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INTRODUCTION

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.