effect of pre and post fermentation treatments …...where india stands at 12 th position in its...
TRANSCRIPT
EFFECT OF PRE AND POST FERMENTATION
TREATMENTS ON QUALITY OF RED WINE
PREPARATION FROM PUNJAB PURPLE GRAPES
Thesis
Submitted to the Punjab Agricultural University
in partial fulfillment of the requirements
for the degree of
INTEGRATED MASTER OF SCIENCE (Hons.) in
MICROBIOLOGY (Minor Subject: Biochemistry)
By
Maninderjeet Kaur (L-2008-BS-09-IM)
Department of Microbiology College of Basic Sciences and Humanities
© PUNJAB AGRICULTURAL UNIVERSITY
LUDHIANA – 141 004
2013
2
CERTIFICATE I
This is to certify that the thesis entitled, “EFFECT OF PRE AND POST
FERMENTATION TREATMENTS ON QUALITY OF RED WINE PREPARATION
FROM PUNJAB PURPLE GRAPES” submitted for the degree of Integrated Master of
Science (Hons.) in the subject of Microbiology (Minor subject: Biochemistry) of the Punjab
Agricultural University, Ludhiana, is a bonafide research work carried out by Maninderjeet
Kaur (L-2008-BS-09-IM) under my supervision and that no part of this thesis/dissertation
has been submitted for any other degree.
The assistance and help received during the course of investigation have been fully
acknowledged.
_______________________
Dr. G.S Kocher
Major Advisor
Microbiologist
Department of Microbiology
Punjab Agricultural University
Ludhiana-141004
3
CERTIFICATE II
This is to certify that the thesis entitled, “EFFECT OF PRE AND POST
FERMENTATION TREATMENTS ON QUALITY OF RED WINE PREPARATION
FROM PUNJAB PURPLE GRAPES” submitted by Maninderjeet Kaur (Admn No. L-
2008-BS-09-IM) to the Punjab Agricultural University, Ludhiana, in partial fulfillment of the
requirements for the degree of Integrated Master of Science (Hons.), in the subject of
Microbiology (Minor subject: Biochemistry) has been approved by the Student’s Advisory
Committee along with Head of the Department after an oral examination on the same.
___________________________ ___________________________
(Dr. G.S. Kocher) (Dr. S.K. Soni)
Major Advisor External Examiner
Professor
Deptt. of Microbiology
Panjab University
Chandigarh
___________________________
(Dr. (Mrs.) Parampal Pal Sahota) Head of the Department
___________________________
(Dr. Gursharan Singh) Dean, Postgraduate Studies
4
ACKNOWLEDGEMENTACKNOWLEDGEMENTACKNOWLEDGEMENTACKNOWLEDGEMENT
I would like to sincerely express my gratitude to all who have supported me through guidance, prayers and
encouragement to successfully complete a work of this kind. I am particularly thankful to the Lord God Lord God Lord God Lord God
AlmightyAlmightyAlmightyAlmighty for His mercy and grace upon my life.
First of all I pay homage to my Major Advisor Dr. G S KocherDr. G S KocherDr. G S KocherDr. G S Kocher, Microbiologist, Department of
Microbiology, Punjab Agricultural University, Ludhiana, who painstakingly edited this work and provided
constructive criticism for writing up this thesis. I really appreciate for his expert guidance, encouragement,
inspiration and advice throughout my research work. This work would not have been possible without her
guidance, support and encouragement. Under his guidance I successfully overcame many difficulties and
learned a lot.
I am also grateful to all the esteemed members of my advisory committee Dr.Dr.Dr.Dr. Maninder AroraManinder AroraManinder AroraManinder Arora,
Senior Food Microbiologist, Department of Microbiology, Dr.SuchDr.SuchDr.SuchDr.Sucheta Sharma, eta Sharma, eta Sharma, eta Sharma, Associate Professor,
Department of Biochemistry, Dr. (Mrs.) Parampal Pal Sahota, Dr. (Mrs.) Parampal Pal Sahota, Dr. (Mrs.) Parampal Pal Sahota, Dr. (Mrs.) Parampal Pal Sahota, Sr. Microbiologist cum Head, Department
of Microbiology, (Dean PG Nominee) for their able guidance, constructive suggestions and continuous
support. I express my sincere thanks to Dr. M I S Gill,Dr. M I S Gill,Dr. M I S Gill,Dr. M I S Gill, Sr. Horticulturist, Department of Fruit Science for
their requisite provision of the fruits they made possible, without which this study would have not been
possible.
I owe a lot to my father S. Amarjit Singh S. Amarjit Singh S. Amarjit Singh S. Amarjit Singh who encouraged and helped me at every stage of my
personal and academic life, and longed to see this achievement come true. I thank my mother Ranjit kaur Ranjit kaur Ranjit kaur Ranjit kaur
for her tender, loving care and compassion. Special thanks to my elder brother Amandeep Singh Amandeep Singh Amandeep Singh Amandeep Singh who make
my life joyful and providing the moral and emotional support I needed to complete my thesis.
As Life is incomplete without friends, so my greatest appreciation goes to my lovely friends
Shiveta Raina, Amandeep Sidhu, Harpreet Kaur, Ritu Didi Shiveta Raina, Amandeep Sidhu, Harpreet Kaur, Ritu Didi Shiveta Raina, Amandeep Sidhu, Harpreet Kaur, Ritu Didi Shiveta Raina, Amandeep Sidhu, Harpreet Kaur, Ritu Didi for supporting me in all my struggles and
frustrations during thesis writing.
My sincerest thanks to Harminder didi Harminder didi Harminder didi Harminder didi for their invaluable help, guidance and constructive ideas
in the laboratory. Thanks to my supportive friends and labmates, Jyoti BalaJyoti BalaJyoti BalaJyoti Bala, Anureet Brar and Pooja Didi Anureet Brar and Pooja Didi Anureet Brar and Pooja Didi Anureet Brar and Pooja Didi
who made the lab a friendly environment for working. I would also like to thanks Kewal Uncle Kewal Uncle Kewal Uncle Kewal Uncle and Ranbir Ranbir Ranbir Ranbir
Bhaji Bhaji Bhaji Bhaji for constantly helping me time to time.
I feel proud to be a part of PAU, Ludhiana where I learnt a lot and spent some unforgettable
moments of my life. I am also thankful to all members of the department.
Last but not least, I duly acknowledge my sincere thanks to all who love and care for me.
Everyone may not have been mentioned but none is forgotten.
_________________ ((((Maninderjeet KaurManinderjeet KaurManinderjeet KaurManinderjeet Kaur))))
5
Title of the Thesis : Effect of pre and post fermentation treatments on
quality of red wine preparation from Punjab purple
grapes.
Name of the Student : Maninderjeet Kaur and Admission No. L-2008-BS-09-IM
Major Subject : Microbiology
Minor Subject : Biochemistry
Name and Designation : Dr. G.S. Kocher
of Major Advisor Microbiologist
Degree to be Awarded : Integrated Master of Science (Hons.) in Microbiology
Year of award of Degree : 2013
Total pages in Thesis : 69+ Annexure (iv) + Vita
Name of University : Punjab Agricultural University, Ludhiana 141004,
Punjab, India
ABSTRACT
The effect of pre and post fermentative treatments in production of red wine from
Punjab purple variety of grapes was evaluated. The prefermentation was optimized for skin
contact (0.25Kg/L of skin weight, temperature 20oC and incubation time 16 h) and enzymatic
treatment using combination of enzymes i.e. Amylase+pectinase+cellulase at 45oC for 6h that
produced 78.7 % clarification of juice. The latter was fermented by Saccharomyces cerevisae
strain 35 (5% v/v) at 25oC using 150 mg/100ml of DAHP producing 12.1% (v/v) of ethanol.
A 10% (v/v) of spent yeast inoculum was optimized for recycling of two successive fermentation batches producing significantly similar ethanol concentrations after
fermentation. Post fermentative storage of wine (at 15 oC) for 150 days freed the wine from
viable yeast cells but also led to the reduction of phenols along with decrease in % ethanol
levels. The free and total sulphur dioxide in the fresh yeast culture wine was 22.1 ppm and 8.2
ppm, respectively. The qualitative analysis of red wine w.r.t amino acids carried out by TLC
revealed the presence of Histidine/Arginine/Lysine, Threonine, Methionine/Alanine/Valine/
Tyrosine, and Methionine/valine. The prepared wines from Punjab purple under optimized
conditions with fresh and recycled yeast inocula were found to be of standard quality with
mean sensory scores of 65.9±8.01 and 63.3±8.52, respectively at 150 days of storage
compared to 56.2±9.47 of a commercial wine.
Keywords: Punjab purple, red wine, recycling, enzymatic treatment, sensory
___________________________ _______________________
Signature of the Major Advisor Signature of the Student
6
Koj pRbMD dw isrlyK : lwl AMgUrW dI vweIn "pMjwb prpl" iksm
au~pr KmIrIkrx qoN pihlW Aqy bwAd dy Asr[
ividAwrQI dw nW : minMdrjIq kOr Aqy dw^lw nMbr (AYl-2w008-bI.AYs-09-AweI.AYm)
mu`K ivSw : sUKm jIv ivigAwn
ishXogI ivSw : jIv rswiexk
mu`K slwhkwr dw nwm Aqy Ahu`dw : fw. jI.AY~s. kocr mweIkrobwieEloijst
id`qI jwx vwlI ifgrI : ieMtIgRytf AY~m.AY~s.sI. (AOnrz) mweIkrobwieElojI
ifgrI imlx dw swl : 2013
Koj pRbMD iv`c ku`l pMny : 69 + AnYgzr (iv) + vItw
XUnIvristI dw nwm : pMjwb KyqIbwVI XUnIvristI, luiDAwxw-141004 pMjwb, Bwrq [
swrMs
lwl AMgUrW dI vweIn 'pMjwb prpl' iksm au~pr KmIrIkrx qoN pihlw Aqy bwAd dy Asr dw mulWkx kIqw igAw [ KmIrIkrx qoN pihlW cmVI sMprk 0.25 iklo/lItr cmVI dy Bwr, qwpmwn 20°C Aqy qwp smW 16 GMty AwSwvwdI sI Aqy ieMzwiemYNitk ielwj ieMzwiem dy imSrx (AmweIlyz+pYktInyz+sYlUlyz) 45°C qwpmwn qy 6 GMty dy iesqymwl sdkw jUs 78.7% q̀k sw& kIqw igAw [ cmVI sMprk vwly dw KmIr sYkromweIisz sYrwivs 35 (5 pRqISq v/v) 25°C qwpmwn qy 150 imlIgRwm pRqI 100/imlI: fIeyAYcpI dw iesqymwl sdkw 12.1 pRqISq vwlI eIQwnol pYdw hoeI [ 10 pRqISq (v/v) KricAw KmIr eInokulm 2 smUh lgwqwr KmIrIkrx dIAW ivDIAW ausy Gxqw dI eIQynol pYdw krn leI AwswvwdI sI [ AMgUrW dI vweIn dy KmIrIkrx qoN bwAd dw BMfwr 15°C qwpmwn qy 150 idnW leI kIqw igAw ijs nwl AMgUrW dI vweIn nUM KmIr sYlW qoN mukq kr id`qw igAw pr nwl dI nwl PInolz Aqy eIQwnol dw pRqISq sqr G`t igAw [ AMgUrW dI vweIn iv`c Awjwd qy swrI slPrfweIAwksweIf dI mwqrw 22.1 pIpIAYm Aqy 8.2 pIpIAYm kRmvwr sI [ lwl AMgUrW dI Srwb dw guxnwqmk ivSlySx AmIno AYisf dy muqwibk tIAYlsI rwhIN kIqw igAw jo ik ihstIfwien/ArjInwien/lweIsIn, QRIEnwien, mIQIEnwien / AYlynwien/vYlylwien/Twieroswien Aqy mIQIEnwien/vYlIn lwl AMgUrW dI vweIn iv`c vYlwien dI hoNd drswauNdw hY [ imQy hlwqW au~pr 'pMjwb prpl' AMgUrW dI vweIn jo ik qwjw qy Krcy hoey ienokUlm qoN iqAwr krky 150 idn dy BMfwr leI r`KI geI sI [ ausdy svwd dy imAwrW au~pr guxv`qw mulWkx ivaupwrk lwl vweIn nwl kIqw igAw [ ijnHW dy guxv`qw AMk 65.9±8.01, 63.3±8.52 Aqy 56.2±9.47 kRmvwr sn [ mu`K Sbd: pMjwb bYNgxI, ieMzymYitk ielwz, lwl AMgUrW dI Srwb, KmIr auTwauxw, dubwrw
iqAwr krnw, idmwgI guxv`qw [ __________________ ________________ mu`K slwhkwr dy hsqwKr iv`idAwrQI dy hsqwKr
7
CONTENTS
Chapter Topic Page
I. INTRODUCTION 1-3
II. REVIEW OF LITERATURE 4-22
III. MATERIALS AND METHODS 23-33
IV. RESULTS AND DISCUSSION 34-54
V. SUMMARY 55-56
REFERENCES 57-69
ANNEXURE i-iv
VITA
CHAPTER I
INTRODUCTION
Grapes (Vitis spp.) are economically one of the important fruit species in the world
where India stands at 12th position in its production. But, in terms of productivity, India has
the highest productivity of 26.2 tonnes/ha among the grape producing countries (Anonymous
2010). Though grapes are used for table purposes and ready to serve drinks, the prime use of
grapes is for wine production. Grape wine is perhaps the most common fruit juice alcohol. As
much as 82% of the produce is used for wine preparation worldwide but we in India, utilize
just 2% of the produce for wine (Anonymous 2010). Henceforth, per capita consumption of
wine in India is a meager 7 ml compared to 60 and 50 litres in France and Italy, respectively
(Patil 2008). Because of the commercialization of the product for industry, the process has
received most attention of researchers.
Wine is an alcoholic beverage which is produced by fermentation of grape juice
through activity of Saccharomyces cerevisiae and other naturally occurring yeasts such as
Kloeckera apiculata, Hansenula anomala, Candida stellata, (Heard and Fleet 1986, Combina
et al 2005). Besides ethanol, wine contains a number of phenolics, organic acids, minerals,
vitamins etc. of fruit as well as microbial origin. These are responsible for the typical quality
of a wine (Kunkee and Gosewell 1996). Nowadays, wine is an integral component of the
culture in many countries, a form of entertainment in others, and a drink full of nutraceuticals
and health stimulating properties for others (Bisson et al 2002a). Red wine in particular is
known for antioxidant activities due to its phenols, lowering of blood cholesterol by its
resveratol, anti-inflammatory properties etc. (Duck et al 2008). It has been found that 75% of
these health stimulating phenolics reside in skin and seeds of grapes for which it is very
important that these are extracted in the juice destined for fermentation. This is the reason that
a prefermentation treatment in the form of skin juice contact needs to be standardized for its
time, temperature and associated malo-lactic fermentation (Marais and Rapp 1988, Staden et
al 2005).
Wine is one of the functional fermented foods and has many health benefits. These
include anti-ageing effects of red grape skins, improvement of lung function from
antioxidants in white wine, reduction in coronary heart disease, development of healthier
blood vessels in elderly people, reduction in ulcer –causing bacteria, destruction of cancer
cells by proteins present in red grape skins, prevention of stroke by keeping the arteries clean
owing to polyphenols present in red grape skin, decreasing ovarian cancer risk in women and
making the bones stronger. Many wines are made from fruits having medicinal value and
such wines have many additional benefits (Tapsell et al 2006).
2
Most well designed population studies have demonstrated a ‘J curve' relationship
between wine consumption and the risk of cardiovascular diseases (Cleophas 1999, Maclure
1993, Marmot 2001). This indicates that people who do not drink alcohol at all and those who
consume more than 30 g/day of alcohol (approx. 2.5 standard drinks) have an increased risk
of death from all causes, higher blood pressure levels, as well as poorer liver function
(Marmot 2001). On the other hand, moderate alcohol intake (1-2 drinks/day) is associated
with a decreased coronary heart disease (CHD) risk in both men and women. One to two
glasses of red wine (with a meal) for men and 1 glass for women may help to fight heart
disease (Snyder 2005).
Indian wine industry is in its infancy and is restricted presently to Maharashtra,
Karnataka and Goa to some extent but is expected to grow by 25-30% between 2009 to 2012
(Patil 2008, Kocher et al 2009, 2011b). The main constraint in growth of wine industry is lack
of suitable varieties and dependence on imported wine producing yeasts. The selection of
suitable grape varieties having potential to make wines of high quality is a continuous type of
work in all the wine producing countries. This is of particular concern in India where varieties
suitable for wine production and adapted to local climatic conditions are lacking. In Punjab,
the only widely grown variety, Perlette is not recommended for wine. Hence, for the past 6
years, 7 grape varieties/hybrids viz., Perlette, Flame Seedless, Porton, Punjab Purple (H-516),
Muscat Hamburg, H-27 and Chasan B have been evaluated. Of these, Punjab Purple has been
recommended by Research Evaluation Committee, PAU for wine preparation at five liter
scale using indigenous strain Saccharomyces cerevisiae strain 35 (Kocher et al 2011b). This
process needs to be scaled up for possible commercialization of the technology.
In this regard, a number of pre-fermentation parameters need to be standardized such
as skin juice contact (as described earlier) and effect of pectinase treatment on the must. The
latter is an important treatment in terms of stability of the must, extraction of skin color and
clarity of the must (Espejo and Armada 2010, Kocher and Pooja 2011). Skin contact time, as
well as temperature at which the treatment is applied could be critical to the composition and
quality of juices and wine as they may be detrimental to wine quality due to increase in the
concentrations of constituents, such as certain phenolic compounds (Marais and Rapp 1988).
Among the prefermentative treatments, supplementation of nitrogen and phosphorous is
another very important fermentation parameter as grape juice is deficient in these salts. In this
regard, availability of Yeast Assimeable Nitrogen (YAN) in the must is very important to
prevent nitrogen stress, which can lead to incomplete fermentation of sugars and the
production of off-flavours in the wine produced, moreover, the nitrogen content of grape
juice affects both yeast cell growth and fermentation rate (Monteiro and Bisson 1992, Ugliano
et al 2007). A considerable research on optimization of fermentation parameters such as
temperature, pH, inoculum size, supplementation of nitrogen and phosphorus has been carried
3
out in different studies (Erten et al 2006; Asli 2010).
Wines produced after alcoholic fermentation are termed raw/green wines, which are
stored/aged for an extensive period of time for maturation of flavours. This requires
standardization of storage temperature, humidity and light so that organoleptic characteristics
are improved (Jackson and Lombard 1993, Chang et al 2008). This is essential because it has
been found that an increased storage may decrease volatiles (Perez-Prieto et al 2003).
Similarly a very high humidity of 80% may lead to risk of contamination and high light
intensities result in undesirable flavour (Jackson and Lombard 1993, Chang et al 2008).
Keeping in view the paucity of information with regard to different pre and post
fermentation parameters for Punjab purple (Syn H 516) variety of grapes, the present study
was designed to accomplish following objectives-
1. Standardization of pre-fermentation ‘maceration’ process in terms of effective contact
of grape skin with juice.
2. To study the effect of nitrogen and phosphorus supplementation in the ‘must’ and
yeast recycling on quality of wine.
3. Effect of storage on oragnoleptic properties of wine.
4
CHAPTER II
REVIEW OF LITERATURE
The recent review of literature on different aspects of wine production from grapes is
presented under the following headings:-
2.1 Wine as a beverage
2.2. Grapes
2.2.1 Chemical characteristics of Grapes
2.2.1.1 Total soluble solids
2.2.1.2 Titrable acidity
2.2.1.3 Volatiles
2.3 Yeast
2.4 Pre-fermentation treatment
2.4.1 Effect of skin contact
2.4.2 Effect of enzymatic treatment
2.5 Ethanol fermentation
2.6 Factors affecting fermentation
2.6.1 Effect of Temperature
2.6.2 Effect of pH
2.6.3 Effect of initial sugar level
2.6.4 Effect of Nitrogen and Phosphorus supplementation
2.7 Post fermentative treatments
2.7.1 Racking
2.7.2 Effect of fining agents
2.7.3 Ageing
5
2.1 Wine as a Beverage
Wine is one of the world’s most popular alcoholic beverages. It has been produced
and consumed throughout history for cultural, economical, social, religious, and, more
recently, health reasons. Wine produced from grapes is constantly being investigated for its
health benefits. The increased popularity of wine has resulted in an enormous upsurge in
prices of the world’s finest wines, a scarce commodity under the best of circumstances.
According to FAO data (2010), the leading grape producing countries in the world in terms of
production are China (8,651.83 thousand tons), Italy (7,787.80 thousand tons), USA (6,777.73
thousand tons) and Spain (6,107.20 thousand tons) and India’s high productivity in grape has
made it to reach 18th position in the world as far as production (total world production
7,116.25 thousand tons) is concerned.
Wine, a fermented undistilled alcoholic beverage is produced by anaerobic
fermentation of grape sugars to ethanol by the wine yeast (Amerine and Ough 1980, Joshi et
al 2011). The ability to produce palatable effervescent beverage by alcoholic fermentation of
natural fruit juices is a demonstration of inherent ingenuity of man. The nutritional role of
wine is important since its average contribution to total energy intake is estimated to be 10 to
20% in adult males (Macrae et al 1993). During the past few decades, grapes are the main
fruits that have been used for wine production. Traditionally, red wine is produced from grape
varieties that have black or red color and the fermentation is carried out on the skin using
standard wine yeast (Amerine and Ough 1980).
Despite that, several studies have investigated the suitability of other fruits as
substrates for the purpose of wine production (Joshi and Bhutani 1991, Okunowo et al 2005).
Moreover, the non-availability of grapes, which is usually the fruit of choice for wine
production in the tropics, has necessitated the search for alternative fruit source in tropical
countries (Alobo and Offonry 2009). A number of secondary metabolites including
polyphenols (anthocyanins, flavonoids etc.) that are key determinants of wine quality endow
it with antioxidant potential (Chen et al 2009).
The main volatile compounds in wines are secondary products arising from alcoholic
or malolactic fermentation; their presence in young wines, whites, roses and reds, is a quality-
defining factor (Ferrarese 1987, Ribereau-Gayon et al 1982). Wine polysaccharides play an
important role in wine technology, either for their sensory characteristics, their implications
during fermentation or their detrimental role in filtration (Ayestaran et al 2004). Red wine is
characterized by the presence of phenolic compounds that contribute to organoleptic
characteristics such as color, taste, astringency and bitterness and their capacity for aging
(Ribereau-Gayon et al 2006).These compounds occur naturally in grapes of red Vitis vinifera
cultivars and winemakers aim to extract them into the wine through various methods of
maceration. Phenolic compounds are extracted from skin and seeds, and their extraction is
6
influenced by winemaking procedures. Macerating enzymes may help in phenolic extraction
and, at the same time, may modify the stability, taste and structure of red wines, because it is
not only anthocyanins that are released from skins, but also tannins bound to the cell walls
(Bautista-Ortín et al 2007). The advantage of cryomaceration treatments over maceration at
20 °C was in a significantly lower content of extracted phenols, which are subject to oxidation
and negatively contribute to wine quality (Radeka et al 2008).
Red wine possesses several potentially heart-healthy mechanisms, including a
favorable effect on blood clotting, endothelial function and serum lipids (most notably the
ability to raise levels of HDL “good” cholesterol). The cardio-protective effects observed in
red wine drinkers are thought to be attributed at least in part to moderate alcohol intake, but
especially due to the polyphenolic compounds red wine possesses, most notably, a flavonoid
called resveratrol. Laboratory studies have revealed that resveratrol possesses several cardio-
protective actions as well as cancer-protective effects (Snyder 2005).
The fermentation of wine is known to be a complex process with various ecological
and biochemical processes involving yeast strain (Fleet 2003). The fermentation for the
elaboration of beverage is known to depend on the performance of yeast to convert the sugars
into alcohol and esters. Besides, the different species of yeast that develop during
fermentation determine the characteristic of the flavour and aroma of the final product. Also,
because different fruits have different composition, there is the need for yeast strains to adapt
to different environments, such as sugar composition and concentration of acetic acid (Fleet
2003, Duarte et al 2010). Although, tropical fruits and several yeast strains have been
screened for their suitability in wine production, most studies have either focussed only on the
suitability of the fruits or the yeast strains. Infact, the winemaking process can have a large
influence on the final attributes of a red wine as the winemaker has different tools and
enological practices at his disposal to alter the final properties of the wine (Jackson 2000a).
2.2 Grapes
The majority of grapes grown throughout the world are utilized for winemaking. In
all wine producing countries, wines are made from varieties of the Vitis vinifera grapes.
Different grape varieties are used for the preparation of red wine. But, historically grapevine
(Vitis vinifera L.) is grown mostly for wine making in the world over. However, each grape
variety will result in different wine quality (Tontemsup 1996) due to their unique
characteristics.
Approximately 75,866 square kilometers of the world are dedicated to grapes and
71% of world grape production is used for wine, 27% as fresh fruit, and 2% as dried fruit
(FAO 2010). The area dedicated to vineyards is increasing by about 2% per year. There are no
reliable statistics that break down grape production by variety. It is believed that the most
widely planted variety is ‘Sultana’, also known as Thompson Seedless, with at least
7
3,600 km2 (880,000 acres) dedicated to it. The second most common variety is Airen. Other
popular varieties include Cabernet Sauvignon, Sauvignon blanc, Cabernet Franc, Merlot,
Grenache, Tempranillo, Riesling and Chardonnay.
In India, Maharashtra ranks first in wine grape cultivation with an area of 8,000 acres,
which formed a proportion of 91.95 per cent in the country’s wine grape area during the year
2007. It is one of the important states growing different varieties of wine grape. Cultivation of
wine grape is extensively carried out in the belts of Nashik, Pune, Sangli, Solapur, Latur,
Buldhana, Osmanabad and Ahmadnagar districts (Kale 2007). In PAU, Punjab Purple, a
colored grape variety was evaluated for red wine production over a period of three years. A
consistent ethanol production of about 10-11% (v/v) with a mean recovery of 62.4 % was
observed. The red wine produced was found to be of standard quality (mean score of 63.5 out
of 80) on the basis of sensory evaluation and had phenolics content of 2496 mg/L which
endows it with a high antioxidant potential (Kocher et al 2011b).
Grape quality is primarily influenced by specific vineyard practices, climate, and
grape varieties (Jackson and Lombard 1993) and thus the potential of the grapes to give a high
quality wine is both evaluated subjectively (e.g. taste, aroma, and visual inspections) and
objectively from compositional analyses of the grapes (Krstic et al 2003). Red winemaking
begins when grapes are harvested and processed at the winery. The grapes for wine
production are harvested at an appropriate stage of maturity whereby particularly important
are the concentrations of sugars and acids, which are major constituents of the juice and have
an important impact on its fermentation properties (Fleet 1998, 2001). For dry or table wines,
grapes of high acidity and moderate sugar content are desirable, while grapes with high sugar
content and moderately low acid are required for sweet or dessert wines.
Grapes contain 0.3%-0.5% of various organic acids, such as tartaric acid and malic
acid, as well as some free sugars and vitamins (Duck et al 2008). Grape juice concentrate is
also used to make wine in many locations that do not or cannot grow grapes (Morris et al
1996). This is because grape juice concentrates can be easily stored, allowing winemakers to
produce wine throughout the year. Grapes are good source of water (82%), carbohydrates
(12–18%), proteins (0.5-0.6%), and fat (0.3–0.4%). Additionally, the grape contains
significant amounts of potassium (0.1-0.2%), vitamin C (0.01–0.02%), and vitamin A (0.001–
0.0015%) and a small amount of calcium (0.01–0.02%) and phosphorus (0.08–0.01%).
The dietary consumption of grape and its products is associated with its medicinal
properties of grape as its constituents are antioxidant, anti-carcinogenic, immunomodulatory,
antidiabetic, anti-atherogenic, neuroprotective, anti-obesity, anti-aging and anti-infection. In
particular, several biological activities of resveratrol (3, 5, 40-trans-trihydroxystilbene), a
major compound extracted from the skin and seeds of grape is a natural phytoalexin
abundantly found in grapes and red wine, which has potent antioxidant property (Yadav et al
8
2009).
2.2.1 Chemical Characteristics of Grapes
2.2.1.1 Total soluble solids
Soluble solids can be measured directly from the refractive index and refractometers
are calibrated to give per cent total soluble solids (oBrix) values directly. In regard to the
sugar content, grapes contain glucose and fructose in similar amounts, while sucrose
contributes less than 1% and the starch concentration is practically negligible (Nelson 1985,
Conde et al 2007). However, a few high-sucrose content cultivars have been characterized in
Vitis rotundifolia and hybrids between V. labrusca and V. vinifera (Liu et al 2006). Cultivars
with more fructose than glucose can be harvested earlier due to the greater sweetness of this
sugar compared to glucose. As the fruit becomes mature, the fructose to glucose ratio
increases (Winkler et al 1974). Grape sugar content varies depending on the species, variety,
maturity, and health of the fruit. V. vinifera generally reaches a sugar concentration of 20% or
more at maturity. Immature berries contain very little sugars (2.0 mg/g) such as glucose and
fructose, as the berry matures, the total sugar concentration increases to 150-300 mg/g. Grape
sugar content is critical to yeast growth and metabolism. The concentration of glucose and
fructose on grape surfaces increases during berry ripening (Ribéreau-Gayon et al 2000). Total
sugar content on grape skin represents, on an average, 10 ± 5% of the equivalent content in
juice; however, the concentration of sugars associated with skin varies with the grape variety
(Varandas et al 2004). Wine grapes also tend to be very sweet: they are harvested at the time
when their juice is approximately 24% sugar by weight. By comparison, commercially
produced "100% grape juice", made from table grapes is usually around 15% sugar by weight
(Wine grapes 2010). The concentration of sugars which are major constituents of the juice
also has an important impact on its fermentation properties (Fleet 1998, 2001).
2.2.1.2 Titrable acidity
Grapes contain appreciable amounts of various organic acids. Malic acid may
constitute about half of the total acidity of grapes and wine. The malic acid concentration
decreases when grape mature, especially during hot period. Malic acid is one of the indicators
for determining harvest date. This acid leads to a flat taste into wine and this acid
transformation is necessary for wine with high content of malic acid. Tartaric acid is the other
major grape acid. This acid does not decrease during grape ripening but it is metabolized by
few skin microorganisms (Ough and Amerine 1988). Wines often are cooled near the end of
maturation to enhance early tartrate precipitation and avoid crystal deposition in the bottle
(Boulton et al 1996).
The acidity level is a very important quality factor in both table grapes and those used
for wine production. Consumer acceptance of table grapes and grape juice is strongly
influenced by the sweetness to acid balance (Winkler et al 1974). Acidity also determines the
9
suitability of the fruit for wine making. Excessive tartness correlates with low sugar levels
which give poor quality wine (Ruffner 1982). However, in warm climates, grapes with a low
pH and high acidity levels are generally desired for table wines. The brilliance and red
intensity of colored grapes is greater at moderate to high acidity and low pH. With low acidity
and high pH, they tend to be bluish and dull (Winkler et al 1974).
Organic acids are present in small amounts compared to sugars. However, they
contribute significantly to the overall taste (Nelson 1985). In general, organic acids do not
exceed more than 1% of the total juice weight, with tartaric acid usually the most important
acid followed by malic, citric, succinic, and other acids. Organic acids enhance grape flavour
and help to improve mouth-feel of grape (Liu et al 2006).
2.2.1.3 Volatiles
Besides sugars and organic acids, which have been investigated for the last few
decennia, phenolic compounds are extremely important constituents of grapes. Grape is a
phenol-rich and its phenolics are mainly distributed in the skin, stem, leaf and seed of grape,
rather than their juicy middle sections. Total concentration of phenolic compounds is about
2178.8, 374.6, 23.8, and 351.6 mg/g GAE (gallic acid equivalent) in seed, skin, flesh, and
leaf, respectively (Pastrana-Bonilla et al 2003). The compounds mainly include
proanthocyanidins, anthocyanins, flavonols, flavanols, resveratrols and phenolic acids
(Makris et al 2008).
Phenols, which are the principle source of wine color, mouth-feel and taste, make an
important contribution to wine quality (Gawel 1998). The main phenolic compound groups
present in grapes are the anthocyanins and flavan-3-ols. The anthocyanins are the red
pigments responsible for the colour of red grape skins and wine. The two main flavan-3-ol
monomers in grapes and wine are catechin and epicatechin (Gawel et al 2000). The
concentration of grape phenolics increases throughout berry development.
Phytochemicals in grapes are mostly phenolic compounds. According to their
molecular structure, the phenolic compounds are divided into four classes: one phenolic ring
(cinnamic acids and benzoic acids), two phenolic rings (stilbenes), three rings (anthocyanins,
flavonols and flavan-3-ols) and complex ring (ellagic acids) (Hartle et al 2005). Grape skins
contain abundant, widely varied phenolics. These phenolics play an important role in the
sensory properties and nutrition of berries and wines. Many studies demonstrated that these
phenolics could reduce the incidence of serious chronic diseases such as cancer and
cardiovascular diseases, due to their antioxidant abilities (Hartle et al 2005, Pezzuto 2008, Xia
et al 2010, Kocher et al 2011b).
2.3 Yeast
Yeasts occur as natural flora on the surface of grapes. Consequently, grapes are a
primary source of yeasts associated with wine fermentation. During alcoholic fermentation,
10
yeasts species and strains within the genera Hanseniaspora, Candida, Metschnikowia, Pichia
and sometimes Kluyveromyces grow during the early stages, but eventually die off due to
toxicity of the increasing concentration of ethanol, leaving Saccharomyces cerevisiae or
Saccharomyces bayanus as the dominant species to complete the fermentation (Fleet and
Heard 1993, Fleet 1998, 2001, Pretorius 2000, Bisson and Block 2002). Saccharomyces
cerevisiae and S. bayanus have become universally accepted as the principal wine yeasts.
Species that have been investigated for wine production thus far include those from the
Candida, Kloeckera, Hanseniaspora, Zygosaccharomyces, Schizosaccharomyces,
Torulaspora, Brettanomyces, Saccharomycodes, Pichia and Williopsis genera (Jolly et al
2006).
S. cerevisiae strains are recognized as GRAS and are normally used in human diets.
The protein content of yeasts rarely exceeds 60%, but yeasts have a reasonable concentration
of essential amino acids such as lysine, tryptophan and threonine. Yeasts are also rich in
vitamins (B group), and their nucleic acid content ranges from 4 to 10%. Yeasts, being larger
than bacteria, their size facilitate their easy isolation and separation. However, the specific
growth rate of yeasts with a generation time of two to five hours is relatively slow, compared
to that of bacteria (Boze et al 1992).
The growth of yeasts that occur naturally in grape juice has been quantitatively
examined during the fermentation of four wines that were inoculated with Saccharomyces
cerevisae. Although S. cerevisae dominated the wine fermentations, there was significant
growth of the natural species Kloeckera apiculata, Candida stellata, Candida colliculosa,
Candida pulcherrima, and Hansenula anomala (Heard and Fleet 1985). The number of
different species, as well as their endurance during alcoholic fermentation, is conditioned by
both the temperature of the must and the temperature during fermentation. These changes
determine the chemical and organoleptic qualities of the wine (Fleet and Heard 1993).
Yeasts are primarily responsible for the alcoholic fermentation of grape juice into
wine (Combina et al 2005). They originate from the flora of grapes and winery equipment and
from added starter cultures, if used (Fleet 2003). Wine fermentation is either performed
conventionally without inoculation or by the addition of selected wine yeast into grape juice.
In conventional wine making, natural (spontaneous) alcoholic fermentation of grape juice is
conducted by a sequence of different yeast species. At the beginning, non- Saccharomyces
yeasts, Kloeckera (teleomorph Hanseniaspora) and Candida in particular, initiate the
alcoholic fermentation of grape juice. These yeasts progressively die off with an increase in
ethanol concentration, and leave more ethanol tolerant Saccharomyces (S.) cerevisiae to
complete the fermentation (Ciani et al 2006, Clemente-Jimenez et al 2005). Infact, the quality
of wine is a direct consequence of the evolution of the micro-flora of the must during
fermentation. Yeasts play a central role in the fermentation process during winemaking.
11
Saccharomyces cerevisiae, ‘‘the wine yeast’’, is the most important species involved in the
alcoholic fermentation (Fleet and Heard 1993). The non-Saccharomyces yeasts contribute to
the fermentation since they can reach populations of about up 106–10
7 cells/ml, affecting both
the kinetics of growth and metabolism of Saccharomyces (Lema et al 1996). Hence, the
selection of the good strain having desirable properties is a prerequisite for the quality of wine
production (Degree 1993).
It was found that the production of red wines using selected yeasts under the
conditions tested contributed in improving the physical-chemical parameters of the wines
obtained, reducing some undesirable components in the finished product, like the volatile
acidity, ethyl acetate, methanol and acetaldehyde, when compared to the artisanal wine. The
addition of the yeasts promoted a decrease in the fermentation time, a good alcoholic yield,
demonstrating their dominance in the must, tolerates the sulfite added and the high alcoholic
grade (Dorneles et al 2005).
The diversity and the composition of the yeast also significantly contribute to the
sensory characteristics of wine. The growth of each wine yeast species is characterized by a
specific metabolic activity, which determines concentrations of flavour compounds in the
final wine. However, it must be underlined that, within each species, significant strain
variability has been recorded. The wide use of starter cultures, mainly applied to reduce the
risk of spoilage and unpredictable changes of wine flavour, can ensure a balanced wine
flavour, but it may also cause a loss of characteristic aroma and flavour determinants. Thus,
the beneficial contribution from the yeast increases when starter cultures for winemaking are
selected on the basis of scientifically verified characteristics and are able to complement and
optimize grape quality and individual characteristics (Romano et al 2003).
Yeasts impact on the color and pigment profiles of wines, especially red wines. Such
influences vary with the strain of S. cerevisiae and need to be considered in the portfolio of
properties when selecting yeasts for starter culture development (Medina et al 2005,
Hayasaka et al 2007). The impact of non-Saccharomyces yeasts on wine color is however still
relatively unexplored.
Keeping in view, significant role of yeasts in development of wines, strain selection
has become very important, as not all strains within a species necessarily show the same
desirable characteristics. For example, significant variability is found in the formation of
undesirable biogenic amines amongst strains within some non- Saccharomyces yeast species
(Caruso et al 2002). Innovative oenological traits can be introduced or exchanged by
hybridizing strains belonging to different species but with a sufficient genetic affinity for
them to mate. Interspecific Saccharomyces hybrids were found to be stable, vigorous and
possessing the parental oenological traits in novel and interesting combinations (Raineri and
Pretorius 2000).
12
Recently, interspecific hybrid strains between Saccharomyces species have been
described as involved in wine fermentations. Wine yeast hybrids between the species S.
cerevisiae X S.kudriavzevii and S. cerevisiae X S. bayanus as well as a triple hybrid S.
cerevisiae X S.kudriavzevii X S. bayanus were characterized. Several strains selected as
commercial wine yeasts also resulted from Saccharomyces hybrids (Gonzalez et al 2006,
Bradbury et al 2006). Therefore, hybrid strains appear as well adapt to the stress conditions
(Low pH, high sugar concentration and ethanol content) occurring during wine fermentations
(Belloch et al 2008), and their enological characterization confirms their interesting properties
according to the new trends in winemaking (Gonzalez et al 2007).
2.4 Pre-fermentation treatment
2.4.1 Effect of skin contact
Grape skin-contact with juice is an effective technique which can be standardized to
extract additional aroma compounds, such as volatile phenols, flavonoids, terpenes etc. from
grape skins for wine production that enhance the aroma intensity and quality of wines from
cultivars (Hernanz et al 2007). The skin contact technique is thus the most important process
that characterizes the elaboration of rose and red wines. Phenolic compounds from grapes and
wines are also receiving increasing interest because of diverse health benefits attributed to
them (Moreno et al 2007, Parker et al 2007, Katalinic et al 2010). In addition, skin contact
technique frequently displays higher colour intensity and sensitivity to oxidation of white
wines (Cheynier et al 1989, Macheix et al 1991). The total concentrations of free and bound
aroma compounds of the wine increased considerably with the skin contact. The results of the
gas chromatographic data and sensory evaluation showed that skin contact treatment
improved the aroma intensity (particularly terpene-like character) and wine quality
(Cabaroglu and Canbas 2002).
Skin-contact may, however, also result in the extraction of skin compounds
detrimental to wine quality, such as certain phenols and acetamides. The temperature during
skin contact is therefore of critical importance. Harvesting during the night, cooling of the
grapes before or after crushing and skin contact at low temperatures has been found to benefit
the production of wines with higher varietal qualities. Marais and Rapp (1988) reported that
wines produced from juice subjected to low temperature skin-contact are generally of a higher
quality than those wines produced from the free-run juice or juice subjected to skin-contact at
elevated temperatures.
Skin contact by maceration has been used to increase the aroma in white varietal
wines through the extraction of aromatic compounds and nonvolatile sugar-bound glycosidic
conjugates (Selli et al 2003). Further, there is reduction in titrable acidity of the macerated
musts which is due to tartaric acid neutralization by the potassium liberated from the skins
(Ribereau-Gayon et al 2006). It has been found that the composition and organoleptic
13
properties of red wine are predominantly affected by maceration and the extraction of grape
derived phenolic components and hence their subsequent reactions in wine (Basha et al 2004,
King et al 2003, Sims and Bates 1994).
2.4.2 Effect of Enzymatic treatment
Amylase, pectinase and cellulase are the common enzymes used for food purposes
such as clarification of juices and beverages, starch and cellulose hydrolysis etc. These
enzymes account for approximately 20% of the world enzyme market (Mantyla et al 1998).
The first enzymes used in enology were pectinases but, recently, enzyme companies have
modified the pectinase preparations (which mainly contain polygalacturonase, pectin esterase
and pectin lyase activities) by adding small amounts of cellulase and hemicellulase in order to
achieve a more complete breakdown of the cells and to enhance color extraction (Parley 1997,
Gil and Valles 2001, Calre et al 2002). Infact, enzymes are used to increase the grape must
yield during pressing, facilitate the settling of musts, and improve clarification and filtration.
The principal enzyme groups used in winemaking are pectinase, cellulase, hemicellulase,
oxidoreductase, protease and β-glycosidase. Evidently, the enzymatic preparations and their
principal and secondary activities are key factors in the results obtained in the products
(Pimenta-Braz et al 1998, Guerin et al 2009). Commercial pectic enzyme preparations are the
extracellularly produced in controlled fermentations by pure, genetically engineered food
grade strains of Aspergillus niger and/or Trichoderma longibrachiatum fungi.
Pectinase enzyme preparations have only been commonly used in wine production
since the early 1970’s, and mostly to improve juice yield from crushed grapes, and to
facilitate clarification of juice. The addition of exogenous pectinase enzymes is beneficial
because although they occur naturally in the fruit, they exist in such small quantities that they
are of little practical value over the short time and under the conditions of the must and juice
handling processes (Felix and Villettaz 1983).
Enzymes used in must treatments are related to skin maceration as an additional and
complementary process. Commercially available enzymes have been widely used in the
oenological industry in wine- producing countries to improve important characteristics of
wines, such as aroma and color. Furthermore, enzymes are used extensively in the fruit-
processing industry and other food sectors (Kashyap et al 2001). The use of pectolytic
enzymes has been shown to be suitable to improve the extraction of colour in red wines
(Revilla and Jose 2003, Bautista-Ortin et al 2005), aroma compounds (Canal-Llauberes 1990,
Castro-Vazquez et al 2002, Cabaroglu et al 2003) and soluble polysaccharides (Ayestaran et
al 2004, Doco et al 2007) from the skins and pulp of the grapes.
Among a number of studies on enzymatic treatment of grape must, a commercial
pectinase from Aspergillus niger containing various polysaccharases has been found to clarify
the white grape juice to an extent of 98-99% and also degrade the grape mash by 25- 30%.
14
Grapes of three white cultivars treated with a commercial pectic enzyme preparation have
revealed that pectic enzyme treatment increases the higher alcohols, free hydroxycinnamic
acids and volatile phenols, whereas amounts of most esters, herbaceous alcohols and
hydroxycinnamate derivative esters get reduced. When tasted by an expert panel, differences
were reported in sensory characteristics, with wines from untreated judged of higher quality
than those from treated grapes (Lao et al 1997).
The effect of two commercial pectolytic enzymes using “musts” from sun-dried
grapes of the Pedro Ximenez variety on oenological parameters before and after enzyamatic
treatments with pre-fermentative maceration at room temperature for three hours has been
reported. It was found that resulting wines vary significantly in total soluble solids (OBrix)
and in the final sensorial controls. The enzyme treatment showed no effect on the total content
of polyphenols and other chemical characteristics. A higher qualitative level has been
reported with regard to aroma and gustative quality. The results also demonstrated that total
juice yield improved after enzyme addition together with maceration (Espejo and Armada
2010).
2.5 Ethanol Fermentation
The alcoholic fermentation is the primary fermentation, where yeasts transform
sugars in grape juice (mainly glucose and fructose) into ethanol (the main metabolite of wine)
and carbon dioxide. This process can be conducted either as an indigenous/wild fermentation,
or as an induced /seeded fermentation. With indigenous fermentation, yeasts resident in the
grape juice initiate and complete the fermentation. With seeded fermentation, selected yeast
strains, generally those of Saccharomyces cerevisiae or Saccharomyces bayanus, are
inoculated into the juice at initial populations of 106-107 cells/mL.
One molecule of glucose or fructose yields two molecules each of ethanol and carbon
dioxide. The theoretical conversion of 180 g of sugar into 88 g of carbon dioxide and 92 g of
ethanol means that yield of ethanol is 51.1% on a weight basis. In model fermentation starting
about 22 to 24 % sugar, 95 % of the sugar is converted into ethanol and carbon dioxide, 1% is
converted into cellular material, and the remaining 4% is converted to other end products.
This percentage may vary depending upon inoculum size, fermentation temperature and
nutrient availability (Boulton et al 1996). Fermentations are initiated by the growth of various
species of Candida, Debaryomyces, Hanseniaspora ,Hansenula, Kloeckera, Metschnikowia,
Pichia, Schizosaccharomyces, Torulaspora, and Zygosaccharomyces. Their growth is
generally limited to the first two or three days of fermentation, after which they die off.
Subsequently, the most strongly fermenting and more ethanol tolerant species of
Saccharomyces take over the fermentation.
Saccharomyces cerevisiae is used to ferment grape musts in strictly anaerobic
conditions, subjected to a prefermentative treatment of skin maceration and following a short
15
aeration after 48 h of fermentation. It has been reported that the short aeration has no
significant effect on the unsaturation index of the cellular fatty acids, although it increases the
ergosterol/phospholipid ratio. This is reflected by an increase in the growth rate, viability and
fermentative capacity of the yeasts (Fleet and Heard 1993).
The risk of stuck fermentation and the development of several wine faults can also
occur during this stage which can last from 5 to 14 days for primary fermentation and
potentially another 5 to 10 days for a secondary fermentation. Fermentation may be done in
stainless steel tanks, which is common with many white wines like Riesling, in an open
wooden vat, inside a wine barrel and inside the wine bottle itself as in the production of many
sparkling wines (Robinson 2006, Kunze 2004).
Fermentation of the juice was traditionally conducted in large wooden barrels or
concrete tanks, but most modern wineries now use sophisticated stainless steel tanks with
facilities for process management. However, some premium-quality white wines (e.g.
Chardonnay) may be fermented in wooden (oak) barrels. White wines are generally fermented
at 10-18oC for 7-14 days or more, where the lower temperature and slower fermentation rate
favour the retention of desirable volatile flavour compounds (Robinson 2006).On the other
hand, red wines are fermented for about 7 days at 20-30oC, where the higher temperature is
necessary to extract colour from the grape skins. In winemaking the temperature and speed of
fermentation is an important consideration as well as the levels of oxygen present in the must
at the start of the fermentation. There are number of factors which affect yeast fermentation
performance like yeast strain employed, fermentation temperature, media composition, pH,
substrate concentration, mode of substrate feeding, osmotic pressure, ethanol concentration,
membrane composition etc (D’ Amore 1992). Some of these important factors are reviewed
below:-
2.6 Factors affecting fermentation
2.6.1 Effect of Temperature
Temperature is one of the most important factor that strongly affects the fermentation
as it directly affects the microbial ecology of the grape must and the biochemical reactions of
the yeasts. Also the number of different species, as well as their endurance during alcoholic
fermentation, is conditioned by both the temperature of the must and the temperature during
fermentation. These changes determine the chemical and organoleptic qualities of the wine.
Some strains are predominant at low temperatures while others at high ones (Fleet and Heard
1993).
The influence of elevated temperatures from 10 to 25°C at 5°C intervals on yeast
growth and fermentation products have been studied in mixed cultures of Kloeckera
apiculata and Saccharomyces cerevisiae in grape juice. In these experiments carried out at 10
and 15°C, K. apiculata grew and survived longer compared to trials conducted above 20°C. In
16
most cases, higher temperatures i.e above 25°C stimulated the production of higher alcohols
but lowered the formation of esters and acetaldehyde (Erten 2006).
Temperature not only affects the fermentation but also the yeast metabolism, and
results in the formation of secondary metabolites such as glycerol, acetic acid, succinic acid,
etc. which determines the chemical composition of the wine (Lafon-Lafourcade 1983). It is
well known that fermentation at 35oC is very restrictive and the effect of higher temperatures
is a premature end of fermentation, which means that fermentation is incomplete and the
ethanol concentration is low (Larue et al 1980).
Temperature can affect the sensitivity of yeast to alcohol concentration, growth rate,
rate of fermentation, viability, length of lag phase, enzyme and membrane function etc.
Because yeast strains differ in response to temperature, the optimum temperature for
vinification can vary widely. A mixed response to fermentation temperature (15- 35°C) on
mixed strain population of Saccharomyces cerevisae has been observed by Torija et al (2001)
whereby alcohol yield was higher at lower temperature while at higher temperature secondary
metabolites were increased.
White wines are often fermented in the range of 10- 20°C. Nevertheless, some
European wineries still prefer fermentation temperatures between 20 and 25°C. As yeast
strains differ in response to temperature, the optimum temperature for vinification can vary
widely (Jackson 2000b). With the effective control of fermentation temperature by the wine
industry, low temperature alcoholic fermentations are becoming more frequent with the aim
to produce white and rose wines with more pronounced aromatic profile. Wines produced at
low temperatures (10-15ºC) are known to develop certain additional characteristics of taste
and aroma (Llauradó et al 2002, Torija et al 2003).
Typically, white wine is fermented between 64-68°F (18-20°C) though a wine maker
may choose to use a higher temperature to bring out some of the complexity of the wine. Red
wine is typically fermented at higher temperatures up to 85°F (29°C). Fermentation at higher
temperatures may have adverse effect on the wine in stunning the yeast to inactivity and even
"boiling off" some of the flavors of the wines. Some winemakers may ferment their red wines
at cooler temperatures, more typical of white wines, in order to bring out more fruit flavors
(Robinson 2006). Further, increasing of fermentation temperature from 15 to 30°C enhances
wine color, reduces herbaceous flavor, increases black current flavor and increases perceived
acidity.
2.6.2 Effect of pH
pH of the fruit wines throughout the period of fermentation ranges from 3.0 to 4.8.
The pH strongly affects the malolactic activity of the cell. The rate of malic acid degradation
is highest at pH 3.2- 3.4. According to Fleet (1998), pH directly affects wine stability. This
may be due to the fact that at a pH close to neutral (7.0) most micro organisms like bacteria
17
and molds including some yeasts become more active for fermentation and subsequent
spoilage of wine, whilst a pH of 3.5 eliminates most of the microbes, and favors only few of
the micro organisms for fermentation. Yeasts can grow in a pH range of 4 to 4.5 and molds
can grow from pH 2 to 8.5, but favour low pH (Mountney and Gould 1988).
Yeast cell growth rate and grape juice fermentation rate have been shown to be
dependent on initial Brix and pH as well as on fermentation temperature. Variation between
pH 3.5 and 4.0 caused small but significant fermentation rate changes with more variation at
lower pH. Optimum fermentation rates for grape juice occur between 15 and 20° initial Brix,
and so does optimum yeast growth rate with these media. Temperature interactions with pH
have been demonstrated on rate of fermentation and yeast growth rates. The lower the pH
with increasing temperature, greater effects are observed on rate of fermentation and rate of
yeast growth (Ough 1966).
Grape juice pH largely determines the pH of the wine after fermentation. High wine
pH has a negative impact on wine colour, stability and taste (Somers 1977). Adjustment of pH
with tartaric acid during vinification is routinely applied to protect against such impacts
whereby the aim is generally to bring pH to 3.0-3.3 for white wines and to 3.3-3.5 for red
wines (Godden and Gishen 2005).
pH also plays an important role in aging, clarifying or fining. As the strength of the
relative charge of suspended particles decreases in the wine, the pH of the wine increases. At
high pH, organic protein fining agents may possess a positive charge insufficient to bind to
the negatively charged particulates, thus potentially increasing the turbidity of the wine. This
phenomenon is called "overfining" (Rotter 2008).
The effect of the interaction between pH (2.9-3.5), alcohol concentration (12.5-14.5%
v/v) and wine matrix (Chardonnay, Grenache blend and Cabernet Sauvignon) on malolactic
fermentation (MLF) induced by two commercially available directly inoculated Oenococcus
oeni starter cultures has been reported while wine matrix has greatest impact on the rate of
MLF, it is followed by pH and alcohol; the matrix effect was shown to be significantly
modified by pH and the bacterial culture (Paul and Hoger 2003).
2.6.3 Effect of initial sugar level
Sugar is the main substrate for fermentation of fruits juice into alcohol (Keller 2010);
although, other food nutrients such as protein and fats can be broken down by some micro
organism in some cases where sugar is limited, but as long as sugar is present yeast cells will
continue the process of fermentation until other factors that affect the growth of yeast become
unfavourable (Dickinson 1999). According to Garrison (1993) sugars are the most common
substrate of fermentation to produce ethanol, lactic acid, hydrogen and carbon dioxide.
The concentration of fermentable sugars in grape musts ranges between 125 and 250
g/L (Fleet and Heard 1993). It is likely that the initial concentrations of glucose and fructose
18
(main grape sugars) will selectively influence the species and strains of yeast present during
fermentation. Musts with lower concentrations of sugar start to ferment fast and the sugar is
fermented to completion, whereas musts with high sugar content ferment slowly and may be
incomplete. High sugar concentrations inhibit fermentation by high osmotic pressure, which
draws water from the yeast cells in particular (Margalit 1997).
The effect of initial sugar concentration on time of fermentation has also been
observed as higher sugars tend to prolong fermentation (Borzani et al 1993). Higher initial
sugars also possess better retention of ascorbic acid, increase in concentration of total esters
and phenols thus improving the wine quality (Attri 2009).
The various yeast species and strains that develop during the overall fermentative
process metabolize grape juice constituents, principally the sugars, to a wide range of volatile
and non-volatile end-products, which influence and determine the types and concentrations of
many products that contribute to the aroma and flavour characteristics of the wine. Although
sugar is an important substrate of fermentation, higher sugar concentration inhibits the growth
of micro-organisms (FAO 2010). However, Yeasts are fairly tolerant of high concentrations
of sugar and grow well in solutions containing 40% sugar (Steinkraus 1992). At
concentrations higher than this, only a certain group of yeasts – the osmophilic type – can
survive. There are only a few yeasts that can tolerate sugar concentrations of 65-70% and
these grow very slowly in these conditions (Board 1983). High sugar musts can also lead to
higher acetic acid production by yeast during fermentation (Monk and Cowley 1984).
Stuck and sluggish fermentations are more frequently observed in vintages of well
matured grapes with high sugar concentrations (Gafner and Schütz 1996). High osmotic
pressure caused by high sugar concentrations can contribute to stuck fermentations (Kunkee
1991).Yeast strains also differ in their ability to ferment musts containing high levels of sugar
(Salmon et al 1993). Higher inoculation levels of yeast should be used to ferment juice with
high sugar concentrations. Strains of Saccharomyces cerevisiae, such as L2226, have been
recommended in high sugar fermentations as they can be more tolerant to high sugar levels
than other yeast strains (Lallemand 1997).
2.6.4 Effect of Nitrogen and Phosphorus supplementation
The nitrogen content of grape juice affects both yeast cell growth and fermentation
rate. The Yeast Assimilable Nitrogen (YAN) content of grape must regulates the growth and
metabolism of fermentation yeast. Yeast cells are subjected to stress during alcoholic
fermentation by non-optimum YAN availability. It has been found that low YAN is
associated with lagging and incomplete fermentation, and sulfide evolution, whereas excess
of YAN is associated with unbalanced production of some aromatic compounds (Bell and
Henschke 2005). Nitrogen supply strongly influences yeast growth and metabolism during
fermentation (Beltran et al 2005). The concentration and ratio of nitrogenous compounds
19
present in grape juice depend on grape variety, harvest time, and vineyard management
factors. Yeast assimilable nitrogen (YAN) includes ammonium and the α-amino nitrogen of
amino acids (excluding proline, which is not used by yeast as a nitrogen source under
anaerobic conditions). Proline and arginine are usually the most abundant nitrogenous
compounds in grape juice (30–65% of total amino acid content) and ammonium makes up ca
40% of grape juice YAN (Beltran et al 2004).
In the preliminary study with a Chardonnay must in combination with a high nitrogen
demanding yeast, Torrea and Henschke (2004) showed that an intermediate concentration of
YAN produced a wine with most preferred sensory attributes. However, they suggested that
more work is needed to determine the optimal YAN concentration of musts of other varieties
in combination with yeast having different demands for YAN. It has been recommended in
other studies also to supplement deficient musts with diammonium phosphate (DAP) at the
start of fermentation to ensure an adequate population of yeast (Bely et al 1990, Ugliano et al
2007, Kocher and Pooja 2011).
Since nitrogen depletion is often the cause of stuck and sluggish fermentations
(Salmon 1996), ammonium salts such as diammonium phosphate (DAP) are frequently added
to grape must fermentations to alleviate nitrogen deficiency. The timed addition of DAP to
grape must significantly affects the amount of ethyl carbamate (EC) produced in wine. This
effect was found to be yeast strain dependent: Pasteur Red produced less EC when DAP was
added at the onset of fermentation, whereas strain 522 produced less EC when DAP was
added during the later stages of fermentation. EC production by strain EC1118 was less
affected by the timing of DAP addition. The metabolically enhanced yeast strains Pasteur
RedEC-, 522EC-, and EC1118EC-, which constitutively express DUR1,2, significantly
reduced the amount of EC produced regardless of the timing of DAP addition (Adams and
Hennie 2010).
Further, fermentation process not only depends upon the quantity of N and P added,
but also on the quality of these supplements. Various nitrogen and phosphorus sources like
ammonium sulphate, diammonium phosphate, potassium dihydrogen phosphate, di potassium
hydrogen phosphate, amino acids, and diammonium hydrogen phosphate have been used to
carry out fermentation efficiently and rapidly. KH2PO4 (46.1g/L) was found to be better
phosphorus source in comparison with K2HPO4 (45.3 g/L) and (NH4)2SO4 (46.1 g/L) was the
best nitrogen source as compared to NaNO3, (NH4)H2PO4, NH4NO3 for the production of red
wine from ‘Siahe sardarsht’ grape variety (Asli 2010).
For a number of fruits, the nitrogen content is insufficient and supplements are made
by the addition of ammonium phosphate or of diammonium phosphate (DAP) before
fermentation. Glutamate is also another preferred nitrogen source because it is utilized
directly for biosynthesis. Glutamine can generate both ammonium ion and glutamate.
20
Nitrogen–containing compounds in grape juice may meet one of three fates: (1) Utilized
directly in biosynthesis; (2) converted to a related compound and utilized in biosynthesis; or
(3) degraded releasing nitrogen either as free ammonium ion or as bound nitrogen via a
transamination reaction (Boulton et al 1996).
2.7 Post fermentative treatments
Consumer acceptability is the final goal of wine development. The crude wine
obtained after fermentation contains yeast, protein hazes and residual sugars and does not
have complete quality (sensory) attributes. Hence, it requires post fermentative treatments
(finishing) to make wine potable. In fact, there are five goals of “finishing” a wine: clarity,
stability, compositional adjustment, style development and packaging. It is important,
especially in white wines; that the wine at the point of consumption should not be cloudy or
contain haze or precipitate. This includes clarification, filtration, fining, stabilization, ageing
and blending of wines. It is also important to prevent unwanted microbial growth from
occurring in the wine after primary fermentation is complete as this will affect the flavour and
aroma profile. Saccharomyces autolysis will replenish nutrients in the wine making them
available for other organisms. Saccharomyces does not consume all possible bacterial energy
sources. Many spoilage organisms are obligate aerobes so the wine ‘must’ be protected
against exposure to air once the carbon dioxide blanket generated during fermentation has
dissipated.
Post fermentation treatment of crude wine can be accomplished by different means:
2.7.1 Racking
“Racking" of wine is the process of separating wine from its sediment, or lees, and
transferring the wine into another container using a siphon. Repeated racking produces the
clarity required in wine, especially if it is aged in a barrel. The wine is repeatedly racked to
leave behind less and less precipitate. During the repeated pouring, the wine is also given a
chance to rid itself of the excess carbon dioxide from fermentation. As the CO2 escapes,
oxygen enters the wine with each transfer, helping eventually to age. Besides clarification,
racking also provides suitable conditions for oxygen to dissolve in the wine, at a rate varying
from 2.5 to 5 mg/L. Oxygen eliminates certain unpleasant reduction odors (H2S), as well as
iron (ferric casse) and is also responsible for intensifying color of wine (Amerine and
Roessler 1983).
2.7.2 Effect of fining agents
In winemaking, fining is the process where a substance (fining agent) is added to the
wine to create an adsorbent, enzymatic or ionic bond with the suspended particles, producing
larger molecules that will precipitate out of the wine more readily and rapidly. A variety of
fining agents are commercially available to the wine industry, including proteins and
inorganic ion exchangers. These fining agents are essentially used to control the levels of
21
phenolics in wine, but they also have the potential to interact with other wine components,
most often as a side effect. They are therefore expected to influence, at least in part, the
potential for wine protein haze formation. Six common fining agents—casein, egg albumin,
isinglass, chitosan, chitin, and polyvinylpolypyrrolidone (PVPP)— have been analyzed to
assess their effects on wine protein haze-forming potential and on the levels of proteins and
phenolic compounds in a Muscat of Alexandria wine (Chages et al 2012). It was found that
benotonite was not most effective and the fining agents did not significantly affect wine
protein content but removed considerable levels of polyphenols and presented no apparent
effect on protein stabilization of the fined wines. Further, precipitation of combined
particulates is faster at lower temperatures (except for bentonite). However, some fining
agents are less temperature sensitive than others (e.g. isinglass is less temperature sensitive
than gelatin. According to Rotter (2008), wines should be low in dissolved CO2 when fined,
since dissolved CO2 will tend to keep particulates in solution and inhibit settling.
The effect of different fining agents, used at different concentrations, on the
antioxidant status of fined wines was studied by Yildirim (2011).The results demonstrated
that the use of a combination of gelatin and Kieselsol led to the highest total phenol value
(3,491 mg/L GAE) and antioxidant activities (29%) among the tested fining agents. Wines
were mostly negatively affected by the use of egg white as an agent and led to the lowest
value of total phenol (3,038 mg/L GAE) and the lowest rate of antioxidant activity (26%).
Significant differences (p<0.05) were determined between gelatin, egg white, and the control
groups. The results of the grouping of analyzed parameters in n-dimensional space, with
different fining agents at different concentrations, demonstrated the importance of a low
concentration of fining agents for high antioxidant activity and total phenols.
2.7.3 Ageing
The ageing of wine, and its ability to potentially improve wine quality, distinguishes
wine from most other consumable goods. Aged red wines possess significantly different
polyphenolic composition compared with young ones, mainly not only to formation of
polymeric compounds but also because of oxidation, hydrolysis, and other transformations
that may occur in native grape phenolics during aging (Arnous et al 2001).
After fermentation and clearing, wine needs to be aged. Aging can be done by storing
the wine in wooden or the wine bottle. Aging is done in a wooden vat for least six months in
most red wine to add the flavour of the wood to the wine (Wimalsiri et al 1971). Red wines
are commonly matured in oak barrels and this is thought to assist with stabilisation of colour.
The nature of the handling operations of wines in barrels means that they are intermittently
mildly aerated, and this can lead to formation of acetaldehyde bridged polymeric pigments
(Ribereau-Gayon et al 1983). All wines treated with oak showed a trend towards slightly
higher hue values than the unoaked wines.
22
In general, wines with a low pH (such as Pinot noir and Sangiovese) have a greater
capability of aging. The white wines with the longest aging potential tend to be those with a
high amount of extract and acidity. The acidity in white wines plays a similar role that tannins
have with red wines in acting as a preservative. The process of making white wines, which
includes little to no skin contact, means that white wines have a significantly lower amount of
phenolic compounds, though barrel fermentation and Oak aging can impart some phenols.
Similarly, the minimal skin contact with rosé wine limits their aging potential (Robinson
2003). With red wines, a high level of flavor compounds, such as phenolics (most notably
tannins); will increase the likelihood that a wine will be able to age. Wines with high levels of
phenols include Cabernet Sauvignon, Nebbiolo and Syrah (Robinson 2006).
23
CHAPTER III
MATERIALS AND METHODS
3.1 MATERIALS
3.1.1 Yeast culture
The yeast culture Saccharomyces cerevisae strain 35, used in the present study for
fermentation of grape juice was procured from the Department of Microbiology, Punjab
Agricultural University, Ludhiana. The culture was maintained on Glucose Yeast Extract agar
(GYE agar) slants and stored at 40 C. The yeast was subcultured fortnightly on the same
media:-
Composition of Glucose Yeast Extract agar (gL-1
)
Glucose : 10.0
Peptone : 5.0
Yeast extract : 5.0
Agar : 20
Distilled water (to make volume) : 1000 ml
pH : 5.5
3.1.2 Chemicals used
All chemicals used were of AR/GR grade. Pectinase (1000 units/100mg) and
cellulase (10000 units/100mg) were procured from SISCO research laboratories Pvt. Ltd.
(Mumbai) and Amylase from Novozyme.
3.1.3 Grapes
The grapes used in the present study were of Punjab purple (Syn H516) variety which
were procured from Department of Fruit Science, PAU, Ludhiana (Plate I).
3.2 METHODS
3.2.1 Selection of fruit
Healthy ripened grapes were selected after manual sorting and discarding of defective
bunches. Grape berries were destemmed and then washed with boiled and cooled water and
used for extraction of juice. Juice was extracted aseptically in an electric juicer under hygienic
conditions.
3.2.2 Extraction of juice from Grapes
Healthy ripened grapes were destemmed, washed with boiled and cooled water as in
3.2.1. With the help of electric juicer, extraction of juice was carried out and the juice after
supplementation of KMS (0.01%) was stored in flasks (Fig. 1).
24
GRAPES
DESTEM AND WASH
EXTRACTION OF JUICE IN THE JUICER
STORAGE OF GRAPE SKIN AT 4OC
GRAPE JUICE
ADD KMS @0.01%
KEEP IN FLASKS AND STORE AT 4 O
C TILL USE
Fig. 1: Flow diagram for extraction of juice from grapes
3.2.3 Physico-chemical analysis of grape juice
The physico-chemical analysis of Grape juice included the estimation of its TSS, pH,
acidity, Brix acid ratio, ascorbic acid and total phenols.
3.2.4 Preparation of sugar solution
Granulated sucrose (commercial sugar) was procured from the local market. The
stock sugar solution was prepared by dissolving 250g of granulated sucrose in 250ml of water
boiled for 10 min and then allowed to cool at room temperature and used for supplementation
in grape juice to adjust the brix.
3.2.5 Preparation of grape ‘must’ and its ethanolic fermentation
The extracted grape juice was subjected to prefermentative treatment for preparation
of grape ‘must’ before its fermentation by Saccharomyces cerevisae to produce ethanol as:-
3.2.5.1 Pre-fermentation treatment
3.2.5.1.1 Skin treatment
The stored grape juice was subjected to skin treatment to improve the color intensity
and volatiles (Plate II). Response surface methodology (RSM) was adopted in the
experimental design as it emphasizes the modeling and analysis of the problem in which
response of interest is influenced by several variables and the objective is to optimize this
response (Design Expert 8.0). A five-level three-factor Central Composite Rotatable Design
(CCRD) was employed. For the three factors namely temperature, time and skin weight,
CCRD was made up of a full 23 factorial design (eight points) augmented with six replications
of the centre points (all factors at level 0) and six star points (points having one factor at an
axial distance to the centre of ± α, whereas other two factors at level 0) (Table 1). The axial
25
Plate I: Punjab purple (Syn H516) grapes
Plate II: Flasks exhibiting different levels of Skin treatment of juice
25
distance α was chosen to be 1, and a set of 20 experiments (each with 300ml grape juice) were
carried out:
Total no. of experiments= 2 no. of variables
+ 2 x no. of variables + central points
For three variables:
Total no. of experiments= 23 + 2 x 3 +6 = 20
Five different levels for each experiment in coded form were –α, -1, 0, +1, +α
Where,α = [2] no.of variables/4
= [2]3/4
= 1.682
Table 1: Level of different process variables for skin treatment of Grape juice
Levels Independent
variables -1 0 +1
Temperature (oC) 10 15 20
Time (hr) 1 8.50 16
Skin weight (Kg) 0.25 0.55 0.85
The independent variables were skin weight (X1), time of contact (X2), temperature
(X3).The three independent variables were coded as -1.682 (lowest level) -1, 0, 1 (middle
level) and +1.682 (highest level). The experimental design matrix in coded (X) form and at
the actual level (X) of variables is presented in Table 2.
Table2: Experimental design of process variables and values of experiment data for
optimization of skin treatment of Grape juice
Coded values Uncoded values
Experiment
No.
Temperature
(0C)
Time
(hr)
Skin
weight(Kg)
Temperature
(0C)
Time (hr) Skin weight
(Kg)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0
0
0
0
+1
-1
0
+1.682
0
-1.682
+1
-1
+1
+1
-1
0
0
0
0
-1
0
-1.682
+1.682
0
+1
+1
0
0
0
0
+1
-1
-1
-1
+1
0
0
0
0
-1
-1.682
0
0
0
+1
+1
0
0
+1.682
0
-1
+1
+1
-1
-1
0
0
0
0
-1
15.00
15.00
15.00
15.00
20.00
10.00
15.00
23.41
15.00
6.59
20.00
10.00
20.00
20.00
10.00
15.00
15.00
15.00
15.00
10.00
8.50
4.11
21.11
8.50
16.00
16.00
8.50
8.50
8.50
8.50
16.00
1.00
1.00
1.00
16.00
8.50
8.50
8.50
8.50
1.00
0.05
0.55
0.55
0.55
0.85
0.85
0.55
0.55
1.05
0.55
0.25
0.85
0.85
0.25
0.25
0.55
0.55
0.55
0.55
0.25
26
A second degree polynomial equation (Eq.1) was provided for the experimental design by the
statistical software (Design Expert Software) so as to estimate the response of the dependent
variable. The response function (y) was related to the coded variables (xi, i=1,2 and 3) by a
second degree polynomial equation as given below:
y=bo+b1x1+b2x2+b3x3+b12x1x2+b13x1x3+b23x2x3+b11x12+b22x2
2+b33x32+ε (1)
The variance for each factor assessed was partitioned into linear, quadratic and interactive
components. The coefficients of the polynomial were represented by bo (constant), b1, b2, b3
(linear effects); b12, b13,b23 (interaction effects); b11,b22,b33 (quadratic effects); and ε (random
error). The significant of all terms in the polynomial functions was assessed statistically using
F-value at probability (P) of 0.05 so as to determine the fitness of the model. The statistical
analysis of the data and three- dimensional (3D) plotting were performed using Design Expert
software ‘DE-8’. The validation of the model was also carried out at 300ml as well as 5L
levels.
3.2.5.1.2 Enzyme treatment
The skin treated juice was subjected to different enzyme treatment i.e. Pectinase
(0.25ml/100ml), Amylase (0.085ml/100ml), Cellulase (0.125ml/100ml), Amylase +
Pectinase, Pectinase + cellulase, Amylase + Cellulase, Amylase + Pectinase + Cellulase (Fig.
2). The temperature of the enzymatic treatment combinations was adjusted 450C for 6h
(Kocher and Pooja 2011).
GRAPE JUICE (300ml in each flask)
ADD ENZYMES INDIVIUALLY AND IN COMBINATIONS ACCORDING TO
SPECIFIED CONCENTRATION I.E. PECTINASE (0.25ML/100ML), AMYLASE
(0.085ML/100ML), CELLULASE (0.125ML/100ML)
KEEP FLASKS AT 450C TEMPERATURE FOR 6h
CENTRIFUGE THE GRAPE JUICE (6000 rpm for 10 minutes)
COLLECT CLARIFIED GRAPE JUICE, HEAT TO 900C
MEASURE OD 550 nm
CALCULATE % DECREASE IN OD 550 nm
Fig. 2: Flow diagram for enzymatic treatment of skin pretreated Grape juice
27
At the end of the enzyme treatment, the suspension was centrifuged at 6,000 rpm for 10 min
and the clarified grape juice was heated at 90oC for 5 min to inactivate the enzyme. The
decrease in OD 550 nm (Fig. 2) was then calculated using the following expression:
% decrease in OD = OD Initial
OD Final - OD Initial x 100
3.2.5.2 Preparation of inoculum
The inoculum of S. cerevisae strain 35 was prepared in glucose yeast extract broth for
the fermentation where a loopful of slant culture was inoculated in 250 ml Erlenmeyer flasks
containing 100 ml GYE broth. It was incubated at 100 rpm and at 28±2oC for 24 hrs to raise
seed inoculum. From the seed inoculum, starter culture was prepared. For the purpose 2% of
seed inoculum was inoculum was inoculated in pasteurized Grape juice (diluted 1:1 with
sterile water) and incubated at 28 oC for 24 hrs under shaking (100 rpm) conditions.
3.2.5.3 Alcoholic fermentation of Grape juice
The pretreated grape juice (300 ml) was taken in the 500 ml Erlenmeyer flasks and
chaptalized with sugar syrup to adjust sugar level at 20 oB. The ‘must’ so prepared was
supplemented with different concentrations (50-250 mg/100ml) of Diammonium hydrogen
ortho phosphate (DAHP) to study the effect of N and P supplementation on ethanolic
fermentation inoculated using 5% starter culture (prepared above) and incubated at 25 oC. The
periodic samples of fermenting grape juice were taken, spun at 6000 rpm for 5 minutes and
analyzed for TSS, pH and ethanol content (as per procedure in 3.3) till no further decrease in
oBrix level was noted.
The fermentation efficiency of different experiment treatments was calculated as:
Fermentation efficiency = 100x(%v/v) ethanol lTheoretica
(%v/v) produced ethanol Actual
Theoretical Ethanol % v/v = sugar utilized (g) X 0.64
Sugar utilized=Available sugar- sugar present after fermentation
3.2.5.4 Effect of Recycled yeast
The effect of recycled yeast (yeast obtained from previous lots) was studied on
ethanolic fermentation of subsequent lots at 300ml fermentation level in 500ml Erlenmeyer
flasks. The fermented grape must of previous lot was partially removed after cessation of
fermentation to form 5-20% of the inoculum i.e. 15ml to 60ml to report four different
inoculum levels were used i.e. 5%,10%,15%,20%. The latter was used to inoculate fresh
‘must’ at similar fermentation conditions in duplicate as in 3.2.5.2. Periodic samples were
taken and analyzed for ethanol content, pH and Brix as described earlier. The recycled yeasts
were used upto 2nd level of recycling.
28
3.2.6 Post fermentative treatments
3.2.6.1 Siphoning
The flasks/ bottles containing prepared wine were stored at 15 o
C and debris (‘lees’)
was allowed to settle. No settling or fining agent was added. The siphoning (racking) was
repeated after every 15 days till there was no further settling of debris in the bottles. At the
time of each siphoning, 0.01% (v/v) KMS was added to prevent any microbial contamination.
3.2.6.2 Bottling and storage
The cleared wine was stored in glass bottles (washed earlier with boiling water) for
upto 4 months (with decantation treatment every 2 weeks) under refrigerated conditions.
3.2.6.3 Shelf life study
Shelf life of grape wine of variety Punjab purple stored at refrigerated temperature (4
oC) was studied. The refrigerated stored wine was analyzed for total microbial count using
plate count method on GYE media at different periods of time upto 3 months. The clarified
aged wine was tasted and evaluated by a panel of 10 judges on a 80 point scale Modified
Davis Score Card (Annexure, Table I) in comparison to commercial wine (‘Galaxy’ brand of
N.D wines Pvt. Ltd., Nasik).
3.3 Analytical Techniques
3.3.1 pH
The pH of the periodic samples was determined using pocket sized pH meter (Hanna
HI96107).
3.3.2 Total Soluble Solids
Total Soluble Solids (%TSS) in juice was determined by using Erma hand
refractrometer of 0-32 oB and by using Brix Hydrometer (1-10 oB, 0-20 oB). The prism of
refractrometer was cleaned and dried with soft tissue paper. A drop of distilled water at 20 oC
was placed on clean and dry prism and calibrated by reading the zero line of demarcation on
the scale. For Brix determination with Brix hydrometer, the sample (juice/ fermenting juice)
was taken in a measuring cylinder and whole Brix hydrometer was gently dropped in it. The
level of TSS was studied by dipped level of Brix hydrometer.
3.3.3 Titrable Acidity
It was expressed as percent acidity and analyzed using the method of Amerine et al
(1967). Titrable acidity was determined by titrating known amount of juice or wine sample
(5ml) against 0.2 N NaOH using a few drops of 1% phenolphthalein solution as indicator. The
end point was appearance of pink/ Purple color which should persist for 15 seconds.
Titrable Acidity (w/v, %) = taken)sample of (volume 5ml
6 X 0.2 X used NaOH of Volume x 100
3.3.4 Brix-Acid Ratio
Brix- Acid ratio was calculated by dividing TSS value with total acidity of the juice.
29
3.3.5 Ethanol estimation
The estimation was performed by the chemical oxidation method of Caputi et al
(1968).
Reagent
1. Potassium dichromate solution: The solution was prepared by dissolving 33.37 g of
K2Cr2O7 in distilled water to which 325 ml of H2SO4 was slowly added by keeping the flask in
a cold water bath and the final volume was made 1L with distilled water. The reagent was
kept in dark colored bottle for overnight, before use.
Procedure
One ml of the fermented wash was added to 500ml distillation flask containing
approximately 20 ml distilled water. The distillate was collected in a 50 ml flask containing
25 ml K2Cr2O7 solution while keeping the condenser tip submerged in the solution
throughout. About 20 ml distillate including the rinsed water from the tip of the condenser
was collected. The distillate containing flask was then kept in a water bath maintained at
62.5oC for 20 min and was cooled to room temperature, thereafter. The final volume of the
flask was raised to 50 ml with distilled water. Five ml of this solution was diluted with five ml
distilled water and used for reading on spectrophotometer (Systronics 106) at 600 nm. The
standard curve of ethanol was prepared by taking 0-20% absolute alcohol (Fig.3).
Fig. 3: Standard curve for ethanol
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0 5 10 15 20
Ethanol % (v/v)
Abso
rban
ce a
t 6
00
nm
30
3.3.6 Ascorbic acid
The titrametric method using 2,6-dichlorophenol indophenols dye was used to
estimate ascorbic acid (AOVC 1996). Dye factor (i.e. mg of ascorbic acid per ml of dye) was
determined by taking 5 ml standard ascorbic acid solution and 5 ml 0.4% oxalic acid against
dye solution to a pink color. Known weight of crushed sample (10mg) or known volume of
wine (10ml) was taken and 100 ml of volume was made up with 0.4% oxalic acid solution.
The mixture was filtered through Whatman filter paper no. 4. To a measured volume of
aliquot (10ml), 15 ml of oxalic acid (0.4%) was added followed by titration against
standardized dye (0.4%) to a pink end point which should persist for at atleast 15 sec.
Fresh mg of Ascorbic acid/ 100mg = (mg) sample ofht ken x weigAliqout ta
made Volumefactor x dye x Titre x 100
3.3.7 Reducing sugars
Reducing sugars were estimated by the method of Miller (1959). Test tubes
containing 3 ml sample and 3 ml DNS reagent (10 g of 3, 5 dinitrosalicyclic acid, 2.0 g
phenol and 0.5 g sodium sulphite solution were dissolved in 500 ml 1% sodium hydroxide
solution and the volume was made 1000ml by adding additional alkaline solution (filtered and
stored in a dark colored bottle) were heated for 15 min in a boiling water bath. One ml of
Rochelle salt solution (40 g sodium potassium tartarate was dissolved in distlled water and the
volume was made to 100 ml) was added to each test tube and the tubes were allowed to cool
to room temperature. The OD of developed color was measured at 575 nm using Systronics
106 Spectrophotometer. The concentration of reducing sugars (as glucose) was calculated
from the standard curve which was prepared by taking 0.1-0.9 mg/ml of glucose (Fig. 4).
3.3.8 Total phenols
3.3.8.1 Extraction of total phenols
A known volume of juice or wine (1ml) was taken in 100 ml conical flask. To this 10
ml HCl (0.3N) in methanol was added and kept on orbital shaker at 150 rpm for 1 hr. After
shaking, crude extract was filtered through Whatman No. 1 filter paper. The filtrate so
obtained was evaporated to dryness in a boiling water bath. To this residue, hot water was
added and final volume was adjusted to 25 ml with distilled water.
3.3.8.2 Estimation of total phenols
Total phenols were estimated by method of Malik and Singh (1971). To 0.5 ml of the
above extract, 1 ml each of Folin-Ciocalteau reagent (diluted 1:2 with distilled water) and
sodium carbonate reagent (35 grams of sodium carbonate dissolved in 60ml distilled water
and final volume made to 100 ml) was added and then mixed. After 10 min, 2 ml of distilled
water was added and intensity of color was recorded at 620 nm against reagent blank.
The concentration of total phenols was calculated from the standard curve (Fig. 5)
prepared by using Gallic acid (10-100 µg/ml).
31
Fig. 4: Standard curve for reducing sugars
Fig. 5: Standard curve for total phenols with Gallic acid as standard
32
3.3.9 Estimation of SO2
SO2 (total as well as free) was estimated by Ripper (1898) method, the principle of
which is based on the following equation:-
H2SO3 + I2 + H2O H2SO4 + 2HI
Reagents
Preparation of 0.02 N I2- 0.1 N: - 2.7 g I2 + 40 g KI in 25 ml Distilled Water (DW) and make
volume 1L. Take one part of 0.1N I2 and four parts of DW.
Preparation of 0.02 N Na2S2O3 -0.2N -:50g Na2S2O3..5H2O + 0.1g Na2S2O3 (anhydrous) and
make volume 1L with DW. Take one part of 0.2 N Na2S2O3 and ten parts of DW.
Starch indicator- 1g starch in minimum DW. Make volume 100ml with boiling water. Cool
and add 0.5 ml CCl4.
Sulfuric Acid solution (1+3): 100ml (concentrated) sulfuric acid plus 300ml DW.
1N NaOH- 4 g in 100ml DW
Total SO2
Pipette 20ml of wine into a 250ml Erlenmeyer flask. Add 25 ml of NaOH solution;
mix, stopper and let it stand for 10 min. Add pinch of Na2S2O3 to expel air and then add 5ml
starch indicator solution; add 10ml sulfuric solution (1+3). Titrate rapidly with 0.02 N iodine
solution till end point as bluish color which persists for 30 sec.
SO2 (total) (mg/L or ppm) = (ml) sample of Volume
100032x x N) (0.02 I2 ofNormality x used I2 of Volume
Free SO2
Pipette 50 ml wine a 250ml Erlenmeyer flask. Add pinch of Na2S2O3 to expel air then
add 5ml starch indicator solution; add 5ml sulfuric solution (1+3). Titrate rapidly with 0.02 N
iodine solution till end point as bluish color which persists for 1-2 min. Temperature should
not exceed 20OC.
SO2 (total) (mg/L or ppm) = (ml) sample of Volume
100032x x N) (0.02 I2 ofNormality x used I2 of Volume
3.3.10 Thin layer chromatography (TLC)
TLC of wine was carried out using glass plate (stationary phase) of 5 x 20 cm size.
Slurry was prepared by taking 2g of silica gel with double amount of water and was spread on
plate using glass rod to be of uniform thickness. The plate was activated at 110 oC for 1 h (it
is done to remove the moisture from the plates).Solvent system (Mobile phase) was also
prepared using Butanol: Acetic acid: water :: 40: 20: 10 (v/v/v).Then, solvent was placed into
solvent chamber containing nearly 100 ml in it and covered with filter paper to ensure the
saturation with solvent vapors. After that, spotting was done using capillary tube atleast 1.5
cm above the end of plate (the side we dip in the solvent) and dried the spot immediately.
33
After spotting, plate was placed in the chamber (Solvent system) and allowed the
solvent to move on plate till it reached atleast 1-2 cm below from top and plate was then
removed and placed in the oven to remove solvent completely. Then, spots were identified
using ninhydrin reagent (mixture of 0.2% solution of ninhydrin in butanol) and 10% acetic
acid was sprayed for detection as pink color appears after heating at 100-110 0C for 10 to 15
min. Rf values were then calculated using formula as below:
Rf = (cm)solvent by travelledDistance
(cm)spot by travelledDistance
3.3.11 Statistical analysis
Besides the Response Surface Methodology (Design Expert 8.0) adopted for
prefermentation treatment of grape juice, statistical analysis of the fermentation data was
carried out using GSTAT04 and CPCS1 software developed by Department of Math, Stat and
Physics, PAU, Ludhiana (Cheema and Sidhu 2007).
34
CHAPTER IV
RESULTS AND DISCUSSION
4.1 Extraction of Grape juice
Healthy fruits of variety Punjab purple (Syn H 516) were selected and juice was
extracted from them. Ten kg of the berries with an initial brix of 16±2.0oB (oBrix depended
upon the maturity stage of the grape fruit), upon juice extraction gave 5.5±0.5 L of brick red
coloured grape juice whereas 1.3 ± 0.1 Kg of grape skin was produced to which 0.01% w/v
KMS was added. The skin was then stored in a deep freezer for further experiments on skin
treatment (Kocher et al 2011a).
4.2 Physiochemical characteristics of grape juice
The physicochemical characteristics of juice were evaluated on the basis of chemical
analysis which is presented in table 3. The analysis revealed TSS (16±2.0), acidity
(0.30±0.06), pH (3.3±0.2), brix acid ratio (58.33 ± 8.33), ascorbic acid (615.38 ± 15.3) and
total phenols (98.49 ± 13.26) mg/100ml in freshly extracted juice.
Table 3: Physico-chemical analysis of Punjab purple juice
Parameters Observations
TSS (oBrix) 16±2.0
Acidity (% w/v) 0.30±0.06
pH 3.3±0.2
Brix-acid ratio 58.33 ± 8.33
Ascorbic acid (mg/100ml) 615.38 ± 15.3
Total phenols (mg/100ml) 98.49 ± 13.26
Since, the parameters like TSS, titrable acidity and brix acid ratio determine the final
sensory quality attributes like appearance, color, aroma, taste, bouquet, body, flavor,
astringency and overall acceptability of the wine, these have been studied by different
research workers prior to fermentation of grape juice. Among the different reports on physico
chemical characteristics of grape juice, Gill and Arora (2009) reported that Punjab Purple had
significantly higher T.S.S. of 17.7%. Topalovic and Mikulic-Petkovse (2010) determined the
sugar content in the grape juice of ‘Cardinal’ cultivar which ranged from 13.1 to 19.4 and its
brix-acid ratio varied between19.4-21.8. Cantos et al (2002) found the content of total
phenolics in range from 115 to 361 mg kg-1
in Dominga and Flame Seedless respectively, of
fresh weight of grapes. Morino-Perez et al (2011) reported 3.18 % acidity, 25.53 oBrix and
3.82 pH in Syrah variety of grapes. Goswami and Ray (2011) found that titratable acidity %
(tartaric acid v/v) and pH was 0.715% and 5.0, respectively in the grape juice concentrate.
35
4.3 Pre-fermentation treatment
4.3.1 Skin treatment
The stored grape skin was used for pretreatment of juice so that skin volatiles like
phenols and anthocyanins are extracted into the juice (Lorrain et al 2013). The results of skin
contact treatment conducted by RSM (as explained in materials and methods) are presented in
table 4 as response in the form of Brix, pH, phenols (mg/100ml) and color intensity at OD
620nm (Optical density/ Absorbance is a reflection of color intensity of the juice).
Table 4: Effect of skin treatment on Grape juice from Punjab purple
Uncoded values Responses
Experiment
No.
Skin
weight(Kg)
A
Temperature
(°C)
B
Time
(hr)
C
(°Brix) pH OD
620nm
Phenols
(mg/100ml)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0.05
0.55
0.55
0.55
0.85
0.85
0.55
0.55
1.05
0.55
0.25
0.85
0.85
0.25
0.25
0.55
0.55
0.55
0.55
0.25
15.00
15.00
15.00
15.00
20.00
10.00
15.00
23.41
15.00
6.59
20.00
10.00
20.00
20.00
10.00
15.00
15.00
15.00
15.00
10.00
8.50
4.11
21.11
8.50
16.00
16.00
8.50
8.50
8.50
8.50
16.00
1.00
1.00
1.00
16.00
8.50
8.50
8.50
8.50
1.00
14.1
15.5
13
15.5
13
13
14
13.6
16
12.5
13
13
15
14.6
13.3
16
13.1
15.6
16
15.5
3.385
3.459
3.27
3.403
3.306
3.308
3.398
3.393
3.389
3.391
3.487
3.405
3.402
3.405
3.403
3.311
3.487
3.499
3.296
3.483
1.641
1.524
1.367
1.352
1.656
1.381
1.321
1.342
1.943
1.291
1.399
1.812
1.741
1.689
1.221
1.381
1.352
1.353
1.321
1.763
98.41
95.72
98.4
97.4
99.4
98.13
97.1
95.43
109.62
87.66
100.18
96.17
98.08
94.62
92.81
97.12
97.43
97.41
97.14
89.11
*Initial values of brix, pH, OD 620nm and phenols (mg/100ml) were 18°B, 3.5, 1.068, 85.3
respectively.
Maximum phenols (mg/100ml) and color intensity of 109.62 and 1.943 were
observed in combination of 1.05 Kg/L skin weight, temperature 15°C and time 8.5 h but this
combination required very high amount of skin which was not possible from the available
quantity of grapes because the skin produced 0.13 Kg/kg of grapes. Hence, the combination
of 0.25 Kg/L skin weight, temperature 20°C and time 16 h was preferred which was having
2nd
highest phenols i.e. 100.18 mg/100ml, color intensity of 1.399 with 13°Brix and 3.487 pH.
There was significant decrease in Brix with skin treatment which is a negative factor and to
compensate it, juice had to be supplemented with sugar to adjust the brix. The results were
36
Table 5: Analysis of variance table for Response 1 (BRIX) [Partial sum of squares]
for response surface quadratic model
Source Sum of
Squares df
Mean
Square
F
Value p-value Prob > F
Model 29.71 9 3.28 77.74 < 0.0001** Significant
A-skin
weight 2.071E-003 1 2.071E-003 0.049 0.8292
B-temp 1.87 1 1.87 44.19 < 0.0001**
C-Time 1.59 1 1.59 37.63 < 0.0001**
AB 0.080 1 0.080 1.89 0.1989
AC 0.045 1 0.045 1.06 0.3264
BC 1.80 1 1.80 42.72 < 0.0001**
A2 4.99 1 4.99 188.02 < 0.0001**
B2 12.79 1 12.79 302.72 < 0.0001**
C2 4.33 1 4.33 102.48 < 0.0001**
Residual 0.42 10 0.042
Lack of Fit 0.089 5 0.018 0.27 0.9128 Not
significant
Pure Error 0.33 5 0.067
Cor Total 29.99 19
R2 0.9859
Adj R-
Squared 0.9732
Pred R-
Squared 0.9708
Adeq
Precision 22.595
** Significant at 5% level
37
Table 6: Analysis of variance table for Response 2 (pH) [Partial sum of squares] for
response surface quadratic model
Source Sum of
Squares df
Mean
Square
F
Value p-value Prob > F
Model 0.088 9 9.778E-003 1293.75 <0.0001** Significant
A-skin
weight 3.457E-005 1 3.457E-005 4.57 0.0582
B-temp 4.091E-007 1 4.091E-007 0.054 0.8207
C-Time 0.078 1 0.078 10367.78 <0.0001**
AB 1.051E-004 1 1.051E-004 13.91 0.0039**
AC 1.250E-007 1 1.250E-007 0.017 0.9002
BC 1.361E-004 1 1.361E-004 18.01 0.0017**
A2 5.563E-004 1 5.563E-004 73.60 <0.0001**
B2 2.845E-004 1 2.845E-004 37.65 <0.0001**
C2 3.136E-004 1 3.136E-004 41.50 <0.0001**
Residual 7.558E-005 10 7.558E-005
Lack of Fit 4.225E-005 5 8.449E-006 1.27 0.4006 Not
Significant
Pure Error 3.333E-005 5 6.667E-006
Cor Total 0.088 19
R2 0.9991
Adj R-
Squared 0.9984
Pred R-
Squared 0.9949
Adeq
Precision 119.599
** Significant at 5% level
38
Table 7: Analysis of variance table for Response 3 (color intensity, OD 620 nm)
[Partial sum of squares] for response surface quadratic model
Source Sum of
Squares df
Mean
Square
F
Value p-value Prob > F
Model 0.82 9 0.091 90.26 <0.0001** Significant
A-skin
weight 0.077 1 0.077 76.13 <0.0001**
B-temp 0.011 1 0.011 11.22 0.0074**
C-Time 0.27 1 0.27 263.90 <0.0001**
AB 1.250E-003 1 1.250E-003 1.23 0.2925
AC 0.012 1 0.012 12.33 0.0056**
BC 0.045 1 0.045 44.16 <0.0001**
A2 0.33 1 0.33 329.76 <0.0001**
B2 3.721E-003 1 3.721E-003 3.68 0.0842
C2 0.085 1 0.085 83.77 <0.0001**
Residual 0.010 10 1.012E-003
Lack of Fit 7.530E-003 5 1.560E-003 2.90 0.1335 Not
Significant
Pure Error 2.593E-003 5 5.187E-004
Cor Total 0.83 19
R2 0.9878
Adj R-
Squared 0.9769
Pred R-
Squared 0.9195
Adeq
Precision 30.856
** Significant at 5% level
39
Table 8: Analysis of variance table for Response 4 (phenols) [Partial sum of squares]
for response surface quadratic model
Source Sum of
Squares df
Mean
Square
F
Value p-value Prob > F
Model 454.16 9 50.46 1007.33 <0.0001** Significant
A-skin
weight 141.20 1 141.20 2818.60 <0.0001**
B-temp 70.95 1 70.95 1416.25 <0.0001**
C-Time 71.55 1 71.55 1428.21 <0.0001**
AB 7.41 1 7.41 147.94 <0.0001**
AC 2.02 1 2.02 40.32 <0.0001**
BC 1.30 1 1.30 25.87 0.0005**
A2 83.99 1 83.99 1676.63 <0.0001**
B2 57.56 1 57.56 1149.04 <0.0001**
C2 1.80 1 1.80 35.89 <0.0001**
Residual 0.50 10 0.050
Lack of Fit 0.37 5 0.074 2.84 0.1380 Not
Significant
Pure Error 0.13 5 0.026
Cor Total 454.66 19
R2 0.9989
Adj R-
Squared 0.9979
Pred R-
Squared 0.9923
Adeq
Precision 137.181
** Significant at 5% level
40
analyzed by multiple regression technique. The analysis of variance tables (Tables 5, 6, 7 and
8) for the four responses Brix, pH, color intensity (OD 620nm) and phenols (mg/100ml)
having model F-values of 77.74, 1293.75, 90.26 and 1007.33, respectively. There was only a
0.01% chance that a "Model F-Value" of this large could occur due to noise. Furthur,values of
"Prob > F" were less than 0.0500 in all the four responses thus indicating that the model
terms wenre significant.
The "Lack of Fit F-value" of 0.27, 1.27, 2.90 and 2.84 for Brix, pH, color intensity
and phenols also implied that the Lack of fit is not significant realtive to the pure error in all
the four models (tables 5, 6, 7 and 8). There are also 91.28%, 40.06%, 13.35% and 13.80%
chances that a “Lack of Fit F-value” of this large could occur due to noise. Since, the lack of
fit P value in all the four models was <0.0500 lack of fit non-significant which was desirable
for the model to be fit.
The R- Squared values for four models i.e. Brix, pH, color intensity and phenols were
0.9859, 0.9991, 0.9878 and 0.9989 respectively. The “Pred R-Squared” values of 0.9708,
0.9949, 0.9195 and 0.9923 for brix, pH, color intensity and phenols respectively were in
reasonable agreement with the "Adj R-Squared" of 0.9732, 0.9984 0.9769 and 0.9979
respectively. "Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is
desirable and ratio of four models were 22.595, 119.599, 30.856 and 137.181 respectively,
indicating an adequate signal. Based upon the above measurable statistical parameter it may
be stated that this model can be used to navigate the design space. The same has been plotted
in Fig. 6,7 ,8and 9 respectively.
By applying multiple regression analysis on the experimental data, the following
second order polynomial equations (eqn. 1, 2, 3, 4) were found to represent the effect of
independent variables (Temperature, Time and skin weight) on Brix, pH, color intensity and
phenols.
(1) Brix
Final Equation in Terms of Coded Factors:
+15.73-0.012*A+0.37* B -0.41* C+0.10 * A* B-0.075*A*C-0.47* B*C-0.59*A2-0.94* B2-
0.72*C2 (1a)
Final Equation in Terms of Actual Factors:
+2.52213+6.42566*skinweight+1.27450*temp+0.37032*Time+0.066667*skinweight*temp-
.033333*skinweight*Time-0.012667*temp*Time-6.53034*skinweight2-0.037651*temp2-
0.012752*Time2 (1b)
41
Fig.6 (a): Response surface of brix as a function of temperature and skin weight
Fig.6 (b): Response surface of brix as a function of time and skin weight
42
Fig.6 (c): Response surface of brix as a function of time and temperature
(2) pH
Final Equation in Terms of Coded Factors:
+3.40+1.591E-003*A +1.731E-004*B-0.091*C +3.625E-003*A*B-1.250E-000*A*C
-4.125E-003*B*C-6.207E-003*A2-4.440E-003 * B2+6.104E-003*C2 (2a)
Final Equation in Terms of Actual Factors:
+3.45550+0.045393*skinweight+4.96792E-003*temp-0.012328*Time+2.41667E-003*
skinweight*temp-5.55556E-005*skinweight*Time-1.10000E-004*temp*Time-0.068970*
skinweight2-1.77582E-004*temp2 +1.08522E-004*Time2 (2b)
Fig.7 (a): Response surface of pH as a function of temperature and skin weight
43
Fig.7 (b) : Response surface of pH as a function of time and skin weight
Fig.7 (c) : Response surface of pH as a function of time and temperature
(3) Color intensity
Final Equation in Terms of Coded Factors:
+1.35+0.075*A+0.029*B-0.17 * C+0.013 *A*B+0.040*A*C+0.075 *B*C+0.15*A2-
0.016*B2+0.10*C2 (3a)
Final Equation in Terms of Actual Factors:
+2.21999-1.88233*skinweight+3.50583E-003*temp-0.092352*Time+8.33333E-
003*skinweight*temp+0.017556*skinweight*Time+1.99333E003*temp*Time+1.68955*skin
weight2-6.42195E-004*temp2+1.78453E-003*Time2 (3b)
44
Fig.8 (a): Response surface of color intensity as a function of temperature and skin
weight
Fig .8 (b) : Response surface of color intensity as a function of time and skin weight
45
Fig.8 (c) : Response surface of color intensity as a function of temperature and time
(4) phenols
Final Equation in Terms of Coded Factors:
+97.29 +3.22*A+2.28*B+2.76*C-0.96*A*B+0.50*A*C+0.40*B*C+2.41*A2-2.00*B2-
0.46*C2 (4a)
Final Equation in Terms of Actual Factors:
+68.09454-11.03548*skinweight+3.11368*temp+0.22341*Time0.64167*skinweight*temp+
0.22333*skinweight*Time+0.010733*temp*Time+26.80028*skinweight2-0.079871*temp2-
8.21629E-003* Time2 (4b)
Fig. 9 (a) : Response surface of phenol as a function of temperature and skin weight
46
Fig. 9 (b) : Response surface of phenol as a function of time and skin weight
Fig. 9 (c): Response surface of phenol as a function of time and temperature
47
The individual prefermentative parameters have been reported in literature. Among
these, Beer et al (2006) investigated that pre-fermentation maceration, especially at 15°C,
resulted in wines with increased vitisin A content. Pre-fermentation skin contact at 10°C
increased the sensory quality of Pinotage wines compared to the control wine for three
separate vintages (Marais 2003). Pre-fermentation maceration at 10 and 15°C enhanced the
colour and anthocyanin content of red (Heatherbell et al 1997, Watson et al 1997, Reynolds et
al 2001, Gomez-Miguez et al 2006) and rose (Salinas et al 2003, Salinas et al 2005) wines.
The use of RSM for optimization of phenolic compounds extraction from cactus pear
(Opuntia ficus-indica) skin has been reported with time of extraction (h), concentration of
ethanol (%v/v) and temperature (oC) as variables (Jorge et al 2013).
4.3.2 Effect of enzymatic treatment on Grape “must” from Punjab purple
Pre-fermentation treatment with pectinase and combination of enzymes viz. Amylase,
Cellulase and Pectinase results in increase in juice recovery and clarity in grape juice (Vaidya
et al 2009). The combination of three enzymes i.e. pectinase (0.25ml/100ml), cellulase (0.125
ml/100ml) and amylase (0.085 ml/100ml) at 45 oC for 6h revealed the best clarity of the grape
juice as 78.7 % which was significantly higher than the individual enzyme and combination
of two other enzymes (Table 9) (Plate III).
Table 9: Effect of enzymatic treatment on Grape “must” from Punjab purple
Enzymes used % clarification (Mean ± SD)
Control 10.0± 1.26
Amylase 41.65±1.06
Cellulase 40.75±1.62
Pectinase 42.5±2.26
Amylase+pectinase 46.5±1.41
Pectinase+cellulose 63.4±1.27
Amylase+cellulose 57.45±0.91
Amylase+pectinase+cellulose 78.7±1.13
Enzymes degrade the polysaccharide gel formed in different fruit pulps and thus
reduce their viscosity and also improve the juice filtration (Qin et al 2005). Earlier, Rai et al
(2004) observed that at constant time and temperature, the clarity increases with increasing
enzyme concentration but at constant temperature and enzymatic concentration, the juice
clarity varies nonlinearly with the duration of treatment.
Earlier, Espedo and Armada (2010) observed decrease in juice turbidity by pectinase
treatment of grape juice from Pedro Ximenez Variety of grapes. They also standardized
increased juice yield by enzyme addition along with dynamic maceration. Mojsov et al (2011)
reported increase in clarity of wine filterability, settling of solids and wine quality when grape
mashes of Smederevka were treated with different pectolytic enzyme preparation. Such effects
48
of pectinase enzyme treatment have been reported in other fruit juices.
Elsewhere, Norjana and Noor Aziah (2011) reported that the juice yield was
increased by 35% using 0.1% pectinase concentration for 3 h of incubation in Durain fruit
(Durio zibethinus Murr) juice. Karangwa et al (2010) optimized conditions for carrot-orange
juice as 0.06% (w/v) of enzyme concentration, 3.6 pH, 49ºC temperature and 91 min of
reaction time. The enzymatic treatment in Kiwifruits studied by Vaidya et al (2009) reported
enhanced juice recovery by 78.46% as compared to control (58.44%). In his study, they used
combination of enzymes (Pectinase 0.025g/kg, amylase 0.025g/kg and Mash enzyme
0.05g/kg for 2 h at 50°C) for clarification of Kiwifruits.
4.4 Ethanol fermentation of pretreated grape juice
A combined source of Nitrogen and Phosphorus viz. Di-ammonium Hydrogen ortho
Phosphate (DAHP) was supplemented (50-250mg/100 ml) in the “must” of grape juice for
optimization under standardized conditions of 20°B, 25°C and fermented by S.cerevisae
strain 35 (Plate IV) inoculum size of (5% v/v).
4.4.1 Effect of DAHP supplementation
The effect of DAHP supplementation (50-250 mg/100ml) on ethanolic fermentation
of Grape juice by S.cerevisae strain 35 was studied. DAHP increases the activity of yeast by
providing the required nitrogen and phosphorus which accelerates the fermentation process
and increases the ethanol production (Ugliano et al 2007). Results presented in table 10 (also
see Table II, Annexure) reveal 150mg/100ml as the optimized concentration of DAHP for
maximum ethanol production from grape juice with a fermentation efficiency of 94.5%
compared to 85.93% in control. The brix values were not zero as the same was measured by
refractrometer in which the produced ethanol interferes (www.moundtop.com).
For a number of fruits, the nitrogen content is insufficient and supplements are made
by the addition of ammonium phosphate or of diammonium phosphate (DAP) before
fermentation. KH2PO4 (46.1g/L) was found to be better phosphorus source in comparison
with K2HPO4 (45.3 g/L) and (NH4)2SO4 (46.1 g/L) was the best nitrogen source as compared
to NaNO3, (NH4)H2PO4, NH4NO3 for the production of red wine from ‘Siahe sardarsht’ grape
variety (Asli 2010). It was observed that higher concentrations of DAHP i.e 400 and 500 mg/
100ml juice produce less ethanol due to reduction in cellular activity of yeast. Ghosh et al
(2010) also found that higher DAHP concentrations produce less ethanol as compared to less
concentration as with higher concentration of nitrogen, cellular activity of yeast gets inhibited
and ethanol production is affected. They also reported that DAHP was better nitrogen source
than urea for fermentation carried out by S.cerevisae. It has been suggested that nitrogen
management is also critical for wine flavour and style and practice of supplementation of
nitrogen, particularly DAHP (a combined source of N & P) has started in wineries (Ugliano et
al 2007).
49
Plate III: Enzyme (Pectinase + Amylase + cellulase) treatment of grape juice,
A-treated juice
B- Untreated juice
Plate IV: Phase contrast microscopic image (45X) of Saccharomyces
cerevisiae strain 35.
A B
50
Table 10: Effect of di-ammonium hydrogen ortho phosphate (DAHP) concentration on ethanol fermentation of Grape juice.
%Eth-Ethanol (%) v/v ; R.S – reducing sugars % Cultural Conditions:
Actual ethanol produced Scale of fermentation : 300 ml
**Fermentation Efficiency % (v/v) = --------------------------------- × 100 Brix : 20°B
Theoretical ethanol produced Temperature : 25°C
Theoretical Ethanol % (v/v) = available sugar × 0.64 Inoculum size : 5% v/v
DAHP concentration (mg/100ml)
Control (0 ) 50 100 150 200 250 Time
(days)
pH Brix (R.S
mg/100ml) %Eth pH
Brix
(R.S) %Eth. pH
Brix
(R.S) %Eth pH
Brix
(R.S) %Eth pH
Brix
(R.S) %Eth pH
Brix
(R.S) %Eth
1 3.5 16 (13.1) 0.1 3.4 16.5
(15.0) 1.1 3.4
17
(15.4) 1.0 3.3
17.5
(14.4) 1.4 3.2
17
(16) 0.96 3.4
16.5
(14.74) 0.55
2 3.4 9 (7.8) 6.8 3.3 10.5
(8.5) 6.45 3.0
10
(7.4) 6.75 3.2
8.25
(6.5) 7.5 3.1
11
(9.4) 6.35 3.3
11.5
(9.1) 6.46
3 3.0 2 (0.42) 11.0 3.1 4.5
(0.80) 11.35 3.0
3.5
(0.45) 11.65 3.0
0
(0.3) 12.1 3.1
5
(0.7) 10.8 3.1
5.5
(0.83) 10.4
**F.E 85.93 88.67 91.0 94.5 84.3 81.2
CD ethanol
(5%)
Fermentation time – 0.379
DAHP conc. – 0.536
49
50
The scale up studies on grape wine at 5L scale validated under the optimized
conditions presented an ethanol production of 12.13±0.25% v/v having total phenols of
111.046 mg/100ml and total ascorbic acid content of 686.23±6.7 mg/100ml. A pictorial
representation of the preparation of red wine from Punjab purple grapes is also presented in
Plate V.
4.5 Effect of recycled yeast
The wort from the first experimental batch where the fermentation had just completed
was used as inoculum (2x108cells/ml) of recycled yeast. Four levels of inoculum sizes i.e. 5,
10, 15 and 20% v/v with direct microscopic counts of 3x109 to 1.2x1010cells/ml were used in
triplicate. The recycling was repeated for upto 2 subsequent lots and fermentation efficiencies
were compared. The results present in Table 11 (also see Table III, Annexure) revealed
increase in ethanol produced with 10% (v/v) inoculum while higher inoculum sizes reported
lower ethanol levels represented 10% (v/v) inoculum (from previous lot) produce statistically
similar ethanol production which is attributed to the fact that higher biomass requires more
sugars for its growth. The same was also reflected in the fermentation efficiencies of different
levels. However, the lots, the first recycled lot (Lot 1) reflected a decrease in ethanol from
fresh inoculum but second lot (Lot 2) produced statistically similar ethanol levels as that of
fresh inoculum lot.
There was no decrease in number of fermentation days for ethanol production,
however, the residual sugars decreased after successive lots. Earlier, Puri et al (2012) also
reported 20% inoculum level for recycling sugarcane fermentation batches. Strehaiano and
Gowa (1983) observed 10% inoculum concentration and Yadav et al 1997 reported 30%
inoculum level as optimum for improved ethanol production and high fermentation efficiency.
The lower efficiencies observed in our studies may be attributed to sustain the initial high
inoculum levels. However, the present study suggests that recycled yeast (10%v/v) is suitable
for wine preparation as the final ethanol concentration of the successive lots is same as that of
fresh yeast inoculum lot.
51
Juice
extraction
Enzymatic treatment
(45ºC,6h and pectinase
(0.25ml/100ml), cellulase (0.125
ml/100ml) and amylase (0.085
ml/100ml)
Optimization
of DAHP and
recylced
inoculum
Grapes
(Punjab purple Syn H516)
0.15% DAHP
(w/v) and 10%
yeast recycled
inoculum
Ethanolic fermentation
Decantation
and storage
Grape wine
Skin treatment
(0.25Kg/L,20oC,16h)
Plate V: Pictorial Diagram for preparation of red wine (Punjab purple,Syn H-516).
52
Table 11: Effect of recycling of yeast on ethanol fermentation of Grape juice.
*Initial number of cells- 2x108 cells/ml Culture conditions:-
%Eth-Ethanol (%) v/v ; R.S – reducing sugars %
Actual ethanol produced Scale of fermentation : 300 ml
**Fermentation Efficiency % (v/v) = -------------------------------------- × 100 Initial Brix : 20°B
Theoretical ethanol produced Temperature : 25°C
Theoretical Ethanol % (v/v) = available sugar × 0.64 Inoculum size : 5%v/v (fresh)
Inoculum level (% v/v)
5 10 15 20
Time
(days) pH Brix (R.S)
%Eth
(F.E) pH Brix (R.S)
%Eth
(F.E) pH Brix (R.S)
%Eth
(F.E) pH
Brix
(R.S)
(F.E)
%Eth
1 3.5 12(10.8) 2.55 3.45 10.5(8.75) 4.6 3.5 11(8.51) 3.2 3.4 9.5(6.1) 3.47
2 3.45 6.75(3.65) 5.1 3.4 3.25(1.9) 8 3.4 4(2.15) 5.6 3.35 3.25(1.45) 4.95
FRESH
LOT
3 3.35 3.25(1.35) 8.92
(69.68) 3.45 2.25(0.43)
9.54
(74.53) 3.4 3.75(1.65)
8.76
(68.43) 3.3 3(1.09)
9.5
(74.21)
1 3.5 13.5(11.77) 3.95 3.5 12(10.55) 4.7 3.5 8(5.56) 5.3 3.5 6(4.56) 5.5
2 3.45 7.75(5.74) 5.45 3.35 5.5(3.93) 6.3 3.35 5(3.81) 5.65 3.5 4(2.50) 6.5
LOT 1
3 3.35 4.25(1.12) 8.4
(65.62) 3.25 3.5(1.11)
8.14
(63.59) 3.25 3.25(0.95)
7.36
(57.5) 3.35 3(0.40)
8.3
(64.84)
1 3.5 10(8.69) 3.25 3.5 11(9.76) 3.25 3.5 9.5(7.4) 2.50 3.5 10.5(7.43) 2.86
2 3.45 5(1.85) 5.55 3.25 5(1.76) 6.50 3.35 4.5(1.68) 4.60 3.35 4(1.47) 4.95
LOT 2
3 3.35 3(0.71) 9.67
(75.54) 3.2 2(0.44)
9.97
(77.89) 3.3 2.5(0.53)
8.53
(66.64) 3.25 3.5(0.57)
6.85
(53.51)
F.E.
(%) 70.28 ±4.98 71.9 ± 7.48 64.18 ± 5.84 64.18 ± 10.36
CD
(5%) Ethanol -0.338
51
52
4.6 Post fermentation treatment
4.6.1 Storage study
The grape wine prepared under the optimized fermentation conditions of Brix (20°B),
temperature (25°C), 5% (v/v) inoculum size of fresh and 10% (v/v) recycled yeast and DAHP
(0.15%) supplementation was subjected to settling for 7 days at refrigerated conditions (4°C)
in glass bottles. Afterwards, the bottles were stored at 15°C and every 15 days upto 6 months,
the process of racking was repeated. During this course, various physiochemical and
microbiological parameters were observed which are presented in table 12 (also see Tables IV
and V, Annexure).
Table 12: Effect of storage time on microbiological and physicochemical properties of
Wine var. Punjab purple
Parameters
% ethanol (v/v) pH Total phenols*
(mg/100ml)
Total yeast count
(cfu/ml)
Storage
Time
(days)
Fresh
yeast
inoculum
With
recycled
yeast
inoculum
Fresh
yeast
inoculum
With
recycled
yeast
inoculum
Fresh
yeast
inoculum
With
recycled
yeast
inoculum
Fresh
yeast
inoculum
With
recycled
yeast
inoculum
0 12.10 9.97 3.4 3.5 222.6 207.1 6.2X106 7.3X10
6
15 11.63 9.86 3.4 3.4 210.9 201.4 1.1X101 2.1X10
1
30 11.49 9.64 3.4 3.4 206.2 180.6 0.1X101 0.3X101
45 11.22 9.53 3.4 3.4 187.4 175.9 0.0 0.0
60 10.89 9.36 3.3 3.4 145.8 155.7 0.0 0.0
75 10.71 9.19 3.3 3.3 138.8 141.4 0.0 0.0
90 10.66 9.10 3.3 3.3 106.6 127.2 0.0 0.0
120 10.45 8.99 3.3 3.3 102.4 112.9 0.0 0.0
150 10.42 8.91 3.3 3.3 100.2 110.4 0.0 0.0
CD
(5%)
CD (days)-0.508
CD (inoculum)-
0.239
NS CD (days)-8.25
CD (inoculum)- NS
-
*% decrease in phenols in fresh yeast inoculum wine- 55%
*% decrease in phenols in recycled yeast inoculum wine- 47%
Total SO2 = 22.1 ppm
Free SO2 = 8.2 ppm
The results revealed that fresh yeast used as inoculum was significantly better than
recycled yeast though a significant decrease in % ethanol was observed in both inoculums.
Moreover, the decrease in ethanol became constant after about 15 days of storage till 150 days
53
upto which storage studies were carried out. pH showed no change in both the treatments. The
decrease in total phenols was significant with storage and total phenols decreased (from fresh
wine) to 55 and 47% in fresh and recycled yeast inoculum, respectively. Zafriella et al (2003)
reported upto 90% decrease of anthocyanins with no change in flavonol content of red wines
during storage. It was also observed that the decrease in phenols stabilized after 90-120 days
of storage. Similar trends have been reported in white and merlot wines. While Kallithraka et
al (2009) observed a decrease in total phenols upto 6 months, Ivanova et al (2009) reported a
continuous decrease of phenols upto 16 months of storage that they studied. Another
important observation was the absence of viable yeast cells after 30 days of storage in the
racked wine suggesting that yeast tends to settle quickly during storage (Table 12).
The free and total sulphur dioxide in the stored wine was also analyzed. The wine
from fresh yeast culture was having 22.1 ppm of total SO2 and 8.2 ppm of free SO2 at 150
days of storage which is less than the recommended value of 150-400 and 25-40 ppm
respectively for red wines (www.morewine.com; www.eco-consult.net). Sims and Morris
(1984) reported that sulfur dioxide levels higher than 25 ppm free SO2 severely bleached the
color of red muscadine wine and lessened browning in high pH wine only. High SO2 levels
also lessened browning of wine stored at 20°C, but not at higher storage temperatures.
4.6.2 Thin Layer chromatography (TLC)
The qualitative analysis of wine w.r.t amino acids carried out by TLC is presented in
table 13. The spots were observed at a distance of 2.5 cm, 4.7cm, 7.6 and 9.9 showing Rf
values of 0.16, 0.30, 0.46 and 0.63, respectively as distance travelled by the solvent was 15cm
(Plate VI). The standard amino acids were also run on thin layer chromatograms and
compared with the spots of wine thin layer chromatograms. Based on the comparison and the
literature (tera.chem.ut.ee; www.reachdevices.com) the Rf values were designated to the
presence of histidine, arginine, lysine, threonine, methionine, alanine, valine, tyrosine,
methionine, valine in red wine (Table 13).
Table 13: Qualitative analysis of Amino acids by Thin Layer chromatography (TLC).
Distance travelled by
spot Rf value* Amino acid**
2.5 0.16 Histidine/ Arginine/Lysine
4.7 0.30 Threonine
7.6 0.46 Methionine/Alanine/Valine/Tyrosine
9.9 0.63 Methionine/Valine
Distance travelled by the solvent-15 cm *Rf = Distance travelled by spot
Distance travelled by solvent
**The ‘/’ indicates similar Rf values of amino acids
54
4.7 Sensory analysis of wine
The red wine samples were subjected to sensory analysis to find out the acceptability
among the tasting panelists. The results of sensory evaluation of 150 days old red wine are
presented in table 14 (also see Tables VI, Annexure). The wines prepared from Punjab purple
under optimized conditions with fresh and recycled yeast inoculums were found to be of
standard quality with mean score of 65.9±8.01 and 63.3±8.52, respectively at 150 days of
storage whereas commercial wine (‘Galaxy’ brand of N.D wines Pvt. Ltd., Nasik) was also
found to be of standard quality but with a lesser mean score value of 56.2±9.47 (Table 14).
Hence, red wine prepared from fresh yeast inoculum was significantly better than commercial
wine whereas the red wines prepared from recycled yeast inoculum and fresh yeast inoculum
were insignificantly different from each other.
In literature, Kocher et al (2009) studied the sensory characteristics of Grape wines
(white and red wines both) prepared from five different grape varieties/hybrids and reported
species specific variation in sensory characteristics of grape wines. Soni et al (2009) observed
that matured Amla wine was better than unmatured wine in terms of its sensory attributes.
They also reported that Amla wine matured in oak wood barrel had higher quantity of
desirable components including ethyl acetate and phenolics etc. and was better in its sensory
characteristics than the wine stored in glass bottles.
Table 14: Sensory evaluation of grape wine (var. Punjab purple) at 150 days of storage
Types of red wine Sensory
analysis
(characters)
Maximum
points Commercial Fresh yeast
inoculum
Recycled yeast
inoculum
Appearance 16 12.2±3.73 13.8±2.27 13±2.04
Taste 24 15.8±2.48 17.9±3.44 17.6±2.28
Aroma 24 16.2±4.48 19.8±3.34 18.3±4.53
Total acidity 8 7.0±1.61 7.8±0.6 7.8±0.6
Overall feel 8 5.0±1.34 6.6±0.9 6.6±0.9
Total 80 56.2±9.47 65.9±8.01 63.3±8.52
CD (5%) 4.076
* The above scoring is mean + standard deviation of evaluation by 10 panelists.
Ratings:
Superior (68-80)
Standard (52-68)
Below standard (36-52)
Unacceptable/ Spoiled (4-36)
55
PLATE VI: Thin Layer Chromatogram of amino acids in red wine. A: Distance
travelled by solvent=15cm; B= Histidine/ Arginine/ Lysine - 2.5cm; C=Threonine -
4.7cm; D=Methionine/ Alanine/ Valine/ Tyrosine - 7.6cm; E=Methionine/ Valine - 9.9cm
A
B
C
D
E
55
CHAPTER V
SUMMARY
Grapes are the foremost substrates used worldwide for wine production. However,
there are no wineries in this part of the country, though central and south Indian states like
Maharashtra, Karnataka have some of them. Further, grape production in Punjab is declining
and there are no wine varieties cultivated in Punjab. The Departments of Microbiology and
Fruit science have been evaluating different grape cultivars/ varieties for their wine potential
for the past about 5 years. In this regard, one variety, Punjab purple (syn H516) has been
identified and a laboratory scale technology standardized for red wine production for it.
However, a number of pre-fermentative and post fermentative parameters still need to be
optimized. Hence, the present study was conducted to study the effect of pre- and post
fermentation treatments on quality of red wine preparation from Punjab purple grapes.
Initially, physico-chemical characteristics of juice in terms of TSS (16±2.0°B), acidity
(0.30±0.06 %, w/v), pH (3.3±0.2), brix-acid ratio (58.33 ± 8.33), ascorbic acid (615.38 ± 15.3
mg/ 100 ml) and total phenols (98.49 ± 13.26 mg/100ml) in freshly extracted juice were
analyzed from the destemmed and crushed berries.
In the present study, an important prefermentative treatment i.e. skin contact of juice
was optimized w.r.t. Temperature, Time and Skin weight by applying Central Composite
Rotatable Design (CCRD) under Response surface methodology (RSM) using Statistical
software “Design Expert software 8.0”.The response provided in terms of pH, Brix, color
intensity and phenols (mg/100ml) revealed a combination of 20oC temperature, 16 hr skin
contact time and 0.25 Kg/L skin weight having phenols 100.18 mg/100ml, color intensity
1.399 at OD620nm with 13OBrix and 3.487 pH as optimum. The juice pretreated under these
optimized conditions was subjected to enzymatic treatment with pectinase (0.25ml/100ml),
cellulase (0.125 ml/100ml) and amylase (0.085 ml/100ml) individually and also in
combinations of enzymes i.e. Amylase+pectinase, Pectinase+cellulase, Amylase+cellulase,
Amylase+pectinase+cellulase at 45 oC for 6h. The treatment with combination of three
enzymes giving 78.7 % clarification was significantly higher than the individual enzymes and
treatments having combination of enzymes. Hence, the dual pretreatment of juice
standardized for its skin contact and enzymatic treatment was used for fermentation.
The ethanol fermentation of pretreated grape juice was carried out at already
standardized conditions of 20oB, 25
oC temperature and inoculum size of 5% (v/v) for the
optimization of DAHP supplementation (50-250 mg/100ml) by S.cerevisae strain 35. The
effect of DAHP supplementation at the rate of 150mg/100ml produced significantly higher
ethanol of 12.10% (v/v) as compared to untreated control 11.0% (v/v) from grape juice with
fermentation efficiencies of 94.5% and 85.93%, respectively. The red wine so produced from
56
Punjab purple was also scaled upto 5L producing 12.13% (v/v) ethanol that also validated the
results obtained at flask level.
Thereafter, experiments were also conducted to study the effect of recycling of yeast
inoculum from previous fermentation lots so that the process may be economized in terms of
fermentation time. The worts from experimental batches where the fermentation had just
completed were used as inoculum (2x108 cells/ml) of recycled yeast. Four levels of inoculum
sizes i.e. 5, 10, 15 and 20% (v/v) with direct microscopic counts of 3x109 to 1.2x10
10 cells/ml
were used and the recycling was repeated for upto two lots. The successive lots of 5 and 10%
inoculum levels represented insignificant changes in final ethanol produced i.e. 9.67 and 9.97
(v/v), respectively but with increase in inoculum size, the overall ethanol production
decreased. The same was also reflected in the fermentation efficiencies in the combined
fermentation efficiencies of three lots of different inoculum levels. Also, there was no
reduction in fermentation time with variable inoculum levels.
The red wine prepared under the optimized fermentation conditions of Brix (20°B),
temperature (25°C), inoculum size 5% (v/v) for fresh and 10% (v/v) for recycled yeast and
150 mg/100ml DAHP supplementation was subjected to settling for 7 days at refrigerated
(4°C) conditions in glass bottles. Afterwards, the bottles were stored at 15°C±5°C and every
15 days for upto 3 months, the process of racking was repeated. The wine (post storage)
prepared from fresh yeast was found to be significantly better than recycled yeast, though the
decrease in % ethanol was also significant. The decrease in total phenols was also significant
with storage as total phenols decreased to 55 and 47% of the initial values in fresh and
recycled yeast, respectively. The free and total sulphur dioxide in the stored wine was also
analyzed. The wine from fresh yeast culture was having 22.1 ppm of total SO2 and 8.2 ppm of
free SO2 at 150 days of storage which is less than the recommended values of 150-400 and
25-40 ppm, respectively for red wines. The qualitative analysis of wine w.r.t amino acids
carried out by TLC revealed the presence of Histidine/Arginine/Lysine, Threonine,
Methionine/Alanine/Valine/Tyrosine, Methionine/valine in red wine.
The prepared wine was subjected to the sensory analysis at 150 days of storage vis-a-
vis commercial wine (‘Galaxy’ brand of N.D wines Pvt. Ltd., Nasik). The red wines prepared
from Punjab purple under optimized conditions with fresh inoculum and recycled yeast
inoculum were found to be of standard quality with mean scores of 65.9±8.01 and 63.3±8.52,
respectively whereas commercial wine was found to be of standard quality but with a lesser
mean sensory score of 56.2±9.47. Thus, the present study successfully optimized the
prefermentation conditions in term of skin contact (0.25 Kg/L), enzymatic treatment
(Amylase+pectinase+cellulase :: 25+50+25 units/ 100 ml). The addition of DAHP
(150mg/100ml) improved the fermentation efficiency and the prepared red wine was found to
be superior than the commercial red wine tested.
57
REFERENCES
Adams C and Hennie J J van Vuuren (2010) Effect of Timing of Diammonium Phosphate
Addition to Fermenting Grape Must on the Productionof Ethyl Carbamate in Wine.
Am J Enol Vitic 61(1): 125-29.
Alobo A P, Offonry S U (2009) Characteristics of coloured wine produced from roselle
(Hibiscus sabdariffa) calyx extract. J Inst Brew 115(2): 91-94.
Amerine M A and Ough C S (1980) Methods for Analysis of Musts and Wines. John Wiley,
New York.
Amerine M A, and Roessler E B (1983) Wines: Their Sensory Evaluation. Pp-432.W. H.
Freeman and Co, San Francisco.
Anonymous (2010) A fruit with tremendous biz potential Focus: Grapes. Agri-Biz &
Commodities-Horticutlure/Fruits & Vegetables. The Hindu Business Line,
www.thehindubusinessline.in/2010/08/16/stories/ 2010081650571300.htm
AOVC (1996) Methods of vitamin assay. Association of Vitamin Chemists Inc. (Ed.)
Interscience Publishers, pp.306-12.
Arnous A, Makris D P and Kefalas P (2001) Effect of Principal Polyphenolic Components in
Relation to Antioxidant Characteristics of Aged Red Wines. J Agric Food Chem
49(12): 5736–42.
Asli M S (2010) A study on some efficient parameters in batch fermentation of ethanol using
Saccharomyces cerevesiae SC1 extracted from fermented siahe sardasht pomace. Afr
J Biotechnol 9: 2906-12.
Attri B L (2009) Effect of initial concentration on the physico-chemical characteristics and
sensory qualities of cashew apple wine. Nat Prod Radiance 8: 374-379.
Ayestaran B, Guadalupe Z, Leon D (2004) Quantification of major grape polysaccharides
(Tempranillo v.) released by maceration enzymes during the fermentation process.
Anal Chim Acta 513: 29–39.
Basha S M, Musingo M and Colova V S (2004) Composition differences in the phenolic
compounds of muscadine bunch grape wines. Afr J Biotechnol 3(10): 523-28.
Bautista-Ortin A B, Fernández-Fernández J I, Lopez-Roca J M and Gomez-Plaza E (2007)
The effects of enological practices in anthocyanins, phenolic compounds and wine
colour and their dependence on grape characteristics. J Food Compos Anal 20:546–
52.
Beer de D, Joubert E, Marais J and M. Manley (2006) Maceration Before and During
Fermentation: Effect on Pinotage Wine Phenolic Composition, Total Antioxidant
Capacity and Objective Colour Parameters. S Afr J Enol Vitic 27(2): 137-50.
Belloch C, Orlic S, Guillamon J M, Mas A and Rozes N (2008) Fermentation stress
adaptation of hybrids within the Saccharomyces sensu strict complex. Int J Food
Microbiol 23:188-95.
Bell S J and Henschke P A (2005) Implications of nitrogen nutrition for grapes, fermentation
and wine. Aust J Grape Wine Res 11: 242-95.
58
Beltran G, Novo M, Rozès N, Mas A, Guillamón J M (2004) Nitrogen catabolite repression in
Saccharomyces cerevisiae during wine fermentations. FEMS Yeast Res 4(6):625–32.
Beltran G, Esteve-Zarzoso B, Rozès N, Mas A, Guillamón JM (2005) Influence of the timing
of nitrogen additions during synthetic grape must fermentations on fermentation
kinetics and nitrogen consumption. J Agric Food Chem 53(4): 996–1002.
Bely M, Sablayrolles J M and Barre P (1990) Automatic detection of assimilable nitrogen
deficiencies during alcoholic fermentation in enological conditions. J Ferm Bioeng
70: 246-52.
Bisson L F and Block D E (2002) Ethanol tolerance in Saccharomyces. In: Ciani M (ed)
Biodiversity and biotechnology of wine yeasts. Pp 85-98. Research Signpost, Kerala,
India.
Bisson L F, Waterhouse A L, Ebeler S E, Walker M A and Lapsley J T (2002) The present
and future of the international wine industry. Nature 418:696-99.
Bradbury J, Richards K, Niederer H, Lee S, Rod D P and Gardner R (2006) A homozygous
diploid subset of commercial wine yeast strains. Antonie van Leeuwenhoek 89: 27-37.
Board R G (1983) A Modern Introduction to Food Microbiology. Blackwell Scientific
Publications, Oxford, UK .
Borzani W, Garab A, Pires M H, Piplovic R, Higuera G A (1993) Batch ethanol fermentation
of molasses: a correlation between the time necessary to complete the fermentation
and the initial concentrations of sugar and yeast cells. World J Microbiol Biotechnol
9: 265-68.
Boulton R.B, Singleton V L, Bisson L F and Kunkee R E (1996) Principles and practices of
winemaking. Pp-75-221. Chapman Hall, New York.
Boze H, Moulin G and Galzy P (1992) Production of food and fodder yeasts. Crit. Review
Biotechnol 12: 65-86.
Cabaroglu T and Canbas A (2002) The effect of skin contact on the aromatic Composition of
the white wine of vitis vinifera l. Cv. Muscat of alexandria grown in southern
Anatolia. Acta Aliment 31(1): 45–55.
Cabaroglu T, Selli S, Canbas A, Lepoutre J P and Gunata Z (2003) Wine flavor enhancement
through the use of exogenous fungal glycosidases. Enzyme Microb Techno 33: 581-
87.
Calre S, Skurray G and Theaud L (2002) Effect of a pectolytic enzyme on the colour of red
wine. Aust N Z Grapegrow Winemake 456: 29-35.
Canal-Llauberes R M (1990) Utilisation des enzymes dans les procedes d’extraction en
oenologie. Rev Fran Oenol 122: 28-33.
Cantos E , Espín J C and Tomás-Barberán F A (2002) Varietal Differences among the
Polyphenol Profiles of Seven Table Grape Cultivars Studied by LC−DAD−MS−MS.
J Agric Food Chem 50(20): 5691–96.
Caputi A J, Ueda M and Brown T (1968) Spectrophotometric determination of ethanol in
wine. Am J Enol Vitic 19:160-65.
59
Caruso M, Fiore C, Contursi M, Salzano G, Paparella A and Romano P (2002) Formation of
biogenic amines as criteria for the selection of wine yeasts. World J Microbiol
Biotechnol 18: 159-63.
Castro-Vazquez I, Perez-Coello M S and Cabezudo M D (2002) Effects of enzyme treatment
and skin extraction on varietal volatiles in Spanish wines made from Chardonnay,
Muscat, Airen, and Macabeo grapes. Anal Chim Acta 458: 39-44.
Chagas R, Monteiro S and Ferreira R B (2012) Assessment of Potential Effects of Common
Fining Agents Used for White Wine Protein Stabilization. Am J Enol Vitic 63(4):
574-578.
Chang H J, Son J H, Park E Y, Kim E J and Lim S T (2008) Effect of vibration and storage on
some physico-chemical properties of a commercial red wine. J Food Compo Anal
21:655-59.
Chen C H, Wu M C, Hou C Y, Jiang C M, Huang C M and Wang Y T (2009) Effect of
Phenolic acid on antioxidant activity of wine and inhibition of pectin methyl esterase.
J Inst Brew 115: 328-33.
Cheynier V, Rigaud J, Souquet J M, Barillere J M and Moutounet M (1989): Effect of
pomace contact and hyperoxidation on the phenolic composition and quality of
Grenache and Chardonnay wine. Am J Enol Vitic 40: 36–42.
Ciani M, Beco L and Comitini F(2006) Fermentation behavior and metabolic interactions of
multistarter wine yeast fermentations. Int J Food Microbiol 108(2): 239–45.
Clemente-Jimenez J M, Mingorance-Cazorla L, Martinez- Rodriguez S, Heras-Vasquez F J L
and Rodriguez-Vico F(2005) Influence of sequential yeast mixtures on wine
fermentation. Int J Food Microbiol 98: 301–308.
Cleophas T J (1999) Wine, beer and spirits and the risk of myocardial infarction: a systematic
review. Biomed Pharmacotherapy 53:417–23.
Combina M, Elia A, Mercado L, Catania C, Ganga A and Martinez C (2005) Dynamics of
indigenous yeast populations during spontaneous fermentation of wines from
Mendoza, Argentina. Int J Food Microbiol 99: 237–43.
Conde C,Silva P, Fontes N, Dias A C P, Tavares R M, Sousa M J (2007) Biochemical
changes throughout grape berry development and fruit and wine quality. Food 1:1-22.
D’ Amore T (1992) Improving yeast fermentation performance. J Inst Brew 98:375-82.
Degree R (1993) Selection and commercial cultivation of wine yeast and bacteria. In: Fleet G.
H. (ed) Wine microbiology and Biotechnology. Pp 421-48. Harwood Academic
Publishers, Chur.
Dickinson J R (1999) Carbon metabolism. In: Dickinson J R and Schweizer M (ed) the
Metabolism and Molecular Physiology of Saccharomyces cerevisiae. Pp 591–95. PA:
Taylor & Francis Philadelphia.
Doco T, Williams P and Cheynier V (2007) Effect of flash release and pectinolytic enzyme
treatments on wine polysaccharide composition. J Agric Food Chem 55:6643-49.
60
Dorneles D, Machado I M P, Chociai M B and Bonfim T M B (2005) Influence of the Use of
Selected and Non-selected Yeasts in Red Wine Production. Braz Arch Biol Technol
48(5): 747-51.
Duarte W F, Dias D R, Oliveira M J, Teixeira J A, Silva, J D, Schwan R F (2010)
Characterization of different fruit wines made from cacao, cupuassu, gabiroba,
jaboticaba and umbu. Food Sci Technol 30: 1-9.
Duck N J, Lee D H, Hwang Y S, Lee S N, Lee D H and Lee J S (2008) Changes of
Physicochemical Properties and Antioxidant Activities of Red Wines during
Fermentation and Post-fermentation. Kor J Microbiol Biotechnol 36(1): 67–71.
Erten H (2006) Relations between elevated temperatures and fermentation behaviour
of Kloeckera apiculata and Saccharomyces cerevisiae associated with winemaking in
mixed cultures. World J Microbiol Biotechnol 18(4): 377-82.
Erten H, Tanguler H, Cabaroglu T and Canbas A (2006) The influence of inoculum level on
fermentation and flavor components of white wines made from cv. Emir. J Inst Brew
112:232-36.
Espejo F and Armada S (2010) Effect of Enzyme Addition in the Making of Pedro Ximenez
Sweet Wines Using Dynamic Pre-fermentative Maceration. S Afr J Enol Vitic
31(2):133-42.
FAO(2010) Fermented Fruits and Vegetables [online]. Available from:
http://www.fao.org/docrop/x056oe14.htm. [Accessed 16th August, 2010].
Felix R,and Villettaz J C (1983) Wine. In: Godfrey, Reichelt T J (ed) Industrial enzymology :
The application of enzymes in industry. pp 410-421.The Nature press, New York.
Ferrarese M (1987) (Alcholic fermentation : In practices in modern oenology). Edagricole,
Bologna.113-34.
Fleet G H (ed ) (1998) Microbiology of alcoholic beverages. In Microbiology of fermented
foods, Vol 1, Pp 217-62. Blackie Academic & Professional, London.
Fleet G H (ed) (2001) Wine: In Food microbiology fundamentals and frontiers. Pp 747-72.
ASM press, Washington DC.
Fleet G H (2003). Yeast interactions and wine flavour. Int J Food Microbiol 86: 11-22.
Fleet G H and Heard G M (1993) Yeast-Growth during fermentation. In: Fleet G H (ed) Wine
microbiology and biotechnology. Pp.27-54. Harwood Academic Publisher, Chur,
Switzerland.
Gafner, J. and Schütz, M. (1996) Impact of glucose-fructose-ratio on stuck fermentations.
Practical experiences to restart stuck fermentations. Vitic Enol Sci 51: 214–218.
Garrison E C (1993) Making Simple Fermented Beverages [online]. Available from:
http://www.homebrew.net/ferment/ [Accessed October, 2010].
Gawel R (1998) Red wine astringency: a review. Austr J Grape Wine Res 4: 74-95.
Gawel R, Ewart A and Cirami R (2000) Effect of rootstock on must and wine composition
and the sensory properties of Cabernet-Sauvignon grown at Langhorne Creek. Wine
Ind J 15: 67-73.
61
Gill M I S and Arora N K (2009) Performance of different grape varieties under north Indian
conditions. Indian J Eco 36(1): 15-17.
Gil J and Valles S (2001) Effect of macerating enzymes on red wine aroma at laboratory
scale: exogenous addition or expression by transgenic wine. J Agric Food Chem 49:
5515-23.
Ghosh S, Chakraborty R and Raychaudhri U (2010) Modulation of palm wine fermentation
by the control of carbon and nitrogen source on metabolism of Saccharomyces
cerevisae. J Food Tecnol 8(5):204-10
Godden P W and Gishen M (2005) Trends in the composition of Australian wine 1984-2004.
In: Blair R J, Francis M E and Pretorius I S (ed) Advances in Wine Science. Pp 115-
139. The Australian Wine Research Institute, Glen Osmond Australia.
Gomez-Miguez M, Gonzales-Miret M L and Heredia F J (2006) Evolution of colour and
anthocyanin composition of Syrah wines elaborated with pre-fermentative cold
maceration. J. Food Eng., in press (doi:10.1016/j.jfoodeng.2006.01.054).
Gonzalez S S, Barrio E, Gafner J and Querol A (2006) Natural hybrids from Saccharomyces
cerevisiae, Saccharomyces bayanus, Saccharomyces kudriavzevii in wine
fermentations. FEMS Yeast res 6:1221-34.
Gonzalez S S, Gallo L, Climent M D, Barrio E and Querol A (2007) Enological
characterization of natural hybrids from Saccharomyces cerevisiae and
Saccharomyces kudriavzevii. Int J Food Microbiol 116:11-18.
Goswami S and Ray S (2011) Studies on the Process Development for the Fermentative
Production of Wine from Grape Juice Concentrate. Internet J Food Safety 13: 367-73.
Guerin L, Sutter D H, Demois A, Chereau M and Trandafir G (2009) Determination of
activity profiles of the main commercial enzyme preparations used in winemaking.
Am J Enol Vitic 60: 322-31.
Hartle D K, Greenspan P, Hargrove J L (ed ) (2005) Muscadine Medicine. Pp. 47–96. Blue
Heron Nutraceuticals, Fullerton, CA, USA.
Hayasaka Y, Birse M, Eglinton J and Herderich M (2007) The effect of Saccharomyces
bayanus yeast on colour properties and pigment profiles of a Cabernet Sauvignon red
wine. Aust J Grape Wine Res 13: 176–85.
Heard G M and Fleet G H (1985) Growth of natural yeast flora during fermentation of
inoculated wines. Appl Environ Microbiol 50:727-28.
Heard G M and Fleet G H (1986) Occurance and growth of yeast species during fermentation
of some Australian wines, Food Technol Aust 38(1):22-25.
Heatherbell D, Dicey M, Goldsworthy S and Vanhanen L (1997) Effect of prefermentation
cold maceration on the composition, color and flavor of Pinot Noir wine. In: Henick-
Kling T, Wolf T E and Harkness E M (eds). Proc. 4th Int. Symp. Cool Climate Vitic.
Enol., Rochester, USA. pp VI 10-VI 17.
Hernanz D, Recamales A F, Gonzalez-Miret M L, Gomez-Miguez M J, Vicario I M and
Heredia F J (2007) Phenolic composition of white wines with a prefermentative
maceration at experimental and industrial scale. J Food Eng 80: 327-335.
62
http://www.moundtop.com/fermentation/RBRIX-ATC-Fermentation-Tables.pdf
http://www.oiv.int/oiv/info/endefinitionproduit
http://www.morewine.com
http://www.eco-consult.com
http://www.reachdevices.com/TLC_aminoacids.html
http://tera.chem.ut.ee/~koit/arstpr/ah_en.pdf
Ivanova V, Stefova M and Vojnosk B (2009) Assay of the phenolic profile of Merlot wines
from Macedonia: Effect of maceration time, storage,SO2 and temperature of storage.
Maced J Chem Chem Eng 28(2): 141–49.
Jackson R S (2000a) Wine Science. Academic Press, USA.
Jackson R S (2000b) Wine Science – principles, practice, perception. Academic Press, San
Diego.
Jackson and Lombard P B (1993) Environmental and management practices affecting grape
composition and wine quality: A Review. Am J Enol Vitic 44: 409-30.
Jolly N P, Augustyn O P H and Pretorius I S (2006) The Role and Use of Non-
Saccharomyces Yeasts in Wine Production. S Afr J Enol Vitic 27(1): 15-39.
Jorge A J, Heliodoro de L G, Alejandro Z C, Ruth B C, Noé A C and Jasso R M (2013) The
optimization of phenolic compounds extraction from cactus pear (Opuntia ficus-
indica) skin in a reflux system using response surface methodology. Asian Pac J Trop
Biomed 3(6): 436-42.
Joshi V K and Bhutani V P (1991) The influence of enzymatic clarification in fermentation
behaviour and qualities of apple wine. Sci Aliment 11: 491-98.
Joshi V K, Thakur N S, Bhat Anju and Garg Chainkya (ed) (2011) Wine and Brandy: A
Perspective. In: Handbook of Enology 1(1), Asia Tech Publication, New Delhi.
Kale K J (2007) Me Draksh Wine Uddoyog Ubharu Shakto ka? Maharashtra Industrial
Development Corporation, Mumbai.
Kallithraka S, Salacha M I and Tzourou I (2009) Changes in phenolic composition and
antioxidant activity of white wine during bottle storage: Accelerated browning test
versus bottle storage. J Food Chem 113:500–05.
Karangwa E, Khizar H , Rao L, Nshimiyimana D S, Foh M B K, Li L, Xia S Q and Zhang X
M (2010) Optimization of Processing Parameters for Clarification of Blended Carrot-
orange Juice and Improvement of its Carotene Content. Adv J Food Sci and Technol
2(5):268-78.
Kashyap D R , Vohra P K, Chopra S and Tewari R (2001) Applications of pectinases in the
commercial sector: a review. Biores Techno 77: 215-227.
Katalinic V, Mozina S S, Skroza D, Generalic I, Abramovic H, Milos M, Ljubenkov I,
Piskernik S, Pezo I, Terpinc P and Boban M (2010) Antioxidative and vasodilatory
63
effects of phenolic acids in wine. Food Chem 119: 715-23.
Keller J B (2010) Pineapple Wine: Directions for Pineapple Wine Baskets [online]. Available
from:http://www.ehow.com/way_5810589_directions-pineapple-wine
baskets.html#ixzz14iXAYA5s. [Accessed 16th November, 2010].
King M C, Cliff M and Hall J (2003) Effectiveness of the mouth-feel wheel for the evaluation
of astringent subqualities in British Columbia red wines. J Wine Research 14(2, 3):
67-78.
Kocher G S, Phutela R P and Gill M I S (2009) Evaluation of grape varieties for wine
production. Indian J Hort 66(3): 410-12.
Kocher G S, Gill M I S and Arora N K (ed) (2011a) Advanced production, post harvest and
processing technology. Punjab Agricultural University, Ludhiana.
Kocher G S, Phutela R P and Gill MIS (2011b) Preparation and evaluation of red wine from
Punjab purple (syn. H 516) variety of grapes. Int. J Food Ferment Technol 1(1):
133-36.
Kocher G S and Pooja (2011) Status of wine production from guava (Psidium guajava L.) A
traditional fruit of India. Afr J Food Sci 5: 851-60.
Krstic M, Moulds G, Panagiotopoulos B and West S (2003) Growing quality grapes to winery
specifications - Quality measurement and management for grapegrowers. Winetitles,
Adelaide.
Kunze W (2004). Technology Brewing and Malting. VLB Berlin, Germany [online].
Available from: http://en wipidia.org/wiki/special. [Accessed 2nd August, 2010].
Kunkee R E(1991) Relationship between nitrogen content of must and sluggish fermentation.
I: Rantz J M (ed) Proc International Symposium on Nitrogen in Grapes and Wine.
Vol.18-19,Pp 148-55. Am Soc Enol Vitic, Seattle, WA, Davis, CA.
Kunkee R E and Gosewell (ed) (1996) Table wines. In: Jay J M (Ed) Modern Food
Microbiology, 325-79, Chapman and Hall, USA.
Lafon-Lafourcade S (1983)Wine and brandy. In: Rehm H J, Reed G (ed) Food and Feed
Production with Microorganisms. Biotechnol. vol. 5 pp. 81– 163.Verlag Chemie,
Weinheim.
Lallemand (1997) Active Dry Wine Yeast Enoferm L2226 Saccharomyces cerevisiae
Technical Information. Lallemand Ink., Montreal.
Lao C, Lopez-Tamames E, Lamuela-Raventos R, Buxaderas S and Del Carmen de la Torre-
Boronat M (1997) Pectic enzyme treatment effects on quality of white grape musts
and wines. J Food Sci 62(6): 1142-49.
Larue F, Lafon-Lafourcade S, Ribereau-Gayon P (1980) Relationship between the sterol
content of yeast cells and their fermentation activity in grape must. Appl Environ
Microbiol 39: 808.
Lema C, Garcia-Jares C, Orriols I, Angulo L (1996) Contribution of Saccharomyces and non-
Saccharomyces populations to the production of some components of Albarino wine
aroma. Am J Enol Viticult 47: 206–16.
64
Liu H F, B H Wu, P G Fan, S H Li and L S Li (2006) Sugar and acid concentrations in 98
grape cultivars analyzed by principal component analysis. J Sci Food Agric 86:1526-
36.
Llauradó J M, Rozes N , Bobet R, Mas A, Constantí M (2002).Low temperature alcoholic
fermentation in high sugar concentration grape must. J Food Sci 67:2684273.
Lorrain B, Ky I, Pechamat L and Teissedre P L (2013) Evolution of Analysis of Polyphenols
from Grapes, Wines and Extracts. Molecules 18: 1076-1100.
Macheix J J, Sapis J C and Fleuriet A (1991) Phenolic compounds and polyphenoloxidase in
relation to browning in grapes and wines. Crit. Review. Food Sci Nutr 30: 441-86.
Maclure M (1993) Demonstration of deductive meta-analysis: ethanol intake and risk of
myocardial infarction. Epidemiology Rev 15:328–51.
Macrae R, Robinson R K, Saddler M J (1993) Wine: In Encyclopaedia of Food Science and
Technology 7, Harcourt Brace, London.
Maiherbe S, Bauer F F and Du Toit M (2007) Understanding Problem Fermentations - A
Review. S Afr J Enol Vitic 28(2): 169-86.
Makris D P, Boskou G, Andrikopoulos N K, Kefalas P (2008) Characterization of certain
major polyphenolic antioxidants in grape (Vitis vinifera) stems by liquid
chromatography-mass spectrometry. Eur Food Res Technol 226: 1075–79.
Mantyla A, Paloheimo M and Suominen P (1998) Industrial mutants and recombinant strains
of Trichoderma reesei. In: Harman G F and Kubicek C P (ed) Enzymes, biological
control and commercial applications. Pp. 291–309. Taylor& Francis, London.
Marais J (2003) Effect of different wine-making techniques on the composition and quality of
Pinotage wine. I. Low-temperature skin contact prior to fermentation. S Afr J Enol
Vitic 24: 70-75.
Marais J and Rapp A (1988) Effect of Skin Contact Time and Temperature on Juice and Wine
Composition and Wine Quality. S Afri J Enol Vitic 9(1):22-30.
Margalit Y (1997) Concepts in wine chemistry. The Wine Appreciation Guild Ltd, 360 Swift
Avenue, South San Francisco, CA 94080.
Marmot M G (2001) Alcohol and coronary heart disease. Intl J Epidemiology 30:724–9.
Medina K, Boido E, Dellacassa E and Carrau F (2005) Yeast interactions with anthocyanins
during red wine fermentation. Am J Enol Vitic 56: 104-08.
Mojsov K, Ziberoski, Jugoslav, Božinović, Zvonimir, Petreska,and Meri (2011) Effects of
pectolytic enzyme treatments on white grape mashs of Smederevka on grape juice
yields and volume of lees. Proc 46 Croatian and 6 International Symposium on
Agriculture, Opatija, Croatia.
Moreno J, Peinado J and Peinado R A (2007) Antioxidant activity of musts from Pedro
Ximenez grapes subjected to off-vine drying process. Food Chem 104: 224-28.
Moreno-Pérez A, Fernandez-Fernandez J I, Martinez-Cutillas A, Vila-Lopez R and Gil-
Muñoz R (2011) Effect of selected enzymes over chromatic parameters during
65
maceration period in syrah and cabernet-sauvignon. J Int Sci Vigne Vin pp 41-50.
Morris J R, Main G and Threlfall R (1996) Fermentations: Problems, solutions and
prevention. Vitic Enol Sci 51: 210-13.
Monk P R, Cowley P J (1984) Effect of nicotinic acid and sugar concentration of grape juice
and temperature on accumulation of acetic acid yeast fermentation. J Ferment
Technol 62:515–21
Monteiro F F and Bisson L (1992) Nitrogen Supplementation of Grape Juice I. Effect on
Amino Acid Utilization during Fermentation. Am J Enol Viti 43(1):1-10.
Mountney G J and Gould W A (1988) Practical Food Microbiology and Technology. AVI
Books, Van Nostrand Reinhold Company, New York, USA.
Nelson K E (1985) Harvesting and handling California table grapes for market: Bull 1913,Pp
72. University of California Press, DANR Publications, Oakland, California, USA.
Norjana I and Noor A A (2011) Quality attributes of durian (Durio zibethinus Murr) juice
after pectinase enzyme treatment. Intl Food Research J 18(3): 1117-22.
Okunowo W O, Okotore R O, Osuntoki A A (2005) The alcoholic fermentative efficiency of
indigenous yeast strains of different origin on orange juice. Afr J Biotechnol 4: 1290-
96.
Ough C S and Amerine M A (2nd ed) (1988) Methods for Analyses of Musts and Wines. Pp
377. John Wiley and Sons, New York.
Ough C S (1966) Fermentation Rates of Grape Juice. II Effect of Initial °Brix, pH, and
Fermentation Temperature. Am J Enol Vitic 17(1): 20-26.
Parker T L, Wang X H, Pazmino J and Engeseth N J (2007) Antioxidant capacity and
phenolic content of grapes, sun-dried raisins, and golden raisins and their effect on ex
vivo serum antioxidant capacity. J Agric Food Chem 55: 8472-77.
Parley P (1997) The effect of pre-fermentation enzyme maceration on extraction and colour
stability in Pinot noir wine. Thesis, University of Lincoln, New Zealand.
Pastrana-Bonilla E, Akoh C C, Sellappan S, Krewer G (2003) Phenolic content and
antioxidant capacity of muscadine grapes. J Agric Food Chem 51:5497–503.
Patil A B (2008) Microbiology-from education to industry. Pro National Conference on
Microbiology-from education to industry 1:41-43, Netaji Subhashchander Bose
College, Nanded, India.
Paul A H and Hoger G (2003) Interaction of pH, ethanol concentration and wine matrix on
induction of malolactic fermentation with commercial "direct inoculation" starter
cultures. Australian J grape and wine research 9:200-09.
Perez-Prieto L J, Lopez-Roca J M and Gomez-Plaza E (2003) Differences in major volatile
compounds of red wines according to storage length and storage conditions. J Food
Comp Anal 16:697-705.
Pezzuto J M (2008) Grapes and human health: A perspective. J Agric Food Chem 56:
6777–84.
66
Pimenta-Braz P N, Ricardo-da-Silva J M and Laureano O (1998) Evaluation of pectolytic
activities of enological interest in industrial enzyme preparations. Z Lebensm Unters
Forsch A 206: 14-20.
Pretorius I S (2000) Tailoring wine yeast for the new millennium: novel approaches to the
ancient art of winemaking. Yeast 16: 675-729.
Puri R, Kocher G S and Phutela R P (2012) Optimization of yeast inoculum for ethanol
production from sugarcane vinegar. J res Punjab agric Univ 49(1&2): 45-47.
Qin L, Xu S Y and Zhang W B (2005) Effect of enzymatic hydrolysis on the yield of cloudy
carrot juice and the effects of hydrocolloids on color and cloud stability during
ambient storage. J Sci Food Agri 85: 505-12.
Radeka S, Herjavec S and Sladonja B (2008) Effect of Maceration on Wine Aroma
Compounds. Food Technol Biotechnol 46(1): 86–92.
Rai P A, Majumdara G C and DasGupta S (2004) Optimizing pectinase usage in pretreatment
of mosambi juice for clarification by Response Surface Methodology. J Food
Engineering 64:397–99.
Rainieri S, Pretorius I S (2000) Selection and improvement of wine yeasts. Ann Microbiol 50:
15-31.
Revilla I and Gonzalez-San Jose M L (2003) Compositional changes during the storage of red
wines treated with pectolytic enzymes: low molecular-weight phenols and flavan-3-ol
derivative levels. Food Chem 80: 205-14.
Reynolds A, Cliff M, Girard B and Kopp T G (2001) Influence of Fermentation Temperature
on Composition and Sensory Properties of Semillon and Shiraz Wines. Am J Enol
Vitic 52(3): 235-40.
Ribereau-Gayon J, Peynaud E, Ribereau-Gayon P and Sudraud P (1982) Science and
technology wine (tr). 1. Dunod, Paris.
Ribereau-Gayon P, Pontallier P, and Glories Y (1983) Some interpretations of colour changes
in young red wines during their conservation. J Sci Fd Agric 34:505-16.
Ribereau-Gayon P, Dubourdieu D and Doneche B (2000) The handbook of Enology : The
microbiology of wine and vinifications. Vol.1 John wiley and sons. New York.
Ribereau-Gayon P, Glories Y, Maujean A and Dubourdieu D (2006) Handbook of enology,
vol. I. John Wiley and Sons, England.
Ripper M (1898) Die Schwelflige Saure im Wein und deren Bestimmung. J Praia Chem 46:
428-73.
Robinson J (ed) (2003) Jancis Robinson's Wine Course. Pp 39–41 ISBN 0-7892-0883-0,
Abbeville Press.
Robinson J (2006) The Oxford Companion to Wine. (Third Ed), Pp 267-269, 779-787.Oxford
University Press.
Romano P, Fiore C, Paraggio M,Caruso M, Capece A (2003) Function of yeast species and
strains in wine flavour. Intl J Food Microbiol 86: 169–80.
67
Rotter B (2008) Fining [online]. Available from: www.brsquared.org/wine. [Accessed 11th
July, 2011].
Ruffner H P (1982) Metabolism of tartaric and malic acids in Vitis: A review-Part A. Vitis
21:346-58.
Salinas M R, Garijo J, Pardo E, Zalacain A and Alonso G L (2003) Color, polyphenol and
aroma compounds in rose wines after prefermentative maceration and enzymatic
treatments. Am J Enol Vitic 54: 195-202.
Salinas M R, Garijo J, Pardo F, Zalacain A and Alonso G L (2005) Influence of
prefermentative maceration temperature on the colour and the phenolic and volatile
composition of rose wines. J Sci Food Agric 85: 1527-36.
Salmon J M, Vincent O,Mauricio J C,Bely M and Barre P(1993) Sugar transport inhibition
and apparent loss of activity in Saccharomyces cerevisiae as a major limiting factor of
enological fermentations. Am J Enol Vitic 44:56-64.
Salmon J M (1996) Sluggish and stuck fermentations: Some actual trends on their
physiological basis. Vitic Enol Sci 51:137-40.
Selli S, Cabaroglu T, Canbas A, Erten H and Nurgel C (2003) Effect of skin contact on the
aroma composition of the musts of Vitis vinifera L. cv. Muscat of Bornova and
Narince grown in Turkey. Food Chem 81: 341-47.
Sims C A and Bates R P (1994) Effects of skin fermentation time on the phenols,
anthocyanins, ellagic acid sediment and sensory characteristics of a red Vitis
rolundifolia wine, Am J Enol Vitic 45(1): 56-62.
Sims C A and J. R. Morris (1984) Effects of pH, Sulfur Dioxide, Storage Time, and
Temperature on the Color and Stability of Red Muscadine Grape Wine. Am J Enol
Vitic 35(1): 35-39.
Snyder R (2005) Wine Basics. Retrieved October 1, 2008. http://winegeeks.com/articles/18
Somers T C (1977) A connection between potassium levels in the harvest and relative quality
in Australian red wines. Australian Wine Brewing and Spirit Review. Pp 32-34.
Soni S K , Bansal N and Soni R (2009) Standardization of conditions for fermentation and
maturation of wine from Amla (Emblica officinalis Gaertn.) Nat Prod Radiance
8(4):436-44.
Staden van J , Volschenk H, Vurran van H J J and Bloom Viljoen M (2005) Malic Acid
Distribution and Degradation in Grape Must During Skin Contact: The influence of
recombinant Malo-ethanolic wine Yeast Strains. S Afri J Enol Vitic 26(1):16-20.
Steinkraus K H (1992) Lactic Acid Fermentations. In: Applications of Biotechnology to
Traditional Fermented Foods. Report of an Ad Hoc Panel of the Board on Science
and Technology for International Development, National Academy Press,
Washington D.C. USA.
Strehaiano P M and Gowa G (1983) Effect of inoculum level on kinetics of alcohol
fermentation. Biotechonol Lett 5: 135-40.
68
Tapsell L C, Hemphill I, Cobiac L, Patch C S, Sullivan D R, Fenech M, Roodenrys S, Keogh
J B, Clifton P M, Williams P G, Fazio VA, Inge K E (2006). Health benefits of herbs
and spices: the past, the present, the future. Med J Aust 185: 4-24.
Tontemsup S (1996) All about wine. Bangkok:Just wine company.
Topalovic A and Mikulic-Petkovsek M (2010) Changes in sugars, organic acids and
phenolics of grape berries of cultivar Cardinal during ripening. J Food Agric Environ
8(3,4): 223- 27.
Torija M J, Beltran G, Novo M, Poblet M, Guillamon J M, Mas A and Rozes N (2003a)
Effects of fermentation temperature and Saccharomyces species on the cell fatty acid
composition and presence of volatile compounds in wine. Int.J.Food Microbiol
85:1274136.
Torija M J, Rozes N, Poblet M ,Gillamon J M and Mas A(2001) Yeast population dynamics
in spontaneous fermentation composition between wine areas during 3 consecutive
years .Automatic van zeeuwenhook 79:345-52.
Torrea D and Henschke P A (2004) Ammonium supplementation of grape juice- Effect on the
aroma profile of a Chardonnay wine. Technical Review 150: 59-60.
Ugliano M, Henschke P A, Herderich M J and Pretorius I S (2007) Nitrogen management is
critical for wine flavour and style. Wine industry journal 22(6): 24-30.
Vaidya A, Vaidya M, Sharma S and Ghanshayam A(2009) Enzymatic treatment for juice
extraction and preparation and preliminary evaluation of Kiwifruits wine. Natural
product radiance 8(4): 380-85.
Varandas S, Teixeira M J, Marques J C, Aguiar A, Alves A and Bastos M (2004) Glucose and
fructose levels on grape skin: interference in Lobesia botrana behavior. Anal Chem
Acta 513: 351-55.
Watson B, Price S, Ping Chen H, Young S, Lederer C and McDaniel M (1997) Fermentation
practices in Pinot Noir: Effects on color, phenols, and wine quality. In: Henick-Kling
T, Wolf T E and Harkness E.M. (eds). Proc. 4th Int. Symp. Cool Climate Vitic. Enol.,
Rochester, USA. pp VI 18-VI 2.
Wimalsiri P, Sinnatamby A, Samaranayake S and Samarasingh C R (1971) Cashew Apple
Wine. Industry Prospect Report 44, Industrial Development Board, Sri Lanka.
Winkler A J, Cook J A, Kliewer W M and Lider L A (1974) General Viticulture. University
of California Press, Berkeley, CA.
"Wine Grapes and Grape-y Wines". Retrieved 03/07/2010.
Xia E Q, Deng G F, Guo Y J, Li H B(2010) Biological activities of polyphenols from grapes.
Int J Mol Sci 11: 622–46.
Yadav B S, Sheoran A, Rani U and Singh D (1997) High ethanol productivity in an
immbolized cell reactor. Ind J Microbiol 37: 65-67.
Yadav M, Jain S, Bhardwaj A, Nagpal R, Puniya M, Tomar R, Singh V, Parkash O, Prasad G
B K S, Marotta F, and Yadav H (2009) Biological and Medicinal Properties of Grapes
and Their Bioactive Constituents: An Update: A review. J Med Food 12(3): 473–84.
69
Yildirim H K (2011) Effects of Fining Agents on Antioxidant Capacity of Red Wines. J Inst
Brew 117(1): 55–60.
Zafrilla P, Morillas J, Mulero J, Cayuela J M, Martínez-Cachá A, Pardo F and López Nicolás
J M (2003) Changes during storage in conventional and ecological wine: phenolic
content and antioxidant activity. J Agric Food Chem 51(16): 4694-700.
Table I: Modified Davis Score Card for Sensory analysis of wine
Wine sample_____________ Name of Panelist_________________
Fruit variety_____________ Date of analysis _________________
Ethanol (w/v) _____________ Titrable acidity __________________
Characteristic Weight Score points
Excellent
(4)
Good
(3)
Average
(2)
Mediocre
(1)
Poor
(0)
Visual
Appearance (clarity, no
suspended material)
2
Colour (pale green & purple rosy
desirable;& off colours
undesirable )
2
Taste
Astringency (pickery feel) 1
Body (full i.e high alcohol
desirable) 1
Sweetness/bitterness (Very sweet
and very bitter undesirable) 1
Sourness (vinegar undesirable) 1
Flavour ( mouth feel when wine
kept in mouth for few seconds) 2
Olfactory
Aroma (wine odour derived from
guava), shouldn’t be too earthy,
stemmy or moldy
3
Bouquet (odour developed
during fermentation, processing
or aging)
Yeasty &burning undesirable
3
Total acidity (titrable) 2
General quality (Overall feel) 2
Total score (Out of 80)
Ratings: Superior- (68-80); Standard- (52-68); Below standard-(36-52);
Unacceptable/ Spoiled(4-36)
ANNEXURES
ii
TABLE II: Analysis of Factorial Experiment in CRD for DAHP (Table 10, Results and Discussion)
No. of reps.= 2 No. of factors= 2
TREAT. COMBINATION MEANS
1 .10000000 6.8000000 11.000000 1.1000000 6.6500000
6 11.350000 1.0750000 6.3500000 11.650000 1.4000000
11 7.5000000 12.100000 .96000000 6.9500000 10.800000
16 .55000000 6.7000000 10.400000
FACTOR MEANS
1 6.1666670 6.6791670 6.0600000
2 .71166680 6.7000000 11.150000 1.0166670 6.9499990 11.283330
ANOVA TABLE
SOURCE d.f. M.S. F-Ratio CD(5%) C.V.
A 2 1.3149620 6.71 .379451
B 5 129.69810 662.08 .536625
AB 10 .42424320 2.17 NS
Error 18 .19589570 7.02
TABLE III: Analysis of Factorial Experiment in CRD for Recycling (Table 10, Results and Discussion)
No. of reps.= 2 No. of factors= 3
TREAT. COMBINATION MEANS
1 2.5000000 4.4500000 3.3500000 3.6850000 3.8600000
6 4.5000000 5.1500000 5.6000000 3.3500000 3.4000000
11 2.6000000 2.8100000 4.9500000 7.9000000 5.7000000
16 4.9800000 5.5500000 6.5000000 5.7500000 6.7500000
21 5.6550000 6.4500000 4.4500000 4.9750000 8.9600000
26 9.6399990 8.8099990 9.6000000 8.2999990 8.2400000
31 7.3100000 8.2000010 9.7700000 9.9850010 8.4950000
36 6.7450000
FACTOR MEANS
1 4.0494440 4.9322220 6.8266660 8.5161110
2 5.2837500 5.9791670 6.9804170
3 6.0600000 6.3162500 5.8670830
ANOVA TABLE
SOURCE d.f. M.S. F-Ratio CD(5%) C.V.
A 3 71.596100 2563.07 .112944
B 2 17.459150 625.02 .978126E-01
AB 6 9.4375170 337.85 .195625
C 2 1.2186690 43.63 .978126E-01
AC 6 1.0220740 36.59 .195625
BC 4 1.2968350 46.43 .169416
ABC 12 2.8804780 103.12 .338833
Error 36 .27933760E-01 2.75
iii
TABLE IV: Analysis of Factorial Experiment in CRD for storage phenols
(Table 12, Results and Discussion)
No. of reps.= 2 No. of factors= 2
TREAT. COMBINATION MEANS
1 222.60000 207.90000 210.90000 201.40000 206.20000
6 180.60000 187.40000 175.90000 145.80000 155.70000
11 138.80000 141.40000 106.60000 127.20000 102.40000
16 112.90000 100.20000 110.40000
FACTOR MEANS
1 215.25000 206.15000 193.40000 181.65000 150.75000
140.10000 116.90000 107.65000 105.30000
2 157.87780 157.04440
ANOVA TABLE
SOURCE d.f. M.S. F-Ratio CD(5%) C.V.
A 8 7390.2420 239.39 8.25056
B 1 6.3402780 .21 NS
AB 8 228.87930 7.41 11.6681
Error 18 30.871530 3.53
TABLE V: Analysis of Factorial Experiment in CRD for storage ethanol (Table 12, Results and Discussion)
No. of reps.= 2 No. of factors= 2
TREAT. COMBINATION MEANS
1 12.100000 9.9700000 11.630000 9.8600000 11.485000
6 9.6450000 11.220000 9.5300000 10.715000 9.3600010
11 10.710000 9.1900010 10.660000 9.1000000 10.450000
16 8.9900000 10.420000 8.9100000
FACTOR MEANS
1 11.035000 10.745000 10.565000 10.375000 10.037500
9.9500010 9.8800000 9.7200000 9.6650000
2 11.043330 9.3950000
ANOVA TABLE
SOURCE d.f. M.S. F-Ratio CD(5%) C.V.
A 8 .93148800 7.96 .508123
B 1 24.453450 208.84 .239532
AB 8 .56233640E-01 .48 NS
Error 18 .11709260 3.35
iv
TABLE VI: Analysis of Factorial Experiment in CRD for sensory evaluation (Table 14, Results and Discussion)
No. OF REP = 3 No. OF TREATMENTS = 6
TREATMENT MEANS
1 13.000 2 17.100 3 18.100 4 7.533 5 6.067
6 61.800
ANOVA TABLE
SOURCE d.f. M.S. F-Ratio F-TABLE(5%)
Treatments 5 1293.3930 246.55 3.11
Error 12 5.2460120
G.M.= 20.600 C.V.= 11.12 CD(5%) = 4.076
v
VITA
Name of the student : Maninderjeet Kaur
Father’s name : S. Amarjit Singh
Mother’s name : Smt. Ranjit Kaur
Nationality : Indian
Date of birth : 29.11.1991
Permanent home address : Ward No. 1, Street No.9, House no. 1760,
Mansa-151505, Punjab.
EDUCATIONAL QUALIFICATION
Master’s degree : Integrated Master of Science (Hons.) in
Microbiology
University and year of award : Punjab Agricultural University, Ludhiana
(2013)
OCPA : 8.03/10.00
Title of Master’s Thesis : Effect of pre and post fermentation
treatments on quality of red wine preparation
from Punjab purple grapes.