The most known and traditional vinegar is the one that is made from wine. For its production, the grape must undergo alcohol fermentation and the posterior oxidation of ethanol to acetic acid. Yeasts and acetic acid bacteria (AAB) carry out the biochemical processes in sequence. The process of wine acetification can be achieved by slow traditional processes (the Orléans or French methods) or by a quick submerged industrial process. High-quality vinegar is usually produced by traditional methods using oak casks, once the wood allows the continuous aeration of the acetic bacteria culture. Sour–sweet vinegar presents a balance of both bitter/sour and sweet flavors. The sourness typically comes from acetic acid, while the sweetness can come from the type of fruit or the amount of sugar present at the end of vinegar production. In general, sour-sweet vinegar has a more complex and nuanced flavor profile compared to regular vinegar, which is often simply sour. One kind of vinegar produced by wine acetification where yeasts and bacteria co-exist and produce savory vinegar is traditional balsamic vinegar (TBV).
Vinegar production dates back to at least 200 BC and is an example of microbial biotransformation. Vinegar can be made from any fermentable sugary substrate and is a globally famous acetic acid condiment produced with dual fermentation (alcoholic and acetic). It possesses a pleasantly sour flavor and has been used as a diet condiment, food preservative, and remedy for people and animals 
The most known and traditional vinegar is the one that is made from wine. In the old days, wine vinegar production was considered a chemical process. In 1732, the Dutchman Boerhaave specified that the “mother of vinegar” was a living organism 
. However, it was only in 1864 that Pasteur claimed that the conversion of wine into vinegar was due to the development of the veil of Mycoderma aceti
on the wine’s surface 
The methods for producing wine vinegar involve the use of grape must, which goes through alcohol fermentation followed by ethanol oxidation to acetic acid 
. These biochemical processes are carried out in sequence by yeasts and acetic acid bacteria (AAB), which produce acetic acid, and several metabolic compounds responsible for the taste and flavor of vinegar, such as nonvolatile compounds (sugars, organic acids, amino acids) and volatile compounds (esters, aldehydes, among others) 
Wine vinegar can be differentiated by its production systems and technologies; there are two types of techniques used to produce vinegar. The slower traditional processes and the quick submerged (industrial purpose) methods. In traditional vinegar, produced by the so-called Orléans or French method, the oxidation of ethanol into acetic acid occurs due to a static culture of AAB at the interface between the liquid and air. Usually, oak casks are used to produce this kind of vinegar, once the wood allows for continuous aeration of the acetic bacteria culture. This is the case with vinegars such as Jerez/Xérès/sherry, where the oak used is Quercus alba
(American oak), and it takes a minimum of six months in wood for the vinegar to be considered a sherry vinegar 
. The wood barrels are filled to 2/3 capacity, have holes on the sides for air passage, and a funnel with an extension to the base of the barrel, allowing the wine to be added at the bottom and preventing the alteration of the biofilm formed by the AAB on the surface 
In the 18th−19th century, Schüzenbach (1793–1869) developed a dynamic acetification system called the “German rapid acetification system.” This process of acetification was industrialized and was named “Rundpump” (round pump) because the wine passed through wood shavings that were continuously sprinkled with the mash by a circulation pump and a rotating sprinkler. The wood shavings served as a support for the bacteria. Alternative materials such as bines, maize cobs, or rushes are cheaper than beechwood but not as efficient 
Advances in technology allowed the development of the submerged fermentation process, first used to produce antibiotics and, subsequently, vinegar, thanks to computerization. The Frings Acetator is an example of this advance. Annual production is half a million liters of vinegar at 10% acidity 
The submerged culture technique involves growing AAB in a liquid medium containing ethanol or other suitable carbon sources. This method is often used in laboratory-scale studies and industrial settings for large-scale acetic acid production. In submerged culture, the liquid medium is typically agitated to enhance oxygen transfer, which provides fast and efficient aeration 
. According to Schlepütz and co-workers 
, in this system, a non-rotating stator supports a hollow-body turbine. Radial holes, which open in the opposite direction of rotation, allow air from outside to be released inside, resulting in very fine air bubbles. A homogenous air–liquid emulsion is formed, which is pushed upwards and diverted by deflectors. All of the mass is maintained in a continuous state of agitation, allowing an easy transfer of oxygen from the medium to the bacteria. This prevents Acetobacter
cell death and promotes efficient microbial growth 
Hence, the necessities for submerged processes are the availability of ethanol, continuous aeration, and acetic acid bacteria strains tolerant to high concentrations of acetic acid and ethanol that require small amounts of nutrients and are not sensitive to phage infections 
. This last requirement is important once phage infections impact AAB growth and, consequently, acetic acid production. Phages, also known as bacteriophages, are viruses that infect and replicate within bacterial cells. They specifically target and infect bacteria, including AAB, resulting in the lysis (bursting) of cells, leading to a decline in population, a disruption in their metabolic activity, and a decrease in acetic acid production 
. Phages that infect AAB have been isolated from various sources, including vinegar production facilities, fruit juice processing plants, and natural environments. These phages can have a significant impact on industrial vinegar production, leading to decreased productivity, spoiled batches, and economic losses in vinegar production 
One major disadvantage of the submerged method is the amount of heat generated and the lack of temperature control. Acetic acid fermentation is an exothermic process, meaning it releases heat as a byproduct. In submerged systems, heat accumulation can be an issue, leading to increased temperature, which may negatively affect bacterial growth and acetic acid yield. Proper temperature control becomes crucial to maintaining optimal conditions 
In terms of organoleptic drawbacks, in submerged fermentations, due to stirred conditions, the liquid creates foam, leading to the establishment of a reducing environment that compromises the acetification process 
. Also, as acetic acid production progresses, the fermentation medium can become more viscous due to the accumulation of acetic acid and bacterial biomass. The increased viscosity can hinder efficient mixing and oxygen transfer, further impacting the fermentation process 
Contrasting submerged methods, the vinegar produced by traditional methods (Orléans or French method) is generally considered to be of high quality, both chemically and sensorially, due to the balance between organic acids and amino acids achieved through long-term surface culture fermentation 
. However, in the traditional method, there is no microbial control, which delays the alcoholic fermentation (AF) and the acetic acid fermentation (AAF), causing contamination and low acidity. Therefore, it is important to understand the interactions between acetic acid bacteria and yeasts to promote strategies that can allow a faster and higher acidity yield fermentation while being able to maintain quality, regardless of the method used.
One type of vinegar produced by wine acetification where yeasts and bacteria co-exist and produce savory vinegar is traditional balsamic vinegar (TBV), which originated in Modena and Reggio Emilia, Italy. In Spain, fortified sherry, with its distinctive nutty and oxidized flavor 
, has also been used for vinegar production. In Portugal, sour-sweet wine vinegar has recently emerged. Port wine vinegar has been produced by some wine companies since 2018. It has been described as having “a robust and naturally fruity flavor accented by a slight hint of acidity and a deep shade of ruby red.”
2. Traditional Balsamic Vinegar (TBV) Processing
The fabrication of TBV involves a three-stage process: cooking of must; microbiological transformations (two fermentations, alcoholic and acetic), and aging (Figure 1).
Figure 1. Schematic representation of TBV processing. The cooking of must and microbiological transformations are the first steps of the process. The aging involves using a set of 5/7 wood barrels, made from different wood species, arranged in a series of decreasing volumes. A certain volume of the final product is removed from the smallest barrel. AAB—acetic acid bacteria.
2.1. Cooking of the Must
Grape must is obtained from grapes such as Ancellotta, Berzemino, Lambrusco, Occhio di Gatta, Sauvignon, Sgavetta, and Trebbiano, all Modena and Emilia Romano local grape-cultivars 
. After crashing, it is boiled in uncovered vessels, which allows the removal of impurities and coagulated proteins by floating. Afterward, the temperature is kept below 85–90 °C. The cooking process stops when the must reaches 35–60° Brix. In the end, the cooked must will present pigments formed by non-enzymatic browning and 5-hydroxymethyl-2-furaldehyde (5-HMF) due to hexose degradation by dehydration and cyclization 
. The cooking of the must produces a highly concentrated and dark liquid that is very sweet to the palate.
2.2. Microbiological Transformations
The microbiological process of cooking must occur in two steps: first, a natural alcoholic fermentation of sugars, followed by ethanol oxidation to acetic acid (the second step). In the first step, yeasts metabolize the sugars from the grapes into ethanol, carbon dioxide, and a huge number of secondary by-products. The cooked and sterile must is transferred into open wooden vessels already contaminated by yeast. Over a few weeks, alcoholic fermentation occurs due to the rapid increase in yeast population (102
. To perform this fermentation, no dried yeast is used (Figure 1
). The bioconversion of ethanol to acetic acid by AAB is the next microbiological process (second step). Acetic acid bacteria occur naturally, making a thin coating or biofilm on the surface of fermented cooked must. Due to the presence of ethanol (9–10%, v
), biofilm formation may be slow. To accelerate the process, bacterial biofilm can be transferred manually from one vessel to another in active oxidation 
Figure 2. (A) The mother of vinegar is removed from a wine vinegar pot. (B) Bacterial layer growing in red wine vinegar. (C) Mother of vinegar formation in home-made vinegar.
2.3. Ageing Process
At least five casks (wooden barrels) are needed to perform the aging process. They are casks of different sizes and woods, as seen in Figure 1
, arranged in decreasing scalar volumes. Every year, a small volume of the aged vinegar (the smallest barrel) is spilled. This barrel is then refilled with the contents of the previous barrel. This procedure is repeated up to the first and largest cask, which receives newly oxidized and fermented cooked must 
. The operation is called “rincalzo” (refilling), and it must be repeated for at least 12 years for the vinegar to be defined as a traditional DOP.
The sugar concentration increases along the barrel set due to the evaporation through the opening on the top of the barrel and the wood. In this procedure, every barrel will contain a blend of vinegar of different ages, and, naturally, the age increases from the bigger barrel to the smaller one. However, it is not easy to define the age of balsamic vinegar. In 2007, Giudici and Rinaldi 
developed a theoretical model that allows predicting the age of traditional balsamic vinegar, considering the volume of vinegar transferred from barrel to barrel and the amount of newly cooked must added, aiming to provide a way to define the age of traditional balsamic vinegar and verify the requirements of the DOP legislation. The referred authors were able to prove that the age of traditional balsamic vinegar is correlated to the amount of vinegar withdrawn and/or to that of the newly cooked must. With their mathematical model, any producer could be able to validate the age of their vinegar, letting the consumer buy the vinegar at the stated age 
At the end of the process, it is possible to obtain a dark-sugary/acid liquid with more or less 40% (w
) of sugars (glucose and fructose in a 1:1 ratio) and with a pH of 2.3–3.2 and 2–5% (w
) in acetic acid