Fermentation Process of Vinegar | Microbiology

In this article we will discuss about the fermentation process of vinegar, explained with the help of suitable diagrams.

Vinegar is the product of a two-stage fermentation. In the first stage, yeast convert sugars into ethanol anaerobically, while in the second ethanol is oxidized to acetic (ethanoic) acid aerobically by bacteria of the genera Acetobacter and Gluconobacter.

This second process is a common mechanism of spoilage in alcoholic beverages and the discovery of vinegar was doubtless due to the observation that this product of spoilage could be put to some good use as a flavouring and preservative.

The name vinegar is in fact derived from the French vin aigre for ‘sour wine’ and even today the most popular types of vinegar in a region usually reflect the local alcoholic beverage; for example, malt vinegar in the UK, wine vinegar in France, and rice vinegar in Japan.

In vinegar brewing, the alcoholic substrate, known as vinegar stock, is produced using the same or very similar processes to those used in alcoholic beverage production. Where differences occur they stem largely from the vinegar brewer’s relative disinterest in the flavour of the intermediate and his concern to maximize conversion of sugar into ethanol.

In the production of malt vinegar for example, hops are not used and the wort is not boiled so the activity of starch-degrading enzymes continues into the fermentation. Here we will concentrate on describing the second stage in the process, acetification.

Acetification, the oxidation of ethanol to acetic acid is performed by members of the genera Acetobacter and Gluconobacter. These are Gram-negative, catalase-positive, oxidase-negative, strictly aerobic bacteria.

Acetobacter spp. are the better acid producers and are more common in commercial vinegar production, but their ability to oxidize acetic acid to carbon dioxide and water, a property which distinguishes them from Gluconobacter, can cause problems in some circumstances when the vinegar brewer will see his key component disappearing into the air as CO₂.

Fortunately over-oxidation, as it is known, is repressed by ethanol and can be controlled by careful monitoring to ensure that ethanol is not completely exhausted during acetification.

Most acetifications are run on a semi-continuous basis; when acetification is nearly complete and acetic acid levels are typically around 10-14% w/v, a proportion of the fermenter’s contents is removed and replaced with an equal volume of fresh alcoholic vinegar stock.

Since a substantial amount of finished vinegar is retained in the fermenter, this conserves the culture and means that a relatively high level of acidity is maintained throughout the fermentation, protecting against contamination.

It also protects against over-oxidation as it has been found that Acetobacter europaeus, a species commonly found in commercial vinegar fermenters, will not over-oxidize when the acetic acid concentration is more than 6%.

Many of the acetic acid bacteria associated with commercial acetification are difficult to culture on conventional solidified media, although some success has been enjoyed using a double-layer medium which provides colonies growing on the surface with a constant supply of ethanol and moisture from a lower, semi-solid layer.

As a result, vinegar fermentations are usually initiated with seed or mother vinegar, an undefined culture obtained from previous fermentations. Depending on the type of acetification, the culture can be quite heterogeneous and A. europaeus, A. hansenii, A. acidophilum, A. polyoxogenes, and A. pasteurianus have all been isolated from high-acidity fermentations.

Oxidation of ethanol to acetic acid is the relatively simple pathway by which acetic acid bacteria derive their energy. It occurs in two steps mediated by an alcohol dehydrogenase and an aldehyde dehydrogenase (Figure 9.14). Both enzymes are associated with the cytoplasmic membrane and have pyrroloquinoline quinone (PQQ) as a coenzyme.

PQQ acts as a hydrogen acceptor which then reduces a cytochrome. The consequent electron transport establishes a proton motive force across the membrane which can be used to synthesize ATP.

Overall, acetification can be represented chemically by:

From the stoichiometry of the equation it can be calculated that 1 1 of ethanol should yield 1.036 kg of acetic acid and 0.313 kg of water. This leads to the approximate relationship that 1 % v/v ethanol will give 1 % w/v acetic acid, and this is used to predict the eventual acidity of a vinegar and to calculate fermentation efficiency.

It implies that, in the absence of over-oxidation, evaporative losses and conversion to biomass, the sum of the concentration of ethanol (%v/v) and the concentration of acetic acid (%w/v), known as the total concentration or GK (German: Gesammte Konzentration) should remain constant throughout acetifica­tion. The GK yield is the GK of the final vinegar expressed as a percentage of the GK at the start of acetification.

There are a number of techniques for acetification which differ in the means by which the three interacting components, ethanol, bacteria and oxygen, are brought together. Surface culture techniques, where the bacteria form a surface film at the interface between the acetifying medium and air, are the simplest but can be applied with varying levels of sophistication.

In the Orleans process, vinegar stock in partially filled casks drilled with air holes (Figure 9.15) is left to acetify until the acidity reaches the appropriate level determined by the initial GK value. At this point a proportion, typically one-third to two-thirds, is drawn off through the tap, replaced with fresh stock and the process restarted.

The vinegar stock is usually added via a pipe passing through the top of the barrel and resting on the bottom. In this way the surface film of bacteria is not disturbed and the delays and losses that result from having to reform the film are avoided. Usually the time taken to complete one acetification cycle is of the order of 14 days.

Only a small proportion of the world’s vinegar is produced by surface culture today, although it is claimed to produce the finest quality vinegar. More elaborate surface culture techniques based on series of trays have been described but these have received only very limited application.

The quick vinegar process derives its name from the faster rates of acetification achieved by increasing the area of active bacterial film and improving oxygen transfer to the acetifying stock. The acetic acid bacteria grow as a surface film on an inert support material packed into a false-bottomed vat.

The acetifying stock is sprayed on to the surface of the packing material and trickles down against a counter-current of air which is either pumped through the bed or drawn up by the heat of reaction within it.

The packing material normally consists of some lignocellulosic material such as birch twigs, vine twigs, rattan, wood wool, or sugarcane bagasse, although other materials such as coke have also been used. The vinegar stock is collected in a sump at the bottom of the vat and re-circulated until the desired level of acidity is reached.

The faster rate of reaction achieved means that the wash heats up during passage through the bed and, depending on the size of the fermenter, some cooling may be required.

The process is operated semi-continuously to maintain a high level of acidity throughout, and most of the biomass is retained within the packed bed. A well operated quick vinegar process fitted with temperature control and forced aeration can usually acetify a vinegar stock with a GK of 10 and an initial ethanol content of 3% in 4-5 days.

The fastest rates of acetification are achieved using submerged acetification in which acetic acid bacteria grow suspended in a medium which is oxygenated by sparging with air. The most commercially successful technique to have been developed is the Frings Acetator (Figure 9.16) which uses a patented self-priming aerator to achieve very efficient oxygen transfer.

Submerged culture is very efficient and rapid, a semi-continuous run normally takes 24-48 h. It does however require far more careful control than simpler processes. The acetic acid bacteria are very susceptible to interruptions to the air supply, indicating that, in order to survive suspended in a medium with a pH of 2.5 and 10-14% acidity, the bacteria need a constant supply of energy from respiration.

A stoppage of only one minute in a stock with a GK of 11.35 is enough to completely arrest acetification which will not resume when aeration is resumed.

Another possible cause of fermentation failure in submerged acetification is phage infection. The presence of bacteriophage particles has been demonstrated in disturbed vinegar fermentations both in submerged acetifiers and the quick vinegar process.

The performance of quick vinegar generators appears to be less affected as their acetification rate may slow but rarely stops. This is probably due to the greater heterogeneity of the culture present which allows organisms of different phage susceptibility to take over in the event of phage attack.

Where legal definitions of vinegar exist, it is specified as a fermentation product. ‘Artificial vinegars’ made by diluting and colouring acetic acid are thus excluded and, in the UK, have to be known rather laboriously as ‘non-brewed condiment’.

Although vinegar can be made up to 14% acidity, it is usually diluted down to an appropriate strength for bottling. The minimum acetic acid content is usually prescribed to be something between 4 and 6% w/v, but higher strength vinegars are available for pickling.

Though most often thought of in terms of its use as a condiment, vinegar is an important food ingredient. It is used as a preservative and flavouring agent in a large and expanding range of products such as mayonnaise, ketchups, sauces and pickles. In the United States only about 30% of the vinegar produced is sold as table vinegar, the rest being used in food processing.

The antimicrobial action of organic acids such as acetic acid has already been discussed and the use of vinegar in a formulated product usually restricts the spoilage microflora to yeasts, moulds and lactobacilli. Vinegar preserves were one of the earliest areas where predictive models were developed.

Work at what is now the Leatherhead Food Research Association indicated that to achieve satisfactory preservation of a pickle or sauce a minimum of 3.6% acetic acid, calculated as a percentage of the volatile constituents, is necessary.

That is to say:

% acetic acid on whole product = 0.036 x % volatile constituents ………. (9.3)

A different formula has been described specifically for sweet cucumber pickles:

% acetic acid on whole product = (80 – S)/20 ……………………………………..(9.4)

where S is the % sucrose on the whole product.

More elaborate formulae have been produced which apply to emulsified and non-emulsified sauces. These are based largely on work conducted at the laboratories of Unilever in the Netherlands and are known as the CIMSEE code, after the French acronym of the European Sauces Trade Association.

The code consists of two formulae; one to determine the potential for spoilage by acetic acid tolerant yeasts, moulds and LAB, and another derived from inactivation rates of salmonella to assess microbiological safety. Each contains terms for salt, sugar, and acetic acid content and pH.

If, when the relevant values are substituted in the formulae, the result is higher than a specified value then this indicates that the product would be microbiologically stable or safe depending on the formula used.

The formula for safety is:

Where (1—α) is the proportion of un-dissociated acetic acid, a is the proportion dissociated, given by the expression:

where pК = 4.76.

Any sauce based on acetic acid with Σ_s > 63 is regarded as intrinsically safe, since viable numbers of E. coli in it will decline by more than 3 log cycles in less than 72 h at 20 °C.

The formula for stability is:

If Σ > 63, any sauce with this formulation should be microbiologically stable without refrigeration, even after opening.

The mould Moniliella acetoabutens, the micro-organism most resistant to acetic acid, would still grow in such products but can be controlled by good hygienic practices and pasteurization of ingredients containing vinegar. Experience in the pickle and sauce industry indicates that spoilage by Moniliella is rare.

Products meeting the CIMSEE requirement for stability would have a relatively strong taste of acid or salt. If levels of these ingredients are reduced to produce a milder taste then some supplementary preservative measure would be necessary such as sorbate or a final pasteurization step.