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Thesis Report

Center Of Biotechnology And Microbiology

University Of Peshawar

Fermentation of wine to know the activity of fungus

Submitted to :Madam Gul E Sehra

Submitted by : Muhammad Junaid Alam

Roll no: 59

4th Semister

Table of Contents
Abstract ......................................................................................................................................................... 4
Introduction .................................................................................................................................................. 5
Literature Review .......................................................................................................................................... 7
Materials & Reagents .................................................................................................................................... 9
Procedure...................................................................................................................................................... 9
Result & Discussion ..................................................................................................................................... 11
Conclusion ................................................................................................................................................... 11
Acknowledgement ...................................................................................................................................... 12
Refrences .................................................................................................................................................... 13

Abstract
The molecular biology revolution has brought forth significant new
advances with application in microbiological analysis during wine production and
storage. For example, traditional methods for microbial strain identification have
been mostly supplanted in favor of ribosomal RNA-based methods for speciation
of cultured yeast and bacterial populations in wine. Moreover culture-independent
molecular methods now allow for more rapid profiling of complex populations, or
quantification of targeted species, thereby enhancing the information available to

the winemaker. Finally, the availability of microbial genome sequences provides a

wealth of new opportunities to understand and exploit the microorganisms in wine,
as well as identify the key genetic factors underlying wine flavor development or
depreciation. In general, advances in molecular biology are fundamentally changing
how scientists and winemakers assess the microbial ecology of winemaking, providing
new insight into the wonderfully complex conversion of grape juice to wine.

Introduction
The conversion of grape juice to wine is a biotechnological tradition dating back to
the dawn of civilization. Throughout the ages numerous winemaking strategies
were developed resulting in the range of wine products, from champagne to port,
available today. However, since the time of Pasteur (1873) the microbial contribution
to the production of wine has become a subject of research and, often, debate. Wine
composition and quality are functions of many different intrinsic and extrinsic variables, many of
which are microbiologically mediated. A large diversity of microbes
are inherent to winemaking including various yeasts, bacteria and fungi. Prominent
in this process are Saccharomycesspecies (predominantly S. cerevisiae), which dominate the
alcoholic fermentation, and the lactic acid bacteria (LAB), which carry out
the malolactic conversion. Efforts to determine the population size and potential
impact of different microbes on the winemaking process are critical to production
of a flavorful product. Spoilage is considered growth of organisms that are
unwanted at any particular place and time in the winemaking process (Sponholz
1993). Thus the same microorganism can be both beneficial and detrimental to the

winemaking process. For example, growth of S. cerevisiaeis required during the

alcoholic fermentation, but growth can be detrimental if it occurs in a finished, and
bottled, wine.
Interestingly, both academics and winemakers have good reason to be interested
in the microbial ecology of the winemaking process. From an academic perspective
wine represents an ideal landscape in which to study basic concepts of microbial
ecology. Several factors promote this view. First and foremost, wine is a liquid
medium that allows samples to be properly mixed prior to analysis, thus ensuring a
representative sampling. This contrasts with the situation for those studying microbial growth on
solid surfaces (e.g., barrel stave, grape surface or vineyard soil) in
which the microbial populations are heterogeneous and spatially distributed across
the surfaces. As a consequence, representative sampling of microbes on surfaces
becomes a more statistically challenging process than sampling of a liquid medium
like must or wine. A second reason why wine is an attractive platform for microbial
ecology is the diversity of microbes present which enables one to witness a range
of microbial interactions from commensalisms and neutralisms to antagonisms.
From the winemaker perspective, close monitoring of the microbial changes
occurring throughout the winemaking process is beneficial for several reasons: to
promote and guide yeast during the alcoholic fermentation, to verify the growth of
the bacteria during the malolactic conversion, and ultimately to ensure the stability
of the wine before bottling and storage (Delfini and Formica 2001). The evolution

of undesired microbes during different stages of winemaking can produce volatile

acidity, off-flavors and polysaccharide hazes, all of which can diminish the quality
and acceptability of the final product (Sponholz 1993). Even prior to the onset of
fermentation, the grapes themselves can be infected with molds, yeasts and bacteria
that can enter and alter the fermentation in a negative fashion. Improper wine storage and
handling post-fermentation can encourage microbiological faults, which
can negatively impact wine quality. As a result the winemaker must conduct basic
physical, chemical, sensory and microbiological analyses of musts and wines to
assure wine quality.
Whether it is for an investigation of basic ecological concepts or for the applied
goal of predicting possible wine spoilage, one must have accurate and reproducible
methods for enumeration of various microbial constituents at different stages in
wine production. Both indirect and direct approaches can be used to view these
populations. In this review, we will summarize both approaches and comment on
the future use of newer molecular tools to view the microbial diversity inherent in
wine fermentations.

Literature Review
Wine is associate degree ancient potable and has been prized throughout time for its distinctive
and pleasing flavor. Wine flavor arises from a mix of many chemical elements interacting with
our sense organs, manufacturing a neural response that's processed within the brain and leading
to a psychophysical representation that we have a tendency to pronto describe as wine. The
chemical elements of wine area unit derived from multiple sources; throughout fermentation

grape flavor elements area unit extracted into the wine and new compounds area unit fashioned
by various chemical and organic chemistry processes(1)
Wine is that the product of advanced interactions between fungi, yeasts and bacterium that start
within the farm and continue throughout the fermentation method till packaging. though grape
tracheophyte and cultivation offer the foundations of wine flavour, microorganisms, particularly
yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it's
necessary to spot and perceive the ecological interactions that occur between the various
microorganism teams, species and strains. These interactions include yeastyeast, yeast
filamentous fungi and yeastbacteria responses. The surface of healthy grapes incorporates a
predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera),
Cryptococcus and Rhodotorula species counting on stage of maturity. This microflora moderates
the expansion of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts
that contribute to alcoholic fermentation, and therefore the bacterium that contribute to
malolactic fermentation. broken grapes have raised populations of potable and carboxylic acid
bacterium that impact on yeasts throughout alcoholic fermentationYeast interactions and wine
flavor(2)
Wine producers face augmentative competition led to by a widening gap between wine
production and wine consumption, a shift of client preferences removed from basic goods wine
to top of the range wine, and economic economic process. Consequently, they're line for a
complete revolution within the magical world of wine. the method of remodeling the wine
trade from a production- to a market-orientated trade ends up in AN increasing dependence on,
amongst others, biotechnological innovation. Market-orientated wine-yeast strains square
measure presently being developed for the cost-competitive production of wine with decreased
resource inputs, improved quality and low environmental impact(3)
Many factors influencing wine aroma are not entirely defined by their sensory impact, but to
boot by chemical info that in many cases modify a extra differentiated approach. By correlating
sensory and analytical info headway has been created, significantly at intervals the sector of wine
off-odors. triumphant analytical selection differentiation supported wine volatiles indicates the
importance and potentialities of wine aroma for control(4)

Research and production developments with relevancy wine-making yeasts square measure
reviewed. Topics coated include: wine-yeast strains; natural versus inoculated fermentings;
vinification fermentations; ethyl alcohol toxicity and tolerance in wine yeasts; fermentation
flavour components; secondary wine fermentations by yeasts; spoilage yeasts; different yeasts
related to wine-making; and applications of genetic science to enology(5)

Materials & Reagents

Graduated cylinder, Test tubes, Erlenmeyer flasks or bottles, Rubber stoppers, Tygon tubing,
Hydrometer, Balance.
Grape juice,Sucrose,Active dry wine yeast, strains of Saccharomyces ellipsoideus

Procedure
Prepare Starter Yeast Culture

Mix 1 g of dry wine yeast culture in 100 ml of grape juice.

Let the yeast grow in a loosely capped container at room temperature for 24 hours.

Primary Fermentation

Add enough sugar to grape juice to prepare the following 4 substrates, about 1 liter each:
------------------------------------------Run
Conc. of Extra Added Sugar
------------------------------------------A
0.0g/l
B
100.0g/l
C
200.0g/l
D
300.0g/l
-------------------------------------------

Measure the specific gravity and PA value for each of the starting substrates with a
hydrometer. This is the initial PA value which will be used later to estimate the alcohol
content.

Inoculate each bottle with 20 ml of the starter yeast culture prepared in the previous step.

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Plug the juice bottle with a rubber stopper. A piece of Tygon tubing is extended from the
stopper to provide a vent for the evolved carbon dioxide. The other end of the tubing is
dipped in water in a small test tube taped to the bottle. The water prevents the entry of
oxygen, which alters the metabolism of the yeast and spoils the wine. At the same time,
carbon dioxide can escape from the bottle.

Ferment at room temperature for one week.

Secondary Fermentation

At the end of one week, decant the juice from the bottle to clean individual temporary
containers.

Measure the PA values for each of the substrate with a hydrometer. Estimate the alcohol
content by subtracting the present PA value from the initial PA value.

Discard the sediment and wash each bottle with water.

Pour the juice back into the cleaned bottle. Put back the cleaned assembly of rubber
stopper and Tygon tubing.

Ferment slowly for another 4-6 weeks.

Measure the PA values

(Note : Pa Value means Potential Alcohal)

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Result & Discussion

We got the production of carbon dioxide and ,we also got the production of yellow crystals and
a specific odour which surely represents fermentation of wine.
Grapes produces wine through anaerobic process by metabolism of carbohydrates present in
grapes.Wine is popular and important beverage that accompanies and enhances a wide range of
European cuisines, from the simple and traditional to the most sophisticated and complex. Wine
is important in cuisine not just for its value as a beverage, but as a flavor agent, primarily in
stocks and braising, since its acidity lends balance to rich savory or sweet dishes. Natural wines
may exhibit a broad range of alcohol content, from below 9% to above 16% ABV, with most
wines being in the 12.5%14.5% range.Fortified wines (usually with brandy) may contain 20%
alcohol or more.

Conclusion
This experiment taught us several things fermentation. We were able to observe anaerobic
respiration of wine, and we saw how an export but lack of import of gases in anaerobic
environments can add gas to an enclosed environment .We performed the fermentation of wine

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and enhanced it with fungal species which enhanced the fermentation and resulted in odour and
yellow crystals which specifies the productivity of wine. The microbial species of fungus
present in wine contribute to the fermentation process, and therefore contribute to the aromatic
properties of the resulting wine.
Fungal species also encourages to create a desired effect in a particular vintage.. On a small
scale, we were able to see how wine and beer are made.

Acknowledgement
First of all we are grateful to ALMIGHTY ALLAH,for establishing us to complete this thesis.
We have taken efforts in this thesis. However, it would not have been possible without the kind
support of our respected MADAM GUL E SEHRA. We would like to extend our sincere thanks
to her.
My thanks and appreciations also go to my colleagues in developing the thesis and people who
have willingly helped us out with their abilities.

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Refrences
chapter 6 wine fermentation by David A. Mills, Trevor Phister, Ezekial Neeley, and Eric
Johannsen book name: molecular techiniques in microbial ecology of fermented foods
Cocolin,l;Ercolini,D.(Eds.)
Experiments in Microbiology, Plant Pathology and Biotechnology by Aneja, K.R.
http://www.redorbit.com/news/science/1112754493/microbial-fungi-affect-winecharacter
http://en.wikipedia.org/wiki/Wine
Wine fermentation By Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
ENCH485
Origins of Grape and Wine Aroma. Part 1. Chemical Components and Viticultural
Impacts by Anthony L. Robinson , Paul K. Boss, Peter S. Solomon, Robert D.
Trengove,Hildegarde Heymann and Susan E. Ebeler(1)
Yeast interactions and wine flavor by Graham H.Fleet(2)
Wine-yeast strains by Isak S Pretorius, Florian F Bauer(3)
Wine aroma by A. Rapp, H. Mandery (4)
Wine-making yeasts by Kunkee, R. E.; Bisson, L. F.(5)