Okafor N. Modern Industrial Microbiology and Biotechnology

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270 Modern Industrial Microbiology and Biotechnology

Vermouth may be of Italian (sweet) or French (dry) type. Vermouth comes from the

German ‘wermut’ (wormwood, Artemesia absinthium a common herb) a frequent

component of vermouth. The other flavored fortified wines, such as ‘Campari’,

‘Dubonnet’, ‘Byrrh’, like vermouth, contain 15-21% alcohol. In both cases the aromatic

components of the herbs are extracted by immersing the herbs in wine or alcohol, or by

distilling them. The nature and proportion of many of the components of flavored

fortified wines are kept secret.

13.1.8.3 Fruit wines: cider and perry

Often fruits do not contain enough sugar to make a potent alcoholic beverage. Under such

conditions, extra sugar in the form of sucrose is added to encourage fermentation. Fruit

wines are popular in some countries where grapes cannot thrive.

Cider is derived from apples, (Malus pumila) and perry from pears or a mixture of pears

and apples. They differ from other fruit wines in that their alcohol content is low (4-5%

with a maximum of 7-8% v/v) because sugar is not usually added. The basic processes

are similar to those of grape wine: pressing out the juice, fermenting, maturing, and

bottling. Fruit wines have been made from cashew, pineapples, and other fruits.

13.2 PALM WINE

Palm wine is a general name for alcoholic beverages produced from the saps of palm

trees. It differs from the grape wines in that it is opaque. It is drunk all over the tropical

world in Africa, Asia, South America. Table 13.3 shows the palms from which palm wine

is derived in the various areas.

Palm wine is usually a whitish and effervescent liquid both of which properties derive

from the fact that the fermenting organisms are numerous and alive when the beverage is

consumed. More information appears to exist on wine derived from the oil palm, Elaeis

guinieensis than on any other and this will be discussed.

The sap of the palm is obtained from a variety of positions: the stem of the standing

tree, the tip or trunk of the felled tree and the base of the immature male inflorescence.

Which method is favored depends on the country concerned but most studies have

centered on inflorescence wine. The sap produced by this method in Elaeis guiniensis

contains about 12% sucrose, about 1% each of fructose, glucose, and raffinose, and small

quantities of protein and some vitamins and is a clear, sweet, syrupy liquid.

To produce palm wine a succession of microorganisms occurs roughly: Gram-

negative bacteria, lactic acid bacteria and yeasts and finally acetic acid bacteria. Yeasts in

palm wine have been identified as coming from various genera (Table 13.4).

The organisms are not artificially inoculated and find their way into the wine from a

variety of sources including the air, the tapping utensils including previous brews and

the tree. The wine contains about 3% (v/v) alcohol and since the bacteria and yeasts are

consumed live, it is a source of (single cell) protein and various vitamins.

The great problem with palm wine is that its shelf life is extremely short. It is best

consumed within about 48 hours, but certainly not beyond about five days after tapping.

For this reason various methods have been devised to preserve it. Pasteurization has met

with some success, but methods which lower the microbial load of the wine by

Production of Wines and Spirits %

Table 13.3 Palms from which palm wine is obtained

Name of palm Location

Acromia vinifera Oerst Nicargua, Panama, Costa Rica

Arenga pinnata (Wurmb.) Merr.

(Syn. A. sacoharifera Labill.) Far East

Attalea speciosa Mart. Brazil, Guyana

Borassus aethipum Mart. Tropical Africa

Broassus flabelifer Linn. India, Cambodia, Java

Caryota urens Linn. India

Cocos nucifera Linn. India, Sri Lanka, Africa

Corypha umbraculifera L. Sri Lanka

Elaesis guineensis Jacq. Africa

Hyospathe elegans Mart. Brazil, Guyana

Hyphaenae guineensis Thonn. West Africa

Jubaea chilensis (Molina) Baillon Chile

Mauritiella aculeate (H.B. and K.)

Burret (Syn. Lepidococcus aculeatus H.

Wendl and Drude) Brazil, Venezuela

Marenia montana (Hump. and Bonpl.) Brazil

Burret (Syn. Kunthia Montana

Humb. and Bonpl.)

Nypa fruticans Wurmb. Sir Lanka, Bay of Bengal, The Phillippines,

Carolines, Salmonon Islands

Orbignya cohune (Mart.) Dahlgreen ex Hondruras, Mexico, Gautemala

Standley (Sun. Attalea cohune Mart.)

Phoenix dactylifera Linn. North Africa, Middle East

Phoenix reclinata Jacq. (Syn. Phoenix)

Spinosa Schum. and Thonn.) Central Africa

Phoenix sylvestris (L) Roxb. India

Raphia hookeri Mann and Wendl. Africa

Raphia sundanica A. Chev. Africa

Raphia vinifera Beauv. Africa

Scheelea princeps (Marts.) Karsten Brazil, Bolivia

(Syn. Attalea princeps Mart.)

centrifugation or filtration permit the use of milder pasteurization temperatures and

lower quantities of chemicals (Table 13.5). Of the chemicals tried, sorbate and sulphite

were found best. Fully fermented palm wine has 5% to 8% alcohol and is distilled for kai-

kai, a gin with a distinct fruity flavor.

Other African alcoholic beverages

(i) Bouza is an alcoholic beverage produced in Egypt since the time of the Pharoahs.

Formerly drunk by all classes, it is now drunk mainly among the lower income groups.

Egyptian Bouza is prepared either from wheat or maize but the most popular is from

wheat. To prepare bouza, coarsely ground wheat (about 20% of total) is placed in a large

wooden basin and kneaded with water to form a dough which is cut into thick loaves and

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lightly baked. The rest of the wheat (about 80% of the total) is germinated for three to five

days, sun dried and coarsely ground. The malted wheat and the crumbled bread are

mixed in water in a wooden barrel. Bouza from a previous fermentation is added and the

whole mixture is fermented for 24 hours at room temperature. The mash is sieved to

remove large particles and the beverage is ready for drinking. The beverage has a pH of

Table 13.4 Yeasts identified in palm wines

Yeast Type of Wine

Saccharomyces pastorianus Oil palm

Saccharomyces ellipsoids Oil palm,

Saccharomyces cereviside Arenga palm

Saccharomyces cerevisiae Oil palm,

Saccharomyces chevalieri Oil palm,

Pichia sp. Oil palm,

Schizosaccharomyces pombe Oil palm,

Saccharomyces vafer Oil palm,

Palmyra palm

Endomycopsis sp. Oil palm,

Saccharomyces markii Oil palm,

Kloekera apculata Oil palm,

Saccharomyces chevalieri Palmyra palm

Saccharomyces rosei Raphia

Candida spp. Oil palm

Saccharomycoides luduigii Oil palm

Palmyra palm

Table 13.5 Counts of Bacteria on whole palm wine and on supernatant of centrifuged palm

wine after various treatments

Treatment Number Percent Survival

Whole palm wine

Untreated ´ 10

100.00

0.5% potassium sorbate 2.96 ´ 10

1.0571

0.10% 2.32 ´ 10

0.8286

0.15% 2.24 ´ 10

0.8000

0.15% 1.40 ´ 10

0.5000

0.15% sodium metaisulfie 1.20 ´ 10

0.4285

0.10% 4.80 ´ 10

0.00017

0.15%

Supernatant after centrifuging

Untreated 4.00 ´ 10

0.00001

Pasteurized at 60

C for ½ hour 2.00 ´ 10

0.0000007

Pasteurization plus 0.05%

sorbate 2.00 ´ 10

0.00000007

Pasteurization plus 0.05%

sodium metabisulfite 0.4 ´ 10

Virtually sterile

Production of Wines and Spirits %!

about 3.6 and an alcohol content of about 4%: but the pH drops while alcohol increases

with further fermentation.

(ii) Talla (tella) is an Ethiopian small-producer beer with a smokey flavor derived from

inverting the fermentation containers and talla collection pots over smouldering olive

wood. Talla also acquires some smoke flavor from the toasted, milled and boiled cereal

grains. During the toasting the grains are roasted until they begin to smoke slightly. In the

production of talla, of which various types exist, powdered hop leaves and water are put

in a fermentation vessel and allowed to stand for about three days. Ground barley or

wheat malt and pieces of flat bread baked to burning on the outside. are added. On the

fifth day hop stems are added in addition to cereal flour made by milling sorghum which

is first boiled and then toasted. Water is added and the fermentation allowed for two days

(i.e., to the seventh day). It is filtered and is ready to drink.

(iii) Busaa is an acidic alcoholic beverage drunk among the Luo. Abuluhya and Maragoli

ethnic groups of Kenya. It is porridge-like and light-brown in color and is warmed to 35-

40°C before being consumed. A stiff dough made from maize flour and water is incubated

at room temperature for three to four days. The fermented dough is pounded and then

heated on a metal plate till it turns brown.

Malt is made from millet, allowed to germinate for three to four days, sun dried, and

ground to powder. A slurry is made in water of the millet malt powder and roasted maize

flour dough and left to ferment for two to four days. It is filtered through cloth. The filtrate

is busaa.

The organisms responsible for fermenting the uncooked maize dough include Candida

krusei. Saccharomyces cerevisiae. Lactobacillus helveticus, L. salivarus. L. brevis. L viridescens. L.

plantarurn and Pediococcus damnosus. The final product is the result of alcohol produced

mainly by S. cerevisiae: the lactic acid in the beverage is produced by several lactobacilli:

(iv) Merissa is a sour Sudanese alcoholic (up to 6%) beverage made from sorghum. It has

a pH of about four and a lactic acid content of about 2.5%. Sorghum grains are malted,

dried, and ground into a coarse powder. Unmalted sorghum is milled into a fine powder.

One third of this powder is mixed with a little water and allowed to ferment at room

temperature for 24-36 hours. The resulting fermented sour dough is heated without

further water addition until the product caramelizes to give rise to soorji. The cooled soorji

is allowed to ferment for four in five hours in a mixture of malt, to which previous merissa

is added as inoculum. The two are mixed together and portions of these are allowed to

cool resulting in a product called ‘futtura. Futtura is mixed from with malt flour (about

5%) and added to the bedoba from time to time. Fermentation lasts from 8 to 10 hours after

which it is filtered to give rise to the drink, merissa.

(v) Tej is a mead (i.e., a wine made by fermenting honey) of Ethiopian origin. It is yellow,

sweet, effervescent, and cloudy due to its yeast content. As it is expensive, it is beyond the

reach of most Ethiopians and used only on special occasions. The wine may be flavored

with spices or hops and also by passing smoke into the fermentation pot before it is used.

To prepare Tej the honey is diluted with water by between 1:2 to 1:5 i.e., to a liquid of

between 13 and 27% sugar since honey contains about 80% sugar. Yeasts of the genus

Saccharomyces spontaneously ferment the brew in about five days to give a yellow wine of

7-14% alcohol (v/v).

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(vi) Agadagidi wines are made from bananas and plantains and have the opaque,

effervescent sweet-sour nature typical of African traditional alcoholic beverages. In

Nigeria the best-known agadagidi is found in the cocoa-growing areas of south western

Nigeria where plantains provide shade for the young cocoa trees. The ripe fruits are

peeled and soaked in water where the sugars dissolving from the preparation permit the

development of yeasts and lactic acid and giving rise to a typical opaque effervescent

wine. The alcohol content is about l%.

(vii) Mbege is consumed in Tanzania mainly by those living near Mount Kilimanjaro. It is

not a wholly banana/plantain wine as is the case with Nigerian agadagidi. Rather it is

produced from a mixture of malted millet and fermented banana juice. The juice is

produced by boiling the ripe banana followed by decantation. The banana infusion is

mixed with cooked and cooled millet malt and allowed to ferment for four to five days:

13.3 THE DISTILLED ALCOHOLIC (OR SPIRIT) BEVERAGES

The distilled alcoholic or spirit beverages are those potable products whose alcohol

contents are increased by distillation. In the process of distillation volatile materials

emanating directly from the fermented substrate or after microbial (especially yeast)

metabolism introduce materials which have a great influence on the nature of beverage.

The character of the beverage is also influenced by such post-distillation processes as

ageing, blending, etc. The components of spirit beverages which confer specific aromas

on them are known as congeners.

13.3.1 Measurement of the Alcoholic Strength of

Distilled Beverages

(i) Proof: In English-speaking countries, such as the United States, Canada, the United

Kingdom, and Australia, the alcoholic content of spirit beverages (and also of non-

potable alcohol) is given by the term ‘proof’. The reason for the term is historical. Before

the advent of the use of the hydrometer in alcoholic measurements, an estimate of the

alcoholic content of a spirit beverage was obtained by mixing it with gunpowder. If the

gunpowder still ignited then it was satisfactory because it contained less than 50% (v/v)

of alcohol. If it did not ignite it was ‘under proof’ because it contained less than 50%

water. Proof has nowadays become more clearly defined, although slight differences

occur among countries. In the United States proof spirit (i.e., 100 degrees proof, written

100°) shall be held to be that alcoholic liquor which contains one half its volume of

alcohol of a specific gravity of 0.7939 at 15.6°C. In other words the proof is always exactly

twice the alcoholic content by volume. Thus, 100 proof spirit contains 50% alcohol. In the

British system proof spirit contains 57.1% by volume and 49.28% by weight of alcohol at

15.6°C.

A conversion factor of 1.142

Volume of alcohol of British oof at C

Volume of alcohol of United States oof at C

Pr .

15 6

57 1

114

is applied to convert United States proof to a British proof.

Production of Wines and Spirits %#

It is customary to quantify large amounts of distilled alcoholic beverages or alcohol in

proof gallons for tax and other purposes. This term simply specifies the amount of alcohol

in a gallon of spirits. Thus a United States proof gallon contains 50% ethyl alcohol by

volume; a gallon of liquor at 120° proof is 1.2 proof gallon and a gallon at 75° proof is 0.75

proof gallon. However, the ordinary United States gallon is 3.785 liters whereas the

British (imperial) gallon is larger, 4.546 liters. The British proof gallon is multiplied by

1.37 to convert it to a United States gallon:

Volume of British gallon

Volume of United States gallon Conversion factor´

4 546

3 785

´ 1.142 = 1.375

The proof is read off a special hydrometer.

(ii) Percentage by weight: This is used in Germany for the ethanol content of a beverage or

other liquid. The hydrometers are graduated at 15°C and the reference is with

(Mendeleaf’s) table of density. This results in a scale independent of the ambient

temperature.

(iii) Percentage by volume: Many countries especially in Europe use the percentage by

volume system. For most of them the hydrometer is calibrated at 15°C. France, Belgium,

Spain, Sweden, Norway, Finland, Switzerland (which also used weight %), Brazil and

Egypt, Russia use 20°C, Denmark and Italy 15.6°C whereas many South American

countries use 12.5°C. In many of these countries the specific gravity of alcohol used as

reference differ slightly.

13.3.2 General Principles in the Production of

Spirit Beverages

In general the following steps are involved in the preparation of the above beverages. The

details differ according to beverage.

(i) Preparation of the medium: In the grain beverages (whisky, vodka, gin) the grain

starch is hydrolyzed to sugars with microbial enzymes or with the enzymes of

barley malt. In all the others no hydrolysis is necessary as sugars are present in the

fermenting substrate as in brandy (grape sugar) and rum (cane sugar).

(ii) Propagation of yeast inoculum: Large distilleries produce hundreds of liters of

spirits daily for which fermentation broths many more times in volume are

required. These broths are inoculated with up to 5% (v/v) of thick yeast broth.

Although yeast is re-used there is still a need for regular inocula. In general the

inocula are made of selected alcohol-tolerant yeast strains usually Sacch. cerevisiae

grown aerobically with agitation and in a molasses base. Progressively larger

volumes of culture may be developed before the desired volume is attained.

(iii) Fermentation: When the nitrogen content of the medium is insufficient nitrogen is

added usually in the form of an ammonium salt. As in all alcohol fermentations the

heat released must be reduced by cooling and temperatures are generally not

permitted to exceed 35-37°C. The pH is usually in the range 4.5-4.7, when the

buffering capacity of the medium is high. Higher pH values tend to lead to higher

glycerol formation. When the buffering capacity is lower, the initial pH is 5.5 but

this usually falls to about 3.5. During the fermentation contaminations can have

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serious effects on the process: sugars are used up leading to reduced yields;

metabolic products from the contaminants may not only alter the flavor of the

finished product, but metabolites such as acids affect the function of the yeast. The

most important contaminants in distilling industries are lactic acid which affects

the flavor of the product.

(iv) Distillation: Distillation is the separation of more volatile materials from less

volatile ones by a process of vaporization and condensation. Three systems used

in spirit distillation will be discussed.

(a) Rectifying Stills: If the condensate is repeatedly distilled, the successive

distillates will contain components which are more and more volatile. The

process of repeated distillation is known as rectification. Rectification is done

in columns, towers, or stills containing a series of plates at which contact

occurs i.e., returned to the system. The alcohol-water mixture flows

downwards and is stripped of alcohol by steam which is introduced from the

bottom and flows upwards. Alcohol-rich distillate is withdrawn at the top of

the column. Fusel oils higher alcohols separate out just above the point of

entry of the mixture and are drawn off to another column. Volatile fractions

are composed of esters and aldehydes. Whisky and brandy may be distilled

successfully in a two-column still, but for high-strength distillates, at least

three columns and possibly four or five may be required. The above

description is of the modern still a modification of which is also used for

producing industrial alcohol. Much older-type stills, such as are described

below, continue to be used in some parts of the world.

(b) Pot Still: These are traditional stills, usually made of copper. They are

spherical at the lower-portion which is connected to a cooling coil. They are

operated batchwise. The first portion, or ‘heads’ and the latter portion, ‘tails’,

of the ‘low wines’ are usually discarded and only the middle portion is

collected. Malt whisky, rum, and brandy are made in the post still. Its

advantage is that most of the lesser aroma conferring compounds are

collected in the beverage thereby conferring a rich aroma to it.

modifications since then have not added much to the original design of the

still. Its main feature is that it has a rectifying column besides the wash

column in which the beer is first distilled.

(v) Maturation: Some of the distilled alcoholic beverages are aged for some years, often

prescribed by legislation.

(vi) Blending: Before packaging, samples of various batches of different types of a given

beverage are blended together to develop a particular aroma.

13.3.3 The Spirit Beverages

The beverages to be discussed are whisky, brandy, rum, vodka, kai-kai (or akpeteshi),

schnapps, and cordials.

Production of Wines and Spirits %%

13.3.3.1 Whisky

Whisky is the alcoholic beverage derived from the distillation of fermented cereal.

Various types of whiskies are produced; they differ principally in the cereal used.

Although many countries including Japan and Australia now produce whisky for

export, the countries best associated with whisky are first and foremost, Scotland

followed by Ireland, the USA and Canada.

In all whisky-producing countries the alcoholic content, the materials and the method

of preparation are controlled by government regulations. Whiskies from various

countries differ. In Scottish Malt Whisky the barley is malted just as in beer making, but

during the kilning smoke from peat is allowed to permeate the green (fresh) malt, that the

whisky made from the malt has a strong aroma of peat smoke, derived mainly from

phenol. In the United States the principal types of whisky are rye and bourbon whiskies.

Rye whisky is prepared from rye and rye malt, or rye and barley and barley malt. Bourbon

whisky is prepared from preferably yellow maize, barley malt or wheat malt. A typical

mash which will contain 51% corn, may have a composition of this type: 70% corn, 15%

rye, and 15% barley malt.

The unmalted rye or corn is cooked to gelatinize it and hence to facilitate

saccharification (or conversion to sugar) by the enzymes in the malt. The solids are not

removed from the mash and the inoculating yeast sometimes contains Lactobacillus,

whose lactic acid is said to improve the flavor of the whisky. Fermentation is usually in a

two-column coffey-type still.

All whisky is matured in wooden casks for a number of years, usually three or more.

They may then be blended with various types (usually controlled by law) before bottling.

13.3.3.2 Brandy

Brandy is a distillate of fermented fruit juice. Thus, brandy can be produced from any

fruit-strawberries, paw-paw, or cashew. However, when it is unqualified, the word

brandy refers to the distillate from fermented grape juice. It is subject to a distillation

limitation of 170° proof (85%). The fermented liquor is double distilled, without previous

storage, in pot stills. A minimum of two years maturation in oak casks is required for

maturation.

Some of the best brandies come from the cognac region of France. Brandies produced in

other parts of France are merely eau de vie (water of life) and are not called brandy. Certain

parts of Europe (e.g., Spain, where brandy is distilled from Jerez sherry) and South

America as well as the USA produce special brandies.

13.3.3.3 Rum

Rum is produced from cane or sugar by products especially molasses or cane juice. Rum

production is associated with the Carribean especially Jamaica, Cuba, and Puerto Rico. It

is also produced in the eastern USA. Rum with a heavy body is produced from molasses;

while light rum is produced from cane syrup using continuous distillation.

During the fermentation the molasses is clarified to remove colloidal material which

could block the still by the addition of sulphuric acid. The pH is adjusted to about 5.5 and

a nitrogen source ammonium sulphate or urea may be added. For the heavier rums

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Schizosaccharomyces pombe is used while Saccharomyces cerevisiae is used for the lighter

types. For maturation rum is stored in oak casks for two to fifteen years.

13.3.3.4 Gin, Vodka, and Schnapps

These beverages differ from whisky, rum and brandy in the following ways:

(a) Brandy, rum and whisky are pale-yellow colored straw to deep brown by

extractives from wooden casks in which they are aged and which have sometimes

been used to store molasses or sherry. To obtain consistent color caramel is

sometimes added. Gin, vodka, and schnapps are water clear.

(b) The flavor of brandy and whisky is due to congenerics present in the fermented

mash or must. For gin, vodka, and schnapps the congenerics derived from

fermentation are removed and flavoring is provided (except in vodka) with plant

parts.

may be used. For gin, maize is used, while for vodka rye is used. The cereals are

gelatinized by cooking and mashed with malted barley. In recent times amylases

produced by fungi or bacilli have been used since the flavor of malt is not necessary

in the beverage. Congeners are removed by continuous distillation in multi-

column stills.

In gin production, the grain-spirits (i.e., without the congeners) are distilled over

juniper berries, Juniperus communis, dried angelica roots, Angelica officinalis and others

including citrus peels, cinnamon, nutmeg, etc. Russian vodka is produced from rye spirit,

which is passed over specially activated wood charcoal. In other countries it is

sometimes produced from potatoes or molasses. Schnapps are gin flavored with herbs.

13.3.3.5 Cordials (Liqueurs)

Cordials are the American name for what are known as liqueurs in Europe. They are

obtained by soaking herbs and other plants in grain spirits, brandy, or gin or by distilling

these beverages over the plant parts mentioned above. The are usually very sweet, being

required to contain 10% sugar. Some well-known brand names of cordials are Drambui,

Crème de menthe, Triple Sac, Benedictine, and Anisete.

13.3.3.6 Kai-kai, Akpeteshi, or Ogogoro

Kai-kai is an alcoholic beverage widely drunk in West Africa. It is produced by distilling

fermented palm-wine. It is the base for preparing some of the better known brands such as

schnapps.