Okafor N. Modern Industrial Microbiology and Biotechnology

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& Modern Industrial Microbiology and Biotechnology

Vinegar is a product resulting from the conversion of alcohol to acetic acid by acetic acid

bacteria, Acetobacter spp. The name is derived from French (Vin = wine; Aigre-sour or

sharp). With the ubiquity of acetic acid bacteria and the consequent ease with which

wine is spoilt, vinegar must have been known to man for thousands of years since he

apparently learnt to produce alcoholic beverages some 10,000 years ago. The Bible has

many references to vinegar both in the Old and New Testaments, the best known of

which, probably is: “It is consummated” which according to John (19, 29-30), was uttered

by Christ before He bowed his head and died, after he had been offered vinegar while he

was crucified on the cross. Vinegar may be regarded as wine spoilt by acetic acid bacteria,

but for which other uses have been found.

Although acetic acid is the major component of vinegar, the material cannot be

produced simply by dissolving acetic acid in water. When alcoholic fermentation occurs

and later during acidifications many other compounds are produced, depending mostly

on the nature of the material fermented and some of these find their way into vinegar.

Furthermore, reactions also occur between these fermentation products. Ethyl acetate, for

example, is formed from the reaction between acetic acid and ethanol. It is these other

compounds which give the various vinegars their bouquets or organoleptic properties.

The other compounds include non-volatile organic acids such as malic, citric, succinic

and lactic acids; unfermented and unfermentable sugars; oxidized alcohol and

acetaldelyde, acetoin, phosphate, chloride, and other ions.

14.1 USES

(i) Ancient uses: The ancient uses of vinegar which can be seen from various records

include a wide variety of uses including use as a food condiment, treatment of

wounds, and a wide variety of illnesses such as plague, ringworms, burns,

lameness, variocose veins. It was also used as a general cleansing agent. Finally, it

was used as a cosmetic aid.

(ii) Modern uses: Vinegar is used today mainly in the food industry as; (a) a food

condiment, sprinkled on certain foods such as fish at the table; (b) for pickling and

preserving meats and vegetables; vinegar is particularly useful in this respect as it

can reduce the pH of food below that which even sporeformers may not survive; (c)

It is an important component of sauces especially renowned French sauces many

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of which contain vinegar; (d) Nearly 70% of the vinegar produced today is

supplied to various arms of the food industry where it finds use in the manufacture

of sauces, salad dressings, mayonnaise, tomato productions, cheese dressings,

mustard, and soft drinks. Most of the vinegar used in industry is the distilled or

concentrated type (see below).

14.2 MEASUREMENT OF ACETIC ACID IN VINEGAR

Just as the alcoholic content of distilled alcoholic beverages is measured in proof, the

acetic acid content is usually measured in ‘grain’. Originally the strength of acetic acid

was expressed in terms of the grains of sodium bicarbonate neutralized by one fluid

ounce measure of vinegar and this was measured by the CO

evolved during the reaction

of the two substances. The ‘grain strength’ is now measured differently and one-grain

vinegar is now defined as that containing 0.1 gm of acetic acid in 100 ml at 20°C. Grain is

derived by multiplying the acetic acid content (w/v) of a sample of vinegar by 10 or by

moving the decimal point one place to the right. Thus, vinegar containing 8% acetic acid

is 80 grain. Sometimes the percentage (w/v) is used.

14.3 TYPES OF VINEGAR

Vinegar is normally a product of two fermentations: alcoholic fermentation with a yeast

and the production of acetic acid from the alcohol by acetic acid bacteria (Chapter 2).

There is no distillation between the two fermentations, except in the production of spirit

vinegar, which is described below. The vinegar may or may not be flavored. The substrate

for the alcoholic fermentation for vinegar productions varies from one locality to the

other. Thus, while wine vinegar made from grapes is common in continental Europe and

other vine growing countries, malt vinegar is common in the United Kingdom; the United

States on account of its great variety of climatic regions uses both malt and wine vinegars.

Rice vinegar is common in the far Eastern countries of Japan and China and pineapple

vinegar is used in Malaysia. In some tropical countries vinegar has been manufactured

from palm wine derived from oil or raffia palm.

The composition and specifications of various types of vinegars are defined by

regulations set up by the governments of different countries . In the United States, for

example, vinegar should not contain less than 4.0% (w/v) acetic acid and not more than

0.5% ethanol (v/v). The various major vinegars are defined briefly

(i) Cider vinegar, apple vinegar: Vinegar produced from fermented apple justice (US)

and non-grape fruits.

(ii) Wine vinegar, grape vinegar: Fermented grape juice malt.

(iii) Malt vinegar: Produced from a fermented infusion of barley malt with or without

adjuncts.

(iv) Sugar, glucose, dried fruits: In the US vinegar from sugar syrup or molasses should

be labeled sugar vinegar, while that from glucose (which should be dextrose-

rotatory) and dried fruits should be labeled with ‘glucose’ or the particular fried

fruit involved.

(v) Spirit vinegar: Vinegar made from distilled alcohol. In the US synonyms for spirit

vinegar are ‘white distilled vinegar’ and ‘grain vinegar’. The alcohol used in the

& Modern Industrial Microbiology and Biotechnology

distillation is denatured for tax reasons with ethyl acetate. One gallon of ethyl

acetate is usually added to 100 gal of 95% alcohol. The ethyl acetate is not

deleterious and in any case is present in vinegar by the alcohol acetic acid reaction.

It should be noted that in the Unites States the term ‘distilled’ refers to the ethanol

used; in the United Kingdom, however, ‘distilled’ vinegar refers to a distillate of

malt vinegar.

(vi) Some specialty vinegars: Specialty vinegars make up a category of vinegar

products that are formulated or flavored to provide a special or unusual taste when

added to foods.

Specialty vinegars are favorites in the gourmet market:

(a) Herbal vinegars: Wine or white distilled vinegars are sometimes flavored with the

addition of herbs, spices or other seasonings. Popular flavorings are garlic, basil,

and tarragon, but cinnamon, clove, and nutmeg flavored vinegars can be tasty and

aromatic addition to dressings.

(b) Fruit vinegars: Fruit or fruit juice can also be infused with wine or white vinegar.

Raspberry flavored vinegars, for example, create a sweetened vinegar with a sweet-

sour taste.

white and sugary Trebbiano grapes grown on the hills around Modena. The

grapes are harvested as late as possible to take advantage of the warmth of the

weather. The traditional vinegar is made from the cooked grape ‘must’ (juice)

matured by a long and slow process of natural fermentation, followed by

progressive concentration by aging in a series of casks made from different types of

wood and without the addition of any other spices or flavorings. The color is dark

brown and the fragrance is distinct. Production of traditional balsamic vinegar is

governed by the stringent standards imposed by the quasi-governmental

Consortium of Producers of the Traditional Balsamic Vinegar of Modena.

(d) Raspberry red wine vinegar: Natural raspberry flavor is added to red wine vinegar,

which is the aged and filtered product obtained from the acetous fermentation of

select red wine. Raspberry red wine vinegar has a characteristic dark red color and

a piquant, yet delicate raspberry flavor.

(e) Other specialty vinegars: Coconut and cane vinegars are common in India, the

Phillipines and Indonesia with date vinegar being popular in the Middle East.

International definitions and standards are set by the joint efforts of the Food and

Agriculture (FAO) as well as the World Health Organization (WHO) of the United

Nations. Apart from these, various professional bodies such as the Vinegar Institute (a

manufacturing association) also set standards.

14.4 ORGANISMS INVOLVED

Although vinegar had been known to man from time immemorial, like many other

fermentative processes, the identity of the organism concerned is recent. Even then much

more needs to be known about them, mainly because of the difficulty of cultivating them.

The bacteria converting alcohol to acetic acid under natural conditions are film-

forming organisms on the surface of wine and beer. The film was known as ‘mother of

Production of Vinegar &!

vinegar’ before its bacteriological nature became known. The bacteria were first described

as Mycoderma (viscous film) in 1822. Later other workers classified them in M. vini

(forming film on wine) an M. acetic (forming film on beer). Pasteur confirmed that acetic

acid is produced only in the presence of the bacteria, but he did not identify them. The

genus name Acetobacter was put forward by Beijerinck in 1900. Suffice it to state that

although Acetobacter spp are responsible for vinegar production, pure cultures are hardly

used, except in submerged fermentation because of the difficulty of isolating and

maintaining the organisms. The only member of the genus which is not useful, if not

positively harmful in vinegar production is Acetobacter xylinum which tends to produce

slime (Chapter 2). Recently a new species, Acetobacter europaeus, was described. Its

distinguishing features are its strong tolerance of acetic acid of 4 to 8% in A–E agar, and

its absolute requirement of acetic acid for growth.

Strains of acetic acid bacteria to be used in industrial production should a) tolerate

high concentrations of acetic acid; b) require small amounts of nutrient; c) not over-

oxidize the acetic acid formed; and d) be high yielding in terms of the acetic acid

produced.

The biochemical processes are simple and are shown below:

OH + (O) CH

CHO + H

Ethyl alcohol oxygen Acetaldedyde Water

CHO + H

O CH

CH + (OH)

Hydrated acetaldehyde

(Aldehyde)

CH(OH)

+ (O) CH

COOH + H

Dehydrogenase Acetic acid

Theoretically, 1 gm of alcohol should yield 1.304 gm of acetic acid but this is hardly

achieved and only in unusual cases is a yield of 1.1 attained. From the reactions one mole

of ethanol will yield one more of acetic acid and more of water. It can be calculated that

1 gallon of 12% alcohol will yield 1 gal. of 12.4% acetic acid.

Over-oxidation can occur and it is undesirable. In over-oxidation acetic acid is

converted to CO

and H

O. It occurs when there is a lack or low level of alcohol. It occurs

more frequently in submerged fermentations that in the trickle processes.

14.5 MANUFACTURE OF VINEGAR

The three methods used for the production of vinegar are the Orleans Method (also

known as the slow method), the Trickling (or quick) Method and Submerged

Fermentation. The last two are the most widely used in modern times.

14.5.1 The Orleans (or Slow) Method

The oldest method of vinegar production is the ‘let alone’ method in which wine left in

open vats became converted to vinegar by acetic acid bacteria entering it from the

atmosphere. Later the wine was put in casks and left in the open field in the ‘fielding

process’. A small amount of vinegar was introduced into a cask of wine to help initiate

fermentation. The introduced vinegar not only lowered the pH to the disadvantage of

many other organisms but also introduced an inoculum of acetic acid bacteria.

&" Modern Industrial Microbiology and Biotechnology

The casks were wooden and of approximately 200 liter capacity. It was never filled

beyond about two-thirds of its capacity so that there was always a large amount of air

available above the wine. A thick film of acetic acid bacteria formed on the wine and

converted it in to vinegar in about five weeks. About 10-20% of the vinegar was drawn off

at weekly intervals and replaced with new wine. The withdrawal and replenishment

were done from the bottom of the cask so that the film would not be disturbed. Often a

series of casks was present and the transfer was done from one cask to another.

Often due to its thickness and consequent weight and sometimes due to disturbance,

the film sank. When this happened the whole process had to be restarted, since acetic

acid bacteria are aerobic. Following Pasteur’s (1868) suggestion, the film of bacteria often

developed in wooden rafts is placed in the cask for this purpose. Later on the casks were

stored, especially in the Orleans district of France, in a heated building or in an

underground cellar to speed up the process. The process now derives its name from the

district. The process had a number of disadvantages:

(a) It was slow in comparison with later methods, taking up to five weeks sometimes

as against days, hence it is also known as the slow method.

(b) It was inefficient, yielding 75-85% of the theoretical amount.

process.

Despite these disadvantages the product was of good quality and it continued to be

used in many European countries long after the introduction of the Quick Process,

described below. Modern vinegar production uses mainly the Trickling (quick) and

submerged methods to be described below. There are fewer and fewer of the Orleans

equipment in use today.

14.5.2 The Trickling Generators (Quick) Method

Credit for devising the fore-runner of the modern trickling generator is usually given to

the Dutch Boerhaave who in 1732 devised the first trickling generator in which he used

branches of vines, and grape stems as packing. Improvements were made by a number of

other people from time to time. Later ventilation holes were drilled at the bottom of the

generator and provided a mechanical means for the repeated distribution of the alcohol

acetic acid mixture over the packing. The heat generated by the exothermic reaction in the

generator caused a draft which provided oxygen for the aerobic conversion of alcohol to

acetic acid. This latter model of the quick method (sometimes called the German method)

enabled the production of vinegar in days instead of in weeks. It remained in vogue

unmodified for just over a century when several modifications were introduced in the

Frings method, including: (a) forced aeration, (b) temperature control, and (c) semi-

continuous operation.

The modern vinegar generator consists of a tank constructed usually of wood

preferably of cypress and occasionally of stainless steel. A false bottom supports the coils

of birchwood shavings and separates them from the collection chamber which occupies

about one fifth of the total capacity of the generator (Fig. 14.1). A pump circulates the

alcohol-acetic acid mixture from the reservoir through a heat exchanger to the top of the

generator where a spray mechanism distributes it over the packing in much the same way

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as a trickling filter functions in waste-water treatment (Chapter 29). Air is forced through

the false bottom up through the set-up. The cooling water in the heat exchanger is used to

regulate the temperature in the generator so that it is between 29°C and 35°C; this is

determined with thermometers placed at different levels of the generator.

The top of the generator is covered but provision exists for exhaust air to be let out.

Meters measure three parameters: (a) the circulation of the mash, (b) the flow of cooling

water through the heat exchange, and (c) the amount of air delivered through the system.

If the air flow rate is too high alcohol and vinegar are lost in effluent air.

Operation of the generator: The trickling or circulating Frings generator is reasonably

efficient, achieving, when operating maximally, an efficiency of 91-92% and it is capable

of producing 500–1000 gallons of 100-grain (i.e. 10%) vinegar every 24 hours. Although

the wood shavings soften with age, well-maintained generators can proceed without

much attention for twenty to thirty years. They are easy to maintain once airflow and

recirculation rates as well as temperatures are maintained at the required level. The level

of ethyl alcohol must be maintained so that it does not fall below 0.3-0.5% at any time.

Complete exhaustion of the alcohol will lead to the death of the bacteria.

When wine and cider vinegar are made no nutrients need be added to the charge (i.e.,

the alcohol-containing material). However, when white vinegar (produced from

synthetic alcohol is used) nutrients e.g. simple low concentration sugar-mineral salts

solution sometimes containing a little yeast extraction may be added. Growth of the

slime-forming Acetobacter xylinum is less with white vinegar (from pure alcohol) than

with wine and cider vinegar. Generators for producing white vinegar therefore become

blocked by slime much less quickly than those used for wine and cider vinegar, and can

last far in excess of 20 to 30 years before the wood shavings are changed.

Fig. 14.1 Trickling Generator for Vinegar Manufacture

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The finished acidity of the vinegar is about 12%; when it is higher, production drops

off. In order not to exceed this level of acidity, when drawing off vinegar, the amount of

alcohol in the replacement should be such that the total amount of alcohol is less than 5%.

14.5.3 Submerged Generators

With knowledge in submerged fermentation gained from the antibiotics and yeast

industry it is not surprising that vinegar production was soon produced by the method.

Several submerged growth vinegar generators have been described or are in operation.

The common feature in all submerged vinegar production is that the aeration must be

very vigorous as shortage of oxygen because of the highly acid conditions of submerged

production, would result in the death of the bacteria within 30 seconds. Furthermore,

because a lot of heat is released (over 30,000 calories are released per gallon of ethanol) an

efficient cooling system must be provided. All submerged vinegar is turbid because of the

high bacterial content and have to be filtered. Some submerged generators will be

discussed below.

14.5.3.1 Frings acetator

First publicized in 1949, most of the world’s vinegar is now produced with this

fermentor. It consists of a stainless steel tank fitted with internal cooling coils and a high-

speed agitator fitted through the bottom. Air is sucked in through an air-meter located at

the top. It is then finely dispersed by the agitator and distributed throughout the liquid.

Temperature is maintained at 30°C, although some strains can grow at a higher

temperature. Foaming is interrupted with an automatic foam breaker. Essentially it is

shaped like the typical aerated stirred tank fermentor described in Chapter 9).

It is operated batchwise and the cycle time for producing 12% vinegar is about

35 hours. Details of the parts of the Frings acetator are shown in Fig. 14.2. It is self-

aspirating, no compressed air being needed. The hollow rotor is installed on the shaft of

a motor mounted under the fermentor, connected to an air suction pipe and surrounded

by a stator. It pumps liquid that enters the rotor from above outward through the channels

of the stator that are formed by the wedges, thereby sucking air through the openings of

the rotor and creating an air–liquid emulsion that is ejected outward at a given speed.

This speed must be chosen adequately so that the turbulence of the stream causes a

uniform distribution of the air over the whole cross section of the fermentor.

The Frings alkograph automatically monitors the alcohol content and signals the end

of the batch when the alcohol content falls to 0.2% (v/v). At this stage about one third of

the product is pumped out and fresh feed pumped to the original level. The aeration must

continue throughout the period of the unloading and loading. The Frings Alkograph is

an automatic instrument for measuring the amount of ethanol in the fermenting liquid.

Small amounts of liquid flow through the analyzer continuously, at first through a

heating vessel and then through three boiling vessels. The boiling temperature of the

incoming liquid is measured in the first boiling vessel. While alcohol is distilled off

continuously from the second and the third boiling vessel, the higher boiling point of the

liquid from which ethanol has been removed is measured in the third boiling vessel. The

difference in temperature corresponds to the ethanol content and is recorded

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automatically. As the flow through the vessels takes some time, there is a delay of about

15 minutes between the beginning of the inflow of a sample and the appearance of the

correct value on the recorder. However, because alcohol concentration is decreasing

slowly during fermentation, this delay has no disadvantage on fermentation control. In

more modern alkographs there is no time gap.

The more recent Frings acetators can be run on a semi-continuous basis. To carry out

the single-stage semi-continuous process at a defined alcohol content, a contact in the

Alkograph activates the vinegar discharge pump. As soon as a preset level has been

reached, the mash pump starts adding fresh mash. This pump is controlled by the

fermentation temperature in order to refill under constant temperature. The pump is

stopped when the desired level is reached and an automatic cooling system is activated.

A fermentation cycle takes 24 to 48 hours Since its first description, improvements and

modifications have been made on the Frings acetator. One in recent operation is shown in

Fig. 14.2 .

Advantages

(a) The efficiency of the acetator is much higher than that of the trickling generator; the

production rate of the acetator may be 10-fold higher than a trickling unit. Values of

94% and 85% of the theoretical have been recorded for both the acetator and the

trickling filter.

(b) The quality is more uniform and the inexplicable variability in quality noted for the

trickling generator is absent.

trickling generator.

a = hollow rotor; b = stator;

c = air suction pipe; d = openings for air exit;

e = wedges to form the channels;

f = channels to form the beams of air–liquid emulsion

Fig. 14.2 The Frings Acetator

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(d) It is easy and cheap to change from one type of vinegar to another.

(e) Continuous production and automation can take place more easily with Frings

acetator than with trickling.

Disadvantages

(a) A risk exists of complete stoppage following death of bacteria from power failure

even for a short time. Automatic stand-by generators have helped to solve this

problem.

(b) It has a high rate of power consumption. Some authors have however argued that

in fact in terms of power consumed per gallon of acetic produced the acetator is less

power consuming.

14.5.3.2 The cavitators

The cavitator was originally designed to treat sewage: it was then modified for vinegar

production. In many ways it resembled the acetator. However, the agitator was fixed to

the top and finely dispersed air bubbles are introduced into the liquid. It operated on a

continuous basis and was quite successful in producing cider and other vinegars as long

as the grain strength was low. It was not successful with high grain vinegar and the

manufacture of the ‘cavitator’ was discontinued in 1969. It is mentioned here only for its

historical interest, although some are still being used in Japan and the US.

14.5.3.3 The tower fermentor

The tubular (tower) fermentor developed in the UK has been used on a commercial scale

for the production of beer, vinegar, and citric acid. The fermentor is two feet in diameter,

about 20 feet tall in the tubular section with an expansion chamber of about four feet in

diameter and six feet high. It has a working volume of 3,000 liters and aeration is

achieved by a stainless steel perforated plate covering the cross section of the tower and

holding up the liquid. The charging wort is fed at the bottom. The vinegar overflows in a

quiet Y-shaped area free of rising gas. The unit can produce up to 1 million gallons

(450,000 liters) of 5% acetic acid per annum. The Acetobacter sp requires a month to adapt

to the new system. The system can be batch, semi or fully-continuous without noticeable

differences in the quality of the product.

14.6 PROCESSING OF VINEGAR

(a) Clarification and bottling: Irrespective of the method of manufacture, vinegar for

retailing is clarified by careful filtration using a filter aid such as diatomaceous

earth. Vinegar from trickling generators are however less turbid than those from

submerged fermentations because a high proportion of the bacterial population

responsible for the acetification is held back on the shavings. After clarification it is

pasteurized at 60-65°C for 30 minutes.

(b) Concentration of vinegar: Vinegar can be concentrated by freezing; thereafter the

resulting slurry is centrifuged to separate the ice and produce the concentrate.

With this method 200° grain (i.e., 20% w/v) acetic acid can be produced.

Concentration is necessitated by two considerations. One is the consequent

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reduction in transportation costs. The other is the need to prevent loss of activity of

the vinegar when cucumbers were picked in it after first being soaked in brine.