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. From the stoichiometry of the equation it can be calculated that 1 1 of ethanol should yield 1.
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.
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. 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. 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. Submerged culture is very efficient and rapid, a semi-continuous run normally takes 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. A stoppage of only one minute in a stock with a GK of 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. 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. 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. However, because entry into the acetate cycle is inhibited by the presence of ethanol, it is essential to maintain a low concentration of ethanol in the presence of acetic acid bacteria to prevent this full oxidation. In fact, ethanol concentrations between 0. Their cultivability is often lower and more irregular than that observed under the microscope, and these differences can be of several log units [ 8 ].
Many strains lose some features e. Species identification has traditionally been performed by physiological and biochemical tests, and only half a dozen species from the genera Acetobacter and Gluconobacter were identified.
These two genera could be differentiated based on their preference for alcohol or glucose as a substrate [ 5 ]. However, the use of molecular methods has improved efforts at taxonomy, and there are currently 14 genera and approximately 70 species described [ 9 ]. Approximately one dozen species and more than 40 strains have been sequenced.
Some of the best-known species in the production of vinegars have been transferred from different genera. For example, three of the oldest species described in the production of vinegar were initially classified as genus Acetobacter, reclassified as Gluconacetobacter [ 10 ], and more recently moved to Komagataeibacter [ 9 ].
These species, Komagataeibacter europaeus, hansenii, and xylinus , now appear in the literature or textbooks under three different genera.
These molecular methods and their adaptation to the conditions of routine studies for the analysis of populations and the control of microbiological processes have been studied by a group at the Rovira i Virgili University. We have developed a number of methods for the routine identification of species by restriction analysis of ribosomal genes or their spacers [ 11 , 12 ], which has allowed us to better understand the process of appearance and resistance during the alcoholic fermentation and vinegar production process.
Likewise, we applied methods for strain-level identification, which allowed us to track acetic acid bacterial populations from grape to wine and during the process of vinegar making [ 13 , 14 ].
However, we routinely applied these methods for the analysis of populations recovered in culture media, which has the disadvantage of low recovery, as mentioned above. In recent years, other molecular applications have allowed us to use independent methodologies such as DGGE culture [ 15 — 18 ] or quantitative PCR [ 8 , 19 — 21 ].
These methods provide us with additional opportunities to follow the acetic acid bacterial populations in wine or vinegar. Focusing on the production of wine vinegar, the use of these techniques has allowed us to observe that the vinegar is produced by a succession of strains and species, depending on the concentration of acetic acid [ 22 ]. At low concentrations of acetic acid, species of the genus Acetobacter predominate. In these cases, we observed that the starter cultures of A.
This can be explained by both the differing acetic acid tolerances of the species and the presence of a contaminating population of acetic acid bacteria in the raw material wine. The final quality of vinegars depends on the selection of appropriate starter cultures generally mixed cultures to lead the process. However, other factors include the quality of the starting material, the production method, and, if applicable, aging. In general, it is relatively easy to appreciate the sensory differences between products made by traditional methods and those manufactured on an industrial scale.
Thorough characterization and quality evaluation requires the determination of the content of a number of compounds and sensory analysis. In recent years, there have been significant advances in the elucidation of the compounds responsible for the sensory quality of the products, and production methods have been changed to obtain vinegars with high acceptance at very competitive prices.
The group at the University of Sevilla has been focusing on the characterization of wine vinegars for the last 20 years. Aromatic compounds have a decisive effect on the quality of vinegars. To date, we have identified more than different chemical compounds in the aroma of wine vinegar, including carbonyl compounds, ethers, acetals, lactones, acids, alcohols, phenols, and volatile esters, all of which are involved to different extents in the final flavor [ 4 ].
During the aging process, the contact with wood produces a substantial increase in the aromatic complexity [ 26 ]. However, not all volatile compounds are responsible for the aroma of the product. They must not only reach odorant receptors but also interact with them in the olfactory epithelium, and not all volatile compounds are active odorants. The use of techniques based on gas chromatography coupled with olfactometry has allowed the contribution of each volatile compound in the final vinegar to be evaluated.
For example, it has been determined that the characteristic aroma of Sherry vinegar involves several volatile compounds, such as diacetyl, isoamyl acetate, isovaleric acid, ethyl acetate, and sotolon [ 27 ]. Polyphenolic compounds, which are ubiquitous in plant products, are of great interest as quality determinants because, in addition to their antioxidant activity, they are responsible for the color and astringency of vinegar.
Acetification is an aerobic process, and oxygen is critical to the growth of the bacteria. The reactivity of phenolic compounds and oxygen is specifically analyzed in winemaking for its relationship to the browning of white wines and the reactions of anthocyanins in red wines. The rate of acetification is also expected to be related to the solubility of oxygen in the medium, a decisive factor in the phenolic composition that can be useful for determining the method by which vinegar is produced.
It should be emphasized that submerged systems use excess oxygen to secure and accelerate the process, whereas oxygen availability is limited in superficial cultures because it is continuously taken up by acetic acid bacteria.
Additionally, oxygen affects the classes of polyphenolic compounds to different degrees. For example, the flavonol content of vinegars is largely influenced by oxygen availability during submerged fermentation.
In contrast, surface acetification vinegars do not affect phenolic aldehydes, which are released from wooden barrels into the product [ 28 ]. The evolution of phenolic compounds during acetification in submerged culture systems has been studied in both laboratory and industrial fermenters. In a laboratory fermenter with Sherry wine as the substrate, the phenolic profile was not significantly altered [ 29 ].
The aging process involves the reaction of compounds over time: both the polymerization and release of compounds from the wood and losses through evaporation. The substances provided by the wood will depend on the type of wood and roasting, the ratio of the contact surface to liquid volume, and the aging time.
As a consequence, significant differences have been observed in the phenolic composition of Sherry vinegars aged two or more years in static or traditional solera systems [ 31 ].
An observation of the evolution of phenolic compounds in Sherry vinegar aged in oak barrels showed that there were significant differences in the compounds vanillin, syringaldehyde, coniferyl aldehyde, and cinnamic acid after 90 days of aging [ 32 ]. Vinegar is a difficult product to taste, due to the intense sensations it provokes. The pungency of the high acetic acid content masks other flavors, and some familiarity with the product is required to proceed with a tasting.
In fact, there is no consensus on how vinegar should be tasted. A vinegar sensory analysis panel requires well-trained tasters, and the specific attributes that are useful for differentiating among samples must be chosen.
To train a vinegar panel, Tesfaye et al. Acetic acid aggressiveness determines the number of samples that can be examined in each session, and each sample is tasted four times. These four replicate samples should be tasted on different days to avoid sensorial saturation of the tasters. A descriptive analysis of the samples is prepared based on previously selected attributes that can be evaluated by the panel. The attributes used to describe the vinegar samples were color, aromatic intensity, woody scent, herbaceous smell, fruity odor of ethyl acetate, wine smell, and pungent feeling [ 34 , 35 ].
Higher sensory thresholds for most compounds were obtained in an acetic acid matrix compared with water solutions. Conversely, high quality vinegars contain a large number of these compounds at concentrations higher than their threshold limits, including vanillin, eugenol, and benzaldehyde, and this characteristic could be therefore selected as an attribute of high quality vinegars [ 36 ].
Additionally, adequate training and a standardized tasting protocol contribute to the reliability of descriptive sensory analyses of a vinegar and of ascertaining the aging period and wood used in its elaboration [ 37 ]. The authors declare that there is no conflict of interests regarding the publication of this paper. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors. Read the winning articles. Journal overview. Special Issues. In these liquids, the bacteria form a film on the surface, since they are aerobic and need good oxygen supply. This film, called mother of vinegar, can be used as a starter culture of acetic fermentation in fresh alcohol liquids. Mother of vinegar can also be found in unpasteurized store brand vinegar. Acetic acid bacteria are transmitted in nature by vectors like fruit flies and Vinegar eels.
This acetic acid fermentation needs oxygenation. If left at room temperature alcohol containing solution with Acetobacter will be converted to vinegar in months. The industrial process can be completed within hours since air is bubbled and mixed through the solution. Vinegar can also be an undesired product in wine production. If the temperature in the fermentation vessel is too high, the Acetobacter will outgrow the yeasts and the produced alcohol will be converted to vinegar.
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