A SEMINAR REPORT

ON

THE ROLES OF MICROORGANISMS IN WINE PRODUCTION

BY

ADEOYE ABIOLA DEBORAH

MATRICULATION NUMBER: 190561012

SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY,

FACULTY OF SCIENCE, LAGOS STATE UNIVERSITY, OJO

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

AWARD OF BACHELOR DEGREE IN SCIENCE,

BSC (HONS) IN MICROBIOLOGY

APRIL, 2023.

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 CERTIFICATION

This is to certify that this work was carried out by ADEOYE ABIOLA DEBORAH with

Matriculation number 190561012, under my supervision in the Department of Microbiology,

Faculty of Science, Lagos State University.

______________________ _______________________

DR. J.A. GRILLO DATE

SUPERVISOR

___________________ ___________________

DR. M.O. FASHOLA DATE

LEVEL COORDINATOR

___________________ _____________________

PROF. A.O.B OYEFOLU DATE

H.O.D

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 DEDICATION

This report is dedicated to God

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 ACKNOWLEDGEMENT

It is my pleasure to acknowledge God for his grace and mercies throughout the period of working

on this seminar report. My sincere gratitude goes to my supervisor, DR. J.A. GRILLO., for

investing his time and wealth of knowledge into this work. My sincere gratitude also goes to MR.

OLAGIDE ANAGUN., for investing his time and contribution of knowledge into this work.

A big thank you to my parents for their love and financial support, MR AND MRS A.A

ADEOYE, my amazing siblings (Blessing, Bukola, Adeola), and my extended family and friends,

for their support and love.

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TABLE OF CONTENT

CONTENT PAGE

TITLE PAGE ...................................................................................................................................1

CERTIFICATION ...........................................................................................................................2
DEDICATION ................................................................................................................................ 3
ACKNOWLEDGEMENT .............................................................................................................. 4
TABLE OF CONTENTS ................................................................................................................ 5
LIST OF TABLES .......................................................................................................................... 6
LIST OF FIGURES ........................................................................................................................ 7
1.0 INTRODUCTION .................................................................................................................... 8
2.0 WINE ................................................................................................................................... 9-10
3.0 TYPES OF WINE ............................................................................................................. 11-12
4.0 THE WINE PRODUCTION PROCESS ............................................................................ 13-23
5.0 GROUPS OF MICROORGANISM INVOLVED IN WINE PRODUCTION ................. 24-25
6.0 ROLES OF EACH GROUP OF MICROORGANISM INVOLVED IN WINE PRODUCTION
.................................................................................................................................................. 26-28
7.0 WINE SPOILAGE ............................................................................................................. 29-30
8.0 MICROBIAL SPOILAGE AND PRESERVATION OF WINE....................................... 31-54
9.0 CONCLUSION ....................................................................................................................... 55
10.0 REFERENCES ................................................................................................................ 56-57

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 LIST OF FIGURES

Fig.1 – The wine production process flowchart………………………………page 16

Fig. 2 – Roles of yeast in wine production………………….………………. page 28

Fig. 3 – roles of bacteria in wine production…………………………………page 30

Fig. 4 – chemical structure of Sulphur dioxide ………………………………. page 48

Fig. 5 – chemical structure of sorbic acid ……………………………………. page 50

Fig.6 – chemical structure of Fumaric acid …………………………………. page 51

Fig. 7– chemical structure of Dimethyldicarbonate…………………………. page 52

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 LIST OF TABLES

Table 1: yeast spoilage in wine …………………………………………page 38

Table 2: Bacteria spoilage microorganism ……………………………...page 45

Table 3: Concentration of Dimethyldicarbonate (Velcorin) required for various

yeasts and Bacteria inoculated at 500CFU/ml………………….…………page 53

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INTRODUCTION

Wine production is a complex process from the vineyard to the winery. On this journey, microbes

play a decisive role (Howell et al., 2019). From the environment where the vines grow,

encompassing soil, topography, weather and climate through to management practices in

vineyards, the microbes present can potentially change the composition of the wine (Howell et al.,

2019). Therefore, quality wine production depends on maintaining proper microbiological control

during the transformation of the grape juice into the finished wine and into the bottle (Romano et

al., 2022).

However, Wine is the product of a complex biological and biochemical interaction between

grapes (grape juice) and different microorganisms (fungi, yeasts, lactic acid bacteria and acetic

acid bacteria) and the mycoviruses and bacteriophages affecting them (Walker & Pretorius, 2022).

The process starts in the vineyard, continues through fermentation and maturation, and concludes

at packaging. It is affected by the various viticultural and oenological practices available to the

grape-grower and winemaker, respectively of the microorganisms involved, it is the yeasts that

play the most important role; they conduct the alcoholic fermentation (conversion of grape sugar

to ethanol and CO2). Furthermore, although wine flavor is directly determined by grape variety,

yeasts also affect wine flavor and quality by the production and excretion of metabolites during

growth and through autolysis (Rana et al., 2021). In some instances, yeasts can also act as spoilage

organisms during wine production (including maturation) and after packaging. Yeasts present

during fermentation are derived from grapes and the vineyard, the equipment used in the cellar,

cellar surfaces and external sources such as selected cultures that are added to facilitate the

fermentation process (Celis et al., 2019).

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WINE

Wine is an alcoholic beverage made from the partial or total fermentation of grapes or other fruit

and plants, such as berries, apples, cherries, dandelions, elder-berries, palm, and rice can also be

fermented (Gade, 2021).

However, the natural chemical balance of grapes lets them ferment without the addition of

sugars, acids, enzymes, water, or other nutrients. Yeast consumes the sugars in the grapes and

converts them into alcohol and carbon dioxide (Shahriar et al., 2019). Different varieties of

grapes and strains of yeasts produce different styles of wine. The well-known variations result

from the very complex interactions between the biochemical development of the fruit, reactions

involved in fermentation, terroir and subsequent appellation, along with human intervention in

the overall process. The final product may contain tens of thousands of chemical compounds in

amounts varying from a few percent to a few parts per billion. Wines made from produce besides

grapes are usually named after the product from which they are produced (for example, rice wine,

pomegranate wine, apple wine and elderberry wine) and are generically called fruit wine (Gade,

2021). The term "wine" can also refer to starch-fermented or fortified beverages having higher

alcohol content, such as barley wine, huangjiu, or sake (Mishra et al., 2016). Grapes belong to the

botanical family Vitaceae, of which there are many species. The species that are most widely used

in wine production are Vitis labrusca and, especially, Vitis vinifera, which has long been the most

widely used wine grape throughout the world.

The theory that wine was discovered by accident is most likely correct because wine grapes contain

all the necessary ingredients for wine, including pulp, juice, and seeds that possess all the acids,

sugars, tannins, minerals, and vitamins that are found in wine (Didur et al., 2017). As a natural

9
process, the frosty-looking skin of the grape, called "bloom," catches the airborne yeast and

enzymes that ferment the juice of the grape into wine. The cultivation of wine grapes for the

production of wine is called "viticulture." Harvested during the fall, wine grapes may range in

color from pale yellow to hearty green to ruby red (Tiefenbacher & Townsend, 2019).

Wine can be made in the home and in small-, medium- or large-sized wineries by using similar

methods. Wine is made in a variety of flavors, with varying degrees of sweetness or dryness as

well as alcoholic strength and quality. Generally, the strength, color, and flavor of the wine are

controlled during the fermentation process (Jeromel et al., 2022).

The name of a wine almost invariably is derived from one of three sources: the name of the

principal grape from which it was made, the geographical area from which it comes, or in the case

of the traditionally finest wines from a particular vineyard or parcel of soil. The year in which a

wine is made is only printed on bottles that have aged for two or more years; those aged less are

not considered worthy of a date. Wine years are known as "vintages" or "vintage years." While

certain wines are considered good or bad depending on the year they were produced, this can vary

by locality (Vezulli et al., 2022).

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CLASSIFICATION OF WINE
Wine is broadly classified based on their color: white, pink or rose, and red, and it can range in

alcohol content from 10 percent to 14 percent (Joshi et al., 2017).

Red wine

Red wine is made from red grapes, which are actually closer to black in color. There are many

different types of red wines (Hunjet et al., 2022). This is considered to be the most classic in the

kingdom of wines, mixing the delicious red grapes with a wide range of aromas, from oak to

eucalypti, chocolate or even mint hints. The juice from most black grapes is greenish-white; the

red color comes from anthocyanin pigments present in the skin of the grape.

White Wine

White wine is not exactly white; it is often yellow, gold or straw colored, depending on whether it

includes the skin of the grape or just the juice (Sawant and Swami, 2014). White wine can be

made by the alcoholic fermentation of the non-colored pulp of green- or gold-colored grapes or

from selected juice of red grapes, produced in Europe, and numerous other places such as

Australia, California, New Zealand and South Africa and so on. It is treated so as to maintain a

yellow transparent color in the final product. White wines often taste lighter, crisper and more

refreshing than a red wine and so they often gain popularity during warmer months of the year.

White wines are typically served alongside white meats and fish (Sawant and Swami, 2014).

Pink or rose wine

A rosé wine gains color from red grape skins, but not enough to qualify it as a red wine (Zhang et

al., 2021). It may be the oldest known type of wine, as it is the most straightforward to make with

the skin contact method. The color can range from a pale orange to a vivid near-purple, depending

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on the varietals used and wine-making techniques. There are three primary ways to produce rosé

wine: Skin contact (allowing dark grape skins to stain the wort), saignée (removing juice from

the must early in fermentation and continuing fermentation of the juice separately),

and blending of a red and white wine (uncommon and discouraged in most wine growing regions)

(Arora et al., 2019). Rosé wines have a wide range of sweetness levels from dry Provençal rosé to

sweet White Zinfandels and blushes (Johnson, 2019). Rosé wines are made from a wide variety

of grapes all over the world

TYPES OF WINE

Aside from the rather broad distinction of classifying wines based on their color. red, white there

are obviously more descriptive means for grouping different types of wine (Hunjet et al., 2022).

Similarly, the procedures used for manufacture of wine have, so far, been described in a mostly

generic manner. These types include:

1. Sweet wines

Sweet wines are simply those that contain unfermented sugar either fructose, glucose, or

sucrose (Colombo et al., 2015). There are several ways to produce a sweet wine. The

easiest (and least expensive) way is to simply add sugar (up to 2% to 4%) or sucrose syrup

to a dry wine. This results in a wine that is definitely sweet, but not necessarily one that

would be acceptable to many consumers (Hunjet et al., 2022). A similar and more common

approach is to add unfermented juice, preferably from the same grapes used to make the

wine. Alternatively, sweet wines can be made by stopping the fermentation before all of

the glucose and fructose have been fermented. In some cases, sugar may be added to the

juice prior to fermentation, such that when the fermentation is complete, residual sugar

(and sweetness) remain. This is one of the more common practices in the United States,

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 and many of the sweet wines from New York state are produced this way. In either case,

arresting the fermentation is the key step. Generally, this is done by rapidly cooling the

wine, and then filtering out the yeast.

2. Fortified wines

Fortified wines are those to which distilled spirits (containing as much as 95% ethanol) are

added to raise the total ethanol concentration to 15% (Abreu et al., 2021). Not only do these

wines contain higher concentrations of ethanol, the source of the ethanol (e.g., brandy) is

also important since they may contribute unique flavor compounds to the finished product

(Etiévant et al., 2017). Aside from this common feature, however, a variety of quite

different fortified wines exist. Included are whites and reds, dry and sweet. Fortification

usually occurs during or just after the fermentation. In some wines, the added ethanol may

inhibit the yeast and prevent the complete fermentation of sugars, resulting in sweet dessert

wines. The most well-known of the fortified wines are sherry and port (Joshi et al., 2017).

3. Sparkling wines

Sparkling wines are those which contain carbon dioxide, providing bubbles and

effervescence (Joshi et al., 2017). For some sparkling wines, CO2 pressures as high as 600

kPa atmospheres can be reached (by comparison, the pressure inside a can of soda pop is

less than 200 kPa). Although sparkling wines are made throughout the world, there are

several manufacturing methods that are used to produce the CO2, and these methods define,

to a certain extent, the type of sparkling wine being produced. Clearly, the most well-

known sparkling wine is Champagne, which is traditionally made, not surprisingly, via the

Champagne method (Hunjet et al., 2022).

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THE WINE PRODUCTION PROCESS

Winemaking also known as vinification is the production of wine, starting with the selection of

the fruit, its fermentation into alcohol, and the bottling of the finished liquid (Joshi et al., 2021).

The science of winemaking is known as oenology. A winemaker may also be called a vintner.

The growing of grapes is called viticulture (Jackson, 2018).

Winemaking is a complex microbial process in which yeast and bacteria play key roles.

After crushing, yeasts, mainly Saccharomyces cerevisiae, consume sugars to produce ethanol

during alcoholic fermentation (Toress et al., 2021). Subsequently, in some highly acidic red wines,

as well as in white wines with aging potential, lactic acid bacteria (LAB), mainly Oenococcus oeni,

convert malic acid into lactic acid during malolactic fermentation (Wojdylo et al., 2020). Both

yeast and bacteria produce aromas responsible for sensorial wine properties. When both types of

fermentations are completed, microbial populations must be reduced because post-fermentation

microbial metabolisms are prejudicial to the wine’s organoleptic qualities. (Mishra et al.,2016)

This is particularly true for volatile phenol synthesis by the yeast Brettanomyces bruxellensis,

which confers off-odors to wine, as well as causing exopolysaccharide, biogenic amine and ethyl

carbamate production by some LAB strains. All of these microbial species are naturally present

on grape skins. Winemaking equipment such as tanks and barrels constitute other natural microbial

sources. All of that underline how important it is to monitor the microbial presence of grape skins,

fermenting must, winery equipment, and wine during aging.

However, there are five basic stages to the wine making process which begins with harvesting or

picking. After the harvest, the grapes are taken into a winery and prepared for primary ferment. At

this stage red wine making diverges from white wine making (Mishra et al., 2016).

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Red wine is made from the must (pulp) of red or black grapes and fermentation occurs together

with the grape skins, which give the wine its color some roses can also be added for good color

and flavor (Mishra et al., 2016).

White wine is made by fermenting juice which is made by pressing crushed grapes to extract a

juice; the skins are removed and play no further role. Occasionally white wine is made from red

grapes; this is done by extracting their juice with minimal contact with the grapes' skins. Rosé. The

five basic stages of wine production process are: (Herstein, et al., 2015).

• Step 1 – Harvesting

• Step 2 – crushing

• Step 3 – fermentation

• Step 4 – clarification

• Step 5 – aging and bottling

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Figure 1-the wine production process flowchart. Source: (Fleet et al., 2013).

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HARVESTING AND PREPARING GRAPES FOR WINE MAKING
According to both viticulturists and enologists, the first step in wine making is considered to be

one of the most important. Grapes must be harvested at just the right level of maturity. This means

that the concentrations of sugars and acids (and the sugar/acid ratio), pH, the total soluble solids,

and even the phenolic constituents must be at just the right level for the particular cultivar and the

type of wine being made (Wilkinson et al., 2015).

In addition, berry size and weight also influence the time at which grapes are harvested. In general,

grapes should be sampled sometime before their expected harvest time and their composition

assessed (at minimum Brix and pH should be measured) to make sure that over-ripening does not

occur (Suter et al., 2021). Unfortunately, there is no exact or objective set of rules to ensure or

predict the optimum time for harvesting grapes. Rather, grapes are frequently harvested based on

more subjective criteria. As grapes ripen on the vine, the sugar concentration, as well as flavor and

color components, increase, and acids usually decrease, so identifying the correct moment for

harvesting can be a real challenge. It is possible, moreover, for grapes to over-ripen, such that the

harvested grapes contain too much sugar or too little acid or be too heavily contaminated with wild

yeast and molds. Once the grapes have been deemed properly mature, it is essential that they be

picked and harvested quickly, since the composition can continue to change. Even in this twenty-

first century, when so much of modern agriculture has become automated and mechanized, a

sizable portion of grapes for wine making is still harvested manually. Only recently has mechanical

harvesting begun to displace manual harvesting. In the United States, the majority of grapes are

now harvested by mechanical means; however, manual picking of grapes is still done for premium

quality American wines and in much of Europe (Jackson, 2018). Manual harvesting is gentler on

the grapes, and bruising and breaking of the grapes is minimized. For certain wines, such as sweet

17
wines made from noble rot grapes, or wines for which grape harvesting methods are regulated

(e.g., French Champagne), manual harvesting is required. Anyone who has seen the odd contours

and steep terrains of some of European vineyards will also appreciate the necessity of manual

grape picking. On the other hand, mechanical harvesters are faster and cheaper, and, unlike hired

laborers, deployable on short notice and available around the clock. Once the grapes are removed

from the vines, they must be transported to the winery. It is important that the grapes not be bruised,

crushed, or otherwise damaged either during harvesting or transport, since this encourages growth

of microorganisms prior to the actual start of the fermentation. For the same reason, transportation

time is also important (Herstein et al., 2015).

CRUSHING

Crushing the whole clusters of fresh ripe grapes is traditionally the next step in the wine making

process. Today, mechanical crushers perform the time-honored tradition of stomping or trodding

the grapes into what is commonly referred to as must. For thousands of years, it was men and

women who performed the harvest dance in barrels and presses that began grape juice's magical

transformation from concentrated sunlight and water held together in clusters of fruit to the most

healthful and mystical of all beverages - wine. As with anything in life, change involves something

lost and something gained (Mishra et al., 2016).

By using mechanical presses, much of the romance and ritual has departed this stage of wine

making, but one need not lament too long due to the immense sanitary gain that mechanical

pressing brings to wine making. Mechanical pressing has also improved the quality and longevity

of wine, while reducing the winemaker's need for preservatives. Having said all this, it is important

to note that not all wine begins life in a crusher (Wilkinson et al.,2015). Sometimes, winemakers

choose to allow fermentation to begin inside uncrushed whole grape clusters, allowing the natural

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weight of the grapes and the onset of fermentation to burst the skins of the grapes before pressing

the uncrushed clusters. Up until crushing and pressing the steps for making white wine and red

wine are essentially the same. However, if a winemaker is to make white wine, he or she will

quickly press the must after crushing in order to separate the juice from the skins, seeds, and solids.

By doing so unwanted color (which comes from the skin of the grape, not the juice) and tannins

cannot leach into the white wine. Essentially, white wine is allowed very little skin contact, while

red wine is left in contact with its skins to garner color, flavor, and additional tannins during

fermentation. (Mishra et al., 2016).

FERMENTATION

Fermentation is a well-known natural process used by humanity for thousands of years with the

fundamental purpose of making alcoholic beverages, as well as bread and by-products (Maicas,

2020). Upon a strictly biochemical point of view, fermentation is a process of central metabolism

in which an organism converts a carbohydrate, such as starch or sugar, into an alcohol or an acid.

For example, yeast performs fermentation to obtain energy by converting sugar into alcohol.

Fermentation processes were spontaneously carried out before the biochemical process was fully

understood. In the 1850s and 1860s, the French chemist and microbiologist Louis Pasteur became

the first scientist to study fermentation, when he demonstrated that this process was performed by

living cells. Fermentation processes to produce wines, beers and ciders are traditionally carried out

with Saccharomyces cerevisiae strains, the most common and commercially available yeast

(Maicas, 2020). They are well known for their fermentative behavior and technological

characteristics which allow obtaining products of uniform and standard quality. Many other

important industrial products are the result of fermentation, such as yogurt, cheese, bread, coffee.

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Yeasts also play a key role in wastewater treatment or biofuel production. Upon a biochemical

point of view, fermentation is carried out by yeasts (and some bacteria) when pyruvate generated

from glucose metabolism is broken into ethanol and carbon dioxide.

Wine fermentation

In wine fermentation, strains with specific characteristics are needed, for instance, highly

producers of ethanol to reach values of 11–13% v/v, typically found in this beverage (Rosa and

Angel, 2019). On the other hand, beers and ciders contain less amounts of ethanol with a balanced

and distinctive sensory profile characteristic of each one. In recent years, new consuming trends

and requirements for new and innovative products have emerged. This situation led to rethink

about the existing fermented beverages and to meet the demands of consumers. Yeasts are largely

responsible for the complexity and sensory quality of fermented beverages. Based on this, current

studies are mainly focused on the search of new type of yeasts with technological application.

Non-Saccharomyces yeasts have always been considered contaminants in the manufacture of wine

and beer (Maicas, 2020). Therefore, procedures for eliminating them are routinely utilized such as

must pasteurization, addition of sulfite and sanitization of equipment and processing halls. In

recent years, the negative perception about non-Saccharomyces yeasts has been changing due to

the fact that several studies have shown that during spontaneous fermentations of wine, these

yeasts play an important role in the definition of the sensory quality of the final product. Based on

this evidence, the fermentative behavior of some non-Saccharomyces yeasts is being studied in

deep with the purpose of finding the most adequate conditions and the most suitable strain to be

utilized in the production of fermented beverages (Maicas, 2020).

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PRIMARY FERMENTATION

After 8-10 hours from inoculation with yeast the primary, alcoholic fermentation starts. This

fermentation generally lasts for 8-10 days and during this period the yeast cells utilize the sugars

in the must and multiply, producing carbon dioxide gas and alcohol. The temperature used for red

wines is typically 25° to 28 °C while for white wines it is 20° to 25 °C (Gonzalez et al., 2016)

However, During fermentation it is important to control the temperature and the oxygen

concentration of the must. During alcoholic fermentation other substances (minor products):

glycerol, acetic acid, higher alcohols, and acetaldehyde are also produced. Wine quality therefore,

is also defined by the quantity of these compounds and particularly by low concentrations of acetic

acid, higher alcohols and acetaldehyde. In the must used for red wine production, skins and seeds

are present and during the alcoholic fermentation the color and tannin must be extracted (Peter et

al., 2018).

In Primary Fermentation, Seeds fall to the bottom of the tank while skins are pushed to the top of

the tank by carbon dioxide (known as "cap of pomace “). Grape skins are richer in flavor and color

(anthocyanin pigments) compounds and contact of the skin with the must allow flavor and color

extraction (Bellon et al., 2013) However, to extract the color and tannin in the skins, this cap must

be broken and the fermenting must is thoroughly mixed several times a day. When the sugar

concentration of the must has reached about 10 g/L or less, usually in 7-10 days, the must is drawn

off the pomace. In some cases, the wine may be allowed to remain with the skins and the seeds for

seven or more days after the fermentation is complete in order to obtain wines with a high tannin

concentration. Pomace and wine are generally separated using either vertical or horizontal presses.

The wine is then placed in a storage tank where the alcoholic fermentation runs to completion.

This process requires about a week (Fleet et al., 2013).

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However, A critical part of wine making involves the choice of whether to produce a dry (no

remaining free sugar) or a sweeter (varying amounts of free sugar) wine. This can be controlled

by regulating the initial must sugar concentration. With higher levels of sugar, alcohol will

accumulate and inhibit the fermentation before the sugar can be completely used, thus producing

a sweeter wine. During final fermentation in the aging process, flavoring compounds accumulate

and influence the bouquet of the wine (Fleet et al., 2013).

MALOLACTIC FERMENTATION

When the sugar is fully utilized, the malolactic fermentation can take place. This process is used

mainly for red wines but also for some white wines. In this process specific strains of the

Bacterium, Oenococcus oeni, convert malic acid to lactic acid (Osborne et al.,2016). The

fermentation reduces the titratable acidity and raises the pH which is advantageous in some wines.

This fermentation is often initiated by inoculation with desired Bacteria but can only be performed

if the pH is higher than 3.2 and the temperature is higher than 20 °C. (Bravo Ferrada et al. 2013).

Malolactic fermentation is performed for 2-4 weeks then the wine is transferred to other tanks for

final wine processing activities (fining, filtration, and aging) (Sumby et al., 2019).

CLARIFICATION
Once fermentation is completed, the clarification process begins. Winemakers have the option of

racking or siphoning their wines from one tank or barrel to the next in the hope of leaving the

precipitates and solids called pomace in the bottom of the fermenting tank. Filtering and fining

may also be done at this stage (Wilkinson et al., 2015). Filtration can be done with everything

from a course filter that catches only large solids to a sterile filter pad that strips wine of all life.

Fining occurs when substances are added to a wine to clarify them. Often, winemakers will add

egg whites, clay, or other compounds to wine that will help precipitate dead yeast cells and other

22
solids out of a wine. These substances adhere to the unwanted solids and force them to the bottom

of the tank. The clarified wine is then racked into another vessel, where it is ready for bottling or

further aging (Mishra et al., 2016).

FILTRATION

Filtration in winemaking is a general operation which encompasses a wide range of conditions

from the partial removal of large suspended solids to the complete retention of microbes by

perpendicular flow polymeric membranes. Microbial growth during the fermentation process

produces sediments, which are removed during racking (Fleet et al., 2013)

RACKING

Racking can be carried out at the time the fermented wine is transferred to bottles or casks for

aging or even after the wine is placed in bottles. Generally, all wines are subjected to fining and/or

filtration processes but only some wines containing high concentrations of ethanol and tannins are

selected for aging (Bellon et al., 2013)

AGING AND BOTTLING

In general aging is used for red wines but some white wines (e.g., Chardonnay) are also subjected

to aging. Aging is a very complex process which has many effects on the wine. It can be performed

in two stages, bulk and/or bottle (Fleet et al., 2013) Bulk storage can be performed with inert

containers like stainless steel or white oak barrels (like French "barriques"). In the latter case,

however, the container reacts with wine. these reactions are more intense when small barrels are

used. The most important reactions that occur during aging in an oak barrel are: wine oxidation,

evaporation of volatile components and reaction between wine and oak components. The aging

period can range from few months to many years and is dependent on the wine type (Wilkinson et

23
al., 2015). At the end of this period wine is generally only filtered and bottled. Aging in the bottle

ranges from some days to many years and is dependent on wine types.

OTHER WINES

Many processing variations can be used during wine production. The wine can be distilled to make

a “burned wine” or brandy. Acetobacter and Gluconobacter can be allowed to oxidize the ethanol

to acetic acid and form a wine vinegar (Fleet et al., 2013) Natural champagnes are produced by

continuing the fermentation in bottles to produce a naturally sparkling wine. However, Sediments

that remain are collected in the necks of inverted champagne bottles after the bottles have been

carefully turned. The necks of the bottles are then frozen and the corks removed to disgorge the

accumulated sediments. The bottles are refilled with clear champagne from another disgorged

bottle, and the product is ready for final packaging and labeling (Bellon et al., 2013).

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GROUPS OF MICROORGANISMS INVOLVED IN WINE PRODUCTION

Yeast and bacteria found in grape must and wine originate from the vineyard, grapes, and winery

processing equipment. This ‘natural microflora’ includes several dozen species of yeast,

with Saccharomyces cerevisiae being predominant (Fleet et al., 2013). Lactic acid and acetic acid

bacteria (AAB) are the only families of bacteria found in grape must and wine. These include four

genera of lactic acid bacteria (LAB), namely:

Lactobacillus,

Leuconostoc,

Oenococcus

and Pediococcus and two genera of AAB, namely:

Acetobacter and Gluconobacter. (Fleet et al., 2013)

The most common yeast generally associated with winemaking is Saccharomyces

cerevisiae which is also used in bread making and brewing. They can be saccharomyces or

nonsaccharomyces (Nyah and Etukudo, 2023).

The Non saccharomyces/bottom yeast include:

• Brettanomyces (Teleomorph Dekkera)

• Candida (Teleomorphs for different species from several genera

including Pichia, Metschnikowia, Issatchenkia, Torulaspora and Kluyveromyces)

• Kloeckera (Teleomorph Hanseniaspora), usually the most common "wild yeast" found

in the vineyard. Some species are known as "killer yeast" that produce inhibitory levels

25
 of ethyl acetate and acetic acid that can kill off sensitive strains of Saccharomyces

cerevisiae

• Saccharomycodes

• Schizosaccharomyces, the only wine yeast that reproduced by fission whereas most

wine yeast reproduce by budding.

• Zygosaccharomyces, very alcohol-tolerant and can grow in wines up to 18% v/v.

Additionally this yeast can survive in extremely high sugar levels (as much as 60%

w/w or 60 Brix) and is very resistant to sulfur dioxide.

• Aureobasidium, particularly the "black yeast" species of Aureobasidium

pullulans found in moist cellars that can contaminate aging wine in barrels (Nyah and

Etukudo, 2023).

The saccharomyces/top yeast include:

• Saccharomyces cerevisae

• Saccharomyces uvarum

• Saccharomyces pastorianus

• Saccharomyces boulardi

• Saccharomyces florentinus (Nyah and Etukudo, 2023).

26
ROLES OF EACH GROUP OF MICROORGANISMS INVOLVED IN WINE

PRODUCTION

1. YEAST

Yeasts are eukaryotic microorganisms that live in a wide variety of ecological niches, mainly in

water, soil, air and on plant and fruit surfaces (Nyah and Etukudo, 2023). Perhaps the most

interesting habitat at this point is the latter, since they directly intervene in the decomposition of

ripe fruit and participate in the fermentation process. In this natural environment, yeasts can carry

out their metabolism and fermentation activity satisfactorily as they have the necessary nutrients

and substrates (Nyah and Etukudo, 2023).

According to technological convenience, yeasts are divided into two large groups namely

Saccharomyces and non-Saccharomyces. Morphologically, Saccharomyces yeasts can be round or

ellipsoidal in shape depending on the growth phase and cultivation conditions (Maicas, 2020). S.

cerevisiae is the most studied species and the most utilized in the fermentation of wines and beers

due to its satisfactory fermentative capacity, rapid growth and easy adaptation. They tolerate

concentrations of SO2 that normally most non-Saccharomyces yeasts do not survive. However,

despite these advantages, it is possible to find in the nature representatives of S. cerevisiae that do

not necessarily have these characteristics (Maicas, 2020).

Non-Saccharomyces yeasts are a group of microorganisms used in numerous fermentation

processes, since their high metabolic differences allow the synthesis of different final products

(Maicas, 2020). Generally, many of these yeasts capable of modifying the sensory quality of wines

are considered as contaminants, so eliminating them or keeping them at low levels was a basic

objective in the past. In order to eliminate their activity in wine fermentation, it is usual to disinfect

27
the tanks and fermentation containers using sulfite. This perception has been modified year after

year, gaining relevance the action of these yeasts in the spontaneous fermentation, since they

contribute positively in the final sensory quality of the wine. These yeasts are the majority in the

initial phase of spontaneous fermentation to the point where the concentration of ethanol reaches

4 and 5% v/v. At that point, between alcohol and the exhaustion of dissolved oxygen, their growth

is inhibited (Maicas, 2020).

Figure 2: roles of yeast in wine production, source: (Nyah and Etukudo, 2023).

28
 2. BACTERIA

Bacteria are part of the natural microbial ecosystem of wine and play an important role in

winemaking by reducing wine acidity and contributing to aroma and flavor (Ding et al., 2023).

Conversely, they can cause numerous unwelcome wine spoilage problems, which reduce wine

quality and value. Lactic acid bacteria, especially Oenococcus oeni, contribute positively to wine

sensory characters, LAB can be homofermentative and producing exclusively lactic acid and CO2

from sugars (glucose and /or fructose) or can be heterofermentative and producing ethanol, acetic

acid and CO2 (Ding et al., 2023). Generally the Malolactic fermentation is conducted by O.oeni,

which presents a heterofermentative metabolism but other species, such as Lactobacillus sp.

and Pediococcus sp can produce undesirable volatile compounds. (Gayan et al., 2016).

Consequences of bacterial wine spoilage include mousy taint, bitterness, geranium notes, volatile

acidity, oily and slimy-texture, and overt buttery characters. Management of wine spoilage bacteria

can be as simple as manipulating wine acidity or adding sulfur dioxide. However, to control the

more recalcitrant bacteria, several other technologies can be explored including pulsed electric

fields, ultrahigh pressure, ultrasound or UV irradiation, and natural products, including

bacteriocins and lysozyme. However, the acetic acid bacteria are considered spoilage

microorganisms during winemaking. Their metabolism is strictly aerobic, and their principal

property is that they can oxidize ethanol into acetic acid by acetaldehyde pathway. (Fleet et al.,

2013).

29
Figure 3: roles of bacteria in wine production: source: (Gayan et al., 2016)

30
MOLDS

Molds are ubiquitous with various genera commonly found on grapes. Common examples include

Aspergillus, Botrytis, and Penicillium, and, to a lesser extent, Phythophthora, Moniliella,

Alternaria, and Cladosporium (Ding et al., 2023). Molds growth plays an important role in the

physical and chemical stability as well as the sensory properties of the future wine. For example,

uncontrolled proliferation of mold on grapes just prior to harvest rapidly leads to growth of

secondary contaminants (yeasts and bacteria), which, in turn, leads to a deteriorative state called

“rot.” Recognizing the importance of mold growth to wine quality, grape contracts generally

include specification for the extent of infections (Ding et al., 2023).

WINE SPOILAGE

Wine spoilage is the change in the physical and chemical properties of the wine so that it becomes

unfit for consumption (Echave et al., 2021). Microbial contamination or spoilage occurs with the

development of micro-organisms whose metabolism can negatively affect wine quality. Grape

juice, rich in sugars and nutrients, is a suitable substrate for the growth of many species of micro-

organisms, including yeasts, bacteria and molds. After alcoholic fermentation, the ethanol

presence reduces the potential for development of many micro-organisms but even under final

wine conditions some yeasts and bacteria can still be active. (Benito, 2019).

There are three stages at which microorganisms can enter the winemaking process and exert an

influencing effect on the quality of the end product. The first stage involves the raw material

(Castro et al., 2022). The grapes are in direct contact with the winery equipment (crushers, presses,

tanks, pipes, pumps, filtration units, etc.), and when not properly sanitized the equipment will serve

31
as an inoculant of the grape juice. The grapes delivered to a winery are not all in a healthy state,

and this will affect the natural biodiversity of the microorganisms present in the juice.

The second stage of spoilage may occur during fermentation. At this point the grape juice contains

the natural flora of the grapes along with the flora harbored by the wine cellar and its equipment

(Castro et al., 2022). The composition of the grape juice (high sugar and acid content, and low

pH) and the addition of Sulphur dioxide (S02) to the juice exerts selective pressure on the

development of yeasts and bacteria during alcoholic fermentation. Saccharomyces cerevisiae is

the dominant yeast during fermentation, and the increase in ethanol concentrations further

suppresses the development of certain fungi and bacteria. In natural fermentation the initiators of

this process are yeast species belonging to the genera Candida, Hanseniaspora, Kloeckera and

Metschnikowia, and less frequently Kluyveromyces and Pichia (Castro et al., 2022).

The third stage at which the product may be susceptible to spoilage is post-fermentation. Here,

spoilage may occur in the bottle or during storage in oak barrels. During this stage, the critical

factors are good cellar sanitation, exclusion of oxygen and the correct dosage of antimicrobial

agents to ensure a stable product that will withstand attack from spoilage yeasts and bacteria. The

wine can also be affected at this stage by fungi and species of Actinomyces and Streptomyces

present in the corks or oak barrels (Castro et al., 2022).

32
MICROBIAL SPOILAGE AND PRESERVATION OF WINE

The winemaking process includes multiple stages at which microbial spoilage can occur, altering

the quality and hygienic status of the wine and rendering it unacceptable (Kumar et al., 2023). The

major spoilage organisms include species and strains of the yeast genera Brettanomyces, Candida,

Hanseniaspora, Pichia, Zygosaccharomyces etc., the lactic acid bacterial genera Lactobacillus,

Leuconostoc, Pediococcus, etc. and the acetic acid bacterial genera Acetobacter and

Gluconobacter. The faults caused include bitterness and off-flavors (mousiness, ester taint,

phenolic, vinegary, buttery, geranium tone), and cosmetic problems such as turbidity, viscosity,

sediment and film formation (Valera et al., 2017). These spoilage organisms can also affect the

wholesomeness of wine by producing biogenic amines and precursors of ethyl carbamate. The

judicious use of chemical preservatives such as Sulphur dioxide (S02) during the winemaking

process decreases the risk of microbial spoilage, but strains vary considerably in their S02

sensitivity. There is, moreover, mounting consumer bias against chemical preservatives, and this

review focuses on the possible use of bio preservatives in complying with the consumers' demand

for "clean and green" products.

SPOILAGE AGENTS

The low pH of juice and wine does not permit the growth of human pathogens which are thus not

a concern in the wine industry (Vicente et al., 2022). Many micro-organisms can however

adversely affect wine quality by producing unwanted chemicals resulting from the degradation of

favorable ones.

33
SPOILAGE BY YEAST

The Yeast, A Taxonomic describes one hundred yeast genera representing over 700 species

(Bhunjun et al., 2022). Only twelve of the yeast genera are associated with grapes or wine,

emphasizing the degree of specialization needed to survive in the hostile wine environment.

Identification of yeast species is of utmost importance to oenologists assessing the risk of potential

spoilage. The term "wine yeasts" applies to those Saccharomyces yeasts which can perform a

complete fermentation of grape juice without the production of off-flavors (Romano et al., 2022).

These yeasts are tolerant to high concentrations of ethanol and sugar. The term "wild yeasts"

applies to those non-Saccharomyces yeasts which can perform a partial alcoholic fermentation,

often with the formation of esters. Both these types of yeasts can bring about spoilage. The yeast

genera that are often found in wine include Brettanomyces and its sporulating form Dekkera,

Saccharomyces, Schizosaccharomyces and Zygosaccharomyces.

Re-fermentation

Saccharomyces is regarded as a spoilage organism only if it is found in the wrong place at the

wrong time (e.g., in a bottle of semi-sweet wine) causing re-fermentation (Mojsov, 2012).

Schizosaccharomyces pombe has been associated with wine spoilage when growing in bottled

wine and forming a sediment at the bottom of the bottle. The yeast Z. bailii is one of the major

wine spoilage yeasts, re-fermenting juice or wine during storage. Saccharomycodes ludwigii,

found in bottled wines, is often regarded as the winemaker's nightmare. This yeast species is highly

tolerant to ethanol and resistant to SO2 and sorbate. It produces high levels of acetaldehyde and

has been isolated as a slimy flocculent mass (Mojsov, 2012).

34
Ester formation
Hansenula anomala (now known as Pichia anomala), Kloeckera apiculata and Hanseniaspora

uvarum are mainly associated with the ester taint of faulty wines, which correlates with large

amounts of acetic acid. These three species are associated with grape juice and result in spoilage

at the early stages of alcoholic fermentation (Fleet, 2013). The ester taint can be linked to the

presence of ethyl acetate and methylbutyl acetate, which are most prominent in wines possessing

this flavor. Wines with concentrations of >200 mg/L ethyl acetate and 0.6 mg/L of acetate are

regarded as spoiled. Growth of Z. bailii may also lead to wine with an increase in acetic and

succinic acid, a decrease of L-malic acid and a concomitant reduction in total acidity and an altered

ester concentration (Mojsov, 2012). Hydrogen sulphide and volatile sulphur compounds Sulphur-

containing compounds play a significant role in the flavor of wine due to their high volatility,

reactivity and potency at low threshold values. These compounds are responsible for off-flavours

that have been described as rotten eggs, rubbery, onion, skunky aroma, garlic and cabbage.

Hydrogen sulphide (H2S) is produced by yeasts during fermentation through the sulphate reduction

pathway and has a flavour threshold of 50-80 mg/L and when exceeding this value will produce

the rotten egg off-flavour. The ability of yeasts to produce H2S varies between strains and is

influenced by environmental factors such as must composition (solids, vitamins and free amino

nitrogen), fermentation temperature, wine pH and the use of fungicides containing elemental

sulphur (Araujo et al., 2017).

35
Volatile acidity

The major volatile acid in wine is acetic acid (> 90%) . Acetic acid has a threshold value of 0.7 to

1.1 g/L depending on the style of wine and above these values it becomes objectionable (Araujo

et al., 2017). High levels of volatile acidity may result from the indigenous wine yeasts and wild

yeasts, as well as lactic acid - and acetic acid bacteria, which will be discussed separately in the

review. Acetic acid is formed as a by-product by yeasts during the early stages of alcoholic

fermentation. Saccharomyces strains displayed variation in their production of acetate and this

phenomenon is influenced by fermentation temperature, pH, juice composition (sugar and nitrogen

levels), levels of acetyl-CoA synthetase enzyme and the presence of other microorganisms (Araujo

et al., 2017).

Formation of volatile phenols

Descriptive words for wines contaminated with Brettanomyces include mousy, barnyard-like,

horsy, wet dog, tar, tobacco, creosote, leathery and pharmaceutical. Contaminated wines often

display an increase in volatile acidity, due to the oxidation of acetaldehyde to acetic acid instead

of ethanol (Mojsov, 2012). Most of the abovementioned descriptors, e.g. phenolic, smoky, horsy,

elastoplast, can be ascribed to the concentrations of volatile phenols such as vinylphenols [ 4-

vinylguaiacol (> 750 µg/L) and 4-vinylphenol (>440 µg/L)] in white wines and ethylphenols [ 4-

ethylguaiacol (> 100 µg/L) and 4-ethyl phenol (>600 µg/L)] produced in red (Mojsov, (2012). .

These volatile phenols are produced by decarboxylation (cinnamate decarboxylase) and reduction

of hydroxycinnamic acids such as p-coumaric- and ferulic acid.

36
Mousiness

The mousy taint resulting from Brettanomyces growth is discussed under spoilage by LAB, as the

mechanism is the same (Bryne, 2020).

Film formation

Some yeasts, called film yeasts, can form a film layer on top of stored wine; species of the genera

Candida, Metschnikowia and Pichia have been associated with this trait (Bryne, 2020). These

yeasts not only create a cosmetic problem, they may also be detrimental to the quality of wine,

imparting an oxidized flavor due to the production of acetaldehyde. The development of these

yeasts is highly dependent on available oxygen and will thus proliferate in wine exposed to air and

in partially filled barrels. The main products formed from ethanol by these film yeasts are acetic

acid, acetaldehyde and acetate esters (Bryne, 2020).

37
Table 1: Yeast spoilage in wine: source: (Cosme et al., 2018).

SPOILAGE EFFECT CAUSAL MICROORGANISM

Ester and aldehyde taints: increased volatile Pichia, Hansenula, Metschnikowia, Dekkera,
acidity. Candida.

Formation of surface films Candida, Pichia

Mousy, horsy tainty Bettanomyces

Refermentation in the bottle Saccharomyces, Zygosaccharomyces bailii

Oxidized taint from aldehyde Saccharomyces ludwigii, Pichia membranafaciens

Deacidification of wine Schizosaccharomyces

38
SPOILAGE BY LACTIC ACID BACTERIA

LAB play a pivotal role in the secondary fermentation of wine by conducting malolactic

fermentation (MLF), but they can also be detrimental to wine quality as spoilage microorganisms

if proliferation of certain LAB occurs at the wrong time during the winemaking process (Bayon,

2012). LAB are Gram-positive, catalase-negative, non-motile, non-sporeforming, rod- and coccus

shaped. They produce mainly lactic acid as the end product of carbohydrate fermentation.

Therefore, the LAB are divided into three groups according to their metabolic activity: obligately

homofermentative, facultatively heterofermentative and obligately heterofermentative. The LAB

associated with grape juice and wine belong to four genera: Lactobacillus, Leuconostoc,

Oenococcus and Pediococcus (Bayon, 2012).

Acid formation

LAB can increase the acid content of wine by producing lactic acid and acetic acid. The D-lactic

acid is associated with spoilage, as the L-lactic acid is produced during MLF (Bryne, 2020). The

homofermentative LAB reduces hexose sugars to lactic acid via the Embden-Meyerhof-Parthway

(glycolytic) pathway. The formation of lactic acid arises from the reduction of pyruvic acid and is

performed by homofermentative species of lactobacilli and pediococci. Heterofermentative

lactobacilli, Leuconostoc and Oenococcus spp. produce D-lactic acid and acetic acid through the

6-phosphogluconate pathway (Bryne, 2020).

Re-fermentation

This is also known as misplaced MLF, and can occur in bottled wine with a pH>3.5 in the presence

of LAB and nutrients (malate or residual sugars) that enhance growth (Bordons, 2021). If

39
secondary growth of LAB occurs in the wine, it will be deacidified and the pH will rise above 3.5.

This problem can be corrected by the addition of tartaric acid (Saranraj et al., 2022).

Ropiness

Wines with an increase in viscosity and a slimy appearance are called "ropy". (Abid and Abadou,

(2021). Viscosity is attributed to the production of extracellular polysaccharide, composed of o-

glucan and the genera Leuconostoc and Pediococcus have been implicated in ropiness The

production of extracellular polysaccharides by Pediococcus damnosus and Pediococcus

pentosaceus isolated from ropy wines was induced by ethanol, and this trait was plasmid mediated

Pediococci associated with ropiness differ from other pediococci in their resistance to ethanol, SO2

and pH, and the ropiness thus only occurs during alcoholic fermentation or after bottling when

ethanol is present. Ropiness can be effectively controlled by lowering the pH to under 3.5 (Saranraj

et al., 2022).

Mousiness

Heterofermentative lactobacilli and the spoilage yeast Brettanomyces have been implicated in

wine that is reminiscent of mouse urine or acetamide (Saranraj et al., 2022). The lactobacilli

associated with this defect are Lactobacillus brevis, Lactobacillus cellobiosus (now synonymous

with Lactobacillus fermentum) and Lactobacillus hilgardii . As in Brettanomyces, the guilty

substances are the ethyl amino acid (lysine) derivatives, 2-acetyl-1,4,5,6-tetra hydropyridine and

its isomer, 2-acetyl-3,4,5,6-tetra hydropyridine. Microbial production of these compounds and

their propionyl analogues is dependent on ethanol or propanol, and are therefore associated with

wine rather than grape juice (Saranraj et al., 2022).

40
SPOILAGE BY ENDO-SPOREFORMING BACTERIA

Rare incidences of Bacillus and Clostridium spp. have been reported in microbiological spoilage

of wines. The genus Bacillus is comprised of aerobic Gram-positive, catalase-negative, endospore

forming rods (Saranraj et al., 2022). The natural habitat of this microorganism is primarily soil,

and will thus secondarily occur in water, which would enable access of this organism into the wine

environment. Clostridium is a Gram-positive, obligate anaerobic, endospore forming rod. Acidity

Gini & Vaughn first reported on Bacillus spoilage in dessert wines in 1962. They isolated Bacillus

subtilis, Bacillus circulans and Bacillus coagulans and demonstrated Koch's postulates by

inoculating the different species into wine. They were able to grow to 106 -10 7 cells/ml, and the

wine showed an increase in volatile and total acidity. spoiled bottled brandy was attributed to the

growth of Bacillus megaterium (Bryne, 2020). Bacillus spp. isolated from wine corks have been

shown to grow when inoculated into wine. More recent reports on Bacillus spoilage have been

from wines produced in Eastern European countries. The spoilage was cosmetic (sediment

formation) and did not present any sensorial changes (Bryne, 2020).

Butyric acid taint

Wines spoiled by Clostridium are even more infrequent than by Bacillus. They have been

implicated in low acid, high pH (>4.0) wine. Growth of clostridia in wine yields nbutyric acid,

acetic acid, CO2, hydrogen peroxide and, depending on the species, varying amounts of butanol,

acetone and propanol (Saranraj et al., 2022). n-Butyric acid is perceived as a taint of rancidness.

Although incidences of Bacillus and Clostridium spoilage are rare, care should be taken, because

under the right conditions they have the potential of significantly lowering wine quality (Saranraj

et al., 2022).

41
SPOILAGE BY ACETIC ACID BACTERIA

AAB belong to the family Acetobacteriaceae and are commonly known as the vinegar bacteria.

AAB are Gram-negative, aerobic, catalase-positive microorganisms and can utilize glucose, with

acetic acid as the end-product (Verma, 2019). According to there are microscopic variations among

pure cultures and their cell morphology may range from spherical, club-shaped, elongated,

swollen, curved rods to filamentous. This makes the preliminary identification of wine-related

AAB with light microscopy difficult for the novice. The habitat of these bacteria is ubiquitous;

they are found on flowers, fruit and vegetables, in wine and beer as spoilage microorganisms, and

in vinegar as the primary fermenter (Verma, 2019).

Volatile acidity

Acetic acid is the major component in wine associated with volatile acidity (Debra et al., 2020).

The legal limit for acetic acid in wine is 1.2- 1.4 g/L, after which the wine becomes objectionable.

However, acetic acid may also modify the perception of other important wine constituents. Tannins

and fixed acids may be intensified. The esters of acetate, especially ethyl acetate, are major

contributors to this defect in wine (Zhang et al., 2022). Ethyl acetate is perceived as "fingernail

polish" and has a detection level of 12.3 mg/L; defective wines can contain levels of 150-200

mg/L.

Dihydroxyacetone

The glycerol produced by yeast and molds serves as carbon source for A. aceti and G. oxydans.

These two species can convert glycerol into dihydroxyacetone under aerobic conditions

(ketogenesis) (Wang et al., 2022). Dihydroxyacetone can affect the sensory quality of the wine

42
with a sweet/etherish property (Campaniello and Sinigaglia, 2017). It can also react with proline

and produce a "crust-like" aroma. Dihydroxyacetone can affect the antimicrobial activity in the

wine, as it has the ability to bind SO2 (Ochando et al., 2020).

Acetaldehyde

Wines containing high amounts of AAB may contain significant amounts of acetaldehyde, an

intermediate metabolite in the production of acetic acid from ethanol under low oxygen

concentrations (Tronchoni et al., 2022). Growth of Acetobacter may produce acetaldehyde at

concentrations exceeding the threshold value of 100-120 mg/L. The descriptors of this defect in

wine range from "classic" nutty and sherry-like to being reminiscent of overripe bruised apples.

Acetaldehyde binds SO2 and will thus affect the antimicrobial activity of the SO2 in wine. This

combined compound may mask the odour of acetaldehyde (Marc et al., 2020).

SPOILAGE BY MOLDS

The infection of grapes by filamentous fungi (molds) before harvest can be disastrous to the quality

of wine if they are not controlled by the use of fungicides (Bano et al., 2023). Molds found on

grapes include species of the genera Alternaria, Aspergillus, Botrytis, Cladosporium, Mucor,

Oidium, Penicillium, Plasmopara, Rhizopus and Uncinula. Molds can affect the wine quality in

one of the following manners: (i) loss in juice yield, (ii) slippery nature of infected grapes prolongs

the pressing process, (iii) alteration of the chemical composition of wine such as the production of

gluconic acid, higher levels of glycerol, oxidation of phenolic compounds, (iv) secretes B-glucan

that will negatively affect clarification, (v) produce off-flavors (such as acetic acid), and (vi)

stimulate the growth of spoilage yeasts and bacteria. Molds are sensitive to ethanol concentrations

of 3%, low pH, S02 and anaerobiosis, and though unable to survive in wine, they alter the chemical

43
composition of the grape juice through the enzymes they secrete (Bano et al., 2023). These molds

can also grow on the surfaces of the wine cellar and on the wooden barrels used for ageing and

give the wine a moldy flavor. suggested evidence that molds produce anti-yeast metabolites that

might affect alcoholic and malolactic fermentations. Molds can also produce mycotoxins, which

are regarded as carcinogens, and thus a matter of great concern. The two genera of molds

associated with infected grapes that can produce these mycotoxins (such as aflatoxins, patulin and

ochratoxin A) are Aspergillus and Penicillium). It seems, however, that the

winemaking/fermentation process inactivates these mycotoxins, as they have not been found in

wine made from grapes containing them (Bano et al., 2023).

Cork taint

Microbiological contamination of corks can affect the quality of the finished wine by producing

off-flavours; corks are used as a substrate by microorganisms, leaching metabolites into the

endproduct. The fungal genera associated with cork taint are Aspergillus, Cladosporium, Monilia,

Paecilomyces, Penicillium and Trichoderma (Saranraj et al., 2022). Yeast and bacteria have been

implicated as part of the natural cork flora, but they occur in numbers. Yeast species associated

with cork are Candida, Cryptococcus, Rhodotorula, Saccharomyces and Sporodiobolus (Saranraj

et al., 2022). Bacterial species implicated in cork are Bacillus, Micrococcus, Streptococcus and

Streptomyces. Cork taint is perceived as a mouldy, earthy or musty off-flavour.

44
 Table 2: Bacteria spoilage microorganism, source: (Saranraj et al., 2022).

Bacteria Spoilage

Lactic acid bacteria:

Lactobacillus brevis Acidification of wine through the production of acetic

and lactic acids, mannitol if formed by the reduction of
fructose, mousy taints.

Lactobacillus Mousy taints from tetrahydropyridine

Cellobiosus Bitterness arising from glycerol metabolism

Lactobacillus trichodes Flocculent growth

Leuconostoc mesenteroides Forms ropiness, bitterness from glycerol metabolism

Pediococcus parvulus Acrolein formation from glycerol contribute to

bitterness

Acetic acid bacteria

Acetobacter aceti Oxidation of ethanolto acetaldehyde and acetic acid

Acetobacter pasteurianus Production of ethyl acetate

Endospore forming bacteria

Bacillus, clostridium spp. Increase in acidity (butyric acid), forms sediment.

45
PRESERVATION OF WINE

Wine preservatives are used to preserve the quality and shelf life of bottled wine without affecting

its taste. Specifically, they are used to prevent oxidation and bacterial spoilage by inhibiting

microbial activity (Lisanti et al., 2019).

Wine contains natural preservatives such as tannin, sugar and alcohol, and can be preserved

through physical preservation methods, such as storage at an appropriate temperature. (González-

Rompinelli et al., 2013). However, bacterial growth is still possible, so chemical preservatives are

added to most wines. Typical chemical preservatives include sulfur dioxide and its related

chemicals, such as sulfites. Despite the alcohol in wine, growth of bacteria is possible, even when

completely fermented. Wine is made from the fermentation of grape juice, which contains sugar.

During the fermentation process, yeast will convert sugar into alcohol. If the fermentation is not

complete, the wine will contain residual sugar content (Lisanti et al., 2019). The sugar acts as

nutrient needed for the growth of bacteria, which can deteriorate the wine or even turn it into grape

vinegar (Pretorius et al., 2017). Because the alcohol content of wine is relatively low, the alcohol

cannot completely inhibit the proliferation of bacteria. Long-term improper storage may accelerate

deterioration.

During the brewing process, microbial spoilage can lead to the deterioration of wine quality and

taste, making it unhygienic and unacceptable to consumers. The main bacterial groups are yeast,

candida and Hanseniaspora (Rodhouse and Carbonero, 2019). In addition, microbial diseases of

wine are mainly caused by yeast, lactobacillus and acetic acid. This is because yeast may re-

ferment a wine with high sugar content, making it cloudy and impure. Lactic acid bacteria can

cause acid spoilage in dry wine with low acidity and lactic acid bacteria disease in sweet wine with

46
low acidity. Acetic acid bacteria may cause the increase of volatile acid in wine, resulting in an

undesirable sour vinegar taste. These fungi are all bad for the safety and flavor of wine.

However, the presence of wine spoilage organisms in the cellar, wine and corks, as

discussed above, illustrates the need for rigorous quality control to assure the microbiological

stability of the winemaking process (Pretorius et al.,2017). Chemical preservatives are used in

general to inhibit specific populations of microorganisms that endanger the quality of the end-

product. The techniques applied in the winemaking process to assure quality and microbiological

safety include: (i) procedures that prevent access of microorganisms in the first instance; (ii)

inactivation of unwanted microorganisms when the first step is unsuccessful; and (iii) procedures

that slow or inhibit their growth in the product.

CHEMICAL PRESERVATION

Preservatives used in the wine industry, such as Sulphur dioxide, Sorbic acid and Benzoic acid,

are most effective in their undissociated form, which is prevalent at a low pH (Lacumin et al.,

2022). Preservatives are more effective against stationary phase yeast and bacterial cultures than

against actively growing cultures producing metabolites that can diminish the effectiveness of the

preservative

Sulphur dioxide:

S02 is one of the oldest compounds used in the food and beverage industries for its antioxidative

and antimicrobial properties (Khatib and Assaf, 2022). The use of S02 in winemaking dates back

to the Egyptians, and later the Romans, who used burning Sulphur fumes to clean their amphora

and other wine vessels. In the centuries that followed S02 became a widely used chemical

preservative in the wine industry through the addition of Sulphite or Bisulphite to inhibit the

47
growth of unwanted yeasts and bacteria. Sulphite is present in three forms in an aqueous solution

and the equilibria are pH dependent. At low pH values Sulphite exists mainly as molecular S02' at

intermediate pH values as Bisulphite ions, and at high pH values as Sulphite ions. It has been

demonstrated that only the molecular form of S02 exerts the antimicrobial activity. At wine pH

values (3.0-4.0) the major proportion is Bisulphite ions (95%), with only 5% in the active

molecular form. Traditionally, S02 is added to the grapes at the crusher, as one of the objectives is

to inhibit or suppress the growth of non-Saccharomyces yeasts so that S. cerevisiae can proliferate

and dominate the fermentation (Khatib and Assaf, 2022). There have been indications that the

pattern of yeast growth in wineries where total S02 levels of 50- 100 mg/L are commonly added

allows for the growth of non-Saccharomyces yeasts at the beginning of alcoholic fermentation.

S02 is an effective antimicrobial agent against LAB associated with must and wine, and they are

more sensitive than yeasts. LAB strains vary in their sensitivity to S02; Lactobacillus and

Pediococcus are more resistant than Oenococcus. Research indicates that free S02 levels of 1-10

mg/L is sufficient to inhibit the growth of wine LAB (Khatib and Assaf, 2022).

Figure 4:, chemical structure of Sulphur dioxide, source : (Khatib and Assaf, 2022).

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Sorbic acid:

Sorbic Acid Sorbic acid (2,4-hexandienoic acid) is a short-chain unsaturated fatty acid that is used

in grape juices and in sweetened, bottled wines to prevent re-fermentation by Saccharomyces

(Reddy and Joshi, 2021). The maximum concentration allowed in the United States is 300 mg/L,

whereas the Office International de la Vigne et du Vin (O.I.V.) places the limit at 200 mg/L. In

practice, concentrations of 100 to 200 mg/L are typically used. At recommended levels, sorbic

acid is generally effective in controlling Saccharomyces, but other yeasts exhibit differential

resistance. For example, Kloeckera apiculata and Pichia anomala (formerly Hansenula anomala)

are inhibited at 156 to 168 mg/L, respectively, whereas Schizosacccharomyces pombe and

Zygosaccharomyces bailii require at least 672 mg/L (Reddy and Joshi, 2021). Mechanisms of

inhibition are not fully understood but probably due to morphological differences in cell structure,

changes in genetic material, alteration in cell membranes, as well as inhibition of enzymes or

transport functions (Reddy and Joshi, 2021). Bacteria are not affected by sorbic acid, and, in fact,

several species can metabolize the acid to eventually yield 2-ethoxyhexa-3,5-diene, a compound

that imparts a distinctive “geranium” odor/tone to wines (Section 11.3.5). Other odor/flavor–active

compounds detected in spoiled wines treated with sorbic acid include l-ethoxyhexa-2,4-diene and

ethyl sorbate the latter of which has been associated with off-flavors in sparkling wines. Whereas

ethyl sorbate was described as possessing a “honey” or “apple” aroma, thought the compound

imparted a very unpleasant “pineapple–celery” odor upon short-term (6 month) storage. Based on

this observation, recommended that sorbates should not be used in sparkling wine production

(Reddy and Joshi, 2021).

49
 Figure 5: chemical structure of sorbic acid, source: (Morata, et al., 2019)

Fumaric acid

Fumaric acid is approved by the United States Alcohol and Tobacco Tax and Trade Bureau for

both controlling growth of lactic acid bacteria and as an acidulant at maximum concentration of

3.0 g/L. Being a relatively strong organic acid, fumaric has received attention as an acidulating

agent in wine, rather than the more expensive tartaric acid. In this regard, fumaric acid additions

of 1 g/L are equivalent to tartaric acid additions of 1.2 g/L. The most important function of fumaric

acid is its ability to inhibit malolactic fermentation. In this regard, Ough and Kunkee (1974)

reported that none of the wines in their study containing 1.5 g/L fumaric acid underwent MLF,

even after 12 months of storage. However, fumaric acid is degraded during alcoholic fermentation

by Saccharomyces forming l-malic acid (Pilone et al., 2023). Fumaric acid might be useful to

reduce initial bacterial populations in musts, such as some species of Lactobacillus. Although it is

not known whether these spoilage bacteria can metabolize fumaric acid, (Pilone et al., 2023) noted

that the acid could be degraded to l-lactic acid by wine leuconostocs (Oenococcus), possibly by

50
the same mechanism of yeast. One concern regarding the use of fumaric acid is its limited

solubility in wine. In fact, fumaric acid is sparingly soluble in water (6.3 g/L at 25ºC/77ºF)

compared with tartaric acid, which is soluble at 1,390 g/L at 20ºC/68ºF . However, the acid is more

soluble at higher temperatures (10.7 g/L at 40ºC/104ºF) or in 95% ethanol (57.6 g/L at 30ºC/86ºF).

Margalit (2004) suggested solubilizing 50 to 80 g/L in hot water and addition to must or wine

while the solution is hot. Like other antimicrobial acids, the efficacy of fumaric acid depends on

pH where less activity is noted with increasing pH (Doores, 2016). Further, fumaric acid can impart

a “harsh” taste (Margalit, 2014). Because of this and solubility concerns, caution should be

exercised when used in wine.

Figure 6: chemical structure of fumaric acid, source: (Liliya, 2021).

51
Dimethyldicarbonate (DMDC)

Sold under the trade name VelcorinTM, the sterilant dimethyl dicarbonate (DMDC) is approved

for use in the United States for table as well as low-alcohol and dealcoholized wines at a maximum

concentration of 200 mg/L over the life of the wine (Ough et al., 2018). The United States Food

and Drug Administration also permits its use in juice drinks and certain non-carbonated juice

beverages. The additive does not possess any residual activity because DMDC will undergo

hydrolysis to yield carbon dioxide and methanol.

Figure 7: chemical structure of Dimethyldicarbonate, source: (Ough et al., 2018).

DMDC has been examined for general control of many wine microorganisms (Ough et al., 2018).

In a wine containing 10% v/v ethanol, a concentration of 25 mg/L was found effective against

Saccharomyces, Brettanomyces, and Schizosaccharomyces (Ough et al., 2018). The additive is

also inhibitory against acetic and lactic acid bacteria. Although a synergy between DMDC and

SO2 against Saccharomyces has been reported (Ough et al., 2018), this effect was not observed.

Rather, these authors noted that DMDC was more effective than combinations of SO2 and/or sorbic

acid and suppressed fermentation of grape juice more effectively at higher temperatures. The

antimicrobial effect of DMDC results from inactivation of microbial enzymes.

52
Table 3. Concentrations of dimethyl dicarbonate (Velcorin) required for control of various yeasts

and bacteria inoculated at 500 CFU/mL. Source: (Costa et al., 2017).

Microorganism Dimethyl decarbonate (mg/L)

YEAST

Candida krusei 100-200

Hansenula anomala 25-50

Kloeckera apiculate 25-50

Rhodotorula spp. 30-300

Saccharomyces spp. 40-200

Torulopsis spp. 75-100

Zygosaccharomyces 50-150

BACTERIA

Acetobacter pasteurianus 190-250

Lactobacillus brevis 200

Lactobacillus buchneri 30

Pediococcus 300

Inoculation at 250 CFU/Ml.

53
Bio preservatives

Although the chemical preservatives outlined above are still widely used, there exists a growing

demand from consumers for alternative methods to preserve products (Schillinger, 2016).

Consumer preferences have shifted to products that are less heavily preserved, less processed, of

higher quality, more natural and healthier. This trend renders the use of chemical preservatives

less acceptable to consumers, and has led to the possible exploitation of natural antimicrobial

compounds from plants, animals and microorganisms as biological preservatives (Schillinger,

2016).

Bacteriocins

Bacteriocin production is a characteristic typical of many LAB (Schillinger, 2016). Bacteriocins

of LAB are ribosomal synthesized antimicrobial peptides that inhibit closely related bacteria by

destabilizing the function of the cytoplasmic membrane. The bacteriocin-producing strain resists

its own bacteriocin by producing a highly specific immunity factor (Quadri et al., 2015). They fall

into three classes, based on their primary structure, molecular mass and heat stability: (Class I)

lantibiotics, which are small, heat-stable, containing lanthionine, e.g., nisin; (Class II) non-

lantibiotics, which are small and heat stable, e.g., pediocin PA-l, leucocin B-Ta11 a, and (Class

III) large and heat labile, e.g., helveticin (Nes et al., 2016). Bacteriocins of LAB have received

considerable attention due to their potential application as natural preservatives. They may provide

a valuable, additional and controllable tool for the inhibition of some deleterious wine-associated

organisms.

54
Nisin

Nisin is the only LAB bacteriocin with GRAS (Generally Regarded as Safe) status and is approved

for usage in 47 countries (Schillinger, 2016). The possible use of bacteriocins, especially nisin, in

winemaking has been, and the results obtained in these studies indicate that most LAB of

importance in winemaking were inhibited by low concentrations of nisin. Wine yeasts were not

affected, nor was the sensorial quality of the wine. However, O. oeni, responsible for MLF, was

affected and nisin would not be applicable in wines having to undergo MLF (Schillinger, 2016).

addressed this problem by developing nisin-resistant mutants of O. oeni, and these strains together

with nisin produced a controlled, pure culture MLF. Although bacteriocins provide the

winemaking community with a safe alternative to chemical preservation.

Plant Extracts

One plant extract that has been proposed as an antimicrobial agent in wine is paprika seed. The

active ingredient(s) remains unknown but was named “paprika seed antimicrobial substance,”

(Yokotsuka et al., 2016). These authors were able to demonstrate that extracts of paprika seeds

exhibited strong antimicrobial activity against S. cerevisiae, even when added at only 16 mg/L.

PSAS appears to be very effective (100% kill) when added during the course of alcoholic

fermentation

Lysozyme

Lysozyme is a low molecular weight protein (14,500 Da) derived from egg white that brings about

lysis of the cell wall of Gram-positive bacteria (Oenococcus, Lactobacillus, and Pediococcus).

Activity toward Gram-negative bacteria (Acetobacter and Gluconobacter) is limited because of

the protective outer layers in this group (Conner, 2016). The enzyme has no effect on yeasts or
55
molds. Given its specificity, lysozyme finds applications among white, rose, and blush wine

producers wanting to prevent malolactic fermentation as well as wineries wanting to reduce initial

populations of lactic acid bacteria before fermentation (Nygaard et al., 2017). Because lysozyme

is a protein, the presence of phenolics as well as the degree of clarification will affect activity.

Plant phenolics are well-known to react with enzymes, thereby decreasing activity. Reflecting this,

lysozyme is more active in white wines than reds, most likely due to the differences in

polyphenolic content (Daeschel et al., 2016).

56
CONCLUSION

wine production is a microbiological process and microorganisms play key important role in the

production processes, from harvesting of grapes down to Aging and bottling. However, these

microorganisms can have both positive and negative effects on wine. Therefore, there is need to

observe good manufacturing practices during the production processes in order to prevent harmful

microorganism from acting on the wine. Also, there are various preservation methods and

preservatives used during wine production to increase the shelf life of the wine and to prevent

microbial spoilage. However, Considerable progress has been made over the last decade in

understanding the roles that yeasts, bacteria, molds and viruses play in the winemaking process.

Crucial questions remain: What can or should be done to control them? What species and strains

grow at the various stages of the process? What substances are produced? These and many others

need further attention. The development of molecular biology techniques, such as DNA probes,

will be a helpful tool in identifying species and strains and following the progress of populations

during the winemaking process. These probes can also be used to assess the risks of spoilage when

certain species are present during vinification. Due to the vast biodiversity present on the grape

berries and the natural populations occurring in grape juice, the selection of novel strains that do

not possess undesirable characteristics should be noted.

57
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