introduction - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/91424/9/09_introduction.pdf ·...
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Wine is a popular drink being enjoyed all over the world. Historians believe that
wine was being made in Caucasus and Mesopotamia as early as 6000 BC. Records to
wine have been found in Egypt and Phoenicia dating as far back as 5000 BC and by 2000
BC, it was being produced in Greece and Crete colonization by Romans spread wine-
making all around the Mediterranean, by 500 BC, it was spread to Sicily, Italy, France,
Spain, Portugal and Northern Africa. Cultivation of the wine also spread in to Balkan
states and the Romans took it into Germany and other parts of the llorthern Europe,
eventually reaching as far as Britain (Robinson 1994).
Production and consumption of fermented beverage like wine is an ancient
practice. However, production and consumption of fruit based distilled alcoholic
beverage is a later development. Different aspects of fruit based alcoholic beverage other
than grapes have been investigated (Barnetl 1980). Rigveda amply testifies that the wine
is perhaps the oldest fermented product known to man. However, still the actual
birthplace of wine is unknown though i t had been prepared somewhere in 350 BC (Joshi
& Devender 2005). European explores in the 16'" century introduced the wine into the
new world (Amerine ei al, 1980). 'The early spreading and world distribution of the
vine and winemaking technology was shown in Figure 1.
Wine has been made in India for as many as 5,000 years. It was the early
European travelers to the courts of the Mughal emperors Akbar, Jehangir and Shah Jehan
in the sixteenth and seventeenth centuries who reported tasting wines from the royal
vineyards. Red wines were made from the arkeshum grape and white wine from urkawati
and bhokry grapes (Joshi & Parmar 2004).
0 2WO BC d) Birlh 01 Chriul tirwce Halkall Sutcn . Crctc . Nonhcrn 1:urnpc
Fig. 1: The early spreading and world distribution of the vine and winemaking technology (Source: Pretorius 2000).
For winemaking, the grapes are crushed immediately after picking and the stems
generally removed. The yeasts present on the skins come into contact with grape sugars
and fermentation hegins naturally. A look at the early days of winemaking makes it
obvious that, the different techniques produced varied styles of wine; the basic principles
changed a very little but the style is very similar (Fig. 2). During the last 150 years or so.
however, the scientific basis of winemaking has gradually become clearer, and many
practices once thought impossible have now become routine. These fundamental
innovations in winemaking practices revolutionized the wine industry, and today the
forces of market-pull and technology-push continue to challenge the tension between
tradition and innovation (Fleet 1993).
Wine production from fruits other than grapes
In European usage, wine and brandy refer to exclusively fermented byproducts of
grapes - member of the genus Vilis, notably cultivars of viniferu. In English, especially
in the new world, wines and brandies may refer to the fermented by-products of any
fleshy fruit or flower. The quality and quantity of grapes depend on geographical,
geological and climatic conditions in the vineyards, and the grape variety and methods of
cultivation (Joshi rr 01. 1999).
In general, grapes are thc main raw materials that have been used for wine
production for the past few decades. However, many research groups havr investigated
the suitability of fruits other than grapes (Table 1) Compared to the quantity of grape
wine produced and consumed in the world, the amount of wine produced from non-grape
fruits is insignificant (Amerine el 01. 1980; Sandhu & Joshi 1995). However in some
countries wine from other fruits like apple (Spain, France, Belgium, Switzerland and
England) plum (Germany) and cashew apple (India) wines have very much demand
(Joshi et a1 1999).
Outline of Wine Production - on ancient art -
Fig, 2: The main steps in wine production (adapted from Walker 1998)
Table 1: Different types of Wine Produced from Different Fruits
F ~ i t s Investigators Apple Sandhu & Joshi 1995
Apricot Joshi el al. 1990
Banana Kotecha el al. 1994
Ber Gautm & Chundawt 1998
Cashew Mandal 1997
Custard apple Kotecha et al 1995
Dates Ali & Dim 1984
Jamun Shukla el ul. 1991
Grape fruit & Kinnow Joshi 61: Thakur 1994
Kiwi fruit Healherbell ef al. 1980;Soufleros ct al.
200 1
Litchi Vyas et ul. 1989
Mandarin Selli il! ul. 2004
Mango Kulkarni el al. 1980; Onkarayya & Singh
1984; Keddy & Reddy 2005
Marula 1:undira et 01. 2002
Muskmelon 'reotia PI ul. 199 1
Orange Selli el ul. 2002 & 2003
Palm sap & Coconut Nathanael 1955
Peach Joshi & Shah 1998
Pear Atrri el 01. 1994
Plum Vyas & Joshi 1982; Joshi & Sharma 1995
Pomegranate Adsule el al. 1992
Sapota & Guava Bardiya el ul. 1974
Strawberry Pilando ct 01. 1985
Wines made from complete or partial alcoholic fermentation of grape or any other
fruit and certain ethyl alcohol as the intoxicating agent, essential elements, vitamins,
sugars, acids, phenolics. Wines from fruits are preferable to distilled liquors for
stirnulatory and healthhl properties (Gasteineau 1979). These beverages also serve as an
important adjunct to the human diet by increasing the satisfaction and contribute to the
relaxation necessary for proper digestion and absorption of food. Joshi el ul. (2002) have
reported the antimicrobial effect of apple wine on pathogenic bacteria.
It is a matter of astonishment to many that mango (Mung;firu indica L.), one of
the most celebrated of tropical fruits, is a member of the family Anacardiaceae. which is
notorious for embracing a number of highly poisonous plants. The mango fruit is one of
the most highly priced desert fruits of the tropics. I t has rich luscious. aromatic flavor and
a delicious taste in which sweetness and acidity delightfully blended. Mango production
has experienced continuous growth in the last decades of the 201h century (Baisya 2004).
The world's total annual mango fruit production was estimated at 22 million
metric tonnes (Mmt). Global production of mangoes is concentrated mainly in Asia and
more precisely in India that produced 12 Mmt per annum. Mangoes are cultivated in 85
countries. Total world production in 2004 was 26,147,900 M'I' (FAOS1'AT 2005). Asia
and the oriental countries produced around 80% ot'the world's total production. Major
mango producing countries are India, Mexico, China and Pakistan.
Mango is the choicest fruit of the country. It has been cultivated in India for the
last 4,000 years. Mango is called as the king of fruits and pride fruit of India. The crop is
significantly important in the fruit economy of India and is the 3Id largest industry in the
country. In India mango is grown in 2 million acres and it occupied@% of total
fruit production (Table 2 and Fig. 3). It is h e most cultivated area occupied crop in India
with 60% of the total area under fruits. Morr: than 25 types of mango cultivars are
available in India, that are widely cultivated allover thr world (Anon 1962).
Andhra Pradesh, being blessed with varied soil types and egro-climatic
conditions, is better placed for cultivation of large varieties of fruit crops and is one of the
largest fruit production states in India. The major fruit crops grown in Andhra Pradesh
are mango, sweet orange, banana, grape, pomegranate, coconut, and cashew. Mango
occupying an area of 3.71akh hectares, with an annual production of 32 lakh mt, has
placed the state in first position with a shart' of 20% of the India's production
coupling with highest productivity (Baisya 2004).
Mango contains a high concentration of sugar (1 6- 18% wlv) and acids with
organoleptic properties, and also contains antioxidants like carotene (as Vitamin A, 4,800
IU). Sucrose, glucose and fructose are the principal sugars in ripened mango, with small
amounts o f cellulose, hemicellulosc and pectin. The green tender fruits are rich in starch,
and during ripening the starch that is present is hydrolyzed to reducing sugars (Anon
1962). The unripened fruit contains citric acid, malic acid, oxalic acid, succinic and other
organic acids, whereas in ripened fruit, the main acid source is malic acid (Giri el ul.
1953). Mango juice along with aromatics is recommended as a restorative tonic; it
contains good concentrations of vitamin A and C, which are useful in heat apoplexy.
Mangoes with higher initial concentration of p-carotene are helpful as cancer-preventing
agents.
Fig. 3: Production of different fruits in India (2004) (Source: FAOSTAT 2005)
4%
3% 3%
I
I Bananas
B Mangoes i 1
o Oranges 1
Apples I Lemons and Limes '
I I a Pineapples
O Grapes I
Table 2: Production of selected fruits in India
Fruit Name Average (199211994) 2002 2003 2004
('000 tonnes)
Banana 9718 16820 16820 16820
Mango 10108 10640 10780 10800
Orange 1743 3120 3070 3070
Apple 1205 1160 1470 1470
Lemons & Lime 863 1440 1420 1420
Pineapple 956 1180 1310 1300
Grape 684 1210 1150 1200
Papaya 470 700 700 700
Pear 127 200 200 200
Peache and Nectarine 83 150 150 IS0
Grapefruit & Pomelo 83 140 142 142
Plum 5 5 80 80 80
Fig 6 I1 11 11
Apricot 7 10 10 10
Cherrie 4 8 8 8
Fig. 4a: Production ( O h ) of fruits in different countries.
Fig. 4b: Production (%) of wines in different countries.
Country Name
(Source: Joshi and Parmar 2004)
Fruits like mango are highly perishable commodities. In developing countries like
India, 20-30% of fruits produced undergo spoilage due to lack of proper utilization and
undeveloped post-harvesting technology (Sandhu & Joshi 1995). The ever-increasing
h i t production needs improvement in preserving technologies. In India, efforts have
been made to utilize the surplus (after export) and small and unattractive mangoes for
various purposes; one of the alternatives is wine production. Fruits are utilized to produce
a variety of alcoholic beverages including different types of h i t wines and their
distillates are known as brandy. RipVeda has also mentioned the medicinal power of
wines (Vyas & Chakravorthy 1971). These beverages have been a part of food of man
ever since his settlement in Tigris Euphrates basins and were used as therapeutic agents.
While fruit wines are produced and consumed throughout world, hard liquors still
constitute the big chunk of alcoholic beverages consumcd in India. The research on wine
production done in lndia reveals that an impressive progress has been made in the
development of technologies for preparation of wines of different rypes from various
fruits. Successful marketing of grapes and apple wines in lndia is an indicator of potential
Indian market waiting for fruit wines.
An alternative and profitable method of using mangoes for winemaking could
become widely accepted. Many investigators have carried out much research on mango
composition, and on cultivation aspects. Anon (1963) first reported that the wine
production from mango pulp and then Czyhrinciwk (1966) have reported the technology
involved in mango wine production. However, Kulkarni el al. (1980); Onkarayya &
Sin& (1984) and Onkarayya (1985; 1986) screened some varieties of mango for
winemaking and found that mango wine had similar characteristics to that of grape wine.
l'hese authors have not given details on vinification techniques and chemical composition
of the wine produced from mango. Their work was inadequate, particularly in the area of
mango wine production and its composition.
PRE-FERMENTATIVE PRACTICES:
The pre-fermentative aspects are different in different places and vintages. But the
main and normal aspects that are used for producing wine are:
a). Crushing/Stemming: crushing wleases the juice and activates enzymes liberated from
grapes. Stemming removes the fruit stalk (rachis and peduncle) and other vine parts;
b). Maceration: Crushing activates a wide range of hydrolytic and oxidative enzymes in
grape cells. Because many enzymes remain bound to cell frtigments, their action
(maceration) can be curtailed hy rapid removal of the juice tiom the pomace (pressing)
(Ramey et al. 1986; Guedes de pinho el (11 1094).
c). Presses and dejuicers: Pressing separates the juice or parlially vinified wine from the
seeds and skins of the must.
d). Clarification: It improves the fermentation performance of the yeast. Bentonite was
preferable for the clarification. in large wineries, centrifugation is commonly used to
speed up the clarification. Filtration and flotation are other options for rapid clarification
and e). Juice adjustment: adjustment of acidity and sugar to desirable level are permitted.
If the acidity of the juice undesirably low (>Sg/l), acids such a tartaric and citric may be
added. If the acidity ofthe juice is high, blending with low acidity juice can be effective.
It can increase sugar concentration, without addition of sugar, do so by the removal of
water by Reverse-osmosis (Duitschaever el al. 1989), cryo-extraction (Chauvet el al.
1986) and entropie concentration (Foment 1993).
FERMENTATION
Yeast diversity associated with grapm and winemaking
Louis Pasteur's simple fermentation of sugars to ethanol and (202. is today more
complex and sophisticated. The production of wine involving the sequential development
of microbial species and biochemical processes, as affected by a particular environment
is established. The process includes the interaction of fungi, yeasts, lactic acid bacteria,
acetic acid bacteria, mycoviruses and bacteriophages and these affecting the grape
associated microorganisms. Of all these, yeasts are at the heart of the biochemical
interaction with the musts derived from the varieties of V. ldnijira and other grape
species (Fleet 1998; Kurtz man & Fell 1998). Of the 100 yeast genera representing over
700 species described in the latest edition of the monographic series, only 15 arc
associated with wine-making. These are, B r e ~ m o m ~ ~ c c ~ s and its sexual equivalent
Dekkara, Candida, Cryptococcus, Debaryom)~ces, Honansia.~poru and its asexual
counterpart Koleckera, Kluyvcro~n"vces, Melschnikowa, Pichia. Rodolorulu,
Soccharomyces, Sacchuromycodes, Schizo.sacchuromyceLs and Zygosucchuromyces
(Kreger-Van Rij 1984; Pretorius el al. 1999). Despite the striking growth of the number
of described yeast species over the last 50 years, it is generally accepted that thc wealth
of yeast biodiversity with hidden oenological potential is still largely untapped (Lodder
1970a & 1970b; Kreger-Van Rij 1984; Kurtzman & Fell 1998).
Wine yeast starter cultures
Spontaneous (natural) versus inoculated wine fermentation
The wine yeast, S, cerevisiae cells are generally ellipsoidal in shape. S. cerevisioe
has a relatively small genome, a large number of chromosomes, little repetitive DNA and
f w ~ introns (Petering et d 1991). Haploid strains contain appmhately 12.13 mega
bases Imb) of nuclear DNA, distributed along 16 linear chromosames. Each chxomosome
is a single deoxyribonucleic acid (DNA) molecule approximately 200-2200 kilo bases
(kb) long. The genome of a laboratory strain of S. cerevhiae has been completely
sequenced and found to contain roughly 6000 protein-encoding genes. The S. cerevjsiae
genome, which is relatively rich in guanine and cytosine content (%WC of 39-41) is
much more compact when compared with the genomes of other eukaryotic cells, Most
laboratory-bred strains of S, cerevisiae are either haploid or diploid. However, industrial
wine yeast strains are predominantly diploid or aneuploid, and occasionally polyploid. It
is not yet clear whether polyploidy in industrial yeast strains is advantageous (Snow
1983). Heterosis rather than ploidy is responsible for improvement of fermentation
performance (Hammond 1996).
Originally, types of wine were made by taking advantage of natural microflora for
spontaneous fermentation; no deliberate inoculation was made to start the process.
Various yeasts found on the surface of grape skins and the indigenous microbiota
associated with winery surfaces participate in this natural wine fermentations. Yeasts of
the genera Koleckera, Hansenimpora and Cundidia predominate in the early stages,
when the ethanol rises to 5.8% (Fleet 1993; Jolly et (11. 2000). The latter stages of natural
fermentations are invariably dominated by the ethanol tolerant strains of S, cerevisiae.
Other yeasts, such as species of Bre!tanomyces, Kluyveromyces, Schizosuccharomyces,
Torulospora and Zygosaccharomyces, may also be present during the fermentation and
subsequently in the wine, some of which are capable of adversely affecting sensory
quality. Selli et al. (2004) produced the wine from orange through spontaneous
fermentation.
With the i m p o m of S. cerevisiae's role in winemaking, there is an ever-
growing strategy for new and impmved wine yeast strains is now tirmly established. In
addition to the primary role of wine yeast to cata ly~ the efficient and complete
conversion of grape sugars to ethanol without development of off-flavours, starter culture
strains of S. cerevisiae must posses a range of other properties (Table 3). Leading
winemakers are now translating the adage "horses for courses" into "special yeasts for
special treats" (Pretorius 2000).
The inoculation of pure starter culture approach has several advantages, such as a
decrease in lag phase, significant reduction of the influence of naturally occurring yeast
strains, rapid and complete grape must fermentation, and hence allows for a higher
degree of wine reproducibility (Bauer & Pretorius 2000).
In spontaneous fermentations, a large diversity of microorganisms participates,
including oxidative and fermentative yeasts, homo- and hetero-fermentative lactic acid
bacteria, and acetic acid bacteria (Fleet Lk Heard 1993). The main drawbacks reported are
variability in the product quality and risk of anomalous fermentation (Beach & Carr
1977; Splitt-Stoisser 1982).
The primary disadvantage of induced fermentations is the cost of purchasing the
yeast inoculum. One technique designed to reduce this expense is cell recycling batch
fermentation (Rosini 1986; Suzzi 1996). The cell-recycling procedure reduces the sulfur
dioxide formation, but it increases the production of acetic acid. However, constant
monitoring is required to check that the strain has not mutated, nor been taken over by
any other contaminated strains.
Table 3: Desirable Cbar~cteristics of wine yeast.
Fermentation & Flavour Technological & Metabolic Properties properties Rapid initiation of fermentation High genetic stability
High fermentation efficiency High sulphite tolerance
High ethanol tolerance Low sulphite binding activity
High osmotolerance Low foam forn~ation
Low temperature optimum Flocculating property
Low biomass production Compacts sediment
Low suphide / DMS / thiol formation Resistance to desiccation
Low volatile acidity production Killer properties
Low higher alcohol production Genetic marking
Liberate glycosylated precursors Proteolytic activity
High glycerol production Low nitrogen demand
Hydrolytic activity Low sulphite formation
Enhance autolysis Low hiogenic amine formation
Modified esterase activity Low ethyl carbamate potential
Stages of Fermentation:
During the first few days, the metabolism of S, cerevisiae is directed towards cell
growth and division. The population increases until reaching about 10' to 1 0 b l l l m l .
This stage is termed the exponential phase. In this phase, cell metabolism primarily
involves in the biosynthesis of amino acids, nucleic ucids. polysaccharides and lipids.
Thus less ethanol is generated during the early stages of fermentation than later.
Oxidation of acetaldehyde to acetic acid supplies need-reducing power to yeast. After
cessation yeast at growth, some of the released acetic acid and acetaldehyde are
transported back in to cell.
As the ethanol content increases, the sugar transport across the cell membrane (at
concentration >2%) becomes increasingly disrupted (Casey & Ingledewl986). Increased
ethanol content decreases the availability of unsaturated fatty acids for cell growth.
Scavenging sterols from metabolically inactive mitochondria1 membranes help in limited
further growth. Cell division was essentially stops when half of the fermentable sugars
have been consumed by yeast cell. At this point, the cell division just balances the cell
death; this stage is called as the stationary phase. This stage is comparatively short in
duration, quickly followed by a prolonged decline phase, where high cell death occurs.
During this phase also, fermentation was continuous at a slower pace and the yeast
population was stabilized at about lo4 to 10\ells/ml. The remaining hall' of the
fermentable sugars was consumed during this stage only. Surprisingly, about half the
yeast population (1 o4 to 10' cellslml) remains viable, dying slowly over the next several
months (Bisson & Block 2002; Verstrepen el al. 2004).
Facton influencing the fermentation
1) CarbonIEnergv Sources: The primary factor affecting the progress of fermentation is
undoubtedly the supply of soluble sugars in the juice. Succhuromyces cerevisiac: can
ferment only some sugars such as glucose and fructose. When sucrose is added during
chaptalization it is rapidly hydrolyzed to glucose and fructose. Acetic acid and ethanol
can be metabolized, but only through respiration. The usual sugar content in wine
fermentations is 20-25%. High sugar contents increasingly cause difficulty, initially due
to the osmolarity of the juice and later, due to the combined effects of ethanol and
osmolarity of the must. Consequently, very sweet musts are prone to inconlplete
fermentation and retention of sweet finish (Vcrstrepen 2004).
2) Alcohol content: the increasing content of alcohol eventually inhibits the yeast
metabolism, even in the presence of fermentable sugars. Ethanol disrupts the transport of
sugars across the cell membrane (Salmon et al. 1993). I'he uptake of ammonium and
several amino acids is adversely affected by alcohol. Fermentation usually ceases at
concentrations between 13 and 15% of ethanol, while yeast growth generally stops at
about half this value (Casey & lngledew 1986)
3) Nitrogen Content: The nitrogen content in the musts immensely affects the
fermentation after sugar. With healthy grapes. there is usually ample ammonium or
amino nitrogen to complete the fermentation. I'he form in which nitrogen is translocated
into cell (ammonium vs. amino acids) may affect the aromatic character of the wine
indirectly. This influence is most well known and related to the fuse1 alcohol content of
wine- ammonium limits the content, while amino acid nitrogen increases the synthesis
(Joshi et al. 1990; Ter Schure ei a!.. 2000).
4) Lipidr: They play important role in yeass, including nutrient storage and regulation.
However, in the fermentation process. their main significance involves cell membrane
function. In presence of oxygen, yeasts are ahle to synthesize their lipid requirements.
During fermentation, yeasts release fatty acids. Among of these. notably octanoic acid
and decanoic acids can accumulate to levels that increase ethanol-induced nutrient
leakage (Sa Correia el a/. 1989).
5) Phenolic compound.^: These compounds arc more important in the fermentation of red
wine and white wine. In red wine, anthocyanin pigments appear to stimulate
fermentation, while the procyanidins of while grapes may be slightly inhibitory
(Cantarelli 1989). In the second fermentation of sparkling wine. phenolic constituents are
typical inhibitors. Some yeast strains can enzymatically modify the phenols found in the
grape juice. Of particular sensory impact is the decurboxylation of' fcrulic acid and
p-coumaric acids to the aromatic con~pounds 4-vinyl guaiacol and 4-vinyl phenols,
respectively (Howell et 01. 2004).
6) Oxygen: It is not required for fermentation. Nevertheless, oxygen uptake during
crushing favours the production of essential sterols (ergosterol and lanosterol) and
unsaturated fatty acids, such as linoleic acid and linolenic acids. Traces of oxygen also
permit yeast synthesis of the nicotinic acid (Bafrncova 1999). Oxygen also favours the
accumulation of urea, associated with the production of ethyl carbarnate (Henschke &
Ough 1991).
7) Temperature: To some extent temperature increases the yeast growth; the speed of
enzyme action approximately doubles with every 10°C rise. Temperature can influence
fermentation by affecting the rate of enzyme action (Ough 1966; Torija et al.. 2002).
Cell sensitivity to the toxic effect of alcohol increases with temperature, presumably due
to increased membrane fluidity. This may partially explain the rapid decline in yeast
viability at temperatures above 20°C during wine fermentation. The enhanced production
of glycerol at warm temperatures counters the bitterness of tannins, and generates a
smoother mouth-feel. The fermentation at low temperatures such us <15"C' leads to more
aromatic and paler wines (Bauer & Pretorious 2000). The musts, which contain high
sugar density and low nitrogen at low temperatures, sliow stuck fermentations. Such
enological influences should bc reflected in the chemical composition nnd sensory
properties of the wine (Lambrechts & Prctorious2000; 'I'urija ei ul. 2002).
8) Carbon dioxide: Equal proportion of carbon dioxide is formed in the fermentation
process as a major by-product. Except for sparkling wines, its retention is not desired. To
avoid slowing fermentation, gas is allowed to escape freely. Yeast growth ceases at
pressures above 700kPa (7atm). albeit fermentation may continue. slowly, up to 3000
kPa. The volume of carbon dioxide produced often approximates fiAy times that of the
juice. The C 0 2 gas also removes ethanol and aromatics from wine (Miller el 01. 1087) to
some extent.
9) Pesticide Reuidues: The most commonly used fungicides are contact type, particularly
those that remain and have their protective action on fruits and leaf surfaces. These
compounds rarely cause problems during fermentation. Besides affecting the start of
fermentation, some fungicides such as elemental sulfur and some insecticides can
promote the production of hydrogen sulfide and sulfur dioxide (Kundu et ul. 1981).
Enzymes in Winemaking:
Winemaking is a complex biochemical process in which the fruit juice is
converted into wine by the action of several enzymes which either by juice and yeast or
exogenous industrial enzymes. There is a glut of research-based information on the
endogenous enzymes in the grape juice or rhal extracted from microorgcu~isms associated
with fermentation (Amerine er 01. 1980; Villettaz 1986). A number of enzynles like
pectinases, oxido-nductases, proteases, glucosidases. lip-oxigenases have originated
fiom grape juice (Pelnik & Rembouts 1981; Cayrel 1.1 al. 1983; Freuillat 1980). Due to
lack of sufficient endogenous enzymes, now a days addition of exogenous enzymes has
become a general practice in winemaking. 'The most frequently used enzymes in the wine
production are protease, pectinase and glucanase. Application of enzymes in food and
beverages has been reviewed by Williams ijr (11. (1982). Though there are several
investigations have been carried out with reference to the grape wine fermentation the
information available with other fruits is scnnty.
Production and role of enzyme protease in wine fermentation was studied in well
manner (Freuillat 1980). It plays very important role in the autolysis process normally
employed in the production of sparkling wine and in reduction of haze in wine (Freuillat
1980, Nelson & Young 1986). The presence of cell wall endo fi ( I , 3) glucanase activity
in the strains of dried yeasts has also been demonstrated (Lyauberes el ul. 1987) and is
responsible for release of manno proteins during maturation of wine on lees (Lyauberes
et al. 1987). P-glucosidase is an important enzyme found in many plants, fungi and
yeasts. This enzyme plays significant role in aroma liberation from grape and other fruits,
though the specific yeast strains affect the varietal aroma of wine (Delfini el 01. 2001).
Treatment of mnsh or fruit pulp with pectinase increases the juice yield and better
clarification. Pectin esterase use in clarification of plum juice has been reported (Bhutani
& Joshi 1995). Addition of pectinase is recommended in the preparation of cider and
apple wine for improved fermentability. clarity and sensory quality (Flares & Heatherbell
1984; Kotecha et a!. 1995). Addition of pectinase changes the physico-chemical
characteristics of apple wine (Joshi 6 Bhutani 1990). Nevertheless, the use of industrial
enzyme preparation is well established norn~al practice (Joshi & Bhu~ani 1991: Fundira el
a/. 2002b) and is a processing aid to improve the wine quality.
Kukarni et al. (1980) suggested that the pectinase (0.5%) treatment could improve
the fermentation rate and quality ofmango wine.
Types of Fermentation:
Batch Fermentation:
Saccharomyces cerevisiue is used extensively in batch renncntations to convert
sugars to ethanol for the production of beverages and biofuels (Dombek & Inyram 1987).
Wine-making is generally carried out by conventional bntch fermentations. Batch culture
is a closed culture system, which contains an initial, limited amount of nutrients. After
inoculation, the yeast takes some time for adapting to the new conditions. l'his culture
will pass through a number of phases such as lag, log and stationary phaqes.
The conventional system for winemaking in batch mode used to be 225-228 L
barrels or 6-12 vats made of wood or cement. These vessels have now been replaced with
well-designed stainless steel fennenters of various shapes like barrel, vat, cylindro-
conical, cylindrical, sphero-conical and tower (Maule 1986; Moresi 1989).
Continuous Fermentation:
The continuous winemaking process was economical leading to 50% reduction in
the space needed for juice and more than 20% duc t ion in salary bill. But the continuous
winemaking process is not suitable for the preparation of vintage wine, especially at the
small-scale level. In order to prevent contamination by lactic acid bacteria higher
quantity of sulphur dioxide (80-100 mdl) and routine testing of must far lactic acid
bacteria are needed for continuous fermentation of red wine (Kunkee & Goswell 1977).
Although continuous fermentation has the economy associated with constant use and is
usually offset by several disadvantages. Continual supply of must, requirement of sterile
and oxygen-free must for many months together. This is both difficult and expensive. In
comparison with batch fermenters continuous fermenters require complex design, which
costs more than batch fermenters. Finally the quality of wine is generally imperfect in
continuous fermentation. Wine quality partially involves the production of many
compounds through the fermentation cycle - not single compound produced during a
particular physiological stage of yeast. Thus, the continuous fermentation of wine is
appropriate in producing biochemically complex beverages like wine (Davies1988).
Yeast Cell Immobilization:
Although wine fermentation has an old tradition, it is in the forefront of
biotechnological development. Today, winemaking research is performed on many
technical, biochemical, microbiological and genetic topics. Some of the possibilities, such
as gene manipulation of grape or yeast are not easily commercialized because of the
uncertainty of consumers toward them (Loureiro 1990). However, immobilization of
microbial cells by active entrapment within natural polymers or passive adsorption on
solid materials has become a rapidly expanding research area.
Many advanced processes are advantaged by immobilization techniques, and
therefore several such techniques and supports have been proposed. These techniques can
be divided into four major categories based on the physical mechanism employed (Fig.5):
a) adsorption or attachment on solid carrier surfaces- examples of solid carriers used in
this type of immobilization are cellulosic materials (DEAE-cellulose, wood, sawdust nnd
delignified sawdust), inorganic materials (polygorskite. montmorilonite, hydromica,
porous glass); b) entrapment within a porous matrix-characteristic exan~ples of this type
of immobilization are the entrapment into polysaccharide gels like alginates, k-
carrageenan, agar, chitosan and polygdacturonic acid or other polymeric matrixes like
gelatin, collagen and polyvinyl alcohol moton & D'Amore 1994; Park L C'hang 2000);
C) self aggregation by flocculation which is a natural phenomena or cross-linkage with
the help of chemical substances- the ability to form aggregates is mainly observed in
moulds, fungi and plant cells. Yeast flocculation is an important property for the brcwing
industry which affects the quality of beer; and d) cell containment behind barriers- it can
be attained either by use of microporus membrane filters or by entrapment of cells in a
microcapsule or by cell immobilization on to an interaction surface of two immiscible
liquids (Pilkington el al. 1998).
In contrast to potable ethanol fermentation, winemaking has additional prerequisites:
final ethanol content at least 11.5% (vlv) and a system of food grade purity. 'l'he
biocatalyst prepared for winemaking by immobilization of yeast cells on solid supports
has to be ethanol resistant. Fruit and sparkling wine production by yeast immobilized on
alginates has also been reported (Mori 1987). White wine also produced from
immobilized yeast cells in batch mode by S. cervisiae OC-2 (Nakanishi &
Yokotsuka1987).
Entrapment within a Natural Roaulatlon Artlflclal fiocculetlon porous matrix (~ggregallon) (mas-llnklng)
Interfacial mlcraencapsulaUon
Contninrnent between micropomus
membranes
Insoluble carrier a. IMMOBILIZATION ON THE
Llquld phase SURFACE OF A SOLID CARRIER
~musmetrix b. ENTRAPMENT WITHIN A POROUS MATRIX
a . . Micropomus membrane - Blfunclbnal reagent c. CELL FLOCCULATION
(cross-Bnkar) (AGGREGATION)
2 2 & & 3 E~ectrosbtlc foC.38 d. MECHANICAL CONTAINMENT BEHIND A BARRIER
Fig. 5: Types of immobilization by using different methods. (Source: Kourkoutas el ul. 2004)
White wine also produced From immobilized yeast cells in batch mode by S cervisiae
OC-2 Wakanishi t YokotsukalY87). S. cerevisioe immobilized on glass beads and
minerals such as polygorskite, montmorilo~~ite and hydromica produce wine in a rapid
fermentation (Hamdy 1990; Ageeva el a/. 1985). Continuous fermentation for wine
production has also been carried out using sodium alginate and DEAE cellulose as
supports (Lomini and Advenainenl990). Various low-cost supports that aw abundant,
and have unlimited reuse, have been proposed for potential use in alcohol or malolactic
acid fermentation in wine. Inorganic supports, such as mineral Kissiris (Bakoyianis el ul.
1992) and gama-alumina have been investigated (Loukatos er a/ . 2000). However, they
do not meet the prerequisites for food grade purity, due to mineral residues Sound in the
final product (Tsakiris rt al. 2004). Food gradc natural products, such as delignified
cellulose materials (Bardi & Koutinas 1994) and gluten pellets (Bardi el ul. 1996) were
successfully used as immobilization supports for ambient and low-temperature wine-
making, producing wines by rapid fermentation and with improved characteristics
compared to wines produced by free cells. In order to satisfy the demand for food grade
purity and combine it with consumer acceptance, some researchers hove proposed the use
of fruit pieces (apple, quince and pear) as cell immobilization carriers for wine beer
production (Kourkoutas el al. 2001; Mallios el al. 2004)) and reported producls with fine
taste and aroma and a distinct fruity character.
Taking into account that raw materials for winemaking is grapes, it was thought
that it would be interesting to use grape products, such as residual grape skins
(Mallouchos el 01. 2002), as a support for the immobilization in wine-making. Tsakiris et
01. (2004) reported the use of raisins (dried seedless grapes) as a support for the
immobilization of yeast cells in wine -making.
Post-Fenneotative Aspe-:
The post fermentative processing is divided into three groups - the procedures that
are helping in getting clear and spoilage-ke product after bottling; adjustment
procedures like colour. taste and flavour characteristics; and those designed to promote
maturation and proper aging. The procedures are: a) SiphoninglKacking; b) Maturation;
c) Clarification and Stabilization; d ) Physico-Chemical Stabilization; e) Blending and f)
Pasteurization.
Characterization of wine:
The most important factor in winemaking is the organoleptic quality of the final
product. A wine's bouquet is determined by the presence of' desirable flavour compounds
and metabolites in a well balanced way, and absence of off-flavours (flretorius 2000).
Volatile compounds are responsible for aroma, because they have greater vapor pressure.
Many substances and compounds in various proportions have contributed to the
distinctive flavour of wine, brandy and other alcoholic beverages. Variety of fruit can
affect the composition of wine (Cole & Nobel 1995; Nobel 1994). Flavour is a
combination of taste and aroma, which is of particular importance in determining food
preferences. Flavour depends on a number of factors, the most important of which is
chemical constitution. Oenological practices. including use of a selected ycast strain and
fermentation conditions, have a prominent effect on primary flavours of wines. Yeast
metabolism makes an importan1 contribution to flavour. High temperatures increase the
rate of yeast metabolism but the quantitative influence of temperature change will be
different for each biochemical reaction, changing the balance of flavour compounds. The
compounds that are formed in the starting of fermentation can dominate in the flavour,
because these compounds are present in the highest concentration (Gomez el al. 1990).
During aging the flavour components move to their equilibrium, resulting in
gradual changes in flavour. The harmonious complexity of wine and brandy can
subsequently be further increased by volatile extraction during oak ham1 aging
(Cantagre1 el 01. 1995). In wines and brandies, the major products of yeast fermentation,
esters and alcohols, contribute to a generic background flavour, whereas, subtle
combinations of trace components derived from the tiuil juices usually elicit the
characteristic aroma notes of these complex beverages. (Schreier 1979; Cole and Nobel
1995; Nobel 1994). Diagrammatic representation of formation lane of flavour compounds
was showed in Fig.5
Monitoring the volatile compounds during and after completion of wine
fermentation is very important in understunding their synthesis and the hctors that atTect
their production (Mallouchos el a/. 2002). The volatile compounds formed during wine
fermentation were determined by Gas Chromatograph. With advances in analytical
chemical research, many different specific instrunlents were entered into the field of
analysis of wines; GC coupled with Solid Phase Micro extraction (SPME) head space,
Gas Chromatography- Mass Spectrometry (GC -MS), which play vital role in
determining the wine flavour and complexity (Vianna & Ebeler 2001; Vas el al. 1999;
Flamini 2003; Hayasaka 2005). The advnntages of these techniques are simplicity and
versatility that provide linear results over a wide concentration of analytes.
Acidity:
The acidity of grape and wine plays an important role in many aspects of
wine production. The juice and wine acidity, (that is 4 5.0), has a profound influence on
the survival and growth of all microorganisms (Cupati B Ryan 1996).
Fig.6: Schematic representation of derivation and synthesis of flavour- activecompounds from sugar, amino acids and sulfur metaholiam by wine yeast 5'. cerevisiae (Source: Swiegers el ul. 2005).
Wine contains a larg number of organic acids. Among them, tartaric acid and
n~alic acids account for 90% of titrable acidity. In mango wine also the main titrable acid
is malic acid (Kulkami et al. 1980). When the acidity was high the higher acidity can be
~ ~ ~ o v e d by using Schizosaccharomyce.~ pomhv (Joshi er 01. 1991).
Volatile acidity (VA) describes a group of volatile organic acids of short carbon
chain-length. The volatile acid content of wine is usually between 500 and I000 m g .
that are 10-15% of the total acid content of wine, of which, acetic acid usually constitutes
about 90% (Bely 2003; Erasmus cr ul. 2004).
Terpenes:
Fruit aroma including volatile-free odorous substances, especially terpenes
(linalool, terpeneol, citronellol, nerol and geraniol) and bound glycosides (nonvolatile
precursors as terpenglycosides). They are the precursors of the flavouring aglycons when
hydrolytic reaction takes place and suggested that increased hydrolysis of aroma
precursors present in juice which can liberate the aylycone to intensify the varietal
character of wines (Cana-Llauberes 1993). 'I'erpenols such as geraniol and nerd can be
released from terpenyl-glycosided by fruit derived P -D-glycouidase activity present in
must.
In general, exogenous glycosidase (commercial enzyme) preparations arc added
to fermented juice (as soon as glucose has been consumed by yeast) or to young wine
(Cana-Llauberes 1993; Joshi & Sandhu 2003). This had led lo renewed interest in the
more active P-glucosidases produced by certain strains of S. crrevisiue and other wine-
associated yeasts such as Cbndida, and Pichia species. Since these P-glucosidases are
absent in most of the yeast species, many investigators (Van Rensberg el ul. 1998)
functionally expressed the fbglucosidase gene (BGLI) of the yeast Saccharomycopsis
fibuligera in S. cerevisiae.
Alcohols
Ethanol: The presence of ethanol, which is a major component of wine, is essential to
enhance the sensory attributes of other wine components. Excessivv ethanol. however.
can produce a perceived 'hotness' and masks the overall aroma and flavour of the wine
(Guth & Sies 2002). The wines particularly from warm climates where grape sugar
content is high, the ethanol concentration in them reach above 15% (vlv) and may be
dangerous to health (de Barros Lopes c.1 ol 2003). Glucose oxidase (GOX) provides one
of the approaches for reducing the glucose content of juice and hence reducing the
alcohol content of wine during the fermentation (Pickering 1999; Mnlherbe el al. 2003).
The ethanol and secondary metabolite l'orniation was shown in Figure 6 .
Kulkami el al. (1980) reportcd tha~ the cthanol in fruit wines like apple and
mango was less than desired concentration and he suggested an alternative of adding
sugar to the fruit juices before fermentation for getting desirable ethanol level.
Higher Alcobols:
Alcohols with carbon numbers greater than that of ethanol such as isobutyl,
isoamyl and active amyl alcohol are termed as fusel alcohols or fusel oils. Wine yeasts
produce these higher alcohols during fermentation from intermediates in the branched
chain amino acids pathway leading to the formation of isoleucine, leucine and valine by
decarboxilation, transamination and reduction (Webb 6t Ingram 1963). Schematic
representation of the higher alcohol formation in wine yeast was shown in Figure 7h. At
higher concentrations these alcohols have undesirable flavour and odour characteristics
(Giudici et 1990). The presence of these higher alcohol levels below their threshold
value does not affect the taste of wine. In some cases, they may contribute to wine quality
(Swiegers & I'retorius 2005). However. higher alcohols can be concentrated by
distillation and their reduction in wines for brandy prtduction is of great importance
(Aragon el a/. 1998). 2-phenylethanol is the only higher alcohol describcd with pleasant
terms such as old rose, sweetish and perfumed (Eteivanl 199 1 ). 11 has been reported that
concentrations below 300mg11 add a desirable level of complexity to wine, whereas
above 400 mg/l can have a detrimental effect (Rapp & Versini 199 1 ).
Non-Sachharomyces yeast can also contribute to the levels of higher
alcohols. Clemente-Jimenez et a1 (2005) reported concentration improvement in mixed
fermentation with Pichia,fermrfi/ans and S ce rn i s iu~ when compared with S crrc~\lisiae
alone.
Esters:
During the alcoholic fermentation of sugars, wine yeast produccs ethanol, carboll
dioxide and number of by-products including csters, of which alcohol ucetates and C~-CIIJ
fatty acid ethyl esters are found in highest concentration in wine and brandy (Soles 1982;
Stashemko 1992; Herraiz & Ough 1993). Schematic representation of' the esters and
formation in wine yeast was shown in figure 7a. The characteristic fmity odour of the
wine is primarily due to a mixture of hexyl acetate, ethyl caproate and caprylate (apple-
like aroma), isoamyl acetate (banana-like aroma) and phenylethyl acetate (fruity, flowery
with a honey note). Although these compounds are ubiquitous in wines and brandies, the
of esters formed varies significantly. In addition to variety of fruit (grape)
rootstock and maturity, the ester concentration produced during fermentation is
dependent Won Yeast strain, fernlentation temperature, insoluble material in the must,
vinification methods. must pH, the amount of sulphur dioxide, amino acids present in the
must and the malolactic fermentation (Cole b Nobel 1995 Houtman 1980; Cabrem el al.
1998; Fundira et al. 2002).
The synthesis of acetate esters like arnyl acetate and ethyl acetate in S. cercvisiae
is ascribed to at least three acetyltransferasc activity: alcclhol acetyltranferase (AAT).
ethanol acetyltransferase (EAT) and iso-uniyl ulcohol acetyltrasferase (IAT) (Malcorps &
Dufour 1987; Malcorps et a1 1991). The gene .4 TFl from widely used conlmercial wine
yeast strain (VIN13) was cloned and placed it under control of the cclnslitutive yeast
phosphoglycerate kinase gene (PGKl ) promoter und terminator (!illy cf ol 2000 h
2004). The over expression of ATFI gene resulted in the increased levels of ethyl acetate,
iso-my1 acetate and 2-phenylethyl acetate in wine and distillates. This shows that the
over expression of acetyltransferase genes could profoundly affect the flavour profiles of
wines and distillates.
Glycerol:
Glycerol has no direct effect on wine quality due to its non-volatile nature but this
trio1 imparts certain other sensory qualities. I t has a slightly sweet taste; and owing to
viscous nature, it contributes to the smoothness, consislency and overall body of wine
(Scanes el a], 1998; Remize el ul. 1999). The pn)duction of glycerol has multifactorial
dependency like variety of grapes, pH, initial sugar concentration, fermentation
temperature, aeration, choice of starter culture and its inoculation level (Kemize1999;
Scanes el al. 1998). Its concentration was higher in red wines compared to that in white
wines. Generally the concentration varies from 1-15 d l in wines and the wine yeast
strains which overproduce glycerol would improve organoleptic quality of wine.
Fig. 7a. Schematic representation of the esters and formation in wine yeart (source: Swiegers & Pretorius 2005)
Fig.7b. Schematic representation of the higher alcohol formation in wine yeast (source: Swiegers % Pretorius 2005)
The threshold level of glycerol in wines is obsenjed to be 5.2 g/l whereas a change in the
viscosity is perceived only at a level of 25 pfl. (Michinick rt al. 1997; Scanes el ai. 1998:
Remize el ai. 1999). The main role of gtycerol synthesis during fermentation is to supply
the yeast cell with an osmotic stress responsive solute arid equilibrate the intracellular
balance (Scanes et al. 1998).
Sulphite and Sulphide:
Due to their high volatility and reactivity. sulphur containing con~pounds have a
profound effect on the flavour of wine. The threshold values of these cclnipounds in wine
are also low. During the fermentation of wine, sulphite is deliberately used as nn
antioxidant and antimicrobial agent. Iiealth concerns and unfivorahle public perception
of suphite have led to demands for restriction of its use in wine and reassessnient of all
aspects of sulphite accumulation in wine (Pretorius 2000).
Sulphur is an essential conlponent for yeast growth. S. ccrnfisiae can use
sulphate, sulphite and eleniental sulphur as sole sources. The formation of sulphite and
sulphide is also affect the quality of wine. In addition, the yeast metabolite H2S has
prominent effect on wine quality and odour because above the threshold level of 50-80
g/], it shows an off-flavour reminiscent of rotten eggs (Snow 1983). lormation of lhese
compounds is greatly affected by pH and temperature. In c u e of red wine production,
yeast cells use more nitrogen during rapid fermentation due to higher fermentation
temperatures. This tends to develop sulphidic smells (Rauhul 1993).
Mango Peel Fermentation:
The optimum level of ethanol in a commercial wine is 12- 13% (wlv). However.
the wines from fruits like mango and apple contain low ethanol, The ethanol percentage
can be adjusted to the appropriate level try two ways: one is the addition of pure ethanol
produced from molasses and another is anielioration of pulp with glucose. Both the ways
increase the cost of wine production from fruits. Utilimtion of the fruit wastes produced
from fruit pulp industries for ethanol production is onc of the alternative strategies
adopted to economise the improvement of ethanol levels in wine.
Mango peel is generally termed as a total waste. I f a factory is pn)cessiny 40 tons
of Totapuri mangoes per day (8 h work) about 6 tons of pccl would be available as waste.
This waste is either used as cattle feed or dumped in open areas, where it adds to
environmental pollution. Use of mango peel as a source of pectin and iiber has been
suggested by a number of researchers (Pandia el crl. 2004). Ilthanol from orange peel has
been reported by Grohmann el ul. (1996). Mango pecl is difficult to decompose, as it
takes a very long time, because of its complex lignocellulosic composition. Suitability of
mango peel for biogas production has been investigated by Mtldhukara el a/. (1993).
However, ethanol fermentation of fruit and vegetable wastes, like mango pecl, appears to
give better returns. Uf lization of this waste is not only a necessity but also a challenge.