Okafor N. Modern Industrial Microbiology and Biotechnology

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$ Modern Industrial Microbiology and Biotechnology

SUGGESTED READINGS

Amerine, M.A., Berg, H.W., Cruess, W.V. 1972. The Technology of Wine Making 3

Ed. Avi

Publications. West Port, USA, pp. 357-644.

Aniche, G.N. 1982. Brewing of Lager Beer from Sorghum. Ph.D Thesis, University of Nigeria,

Nsukka, Nigeria.

Ault, R.G., Newton, R. 1971. In: Modern Brewing Technology, W.P.K. Findlay, (ed) Macmillan,

London, UK. pp. 164-197.

Battcock, M., Azam-Ali, S. 2001. Fermented Fruits and Vegetables: A Global Perspective. FAO

Agric. Services Bull. 134, Rome, Italy, pp. 96.

Doyle, M.P., Beuchat, L.R., Montville, T.J. 2004. Food Microbiology: Fundamentals and Frontiers,

2nd edit., ASM Press, Washington DC, USA.

Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,

Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York, USA.

Gastineau, C.F., Darby, W.J., Turner, T.B. 1979. Fermented Food Beverages in Nutrition.

Academic Press; New York, USA, pp. 133-186.

Gutcho, M.H. 1976. Alcoholic Beverages Processes. Noyes Data Corporation New Jersey and

London. pp. 10-106.

Hammond, J.R.M., Bamforth, C.W. 1993. Progress in the development of new barley, hop, and

yeast variants for malting and brewing. Biotechnology and Genetic Engineering Reviews 11,

147-169.

Hanum, H., Blumberg, S. 1976. Brandies and Liqeuers of the World. Doubleday Inc. New York

USA.

Hough, J.S. 1985. The Biotechnology of Malting and Brewing. Cambridge University Press;

Cambridge, UK. pp. 15-188.

Hough, J.S., Briggs, D.E., Stevens, R. 1971. Malting and Brewing Science, Chapman and Hall,

London, UK.

Hoyrup, H.E. 1978. Beer Kirk-Othmer’s Encyclopaedia of Chemical Technology, Wiley, New

York, USA. Microbiology 24: 173-200.

Okafor, N. 1972. Palm-wine yeasts of Nigeria. Journal of the Science of Food and Agriculture 23,

1399-1407.

Okafor, N. 1978. Microbiology and biochemistry of oil palm wine. Advances in Applied.

Okafor, N. 1987. Industrial Microbiology. University of Ife Press Ile-Ife: Nigeria. pp. 201-210.

Okafor, N. 1990. Traditional alcoholic beverages of tropical Africa: strategies for scale-up. Process

Biochemistry Intemational 25, 213-220.

Table 12.6 Properties of South African sorghum beer

Properties Small scale Factory

pH 3.5 3.4

Alcohol 0.1 3.0

Solids (%w/v)

Total 4.9 5.4

Insoluble 2.3 3.7

Nitrogen (%w/v)

Total 0.084 0.093

Soluble - 0.014

Production of Beer $

Okafor, N., Aniche, G.N. 1980. Lager beer from Nigerian Sorghum. Brewing and Distilling

International. 10, 32-33, 35.

Packowski, G.W. 1978. Distilled Alcoholic Beverages, Encyclopadeia of Chemical Technology, 3

Edit. Wiley, New York, USA. pp. 824-863.

Singleton, V.L., Butzke, C.E. 1998 Wine. Kirk-Othmer Encyclopedia of Chemical Technology.

John Wiley & Sons, Inc. Article Online Posting Date: December 4, 2000.

Soares, C. 2002. Process Engineering Equipment Handbook Publisher. McGraw-Hill.

Taylor, J.R.N., Dewar, J. 2001. Developments in Sorghum Food Technologies. Advances in Food

and Nutrition Research 43, 217-264.

Vogel, H.C., Tadaro, C.L. 1997. Fermentation and Biochemical Engineering Handbook -

Principles, Process Design, and Equipment. 2nd Edit Noyes.

Zeng, A. 1999. Continuous culture. In: Manual of Industrial Microbiology and Biotechnology.

A.L. Demain, J.E. Davies (eds) 2

Edit. ASM Press. Washington, DC, USA. pp. 151-164.

$ Modern Industrial Microbiology and Biotechnology

13.1 GRAPE WINES

Wine is by common usage defined as a product of the “normal alcoholic fermentation of

the juice of sound ripe grapes”. Nevertheless any fruit with a good proportion of sugar

may be used for wine production. Thus, citrus, bananas, apples, pineapples,

strawberries etc., may all be used to produce wine. Such wines are always qualified as

fruit wines. If the term is not qualified then it is regarded as being derived from grapes,

Vitis vinifera. The production of wine is simpler than that of beer in that no need exists for

malting since sugars are already present in the fruit juice being used. This however

exposes wine making to greater contamination hazards.

Wine is today principally produced in countries or regions with mild winters, cool

summers, and an even distribution of rainfall throughout the year. In North America, the

United States is the leading producer, most of the wine coming from the State of California

and some from New York. In Europe the principal producers are Italy, Spain and France.

In South America, Argentina, Chile, and Brazil are the major producers; and in Africa,

they are Algeria, Morocco, and South Africa. Other producers are Turkey, Syria, Iran, and

Australia.

13.1.1 Processes in Wine Making

13.1.1.1 Crushing of Grapes

Selected ripe grapes of 21° to 23° Balling (Chapter 12) are crushed to release the juice

which is known as ‘must’, after the stalks which support the fruits have been removed.

These stalks contain tannins which would give the wine a harsh taste if left in the must.

The skin contains most of the materials which give wine its aroma and color. For the

production of red wines the skins of black grapes are included, to impart the color.

Grapes for sweet wines must have a sugar content of 24 to 28 Balling so that a residual

sugar content is maintained after fermentation. The chief sugars in grapes are glocuse

and fructose; in ripe fruits they occur in about the same proportion. Grape juice has an

acidity of 0.60-0.65% and a pH of 3.0-4.0 due mainly to malic and tartaric acids with a

little citric acid. During ripening both the levulose content and the tartaric acid contents

rise.

Production of

Wines and Spirits

+0)26-4

Production of Wines and Spirits 263

Nitrogen is present in the form of amino acids, peptides, purines, small amounts of

ammonium compounds and nitrates.

13.1.2 Fermentation

(i) Yeast used: The grapes themselves harbor a natural flora of microorganisms (the

bloom) which in previous times brought about the fermentation and contributed to the

special characters of various wines. Nowadays the must is partially ‘sterilized’ by the

use of sulphur dioxide, a bisulphate or a metabisulphite which eliminates most micro-

organisms in the must leaving wine yeasts. Yeasts are then inoculated into the must. The

yeast which is used is Saccaromyces cerevisiae var, ellipsoideus (synonyms: Sacch. cerevisiae,

Sacch. ellipsoideus, Sacch, vini.) Other yeasts which have been used for special wines are

Sacch. fermentati, Sacch. oyiformis and Sacch. bayanus.

Wine yeasts have the following characteristics: (a) growth at the relatively high acidity

(i.e., low pH) of grape juice; (b) resistance to high alcohol content (higher than 10%); (c)

resistance to sulfite.

(ii) Control of fermentation

(a) Temperature: Heat is released during the fermentations. It has been calculated

that on the basis of 24 Cal per 180 gm of sugar the temperature of a must containing

22% sugar would rise 52°F (11°C) if all the heat were stopped from escaping. If the

initial temperature were 60°F (16°C) the temperature would be 100°F (38°C) and

fermentation would halt while only 5% alcohol has been accumulated. For this

reason the fermentation is cooled and the temperature is maintained at around

24°C with cooling coils mounted in the fermentor.

(b) Yeast Nutrition: Yeasts normally ferment the glucose preferentially although some

yeasts e.g. Sacch. elegans prefer fructose. To produce sweet wine glucose-fermenting

wine yeasts are used leaving the fructose which is much sweeter than glucose.

Most nutrients including macro- and micro-nutrients are usually abundant in

must; occasionally, however, nitrogeneous compounds are limiting. They are then

made adequate with small amounts of (NH

)

SO4 or (NH

)

HPO4.

yeast multiplication is occurring. In the second stage when alcohol is produced the

growth is anaerobic and this forces the yeasts to utilize such intermediate products

as acetaldehydes as hydrogen acceptors and hence encourage alcohol production.

(iii) Flavor development: Although some flavor materials come from the grape most of it

come from yeast action. The flavor of wine has been elucidated with gas chromatography

and has been shown to be due to alcohols, esters, fatty acids, and carbonyl compounds,

the esters being the most important. Diacetyl, acetonin, fusel oils, volatile esters, and

hydrogen sulfide have received special attention. Autolysates from yeasts also have a

special influence on flavor.

13.1.3 Ageing and Storage

The fermentation is usually over in three to five days. At this time ‘pomace’ formed from

grape skins (in red wines) will have risen to the top of the brew. As has been indicated

$" Modern Industrial Microbiology and Biotechnology

earlier for white wine, the skin is not allowed in the fermentation. At the end of this

fermentation the wine is allowed to flow through a perforated bottom if pomace had been

allowed. When the pomace has been separated from wine and the fermentation is

complete or stopped, the next stage is ‘racking’. The wine is allowed to stand until a major

portion of the yeast cells and other fine suspended materials have collected at the bottom

of the container as sediment or ‘lees’. It is then ‘racked’, during which process the clear

wine is carefully pumped or siphoned off without disturbing the lees.

The wine is then transferred to wooden casks (100-1,000 gallons), barrels (about

50 gallons) or tanks (several thousand gallons). The wood allows the wine only slow

access to oxygen. Water and ethanol evaporate slowly leading to air pockets which

permit the growth of aerobic wine spoilers e.g. acetic acid bacteria and some yeasts. The

casks are therefore regularly topped up to prevent the pockets. In modern tanks made of

stainless steel the problem of air pockets is tackled by filling the airspace with an inert gas

such as carbon dioxide or nitrogen.

During ageing desirable changes occur in the wine. These changes are due to a

number of factors:

(a) Slow oxidation, since oxygen can only diffuse slowly through the wood. Small

amounts of oxygen also enter during the filling up. Alcohols react with acids to

form esters; tannins are oxidized.

(b) Wood extractives also affect ageing by affecting the flavor.

malic acid is first converted to pyruvic acid and then to lactic acid. (Fig. 13.1)

Fig. 13.1 Chemical Reactions Involved in Flavor Development in Grape Wines

The reaction is responsible for the rich flavor developed during the ageing of some

wines e.g. Bordeaux. Cultures which have been implicated in this fermentation are

Lactobacillus sp and Leuconostoc sp. A temperature of 11-16°C is best for ageing wines,

High temperature probably functions by accelerating oxidation.

13.1.4 Clarification

The wine is allowed to age in a period ranging from two years to five years, depending on

the type of wine. At the end of the period some will have cleared naturally. For others

Production of Wines and Spirits 265

artificial clarification may be necessary. The addition of a fining agent is often practiced

to help clarification. Fining agents react with the tannin, acid, protein or with some

added substance to give heavy quick-settling coagulums. In the process of setting various

suspended materials are adsorbed. The usual fining agents for wine are gelatin, casein,

tannin, isinglass, egg albumin, and bentonite. In some countries the removal of metal ions

is accomplished with potassium ferrocyanide known as ‘blue fining’; it removes excess

ions of copper, iron, manganese, and zinc from wines.

13.1.5 Packaging

Before packing in bottles the wine from various sources is sometimes blended and then

pasteurized. In some wineries, the wine is not pasteurized, rather it is sterilized by

filtration. In many countries the wine is packaged and distributed in casks.

13.1.6 Wine Defects

The most important cause of wine spoilage is microbial; less important defects are acidity

and cloudiness. Factors which influence spoilage by bacteria and yeasts include the

following (a) wine composition, specifically the sugar, alcohol, and sulfur dioxide

content; (b) storage conditions e.g. high temperature and the amount of air space in the

container; (c) the extent of the initial contamination by microorganism during the bottling

process.

When proper hygiene is practiced bacterial spoilage is rare. When it does occur the

microorganisms concerned are acetic acid bacteria which cause sourness in the wine.

Lactic acid bacteria especially Leuconostoc, and sometimes Lactobacillus also spoil wines.

Various spoilage yeasts may also grow in wine. The most prevalent is Brettanomyces, slow

growing yeasts which grow in wine causing turbidities and off-flavors. Other wine

spoilage yeasts are Saccharomyces oviformis, which may use up residual sugars in a sweet

wine and Saccharomyces bayanus which may cause turbidity and sedimentation in dry

wines with some residual sugar. Pichia membanaefaciens is an aerobic yeast which grows

especially in young wines with sufficient oxygen.

Other defects of wine include cloudiness and acidity.

13.1.7 Wine Preservation

Wine is preserved either by chemicals or by some physical means. The chemicals which

have been used include bisulphites, diethyl pyrocarbonate and sorbic acid. Physical

means include pasteurization and sterile filtration. Pasteurization is avoided when

possible because of its deleterious effect on wine flavor.

13.1.8 Classification of Wines

Grape wines may be classified in several ways. Some of the criteria include place of

origin, color, alcohol content and sweetness. The one being adopted here is primarily

used in the United States and is shown in Table 13.1. This system classifies wine into two

groups: natural wines and fortified wines.

266 Modern Industrial Microbiology and Biotechnology

13.1.8.1 The natural wines

These result from complete natural fermentation. Further fermentation is prevented

because the sugar is to a large extent exhausted. Spoilage organisms such as acetic acid

bacteria do not grow if air is excluded. Owing to the natural limit of sugar in grapes, the

alcohol content does not usually exceed 12%. They are sub-divided into still (without

added CO

) and sparkling (with added CO

). Color and sweetness also subdivide the

wines. The color, pink or red, is derived from the color of the grape; white wine comes

from a grade whose skin is light-green, but whose juice is clear. White wine can also

result from black (or deep-red) grapes if the skin is removed immediately after pressing

before fermentation. Red wine results when the skin of black grapes are allowed in the

fermenting must. Pink wine results when the skin is left just long enough for some

material to extract some skin coloring.

Table wines: In general the natural wines are usually consumed at one sitting once they

are opened. For this reason they are called ‘table’ wines and intended to be part of a meal.

Table 13.1 Broad classification of grape wines

A. Natural wines: 9-14% alcohol; nature and keeping quality mostly dependent on

‘complete’ yeast fermentation and protection from air

1. Still wines (known as ‘Table’ wines intended as part of meal); no carbon

dioxide added.

(a) Dry table wines: (no noticeable sweetness)

(i) White; (ii) Rose (pink); (iii) Red

(b) Sweet table wines

(i) White (ii) Rose

Further naming of above depends on grape type, or region of origin.

2. Sparkling wines (appreciable CO

under pressure)

(a) White (Champagne); (b) Rose (Pink Champagne); (c) Red (Sparkling

burgundy; cold duck)

B. Fortified (Dessert and appetizer) wines: Contain 15 to 21% alcohol; nature and

keeping quality depends heavily on addition of alcohol distilled from grape wine.

1. Sweet wines

(a) White (Muscatel, White port, angelica)

(b) Pink (California tokay, tawny port)

2. Sherries: (White sweet or dry wines with oxidized flavors)

(a) Aged types

(b) Flor types

3. Flavored specialty wines (usually white Port base)

(a) Vermouth (pale dry, French; Italian sweet types)

(b) Proprietary brands

Production of Wines and Spirits $%

They are usually served in generous amounts, partly because they contain less alcohol

than the desserts and appetizers and partly because they do not have a high keeping

quality once opened compared with appetizers and dessert wines.

Dessert and appetizer wines: The second broad group of wines are dessert or appetizer

wines. As can be seen from their names they are served at the beginning (appetizers) or at

the end (dessert) of meals. They contain extra alcohol from distilled wines, partly to make

them more potent, but also to preserve them from yeast spoilage. These are divided into

three categories: (a) Sweet e.g. port; (b) Sherries – sweet or dry, they originated from

Portugal and are characterized by flavors induced by various degrees of oxidation; (c)

Flavored wines e.g. vermouth; these are flavored with herbs and other components which

are secrets of the producing firms (Table 13.1).

Sparkling wines (especially champagne), sherry, and flavored wines will be

discussed briefly.

Sparkling Wines: Sparkling wines contain CO

under pressure before they are opened.

They are called sparkling because the gentle release of carbon dioxide from the wine after

the bottle is opened gives the wine a sparkle. The best known of the sparkling wines is

produced in Champagne region in northern France which has given its name to the wine.

Champagne is produced either in a bottle or in bulk.

(a) Bottle Champagne: Champagne is usually a clear sparkling wine made from white

wine. Pink or red champagne made from wines of the same color are preferred by

some. Champagne is produced by a second fermentation in the bottle of an already

good wine. Not only does its production take a long time, it also requires a

complicated method which is difficult to automate and hence must be handled

manually. For these reasons the drink is expensive. The usual process is described

below. The parlance of champagne manufacture is understandably French. The

method Champenoise to be described is the one used for making the best sparkling

wines. After the must is fully fermented, the wines to be used for champagne are

racked, clarified, stabilized and fined. A blend is then made from several different

wines to give the desired aroma. The blend is known as cuvèe. The cuvèe should

have an alcohol content of 9.5 to 11.5%, adequate acidity (0.7 to 0.9% titrable as

tartaric acid) and light straw or light yellow color. The SO

content should be low

otherwise SO

odor would show when the wine is poured, or worse still, the yeasts

might convert SO

to forms of hydrogen sulphide, which would give a rotten egg

odor.

The cuvee is placed into thick walled bottles able to withstand the high pressure

of CO

to be built up later in the bottle. For the secondary fermentation in the bottle,

more yeast, more fermentable sugar, usually sucrose and nowadays sometimes a

small amount (0.05 to 0.1%) of ammonium phosphate is added. The yeast usually

used is Saccharomyces bayanus. It is selected because it meets the following

requirements encountered in secondary fermentation for champagne production:

it must grow at a fairly high alcohol content (10-12%), at high pressures (see below)

and relatively low temperatures; the yeast must die or become inactive within a

short time in order to prevent further fermentation after sugar (known as the dosage)

is added once again before the final corking. Finally the yeast must be able to form

a compact granulated sediment after fermentation.

$& Modern Industrial Microbiology and Biotechnology

The amount of sugar added depends on the expected CO

pressure of the

sparkling wine. Sparkling wines have a pressure of about six atmospheres but not

less than four. As a rough rule, 4 gm of sugar per liter will produce one atmosphere

of CO

pressure. Therefore the sugar added is about 24 gm/liter. Account is taken

of any sugar present in the wine. Although the bottles are thick, if the fermentation

is too rapid, temperature too high or sugar too high the bottle may burst.

Champagne bottles are therefore not reused since a scratched bottle may burst.

The bottle with its mixture of wine, sugar and yeast is placed horizontally and

allowed to ferment at a temperature of about 15-16°C; this secondary fermentation

takes two to three months. The secondarily fermented wine now known as triage is

then stored still horizontally at a temperature of 10°C and remains undisturbed in

that position for at least a year and sometimes up to five years. Much of the aroma

of well-aged champagne appears to come from the reactions involving materials

released from yeast autolysis.

The next stage is getting the sediment from the side to the neck of the bottle.

Several methods are used by individual handlers. This transfer or remuage is

achieved by first placing the bottle neck downwards at an angle in a rack with

varying degrees of jolting. The bottles are next rotated clockwise and anti-

clockwise on alternate days during which the bottle is gradually straightened to

the perpendicular. The process may take anything from two or six weeks at the end

of which the sediment finds it way to the neck of the bottle. To remove the sediment

of yeast cells, the neck of the bottle is frozen to 1°C to 15°C; an ice plug which

includes the sediment forms therein. The bottle is turned to an angle of about 45°

and opened. The pressure in the bottle forces out the ice plug. During this process

of disgorging some CO

is lost but sufficient is left to give the usual pop which

launches a bride, a ship or a graduating student into the future! The lost wine is

replaced from another bottle and the dosage is added. The dosage is a syrup

consisting of about 60 gm of sugar in 100 ml of well-aged wine. All champagnes,

even those labeled dry contain dosage; otherwise it would taste sour. Sweet

champagne contains up to 10% sugar.

(b) Bulk production of Champagne: Champagne is sometimes produced in bulk in a

large tank rather than in individual bottles. Bulk production is known as the

Charmat process named after its inventor. When this is so produced it is usually

declared on the label to save the more labor-intensive bottle-made versions from

unfair competition. The tank which has a lining of a inert material such as glass

holds 500 to 25,000 gallons and is built to withstand 10-20 atmospheres as a safety

measure. Valves control the pressure and cooling coils the temperature. Since the

tanks are aerated a rapid turnover is possible and 6-12 fermentations per year are

made. Another reason for the rapid turnover is that a heavy yeast growth occurs

which could lead to the production of off flavors especially H

S if allowed

prolonged contact with the wine.

Tank fermented champagne is usually given a cold-stabilizing treatment to

remove excess tartarates. It is filtered still cold and under pressure to remove yeast

cells. The wine is then filled into bottles with dosage of the right kind. It is usual to

introduce some sulfur dioxide into it just in case some yeasts were not removed by

filtration. Sulfur dioxide also helps to prevent darkening oxidation which may

Production of Wines and Spirits 269

occur as the wine takes up oxygen during the transfer process. The sulphur

dioxide odor is usually noticeable when Charmat-prepared champagne bottles are

opened. Furthermore, they lack the aroma conferred on well-aged bottle brands by

the autolysis of yeast. Bulk champagnes are amenable to bulk production because

they are more difficult to ferment owing to their higher tannin contents; they may

also require higher fermentation temperatures.

13.1.8.2 Fortified wines

The fortified wines can be divided into three main groups, which derive their names from

the warmer more southern portion of the Iberian (Spain-Portugal) peninsula in Europe,

and an island off that peninsula, where they were originally produced: sherry (Jerez de la

Frontera area in Spain); port (Douro Valley in Portugal); Madeira (the Island of Madeira).

In other wines contact with oxygen no matter how small is undesirable. The fortified

wines are however produced by the deliberate but controlled oxidation of wine. The

oxidation is achieved by prolonged ageing in the pressure of air, by the growth of an

aerobic yeast or by heating. The consequence of this oxidation is a product which has a

dark, reddish-brown color with a characteristic flavor, whether the starting wine is white

or red. For sherry therefore a white wine is used but for port or Madeira a red or a white

wine may be used.

All the three fortified wines have a high alcohol content of ranging from 15-20% (v/v),

derived from added alcohol hence their name. They are usually separated into two

groups: (a) Vermouth (b) Other flavor wines (Special natural wines).

Table 13.2 Flow Sheet for the Production of Sherry, Port, and Madeira

Sherry Port Madeira

1. Type of wine used White White/red White/red

2. Grape sun dried to increase sugar

content of juice + + - -

3. Skin of grapes left in contact with

must during initial wine _ _ + -

4. fermentation + + - -

5. SO

added to juice + + - +

6. Juice fermented to dry wine

Freshly fermented wine fortified to 15% 15% 17% 18%

7. alcohol content 15% given (% v/v)

8. Matured in contact with air for + — — -

flor film _ _ - +

9. Maturation with heating

Adjusted to given alcohol content

10. About six months after fermentation 15% 18% 20% 18%

(% v/v) + + + +

11. Matured in wood for several months Fino, Oloros Port Madeira

Fortified wine Amantillado

Key: + = Yes, — = No.