
75-125 ppm
Fig. 2.2 Flow chart of red table wine preparation
Source: Rout (2014)
2.7.3 Sulphiting/Preservatives
The antiseptic and antioxidant properties of sulphur dioxide are taken advantage of both in
connection with treatment of musts prior to fermentation and later in the winemaking
process. The dosage of SO2 (sulphur dioxide) usually ranges between 100 and 200 ppm
(Douglas et al., 1997).
SO2 (sulphur dioxide) is added before the fermentation process to prevent oxidation of
juice from air and after to prevent conversion of alcohol into vinegar by acetic fermentation.

20
The air has bacteria principally Acetobacter i.e. it is alive in the presence of air of oxygen.
These Acetobacter cannot convert alcohol into vinegar because SO2 (sulphur dioxide) being
hungry for oxygen, takes of the oxygen from the must to create anaerobic condition for wine
yeast to convert the fruit sugar into alcohol. SO2 (sulphur dioxide) also forms a coating on
the surface of juice to prevent the air entering the juice (Andrew, 1980).
2.7.4 Yeast
Wine yeasts are the member of genus of Saccharomyces and consequently of great individual
importance (Austin, 1968). A good quality of wine yeast should have the following
characters (Vernam and Sutherland, 1994).
 Introduction of flocculation and reduction of H2S production.
 Reduction of higher alcohol production.
 Improvement of fermentation efficiency.
 Reduction of foaming.
 Resistance of ethanol.
 Resistance of killer activity.
2.7.4.1 Yeast nutrition
Proper nutrients are ‘must’ for the growth of yeast in cultural media. The cultural medium
used ‘must’ therefore contain all the essential elements for growth, in proportion similar to
those occurring in yeast biomass (Berry and Watson, 1987). The elemental requirement and
the source for yeast nutrition is given in Table 2.2

21
Table 2.2 Elemental requirement and source for yeast nutrition
Element Major source
Carbon Sugar
Hydrogen Water, organic compound
Oxygen Water, dissolved oxygen, organic compound
Nitrogen Inorganic source: NH4Cl, (NH4)2SO4
Phosphorus KH2PO4, Na2HPO4
Sulphur Na2SO4, Na2S2O3 and organic sulfur compound
Potassium KH2PO4
Magnesium MgCl2
Sodium NaCl
Calcium CaCl2
Iron FeCl3, FeSO4
Source: Madigan et al. (1997)
2.7.4.2 Pitch development
Pitch of sufficient quantity is developed before preparation of must. The developing medium
should have low sugar concentration so that the ‘Pasteur effect’ is maintained. Pitching is
done when the culture of the pitch is at its optimum stage of growth. Vigorous agitation is
done after pitching to help distribute the culture and also to help in their initial growth (Karki,
2000).
2.7.5 Fermentation
Fermentation is the soul (heart) of wine making. All the desirable reactions take place during
this step so most of wine makers pay strict attention to this stage. Fermentation is the process
of adding wine yeast (technically termed as S. ellipsoidues) to fresh juice to convert the
natural sugar to ethyl alcohol. In this process, CO2 is simultaneously released making
fermentation violent at first and then slow. The yeast added is 1-3 % of the volume of the

22
juice. Generally 14 days is required for complete alcoholic fermentation. Most of the
fermentation takes place in three stages.
 An initial stage, during this time the yeast cells are multiplying.
 A very vigorous stage accompanied by bubbling and marked rise in temperature.
 Quiet fermentation that can proceed for quite a long time at a lower temperature and
lower rate.
Fermentation time may range from 2-20 days depending upon numerous variables types
and condition of fruits, type of wine being made, climatic condition and others. Temperature
is quite critical to the fermentation process (Douglas et al, 1997).
The optimum temperature for fermentation of red wine is higher than that of white wine.
The optimum temperature is believed to be 21.1-27.4ºC for red wine (Johnson and Peterson,
1974). At temperature above 90ºF (32.2ºC), it is likely that wine flavor and bouquet will be
injured. High temperature also encourages heat tolerant bacteria to produce acid, mannitol
and off flavor (Douglas et al, 1997).
Johnson and Peterson (1974) reported that at the usual of total sugar content of 19-23%,
alcoholic fermentation proceeds rapidly and with alcohol tolerant strains of yeast, to
completion, producing about 10-12.5% alcohol (by volume). If the sugar content is greater
than 23%, the high sugar content may inhibit fermentation and the rate of fermentation will
be slower and may be incomplete. Under special condition of simulation, 16-18% alcohol
can be reached (Johnson and Peterson, 1974).
It is generally agreed that methanol is not produced by alcoholic fermentation for
example, from glycine but is primarily derived from hydrolysis of naturally occurring pectin.
The amount of higher alcohols produced is less when ammonium phosphate is added prior
to fermentation. At very low concentration the higher alcohols may play a desirable role in
sensory quality (Amerine et al., 1980).
Guymon et al. (1961) showed that oxidative conditions during fermentation favor higher
alcohol production. According to Gentillini and Cappelleri (1959), glycerol production is
favored by low temperature, high tartaric content and by addition of SO2. Most of the
glycerol develops in the early stages of fermentation. Most enologists consider that glycerol

23
is of considerable sensory importance because of its sweet taste and its oiliness (Guymon et
al., 1961).
Acetaldehyde is a normal by-product of alcoholic fermentation. Acetaldehyde retention
is much greater when SO2 is added before the fermentation. The primary source of
acetaldehyde is from enzymatic process i.e., in the presence of yeast. Acetaldehyde reacts
with ethyl alcohol to form acetal, a substance with a strong aldehyde like odor, found very
little in wines (Johnson and Peterson, 1974).
The tartaric, malic and citric acids of the must are found in the resulting wines but in
decreased amounts. They are important constituents of wine not only for their acid taste
because they protect the wine from spoilage, maintain the color but are themselves
sometimes attacked by microorganisms (Douglas et al., 1997).
Malic acid disappears during alcoholic fermentation to the extent of 10 to 30 %.
Succininc acid is a product of alcoholic fermentation. Lactic acid has a slight odor and is a
weak acid. It is a constant by-product of alcoholic fermentation 0.04 to 0.75 g/L. Carbonic
acid constitutes a very special case for both still and sparkling wines. It has no odor and very
little taste but it does have a feel and disengagement of the bubbles from the wine probably
brings more oxygen away from the surface of wine (Amerine et al., 1980).
The end of fermentation is signaled by a clearing of the liquid, by a vinous taste and
aroma, drop in temperature and can be confirmed by checking degrees balling (sugar
residual) (Douglas et al., 1997).
2.7.6 Racking
After completion of fermentation, the wine must be separated from the dead cells, which
decompose and give off-flavours and odours to wine. This dead yeast settle at the bottom of
the fermentation vessel and the wine is carefully siphoned to other vessel without disturbing
the dead yeast, leaving some wine at the bottom called lees. The advantages of racking are
as follows (Andrew, 1980).
 It helps to remove CO2.
 It raises O/R potential, which retards the formation of H2S.

24
 It clarifies the wine
2.7.7 Fining and filtration
This is a process of converting cloudy wine into clear wine. This may be done by adding
gelatinous substances such as icing glass, egg white, bentonite and tannin. Pectin
hydrolyzing enzymes are also used in the clarification of wine (Andrew, 1980). Over fining
can cause a permanently cloudy wine (Douglas et al., 1997).
After clarification, the wine is passed through fine filters for filtration. The pad filters are
most common. In order to increase filter life, diatomaceous earths are added to wine during
filtration. These mix with mucilaginous materials maintain the capacity of the filter for
longer times i.e. increase filter capacity. Recently membrane filters have been widely
employed for wines. These have uniform but small pore size so that a very large percentage
of the filter-surface is available for filtration. They also greatly reduce the number of bacteria
(Johnson and Peterson, 1974).
2.7.8 Stabilization of wine
Because of the unknown nature of the wine, it is generally good practice to stabilize them
against microbiological changes by use of antiseptics such as sorbic acid or its potassium
and sodium salts in amount ranging from 300 to 1000 ppm. An alternative is to pasteurize
the wines after bottling. Another alternative may be to flash pasteurize, fill into clean bottles
and seal using clean closures (Chan, 1983).
2.7.9 Maturing and aging of wine
Aging is one of the most interesting and important yet, one of the most complex process in
winemaking. Newly fermented wine is cloudy, harsh in taste, yeasty in odor and without
pleasing bouquet that develops later in its history. This takes place naturally by retaining the
wine in oak barrel for one or two years to gain maturity and pick up soft and mellow
characters from the oak wood (Amerine et al., 1980).
Andrew (1980) found that maturation can be artificially induced by agitation, heating,
refrigeration and electrical impulses. The bouquet and aroma of wine are developed during
aging (Banwart, 1987). Additional racking may be performed and these will be followed by

25
final filtration and stabilization treatments to produce brilliantly clear wines (Potter and
Hotchkiss, 1995).
Aging of wines improves the flavor and bouquet due to oxidation and formation of esters.
These esters of higher acids formed during aging give the ultimate pleasing bouquet to the
well-aged wine. Aged wine may be polished by filtration to give a clear, bright appearance
prior to bottling (Desrosier and Desrosier, 1978).
2.7.10 Bottling
This is done before the blended wine lose its bouquet, fineness, quality and color. Bottles are
cleaned and dried with hot air. Cool and dry weather is chosen for this purpose. Bottles are
closed with a fine, soft supple cork applying pressure with the finger. Corks are finally sealed
with Spanish wax (Andrew, 1980).
2.7.11 Pasteurization
Pasteurization is the process used to kill microorganisms present in the wine so that
fermentation is stopped. Pasteurization is applied in one of the three ways.
 By flash pasteurizing and returning to the storage tank.
 Flash pasteurizing into the final bottles and
 Pasteurization by heating the filled and sealed bottle.
The time temperature relationship for pasteurization of wine is, vegetative yeast cells are
killed at 40ºC while yeast spores are only killed at 57ºC (Desrosier and Desrosier, 1978).
The quality of some wine is reduced by pasteurization while that of other may be improved.
Pasteurization inactivates the enzymes but injure the quality of the product (Johnson and
Peterson, 1974).
2.7.12 Finishing
The traditional method of finishing the wine was to turn the bottles on end, place them in
racks at about 45º angle and turn them to the left and right daily to get the yeast deposit into
the neck of the bottle and on the cork. The process is called riddling “reumage”. The
temperature of the whole bottle is then reduced to about 30º to 40º F. The neck of the bottle

26
containing the yeast deposit is then frozen (by placing in brine or other freezing solution).
When the cork is removed, the solid plug containing the yeast is ejected. This is called
disgorging (Pederson, 1971).
2.7.13 Storage of wine
A wine cellar should be maintained at a uniform temperature of 60ºF (15.5°C) and a humidity
of 50%. When stored, each bottle of wine must be laid in a horizontal position so that the
wine keeps the cork moistened. The room should be darkened, free from drafts and
mechanical or sound vibrations (Smith, 1996).
2.7.14 Yield
Kirk (1969) has found that roughly each percentage of sugar fermented yield 0.55 % of
alcohol by volume. Under special condition of simulation, 16-18 % alcohol can be reached
but normally in commercial operation, 13-15 % is the maximum (Johnson and Peterson,
1974).
2.8 Wine analysis
Throughout the history of wine making, analytical techniques have become increasingly
important with the development of technology and increased governmental regulation.
Analysis of wine is performed for a number of reasons such as quality control, spoilage
reduction, process improvement, blending, export certification and global regulatory
requirements (Fugelsang, 1996).
2.8.1 Physical and chemical analysis
All wines should be subjected to appropriate analyses during their production and storage to
meet the requirements of regulatory agencies and to give the winemaker information to
monitor the operations properly (Fugelsang, 1996).
Experimental wines often require additional analyses to obtain more complete
information and study the specific effects of the experimental conditions. There is no sense
in doing the experiments unless analytical methods are available to evaluate the results.
Planning for these analyses, the labor and timing for them should precede initiation of the
experiments. Some analyses can be done more or less at leisure on the finished wine, others

27
must be done at specific moments if not then the experiment is spoiled. Sometimes interim
samples can be quickly frozen and held for later analyses as a group. Other cases arise where
this is not possible for experimental or logic reasons (Boulton, 1998).
The components of wine and must can be broken into classes and are given in Table 2.3.
Table 2.3 Components of wine.
Soluble solids
Acidity
Alcohols
Carbonyle compounds
Esters
Nitrogen compounds
Phenolic compounds
Chemical additions
Other
Sugar, extract, glucose and fructose
Total volatile, pH, individual acids
Ethanol, methanol, fusel oils, glycerol
Acetaldehyde, HMF, diacetyl
Ethyl acetate, methyl anthranilate (labruscana)
NH3, amino acids, Amines, proteins
Total, phenolic fractions including anthocyanins
SO2, sorbic and benzoic acids, illegals
Common and trace metals, oxygen, CO2, fluoride
Source: Fugelsang (1996)
According to Amerine et al. (1980), the different parameters viz. alcohol, glycerol, ash,
total acids, volatile acids, reducing sugars, proteins, tannins and specific gravity of different
wines were analyzed. According to Pearson (1981), analytical parameters of different wines
were specific gravity, alcohol (g/100 ml), % total solids, % free volatile acids (as acetic acid),
% fixed acid (as acetic acid), % ash and % sugar.
Different dissertations related to wine held in Central Campus of Technology, Hattisaar,
Dharan, have mostly analyzed the parameters such as pH, TSS, alcohol content, acidity, total
sugar, aldehydes, esters, specific gravity, total sugars, ash, methanol and higher alcohols
(Rout, 2014).

28
2.8.2 Sensory evaluation
2.8.2.1 Development of sensory testing
Sensory tests, of course have been conducted for as long as there have been human beings
evaluating the goodness and badness of food, water, weapons, shelters and everything else
that can be used and consumed (Pearson, 1981).
The rise of trading inspired slightly more from sensory testing. A buyer, hoping that a
part would represent the whole, would test a small sample of a shipload. Sellers began to set
their prices on the basis of an assessment of the quality of goods. With time, ritualistic
schemes of grading wine, tea, coffee, butter, fish and meat developed. Some of which
survive to this day. Grading gave rise to the professional taster and consultant to the budding
industries of foods, beverages and cosmetics in the early 1900s. A literature grew up which
used the term “organoleptic testing” to denote supposedly objective measurement of sensory
attributes. In reality, tests were often subjective tasters and interpretations open to prejudice.
Scientists have developed sensory testing then it is very recently as a formalized, structured
and codified methodology and they continue to develop new methods and refine existing
ones (Pfenninger, 1979).
The methods that have been developed serve economic interests. Sensory testing can
establish the worth of a commodity or even its very acceptability. Sensory testing evaluates
alternative courses in order to select the one that optimizes value for money. The principal
uses of sensory techniques are in quality control, product development and research. They
find application not only in characterization and evaluation of foods and beverages but also
in other fields such as environmental odours, personal hygiene products, diagnosis of
illnesses, testing of pure chemicals etc. The primary function of sensory testing is to conduct
valid and reliable tests, which provide data on which sound decisions can be made
(Meilgaard et al., 1999).
2.8.2.2 Sensory evaluation of wine
Even the most sophisticated chemical analysis cannot now and probably never, will define
the subtle flavors that make one wine greater than another in the opinion of observant
consumers. That is as it should be. As a consequence, it is almost always necessary to

29
compare wines by sensory analysis in addition to chemical and physical methods. This is
true of commercial wines but often especially so with experimental wines (Villamor, 2012).
Inspite of opinion to the contrary by wine writers and some wine makers, one person’s
opinion is hardly definitive on any wine’s sensory character and quality. That is not to say
that one tester may not be better than another in natural ability, concentrated effort, amount
of experience and/or comparative memory. In evaluation of the sensory qualities of one or
more wines, a panel of testers is necessary. This panel should be as sensitive and experienced
as possible but each individual is erratic, biased or unobservant on some occasions, hence
the need for panels and statistical evaluation of the testing results (Bisson and Gregory,
1997).The tend to perceive the attributes of a wine in the following order.
 Colour
 Smell
 Taste
 Flavour
 Mouth-feel
 Overall acceptance
However, in the process of perception, most or all of the attributes overlap i.e. the subject
receives a jumble of near-simultaneous sensory impressions and without training, he or she
will not be able to provide an independent evaluation of each. The types of sensory attributes
that exist in terms of the way in which they are perceived and the terms, which may be
associated with them. Flavour is the combined impression perceived via the chemical senses
from a product in the mouth i.e. it does not include appearance and texture. The term
“aromatics” is used to indicate those volatile constituents that originate from food in the
mouth and are perceived by the olfactory system via the posterior nares (Carr et al., 1999).
2.9 Colour of wine
The fermentation of grapes for wines has a marked effect upon the colour of the product.
The final colour may be influenced by the SO2 content and the alcohol content attained at
the time of screening. Maximum colour is attained between 3 and 6 % alcohol and the
amount of colour extracted increases with increasing SO2 content upto 250 ppm.The colour

30
stability during the aging of wines was superior at the higher level of SO2 (Berg and
Akiyoshi, 1962).
Berg and Akiyoshi (1962) noted that wines fortified with alcohol had much higher colour
retention during aging than those produced without fortification. Wine production practices
including the level of SO2 and alcohol content have an influence on the colour equilibrium
between anthocyanogens and anthocyanins (Austin, 1968).
2.10 Nutritional aspects of wine
From a scientific standpoint, much more attention has been given by the researchers to the
non-nutritional aspects of wine than to what substances, in addition to alcohol, it may contain
of tangible value to the consumer. Research concentration has largely been directed to better
understanding such aspects as flavor, bouquet, keeping qualities, better ways to utilize
chemistry and biochemistry etc. in processing (Douglas et al., 1997).
The increasing consumer awareness concerning the nutritional value of all foods and the
fact that wine may be prescribed for a variety of medical treatments. In their research, they
found that the mineral contents of red wine generally exceed those of white wine, notably as
regards potassium, sodium, phosphorus, magnesium, iron, strontium, manganese, zinc,
copper, barium. Thus in terms of total ash, red wines were slightly lower in calcium and
aluminum (Douglas et al., 1997).
As regards to vitamin content of wine, Austin (1968) reported that about 2/3rd
of the
thiamin and riboflavin in grape juice is lost during winemaking but that very little is lost
during aging. They found that white wines contained more riboflavin than red wines.Manay
and Shadashaswaswany (1987) reported that when wines are taken along with a good and
balanced diet, their content of thiamine, riboflavin, pentothenate, niacin and vitamin B6
contribute to total nutrition.
Berg and Akiyoshi (1962) set out to determine the B-vitamin and ascorbic acid contents
of California wines. The thiamine, riboflavin, vitamin B6, vitamin B12, niacin, biotin, folate,
pentothenate and vitamin-C levels in several California wine were determined. They found
that the levels of the eight B-vitamins were higher in the red wines than in white wines.
Nutritionally significant levels of riboflavin, vitamin B6 and niacin were found in the red
wines.

31
According to Manay and Shadashaswaswany (1987), beer and wine contain some
nutrients present in the original malted barley and the fruit juice used in their proportion and
naturally their energy value would be higher than that of distilled liquor, 100 ml of wine
gives about 80 Kcal.
2.11 Wine from grapes with other ingredients
It is clear that wine making from grapes has ancient roots (Rout, 2014). According to Crossen
(2013), non-grape wines or wines made from grapes with other fruits, herbs etc. where grape
were not adequately available were found. Malbec wine is a red wine produced in Argentina
from grapes and berries like black berry, raspberry (Puckette, 2011). Grapple is another
popular wine made from grapes imported from India and apples from Mustang, Nepal
(Bhandari, 1992).
2.12 Wine defects and spoilage
Like beer, wine has its defects from non-microbial causes and its spoilage caused by
microorganisms. Defects include those, due to metals or their salts, enzymes and agents
employed in colouring the wine. Iron, for example, may produce a sediment known variously
on grey, black, blue or ferric-casse and in white wine, it may be responsible for a white
precipitate of iron phosphate termed white-casse. Tin and copper and their salts have been
blamed for cloudiness. White wines may be turned brown and red wines may have their
colour precipitated by peroxidase and oxidizing enzyme of certain moulds. Gelatin used in
clarifying wines, may cause cloudiness (Guymon et al., 1961).
The microorganisms causing wine spoilage are chiefly wild yeasts, moulds and bacteria
of the genera Acetobacter, Lactobacillus, Leuconostoc and perhaps Micrococcus and
Pedicoccus (Frazier, 1989).
2.12.1 Wine defects caused by yeasts
Most of the defects due to yeasts are caused by after fermentations. The most frequent defects
are caused by species of Saccharomyces such as S. cerevisiae, S. buyanus and S. bacilli.
Spoilage yeasts of other genera are principally Saccharomycodes ludwigii and
Brettanomyces species (Prescott and Dunn, 1987). Film yeasts, which can oxidize alcohol

32
and organic acids may grow on the surface of must and wines exposed to air, producing a
heavy pellicle called ‘wine flowers’ (Frazier, 1989).
2.12.2 Wine defects caused by bacteria
In the presence of air, the aerobic acetic acid bacteria, usually Acetobacter aceti of
Gluconobacter oxydane, oxidize alcohol in wine to acetic acid, an undesirable process called
‘acetification’. They also may oxidize glucose in the must to gluconic acid and may give a
‘mousy’ or ‘sweet-sour’ taste to the must (Frazier, 1989). If larger amounts of sugar in must
or wines are fermented by lactic acid bacteria, variable amounts of CO2, ethanol, volatile
acid and mannitol are formed depending on the particular species. Wines which have
undergone undesirable changes in this manner are said to have a “lactic acid flavour’
(Prescott and Dunn (1987). The growth of lactobacilli produces milky cloudiness, increases
lactic and acetic acid and yields CO2. It sometimes gives “mousy” or other disagreeable
flavour and damages the color of the wine (Frazier, 1989).
2.12.3 Prevention of wine spoilage
To prevent microbial spoilage of the finished wine, it is important to deactivate any residual
microorganisms before or after bottling. This can be accomplished by pasteurization,
addition of inhibitors such as SO2 or by filtration. The delicate flavour of some wines is
harmed by heating or by adding SO2. For these wines, filtration is preferred method of
removing microorganisms (Banwart, 1987).

Part III
Material and methods
3.1 Raw materials
3.1.1 Bael
The most common type of bael (15 kg) of good quality was bought from riversides of Tamor,
Dhankuta, Nepal.
3.1.2 Pineapple
Pineapple (5 kg) is also bought from local market of Dharan, Nepal.
3.1.3 Grape
Grapes (5 kg) is also brought from local market of Dharan, Nepal.
3.1.4 Table sugar
Table sugar was also bought from local market of Dharan, Nepal.
3.1.5 Wine yeast
The baker yeast was obtained because viable and active wine yeast was not present in Central
Campus of Technology.
3.2 Methods
The total work was based on the analysis of wine made from different concentration of mixed
juice concentrations which included the determination of fermentation kinetics, ultimately
optimizing the juice concentration and quality parameters were also analyzed.
3.3 Experimental procedure
3.3.1 Preparation of bael juice
Bael was cleaned, sorted and graded. Graded bael was broken by the use of Hammer. The
pulp from the broken bael was scrapped into clean vessel. Then water was added in the ratio
1:4 (bael: water).The prepared juice was then filtered by muslin cloth. According to

34
Zoecklein et al. (1997), 150 ppm KMS (potassium metabisulphite) was added with for
preservation purpose.
3.3.2 Preparation of pineapple
Pineapple was also cleaned with tap water. Then it was peeled with sharp knife. After
peeling, core was also removed by coring machine available at laboratory of Central Campus
of Technology. Then it was cut into pieces to weight easily and for pressing during must
preparation.
3.3.3 Preparation of grapes
Grapes were de-stemmed, cleaned and sorted out in a clean vessel. It was graded for better
result.
3.3.4 Preparation of must
Must were prepared by weighing and mixing of the three different fruits i.e. bael, pineapple
and grapes in the ratio 17:3:1, 16:4:1 and 15:4:1 and named as musts’ M1, M2 and M3. After
putting bael juices by proportion in three different containers, pineapple and grapes were
added into it. Grapes was gently pressed with the help of fingers. Pineapple was also further
cut into small pieces and was pressed by hand then it was mixed well for uniform
distribution. Analysis of TSS, pH and acidity were carried out after three musts prepared in
different container. Sugar was added to maintain TSS of 24o
brix and the fermentation was
carried out around pH 4.0 i.e. 3.9, 4.1 and 4.3 for musts M1, M2 and M3 respectively. The
optimization of must was done according to the bael juice concentrations of 85%, 80%, 75%
and pineapple 10%, 15%, 20% and then 5% grapes was added to all must.
3.3.5 Pitching
Baker yeast (BRAVO), a product of Canada, was used for pitching. It was activated with
mildly heated water at around 40ºC and pitching was done at the rate of 1 g per liter for all
musts. The general flow sheet for procedure is given in the Fig. 3.1.

35
Selection of raw material
Cleaning, sorting, grading, coring, slicing and pressing
Mixing
KMS 150 ppm Must
Optimization of must
Yeast @ 1 g/ltr Primary fermentation for 2 days
Pressing off
Secondary fermentation for 10 days
Racking, blending, finning, malo-lactic fermentation
Aging
Filtration and tartarate stabilization
Polishing
Pasteurization at 70 °C for 1 minute
Bottling, leveling and casing
Mixed fruit wine
Fig. 3.1 Flow-sheet for mixed fruit wine preparation
3.3.6 Fermentation
Musts after pitching were kept in plastic jars for fermentation. After two days of pitching,
when vigorous evolution of CO2 ceased, the primary fermentation was completed. Then the
Bael 85%
Pineapple 10%
Grapes 5%
Grapes 5%
Bael 80%
Pineapple 15%
Grapes 5%
Grapes 5%
Bael 75%
Pineapple 20%
Grapes 5%
M1 M2 M3
Acidity@0.4
pH@3.9
Acidity@0.41
pH@4.1
Acidity@0.43
pH@4.1

36
necks of jars (10 litres of capacity) were closed tightly with cotton plugs for secondary
fermentation. The exact process followed in this study is given in Fig. 3.1
It was necessary to create an anaerobic condition inside the jars during secondary
fermentation for improving the quality of product. The progress of fermentation was
followed by measuring the drop in o
brix. The fermentation was assumed to be completed
after ºbrix ceased to drop further. During the fermentation, the kinetics based on pH, TSS
and acidity were observed in every two day time interval during secondary fermentation for
10 days than racking was done.
3.3.7 Racking, pasteurization and bottling
The wine after secondary fermentation was pasteurized in the glass wine bottles by heating
at 70 °C for 1 minute using water bath and cooled to room temperature. The cooled wine
was racked after secondary fermentation and pasteurization for 15 days. The sediments
known as ‘lees’ were then separated out and discarded. The obtained clear wines were kept
in pre-sterilized wine bottles. The wines were racked again and kept at room temperature
until needed for further analysis.
3.4 Analytical procedure
Although different authors have described different methods and parameters to analyze wine,
only the parameters and related methods, which were feasible in this laboratory, were
determined in this present study. The determination was conducted in triplicates.
The quality of musts were analyzed for chemical composition (TSS, pH and acidity) and
the prepared wines were analyzed for chemical composition and properties (pH, TSS,
acidity, tannin, vitamin-C, total phenol, specific gravity, alcohol content, ester and
methanol). The sensory analysis (smell, taste, flavor, mouth-feel, color and overall
acceptance) was done to select best product.
3.4.1 Determination of total soluble solids (TSS)
The TSS of the fermented mashes was measured by Hand sugar refractometer (Model WYT-
32, Zhongyou Optical Instruments).

37
3.4.2 pH determination
pH was measured by pH meter (digital, portable, Japsin Industrial Instrumentation).
3.4.3 Total acidity determination
10 ml wine was pipetted out and was titrated with 0.1 N NaOH using phenolphthalein
indicator. The total acidity was determined by titrimetric method as per (AOAC, 2005).
3.5 Analysis of prepared wine
3.5.1 Sensory evaluation
Prepared wine by mixing three different fruits such as bael, pineapple and grapes in different
proportion were subjected to sensory evaluation for consumer’s acceptability. The samples
were served in clean wine glasses at silent environment. Sensory attributes (such as smell,
taste, mouth feel, flavour, color and overall acceptance) were evaluated using 9 points
Hedonic scale rating test ranging from dislike extremely (1) to like extremely (9) as
described by Ranganna (2010) with the help of 10 semi-trained panelist who were the
researcher and teacher of Central Campus of Technology, those who were familiar with
alcoholic beverage. The data obtained from the sensory analysis was analyzed by two-way
ANOVA at 5% level of significance to study the difference among them.
3.5.2 Quality analysis
The quality parameters of the all wines prepared were analyzed. Total phenols and tannins
were analyzed by the procedure given in Ranganna (2010), Kirk and Sawyer (1999) and
AOAC (2005).
3.5.2.1 Tannin content
15 ml wine was taken. 20 ml indigo caramine solution was added in the solution and was
titrated with potassium permagnate solution, 1 ml at a time until the color becomes light
green. Again the solution was titrated with same solution until the color change to bright
yellow or faint pink.
Further 50 ml wine was taken in 250 ml volumetric flask and 25 ml gelatin solution was
added. Volume was made by adding acidic sodium chloride solution. Then whole solution

38
was transferred to conical flask and shake for 15 minute and filtered. Further the filtrate was
titrated with potassium permagnate solution as above procedure. Finally tannin content was
determined by volumetric method as per AOAC (2005).
3.5.2.2 Vitamin-C content
20 ml wine was taken and 3% meta phosphoric acid was added to make up 100 ml volume.
Further 2 ml acetone was added. Then 10 ml from that solution was taken for titration with
standard dye solution. Finally vitamin-C content was determined by 2,6- Dichlorophenol
indophenol titration method as per AOAC (2005).
3.5.2.3 Total phenol content
50 ml standarize indigo caramine solution was poured in 500 ml Erlenmeyer flask. 2 ml wine
was added in it and titrated with standarize potassium permagnate solution until the color
become blue to yellow. Finally total phenol content was determined by Permanganate Index
method as described in Ranganna (2010).
3.5.2.4 Specific gravity
150 ml wine was neutralized with 1 N NaOH and was poured in distillation flasks. Then it
was distilled to get 50 ml distillate. Then after the distillate was gently poured in specific
gravity bottle. Finally specific gravity was determined as per AOAC (2005).
3.5.2.5 Alcohol content
Alcohol content was determined by specific gravity method as per AOAC (2005) by using
specific gravity chart.
3.5.2.6 Ester
500 ml wine was taken for distillation and 50 ml distillate was collected. Then it was
neutralized with 0.1N NaOH. Further 5 ml excess 0.1 NaOH was added and reflux for 1
hour. Finally it was titrated with 0.05 M sulfuric acid. Lastly Ester content was determined
as per Kirk and Sawyer (1999).

39
3.5.2.7 Methanol
500 ml wine was taken for distillation and 50 ml distillate was collected. Then alcohol
content of distillate was adjusted to 5% abv. by adding distilled water. Then after 2 ml
potassium permagnate solution was added in three different 50 ml volumetric flask. Chilled
sample, standard methanol solution and blank solution, each 1 ml was added in different
flask and named as “sample”, “standard” and “blank”. Then all three flask was placed in ice
bath for 30 minutes. Further little dry NHSO4 was added. Then after 1 ml chromotopic acid
was added in each flask. Further 15 ml sulfuric acid was added in each flask and was placed
in hot water bath for 15 minutes. Then all flasks were cooled and volume was make up 50
ml by adding distilled water. Finally absorbance of each solution was measured at 575 nm
using spectrophotometer. Finally methanol was determined as per AOAC (2005).
3.6 Statistical analysis
The data were analyzed for one-way and two-way ANOVA using Genstat (Genstat
Discovery Edition 12, 2014) at 5% significance level. The means was compared using LSD
method. Two-way ANOVA was done for fermentation kinetics. Standard deviation and
means were also analyzed from the same statistical tool.

Part IV
Results and discussion
In this experiment, must were prepared by weighing and mixing of the three different fruits
i.e. bael, pineapple and grapes in the ratio 17:3:1, 16:4:1 and 15:4:1 and named as must M1,
M2 and M3. TSS was maintained at 24°Bx by using table sugar. The fermentation was carried
out at room temperature around 27°C using baker yeast. Secondary fermentation was
completed after 10 days and prepared wines were subjected for chemical and sensory
analysis.
4.1 Chemical composition of must
Chemical composition of must was determined including TSS, pH and acidity and is
presented in Table 4.1
Table 4.1 Chemical composition of musts.
Component M1* M2* M3*
TSS, ºBrix 24±0 24±0 24±0
pH 3.9±0.1 4.1±0.173205 4.3±0.2
Acidity, % citric acid 0.40±0.004 0.41±0.004 0.43±0.004
*values are the means of triplicate, figures in the ± are the standard deviation.
M1, M2 & M3 are the must made from three different fruits bael, pineapple and grapes in the
ratio 17:3:1, 16:4:1 and 15:4:1 respectively.
The results obtained for TSS, pH and acidity from this study did not agree with that of
(Panda et al., 2014). The difference may be due to the change in composition of must by the
addition of pineapple and grapes in different proportions. In this study, the bael juice
extraction was difficult to extract so by diluting it in the ratio 1:4 whereas an author had
extracted juice in the ratio 1:1 with water. The raw material collected for this study was

41
collected from Dhankuta District, Nepal whereas raw material used by author was collected
from the Garden of North Odisha University at Baripada, India.
4.1.1 TSS
The TSS of the all three musts were made constant i.e. 24ºbrix. TSS of must for red wine
preparation should be 20-25o
brix (Rai, 2012). TSS maintained for this study was within
range.
4.1.2 pH
The pH of M1, M2 and M3 were 3.9, 4.1 and 4.3 respectively. The optimum pH for wine
production varies from types of the selected fruit but generally 3.8-4.5 is supposed to be
optimum (Prescott and Dunn, 1987). The pH of the must for this study was within the range
for red wine production.
4.1.3 Acidity
The acidity of M1, M2 and M3 were 0.4366, 0.4066 and 0.431 respectively.
4.2 Fermentation kinetics
Fermentation kinetics of musts were studied in every two days of fermentation. All
parameters taken for the study is described as follows.
4.2.1 TSS
A graphical representation of patterns of TSS changes during fermentation is shown in
Fig.4.1. It shows that TSS was decreased with increase in fermentation days up to final day
of fermentation during secondary fermentation. This was the indication of alcoholic
fermentation occurred continuously up to 10 days and then fermentation was stopped. The
LSD test was conducted to identify the significant difference among them.
Two-way ANOVA result (Appendix I) of TSS shows that there was significant difference
(p˂0.05) in TSS with respect to fermentation days but there was no significant different
(p>0.05) among formulations and LSD test was conducted to analyse the individual
difference among them. The result of LSD test is tabulated in Table A.1 and Table A.2.

42
Fig. 4.1 Patterns of TSS change during fermentation
*plotted value are the means of triplicates determination.
Table A.1 shows that the variation in TSS of all fermentation days was significantly
different (p˂0.05) to each other. The change in TSS signifies that alcoholic fermentation is
continuously occurring as the day progress.
Table A.2 shows that there was no significant different (p>0.05) in TSS of all
formulations. It may be due to the composition of must variation is only at 5 % difference so
the comparable change was not seen in TSS with respect to formulations.
4.2.2 pH
A graphical representation patterns of pH changes during fermentation is shown in Fig 4.2
5
10
15
20
25
0 2 4 6 8 10 12
TSSvalue,°Brix
Time (days)
M1 M2 M3

43
Fig. 4.2 Patterns of pH change during fermentation
Fig. 4.2 shows that the changing pattern of pH was biphasic with fermentation days and
was almost same in all formulations. pH value was decreased up to six days of fermentation
and then increased until the final day of fermentation. As describes in beer fermentation, pH
value is decreased up to two days of fermentation and then it remains almost constant until
the complete fermentation (Guymon et al., 1961). Hence, it can be concluded that first phase
of graph in Fig. 4.2 was similar with that of beer fermentation and the reason to decrease the
pH at first stage was that there was always decrease in pH at sugar metabolism except in
alkaline fermentation where protein metabolism occurs. Likewise, increase in pH at second
phase of the graph was due to the utilization of acid compounds by yeast on fermentation
process.
The ANOVA result (Appendix I) shows that there was significant difference (p<0.05) in
pH value among fermentation days but there was no significant difference (p>0.05) in
formulations and LSD test was conducted to analyze the individual difference among them.
The result of LSD test is tabulated in Table A.3 and Table A. 4
2
2.5
3
3.5
4
4.5
0 2 4 6 8 10 12
pHValue
Time (days)
M1 M2 M3

44
Table A.3 shows that pH of all fermentation days was significantly different (p<0.05) to
each other. The least pH value is found in 6th
day.
Table A.4 shows that pH of M1 was significantly different (p<0.05) with pH of M2 and
M3 but pH of M2 resembled similarity (p<0.05) with M3.
4.2.3 Acidity
A graphical representation of pattern of acidity change during fermentation is given in Fig.
4.3
Fig. 4.3 Patterns of acidity change during fermentation
Fig. 4.3 shows that the variation of acidity of all formulations was similar biphasic
pattern. Acidity of all formulation was increased up to sixth day of fermentation and then
acidity started to decrease from 6th
day to the final day of fermentation. A graphical
representation shows almost reverse pattern of a graph shown in Fig. 4.2. This is because of
obvious reason.
Two-way ANOVA result (Appendix I) of acidity shows that there was significant
difference (p<0.05) in acidity with reference to both fermentation days and formulations and
0.35
0.4
0.45
0.5
0 2 4 6 8 10 12
Acidy,%citricacid
Time (days)
M1 M2 M3

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