Exploring Microbial Dynamics: The Interaction between Yeasts and Acetic Acid Bacteria in Port Wine Vinegar and Its Implications on Chemical Composition and Sensory Acceptance

Abstract

Port wine vinegar, a product of the esteemed Port wine, is renowned for its intricate blend of flavors and aromas, a result of complex microbial interactions. This study delves into the fascinating world of yeast and acetic acid bacteria (AAB) interactions during fermentation, which significantly influence the vinegar’s chemical composition and sensory properties. We specifically investigate the role of yeasts in fermenting sugars into ethanol, a process that AAB then converts into acetic acid. The impact of these interactions on the production of secondary metabolites, such as gluconic acid, ketones, aldehydes, and esters, which contribute to the vinegar’s unique sensory profile, is thoroughly examined. Advanced analytical techniques, including GC-MS and e-nose technology, alongside sensory evaluation, are employed to assess these effects. The research underscores the significance of ethanol tolerance in AAB and other production challenges in determining vinegar quality and underscores the importance of optimizing fermentation conditions and sustainable practices. The findings of this study underscore the importance of strain interactions and production techniques, which can significantly enhance the quality and market appeal of Port wine vinegar, providing valuable insights for the industry. This review also identifies exciting and critical areas for future research, inspiring further exploration and proposing strategies for advancing production and application in culinary, health, and industrial contexts.

1. Introduction

Vinegar holds significant historical and cultural importance across civilizations and is revered for its culinary uses and medicinal and preservative properties. From the spontaneous acetification of wine, vinegar’s roots trace back to ancient Babylon around 5000 years ago, where it was first documented as a food preservative [1,2,3]. Initially, vinegar was considered a low-value by-product of wine with limited commercial appeal due to its sour taste and the simplicity of its production process. When exposed to air, wine naturally transformed into vinegar [1].

Hippocrates, the father of medicine, recognized vinegar’s medicinal potential, using it for wound disinfection and treating liver diseases [1,4]. Nowadays, vinegar is appreciated for its antibacterial properties, antioxidant activity, and benefits in managing diabetes, tumors, and cardiovascular health, owing to the various bioactive compounds derived from its raw materials [5,6].

In modern culinary applications, vinegar is a versatile condiment, ingredient, and preservative, prized for its acidic profile [3,5,7]. One notable advantage is its use in preserving foods, which extends shelf life and enhances probiotic qualities [8,9], thereby reducing food waste. The quality of vinegar is intrinsically linked to factors such as production methods, fermentation parameters (temperature, oxygen availability, and pH), microbial composition, storage conditions, and the raw materials used. These factors collectively influence the physicochemical and phytochemical characteristics of vinegars [4,7,10].

Biochemically, vinegar production involves an initial anaerobic process known as alcoholic fermentation, where yeasts convert sugars into ethanol. This is followed by an aerobic process in which acetic acid bacteria oxidize ethanol into acetic acid [2,3,7,11]. The dynamic interaction between yeasts and acetic acid bacteria is fundamental in transforming substrates like wine, fruit juices, or cereals into vinegar, significantly impacting its organoleptic properties [12].

Understanding the complex interactions between yeasts and acetic acid bacteria is crucial for optimizing fermentation processes, ensuring product consistency, and leveraging biotechnological advancements for improved production methods. In the industrial context, these insights can lead to more efficient production processes and higher-quality products, meeting consumer demands and enhancing economic viability [12]. Despite the advances, the production of vinegar, particularly Port wine vinegar, faces challenges such as maintaining the balance of microbial populations and controlling fermentation conditions [7]. Addressing these challenges requires a deep understanding of microbial dynamics and their impact on the final product.

This article aims to comprehensively review the microbial dynamics occurring during wine vinegar production, particularly on Port wine vinegar, a derived product of Port wine. The review will focus on the interactions between yeasts and acetic acid bacteria and evaluate the impact of these interactions on the chemical composition and sensory characteristics of wine vinegars. By exploring these aspects, the article aims to offer insights that can enhance vinegar products’ quality and consumer appeal while identifying potential areas for future research and technological improvement.

2. Historical Development and Traditional Practices

Evolution of Vinegar Production, Raw Materials, and European Legislation

Vinegar production has evolved significantly throughout history, reflecting cultural practices and technological advancements across different civilizations. Ancient civilizations such as the Babylonians, Egyptians, and Romans utilized rudimentary methods to convert wine and other fermentable liquids into vinegar. These early methods primarily involved natural fermentation processes, where exposure to air allowed ethyl alcohol to oxidize into acetic acid, giving rise to vinegar’s characteristic sourness [1,13]. For instance, Babylonian records dating back to 5000 BCE mention the use of vinegar for preserving and flavoring foods, indicating its integral role in ancient culinary practices [1].

During the Middle Ages, particularly in Europe, wine and vinegar production became more refined with the introduction of wooden barrels and clay amphoras for fermentation [14,15]. The Orleans process originated in France and involved a slow fermentation in wooden barrels, allowing for gradual acetic acid production while preserving the wine’s delicate flavors. This method became synonymous with traditional vinegar production and persisted through the centuries [3,7,12,16]. The Orleans method is notable for its reliance on natural microbial populations and carefully maintaining optimal environmental conditions, contributing to the resulting vinegars’ unique flavor profiles.

Significant advancements were made in vinegar production in the 18th and 19th centuries, particularly with the development of the German rapid acetification system. Schüzenbach (1793–1869) pioneered dynamic acetification, a method that drastically increased the efficiency of vinegar production by optimizing the conditions for acetic acid bacteria. This technique allowed for faster fermentation and higher yields, representing a significant leap forward in vinegar manufacturing technology [3,7,12,16].

Modern industrialization has brought about significant changes in vinegar production techniques. Innovations such as submerged fermentation in stainless steel tanks and controlled environments for microbial growth have streamlined production processes while maintaining quality and consistency [1,3,7,12]. Submerged fermentation, in particular, allows for faster production rates and greater control over the fermentation process, which results in higher yields and consistent product quality [7,12,17]. These advancements have expanded the range of available vinegars, catering to diverse culinary preferences and applications. Developing new analytical techniques for monitoring fermentation has also enhanced our understanding of microbial dynamics and their impact on vinegar quality.

Modern technology has played a crucial role in preserving traditional vinegar production methods. The increasing demand for artisanal and specialty wine and vinegars has led producers to combine ancient practices with modern technology, creating unique, high-quality vinegars that appeal to contemporary tastes. For instance, balsamic vinegar from Modena, Italy, is still produced using age-old techniques involving the slow fermentation and aging of grape must in a series of wooden barrels, resulting in its distinctive rich flavor and complexity [12,18].

Vinegar production is steeped in tradition, where specific techniques and carefully selected raw materials play a paramount role in shaping the distinctive flavors and qualities of various vinegar types. Each type of vinegar, whether balsamic vinegar from Italy, sherry vinegar from Spain, Port wine vinegar from Portugal, rice vinegar from Asia, or apple cider vinegar, relies on unique ingredients and traditional methods refined over centuries.

The European Union defines vinegar as a product obtained through the dual fermentation process—alcoholic and acetic—of agricultural products [7,11,19]. Various types of vinegars are highlighted in

Table 1. Classification of vinegars by origin, according to [19].

Classification	Description
Wine vinegar	Exclusively produced from wine through the biological process of acetic fermentation.
Fruit and berry vinegar	Obtained from fruit or berries through the biological process of alcoholic and acetic fermentation.
Cider vinegar	Obtained from cider through the biological process of acetic fermentation.
Spirit vinegar	Obtained from distilled agricultural alcohol through the biological process of acetic fermentation.
Cereal vinegar	Obtained, without intermediate distillation, through the biological process of dual fermentation (alcoholic and acetic) from cereals whose starch has been converted to sugars by malted barley diastase or another method.
Malt vinegar	It is obtained without intermediate distillation through the biological process of dual fermentation (alcoholic and acetic) from malted barley, with or without adding other cereals, where starch has been converted to sugars solely by malted barley diastase.
Distilled malt vinegar	It is obtained by distilling malt vinegar under reduced pressure, containing only the volatile constituents of the malt vinegar from which it is derived.
Other vinegars	Vinegar from other agricultural products through dual fermentation, including honey and beer, is not covered in the previous items.
Flavored and spiced vinegars	From the previous categories to which aromatic plants or parts thereof, spices and flavoring extracts have been added, perceptible organoleptically.

. The choice of raw materials significantly influences vinegar quality. Grapes and wines are primary ingredients for wine vinegars, with different grape varieties imparting unique flavors. Other fruits, such as apples for apple cider vinegar, and grains, notably barley for malt vinegar, also play crucial roles in defining vinegar characteristics [2,3,20].

In the European Union, vinegars’ preparation allows for adding certain ingredients to enhance flavor and quality. Permitted additions include aromatic plants or parts thereof, spices, flavoring extracts, fruit juices or concentrates, honey, sugar, and salt. However, additives such as artificial flavorings, grape seed oils, distillation and fermentation residues, and substances extracted from pomace are strictly prohibited from vinegar preparation. The legislation further stipulates specific quality standards for vinegars. Wine vinegars must have a minimum total acidity of 6 g per 100 mL expressed as acetic acid, while other vinegars must have a minimum total acidity of 5 g per 100 mL. Residual alcohol content, measured at 20 °C, must not exceed 1.5% for wine vinegars and 0.5% for other vinegars [19].

This regulatory framework ensures consistency in vinegar production and quality across the European Union, safeguarding consumer interests and fostering uniform practices among producers. The significant investment required to produce Port wine is reflected in its price. Every step is resource-intensive, from cultivating grapes in the Douro Valley to meticulous winemaking, including wine fortification with “aguardente” and aging processes in oak wood. This investment ensures a high-quality, distinctive product that commands a premium in the market. The less expensive Port wine categories are usually used to make Port wine vinegar, including plain Tawny, Ruby, or White. Moreover, when an excess of wine production occurs, making vinegar is a way to innovate and market wines that could be difficult to sell. Exploring the complex microbial ecosystem that underpins the fermentation processes is essential for gaining a comprehensive understanding of the intricacies involved in the production of wine vinegars.

3. Microbial Ecosystem and Chemical Evolution in Wine Vinegar Production

As mentioned, vinegar production involves two essential phases: alcoholic fermentation and acetic oxidation. These biochemical transformations are driven by microorganisms with functional characteristics that enable them to catalyze organic compounds, producing acetic acid, the most abundant and characteristic organic acid in vinegar. Therefore, a thorough understanding of the microbial ecosystem and the chemical transformations during vinegar production—particularly in Port wine vinegar—is crucial for ensuring the product’s quality and consistency.

3.1. Diversity of Microorganisms and Their Roles

The production and quality of Port wine vinegar are intrinsically linked to the diverse set of microorganisms present in the raw material, each playing a specific role in the fermentation processes. During the alcoholic fermentation phase, yeasts metabolize the sugars in the raw material to produce ethanol, with Saccharomyces cerevisiae being one of the predominant species involved [3,7]. Beyond ethanol production, these microorganisms, alongside other yeasts such as Hanseniaspora, Metchnikowia, Pichia, and Candida (semi-fermentative yeast genera), also generate secondary metabolites. These metabolites can significantly influence the flavor and overall quality of the vinegar, either enhancing it or introducing undesirable characteristics [21,22].

Acetic acid bacteria (AAB), such as Acetobacter and Gluconobacter species, play a pivotal role in the second phase of fermentation. These bacteria oxidize ethanol to acetic acid in the presence of oxygen through aerobic reactions, a fundamental process in developing vinegar’s characteristic acidity [7,23]. The various species and strains of acetic bacteria can produce varying amounts of acetic acid and other metabolites, thereby influencing the quality and flavor profile of the Port wine vinegar. In vinegars derived from non-fortified wines, the microbial ecosystem’s complexity may encompass lactic acid bacteria (LAB) and other microorganisms that can impact the final product’s overall fermentation process and characteristics [24]. LAB in vinegar production contributes to pH regulation and flavor development. They also offer probiotic benefits, promoting a balanced fermentation environment and potentially enhancing the nutritional value of the final product [24,25,26].

3.2. Functions of Yeasts in Vinegar Production

Fermentation is a general term that describes the anaerobic breakdown of glucose or other organic nutrients, which generates energy stored in the form of adenosine triphosphate (ATP) [27]. In alcoholic fermentation, yeasts play a crucial role by metabolizing sugars in the raw material, converting them into ethanol and carbon dioxide (CO₂). Glucose is initially converted to pyruvate through glycolysis, and pyruvate is then transformed into ethanol in a two-step process [2,3,7,27], as illustrated in Figure 1.

Morphologically, yeasts are unicellular fungi, typically single-celled structures varying in shape and size, often spherical or oval [30,31]. These microorganisms thrive in anaerobic environments and are particularly adept at converting sugars into ethanol during the initial stages of Port wine vinegar production [30]. Saccharomyces cerevisiae is one of the most widely used species in this process due to its robust fermentative capabilities and tolerance to varying environmental conditions [3,11,12,32]. Its optimal temperatures are commonly between 28 °C and 33 °C [33].

The diverse epiphytic microbiota found on grape surfaces initiates spontaneous fermentation, encompassing various yeast strains from non-Saccharomyces genera such as Hanseniaspora, Candida, Debaryomyces, Starmerella, Dekkera, Kluyveromyces, Metschnikowia, Torulaspora, Pichia, Zygosaccharomyces, Cryptococcus, and Rhodotorula [34]. Among these, Saccharomyces cerevisiae stands out for its enhanced tolerance to sulfur dioxide compared to other yeasts. This resilience, combined with traits like the Crabtree effect, low oxygen requirements, and tolerance to ethanol, heat, and osmotic stress, positions S. cerevisiae as predominant in spontaneous fermentations [32,34].

Despite nutrient limitations (including thiamine) and high acidity, yeasts thrive in grape-must environments influenced by osmotic pressure and ethanol concentration [32,35]. Sensory-wise, yeasts, notably S. cerevisiae, catalyze the conversion of primary aroma precursors in grapes, such as glycosylated compounds and polyfunctional mercaptans, releasing a spectrum of volatile organic compounds contributing to the secondary aroma profile. These compounds include fusel alcohols derived from amino acid metabolism via the Ehrlich pathway and acetate and ethyl esters [32,36,37,38,39].

In addition to these aromatic contributions, yeast metabolism in wine fermentation yields glycerol, organic acids (including acetic acid), acetaldehyde, mannoproteins, and sulfur-containing compounds (such as SH₂ and SO₂) [32,38,39,40], as shown in Table 2. These compounds collectively enrich the sensory complexity of wine, influencing its flavor, aroma, and overall character.

Overall, wine vinegar, similar to wine, offers a spectrum of health benefits attributable to its diverse array of bioactive compounds formed during fermentation. These compounds contribute to its nutraceutical profile, promoting overall health and well-being.

The review by Marques and colleagues (2023) [41] underscores that alcoholic fermentation is pivotal in generating bioactive compounds with significant health benefits in wine and wine vinegar. Notably, red wine, due to its phenolic composition, is associated with preventing cardiovascular diseases when consumed moderately [42,43]. Similarly, wine vinegar also retains these health-promoting compounds [6]. Compounds such as tyrosol, hydroxytyrosol, and tryptophol are synthesized during fermentation. These compounds are recognized for their nutraceutical properties, including anti-atherogenic, anti-cancer, neuroprotective, anti-diabetic, lipid-regulating, and anti-obesity effects. They contribute significantly to promoting cardiovascular health-related benefits [44,45]. Furthermore, melatonin and serotonin, also formed during fermentation, are bioactive compounds known for their nutraceutical properties. These compounds have beneficial effects on health, including their roles in regulating sleep patterns and mood, among other functions [46,47]. Moreover, wines and their vinegar derivatives contain essential minerals and vitamins. Glutathione, a powerful antioxidant composed of glutamic acid, cysteine, and glycine, is another vital compound in wines. It plays a crucial role in protecting organisms from oxidative damage by scavenging free radicals and reactive oxygen species [48,49,50].

Table 2. Key metabolites produced by Saccharomyces and non-Saccharomyces yeasts during wine fermentation. Data obtained from [12,51,52,53,54,55,56,57,58,59].

Metabolite	Sensory Impact	Health Benefit
Saccharomyces
Acetaldehyde	Fruity, green apple notes	-
Acetic acid	Vinegar-like aroma and taste	Can improve digestion in small amounts
B-complex vitamins (e.g., thiamine)	Nutritional enhancement	Essential for energy metabolism; nerve function
Beta-glucans	Viscosity, mouthfeel	Immunomodulatory effects
Citric acid	Tartness	Antioxidant; may enhance nutrient absorption
Diacetyl	Buttery or butterscotch flavor	-
Esters (e.g., ethyl acetate)	Fruity aromas	-
Ethanol	Contributes to the alcohol content	Antiseptic properties
Glutathione	Antioxidant	Detoxification; supports immune function
Glycerol	Sweetness, fullness	Hydrating properties
Higher alcohols (e.g., isoamyl alcohol)	Solvent-like, fuel oils	-
Lactic acid	Tangy, sour taste	Metabolic acid; contributes to microbiota balance
Minerals (e.g., potassium, magnesium)	-	Essential for enzymatic reactions; muscle function
Phenolic compounds	Spicy, clove-like flavors	Antioxidant properties
Polysaccharides	Mouthfeel enhancement	Prebiotic effects
Succinic acid	Bitterness, acidity	May support cellular metabolism
Non-Saccharomyces
4-Ethylguaiacol	Smoky, clove aroma	-
4-Ethylphenol	Barnyard, medicinal aroma	-
Acetaldehyde	Green apple or ripe persimmon aroma	Acts as an antimicrobial agent
Acetoin	Butter-like aroma	Potential antioxidant properties
Carotenoids	Pigmentation, antioxidant properties	Antioxidant properties
Ergosterol	Contributes to vitamin D synthesis	Supports bone health
Ethanol	Contributes to alcohol content	Antiseptic properties
Ethyl acetate	Fruity, nail polish remover aroma	-
Ethyl butyrate	Pineapple aroma	-
Gluconic acid	Mild acidity	Supports mineral absorption
Isoamyl acetate	Banana aroma	-
Isoamyl alcohol	Solvent-like	-
Polyunsaturated fatty acids	Contributes to flavor and aroma	Supports cardiovascular health
Succinic acid	Bitterness, acidity, saltiness	It may support cellular metabolism
β-Galactosidase	Enhances sweetness from lactose	Potential probiotic effects

Table 2. Key metabolites produced by Saccharomyces and non-Saccharomyces yeasts during wine fermentation. Data obtained from [12,51,52,53,54,55,56,57,58,59].

Metabolite	Sensory Impact	Health Benefit
Saccharomyces
Acetaldehyde	Fruity, green apple notes	-
Acetic acid	Vinegar-like aroma and taste	Can improve digestion in small amounts
B-complex vitamins (e.g., thiamine)	Nutritional enhancement	Essential for energy metabolism; nerve function
Beta-glucans	Viscosity, mouthfeel	Immunomodulatory effects
Citric acid	Tartness	Antioxidant; may enhance nutrient absorption
Diacetyl	Buttery or butterscotch flavor	-
Esters (e.g., ethyl acetate)	Fruity aromas	-
Ethanol	Contributes to the alcohol content	Antiseptic properties
Glutathione	Antioxidant	Detoxification; supports immune function
Glycerol	Sweetness, fullness	Hydrating properties
Higher alcohols (e.g., isoamyl alcohol)	Solvent-like, fuel oils	-
Lactic acid	Tangy, sour taste	Metabolic acid; contributes to microbiota balance
Minerals (e.g., potassium, magnesium)	-	Essential for enzymatic reactions; muscle function
Phenolic compounds	Spicy, clove-like flavors	Antioxidant properties
Polysaccharides	Mouthfeel enhancement	Prebiotic effects
Succinic acid	Bitterness, acidity	May support cellular metabolism
Non-Saccharomyces
4-Ethylguaiacol	Smoky, clove aroma	-
4-Ethylphenol	Barnyard, medicinal aroma	-
Acetaldehyde	Green apple or ripe persimmon aroma	Acts as an antimicrobial agent
Acetoin	Butter-like aroma	Potential antioxidant properties
Carotenoids	Pigmentation, antioxidant properties	Antioxidant properties
Ergosterol	Contributes to vitamin D synthesis	Supports bone health
Ethanol	Contributes to alcohol content	Antiseptic properties
Ethyl acetate	Fruity, nail polish remover aroma	-
Ethyl butyrate	Pineapple aroma	-
Gluconic acid	Mild acidity	Supports mineral absorption
Isoamyl acetate	Banana aroma	-
Isoamyl alcohol	Solvent-like	-
Polyunsaturated fatty acids	Contributes to flavor and aroma	Supports cardiovascular health
Succinic acid	Bitterness, acidity, saltiness	It may support cellular metabolism
β-Galactosidase	Enhances sweetness from lactose	Potential probiotic effects

3.3. Roles of Acetic Acid Bacteria in Vinegar Production

Acetic acid bacteria are crucial in making Port wine vinegar [7]. Their versatility and metabolic adaptability make them subjects of extensive study to optimize the production of diverse products and understand the mechanisms enabling their growth under extreme conditions. The presence of membrane-bound and soluble dehydrogenases provides opportunities to innovate processes leveraging their ability to partially oxidize various substrates. Moreover, the resilience of acetic acid bacteria to adverse conditions, such as low pH, and their adaptation to diverse habitats enhance their competitiveness, underscoring the significance of investigating their interactions with other organisms and plants [60]. Table 3 provides an overview of the characteristics of AAB and their role in vinegar production.

Table 3. Characteristics of acetic acid bacteria. Data sourced from [1,3,7,12,61,62,63].

Characteristic	Details
Size	Typically, 0.4 to 1.0 µm in width and 0.8 to 4.5 µm in length
Morphology	Gram-negative or gram-variable, they exist as single cells, filamentous or rod-shaped, with ellipsoidal to rod morphologies. They are mobile due to peritrichous or polar flagella.
Family and classification	The Acetobacteraceae family comprises 47 genera and 207 species; 20 genera and 108 species are classified under AAB.
Prominent genera in vinegar production	Acetobacter species (e.g., Acetobacter aceti, Acetobacter pasteurianus, Acetobacter xylinum) and Gluconobacter oxydans among Gluconobacter species
Enzymatic abilities	Catalase and oxidase positive; capable of degrading hydrogen peroxide and using oxygen as a terminal electron acceptor in respiration
Metabolic capabilities	Capable of nitrogen fixation; utilize nitrogen gas as a nitrogen source for growth
Optimal growth conditions	pH range of 4.0–6.0; temperature range of 25–30 °C
Substrate requirements	Require ethanol or other alcohols as substrates for growth and energy production.
Metabolic pathways	The primary pathway is oxidative fermentation (AAB oxidative fermentation–AOF), converting ethanol into acetic acid.
Supporting metabolic pathways	Utilize the pentose phosphate pathway (PPP) and Entner–Doudoroff (ED) pathways to provide energy and reduce power for ethanol oxidation.
Pentose phosphate pathway	Generates NADPH and pentose sugars essential for biosynthetic processes and redox balance
Entner–Doudoroff (ED) pathway	Produces pyruvate and NADPH through oxidation of glucose or related compounds, supporting energy production and redox homeostasis

Table 3. Characteristics of acetic acid bacteria. Data sourced from [1,3,7,12,61,62,63].

Characteristic	Details
Size	Typically, 0.4 to 1.0 µm in width and 0.8 to 4.5 µm in length
Morphology	Gram-negative or gram-variable, they exist as single cells, filamentous or rod-shaped, with ellipsoidal to rod morphologies. They are mobile due to peritrichous or polar flagella.
Family and classification	The Acetobacteraceae family comprises 47 genera and 207 species; 20 genera and 108 species are classified under AAB.
Prominent genera in vinegar production	Acetobacter species (e.g., Acetobacter aceti, Acetobacter pasteurianus, Acetobacter xylinum) and Gluconobacter oxydans among Gluconobacter species
Enzymatic abilities	Catalase and oxidase positive; capable of degrading hydrogen peroxide and using oxygen as a terminal electron acceptor in respiration
Metabolic capabilities	Capable of nitrogen fixation; utilize nitrogen gas as a nitrogen source for growth
Optimal growth conditions	pH range of 4.0–6.0; temperature range of 25–30 °C
Substrate requirements	Require ethanol or other alcohols as substrates for growth and energy production.
Metabolic pathways	The primary pathway is oxidative fermentation (AAB oxidative fermentation–AOF), converting ethanol into acetic acid.
Supporting metabolic pathways	Utilize the pentose phosphate pathway (PPP) and Entner–Doudoroff (ED) pathways to provide energy and reduce power for ethanol oxidation.
Pentose phosphate pathway	Generates NADPH and pentose sugars essential for biosynthetic processes and redox balance
Entner–Doudoroff (ED) pathway	Produces pyruvate and NADPH through oxidation of glucose or related compounds, supporting energy production and redox homeostasis

Two key enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), facilitate the fermentative oxidation process of alcohol in acetic acid bacteria (AAB) [7,12]. The oxidation of ethanol begins with ADH, which converts ethanol into acetaldehyde. Subsequently, ALDH oxidizes acetaldehyde into acetic acid, producing NADH (Figure 2). NADH is then utilized in the electron transport chain to generate ATP, involving several carriers, including quinones and cytochromes [12,61].

The efficiency of AAB is influenced by the stability of ADH under acidic conditions. For instance, some Gluconobacter species exhibit an inactive form of ADH, which affects the conversion of ethanol to acetic acid. Additionally, certain AAB species possess the ability to overoxidize acetate. After acetic acid is formed and in low ethanol concentrations, these bacteria can further convert acetic acid into CO₂ and H₂O via the tricarboxylic acid cycle [7,61,64], gradually increasing the product’s pH.

Several factors influence the efficiency and outcome of the AOF process. These include the type and concentration of AAB, pH levels, temperature, and oxygen availability. The primary goal of this process is the production of acetic acid. However, the accumulation of acetic acid can decrease the extracellular pH, inhibiting AAB growth. AAB produces and releases substantial amounts of gluconic acid to counteract this pH decrease and maintain optimal growth conditions. Gluconic acid is a buffer, helping stabilize the surrounding environment’s pH [12,65]. This pH homeostasis is crucial for sustaining the metabolic activity of AAB and thereby enhancing the efficiency of acetic acid production in industrial and natural fermentation processes.

AAB, recognized for its proficiency in acetic acid production, exhibits varying tolerances to high concentrations of acetic acid. For example, Acetobacter species can endure concentrations ranging from 6% to 10%, while Komagataeibacter strains like K. xylinus and K. hansenii tolerate up to 10% to 15%. K. europaeus demonstrates exceptional tolerance, withstanding concentrations from 15% to 20%. This resilience is supported by specific intracellular proteins that mitigate acid stress [7,63]. Moreover, AAB demonstrates significant resistance to ethanol as well. They efficiently metabolize ethanol up to 15% (v/v), with some strains capable of resisting concentrations up to 25% (v/v). However, beyond this limit, they often lose their characteristic metabolic capabilities [66].

While glucose is the primary substrate for AAB metabolism, these bacteria can also metabolize various other sugars, including arabinose, fructose, galactose, mannose, ribose, sorbose, and xylose [1,67,68]. Notably, most AAB species are characterized by a non-functional glycolysis pathway due to the absence of the enzyme phosphofructokinase [1]. Although AABs do not directly metabolize phenolic compounds, the oxidative and acidic environment they create can alter the concentration and composition of these compounds. Additionally, AAB produces a range of secondary metabolites such as gluconic acid, ketones, aldehydes, and esters (Table 4) [23,67]. These compounds contribute significantly to the complexity and sensory profile of the final Port wine vinegar product.

Furthermore, certain AAB species can produce extracellular polymeric substances (EPS) like bacterial cellulose, levan, dextran, and acetan [7]. These EPS have diverse applications in the food industry, including their use as thickeners, stabilizers, and components in biomedical materials. EPS also plays a crucial role in the survival, persistence, and ecological success of AAB in various environments [7,23,64,69].

In summary, AAB are versatile microorganisms with unique metabolic capabilities that contribute to their industrial and ecological significance, particularly in producing vinegar and other fermented products and in various biotechnological applications.

Table 4. Key metabolites are produced by AAB and LAB during wine oxidative fermentation. Data obtained from [7,10,12,62,70,71,72,73].

Metabolites	Details
Acetaldehyde	Intermediate in acetic acid production, which also affects flavor
Acetic acid	The primary product of ethanol oxidation by AAB, responsible for vinegar’s acidity
Acetoin	Intermediate in butanediol fermentation pathway
Acetone	Ketone produced during fermentation
Bacteriocins	Antimicrobial peptides produced by LAB
Butyric acid	Short-chain fatty acids that may affect the flavor profile
Citric acid	Organic acid contributes to flavor complexity and freshness.
Diacetyl	A by-product that can impart a buttery flavor, contributing to the vinegar’s depth
Ethanol	Residual ethanol from incomplete oxidation
Ethyl acetate	Ester that contributes to fruity and solvent-like aromas.
Formic acid	Minor by-products affecting flavor and acidity.
Gluconic acid	Organic acid formed from glucose oxidation
Lactic acid	Organic acid produced by LAB
Minerals	Micronutrients required for bacterial growth
Polyphenols	Extracted from Port wine, affecting flavor and color
Polysaccharides	Exopolysaccharides contribute to viscosity.
Propionic acid	Organic acid contributes to flavor.
Succinic acid	Depending on its concentration, dicarboxylic acid contributes to acidity, bitterness, and saltiness.
Trace elements	Essential for microbial enzyme function
Vitamins	Essential for bacterial metabolism
Water	Formed alongside acetic acid during oxidation

Table 4. Key metabolites are produced by AAB and LAB during wine oxidative fermentation. Data obtained from [7,10,12,62,70,71,72,73].

Metabolites	Details
Acetaldehyde	Intermediate in acetic acid production, which also affects flavor
Acetic acid	The primary product of ethanol oxidation by AAB, responsible for vinegar’s acidity
Acetoin	Intermediate in butanediol fermentation pathway
Acetone	Ketone produced during fermentation
Bacteriocins	Antimicrobial peptides produced by LAB
Butyric acid	Short-chain fatty acids that may affect the flavor profile
Citric acid	Organic acid contributes to flavor complexity and freshness.
Diacetyl	A by-product that can impart a buttery flavor, contributing to the vinegar’s depth
Ethanol	Residual ethanol from incomplete oxidation
Ethyl acetate	Ester that contributes to fruity and solvent-like aromas.
Formic acid	Minor by-products affecting flavor and acidity.
Gluconic acid	Organic acid formed from glucose oxidation
Lactic acid	Organic acid produced by LAB
Minerals	Micronutrients required for bacterial growth
Polyphenols	Extracted from Port wine, affecting flavor and color
Polysaccharides	Exopolysaccharides contribute to viscosity.
Propionic acid	Organic acid contributes to flavor.
Succinic acid	Depending on its concentration, dicarboxylic acid contributes to acidity, bitterness, and saltiness.
Trace elements	Essential for microbial enzyme function
Vitamins	Essential for bacterial metabolism
Water	Formed alongside acetic acid during oxidation

3.4. Interaction between Yeasts and Acetic Acid Bacteria

In Port wine vinegar production, the interactions between yeasts and acetic acid bacteria (AAB) are pivotal to the final product’s fermentation process and quality. These interactions are symbiotic and competitive, involving complex dynamics influenced by various factors such as osmotic stress, temperature, acetic acid concentration, and nutrient availability [12,64]. Yeasts and AAB often colonize similar environmental niches [74], such as fermenting fruits, wines [75], or vinegar production vessels. These environments provide suitable conditions for both microorganisms to thrive and interact, ensuring efficient utilization of substrates and production of desired fermentation products [10,12].

Yeasts are primarily responsible for the initial fermentation, producing by-products such as ethanol, glycerol, acetaldehyde, and organic acids [76,77]. These by-products can affect AAB growth; however, the release of free amino acids and yeast cell autolysis benefit AAB [78,79]. This symbiotic relationship is advantageous for Port wine vinegar production, creating favorable conditions for the growth of both microorganisms and enhancing fermentation efficiency [12,80].

Considering other types of vinegar, such as vinegar made from non-fortified wines, lactic acid bacteria (LAB) may also be present in small quantities, producing lactic acid and other organic compounds that contribute to the vinegar’s flavor and acidity [12]. LAB can also produce bacteriocins that inhibit the growth of other microorganisms, including AAB [81,82]. While yeasts provide essential nutrients, such as vitamins and amino acids, for AAB and LAB growth, these bacteria can compete for nutrients and oxygen, potentially influencing microbial population dynamics and the overall fermentation process [12,24].

Since acetic fermentation is aerobic, AAB requires oxygen to produce vinegar. Oxygen can be supplied through aeration systems or by direct exposure to air. A lack of oxygen reduces the fermentative capacity for AAB [64,83]. However, excessive oxygen exposure can harm yeast by causing oxidative stress and forming reactive oxygen species, which damage cellular components [84,85]. Osmotic stress from high sugar concentrations also affects yeast ethanol production [86,87].

Temperature is another critical factor influencing microbial interactions in Port wine vinegar production. Low temperatures slow fermentation [88], while high temperatures can promote undesirable microorganisms’ growth, affecting vinegar quality [12,65]. Although high acetic acid concentrations are lethal for yeasts [89], AAB typically requires an ethanol concentration of around 5–7% for effective fermentation. Low ethanol levels can result in slow or incomplete fermentation, while high ethanol levels can be toxic to AAB [7,12].

Nutrient availability is crucial for both yeasts and AAB during fermentation. Yeasts provide essential nutrients, such as vitamins and amino acids, that support AAB growth [78]. However, AAB may face competition for these nutrients, particularly after alcoholic fermentation, when nutrient levels become depleted. Depending on the raw material used, supplementation with amino acids, proteins, minerals, and vitamins may be necessary to optimize fermentation conditions and ensure robust microbial activity [90].

The dynamic interactions between yeasts, AAB, and abiotic factors like oxygen availability, temperature, and nutrient levels are essential for maintaining an efficient fermentation process and producing high-quality Port wine vinegar. Managing these interactions and environmental conditions is critical for optimizing fermentation efficiency and achieving the desired sensory profile of the final product.

Recent insights into AAB reveal their remarkable ability to thrive in ethanol-rich environments, a critical trait for their role in vinegar production [7]. AAB strains exhibit diverse and nuanced tolerance levels to ethanol, with some demonstrating exceptional resilience even in concentrations as high as 25% (v/v) ethanol [66]. This adaptability is underpinned by sophisticated metabolic and genetic mechanisms that enable AAB to metabolize ethanol and maintain productivity efficiently under challenging fermentation conditions.

Mechanistically, AAB employs specialized enzymatic pathways to manage ethanol stress effectively. Key enzymes, like ADH and ALDH, are crucial for ethanol metabolism, maintaining cellular redox balance, and mitigating oxidative stress induced by high ethanol concentrations [7,91]. Genetic diversity among AAB strains contributes significantly to their ethanol tolerance profiles. Variations in genetic makeup, particularly in genes encoding ethanol-metabolizing enzymes and stress response proteins, influence the ability of AAB to adapt to different environmental stresses, such as temperature fluctuations and pH variations [7,91,92]. This genetic diversity may enable AAB to thrive in diverse fermentation conditions encountered during Port wine vinegar production, ensuring consistent fermentation outcomes and high-quality vinegar products.

Moreover, AAB’s adaptive capabilities extend beyond enzymatic and genetic factors. These bacteria exhibit plasticity in membrane composition and transport systems, which play crucial roles in maintaining intracellular homeostasis and protecting against ethanol-induced damage [54,55]. Modifications in membrane lipid composition, including higher phosphatidylcholine content and hopanoids like tetrahydoxybacteriohopane, enhance ethanol tolerance by stabilizing membrane integrity and reducing permeability to toxic compounds [7,63,64].

3.5. Chemical Evolution during Fermentation during Port Wine Processing

Producing Port wine vinegar involves several vital steps that influence its final characteristics. Initially, Port wine is diluted with water to lower its alcohol content, making it suitable for the acetic fermentation process [7]. This process may begin with a rapid acetification method using a Frings acetifier, which efficiently converts alcohol into acetic acid through controlled oxygenation [1]. After the initial fermentation, the wine-vinegar mixture is transferred to a wooden barrel, or “pipo”, where acetification continues. This step is crucial for developing traditional Port wine vinegar’s complex flavors and aromas, as contact with wood imparts unique sensory qualities. Each stage of this process, from dilution to fermentation and aging in wood, affects the sensory profile of the vinegar. Dilution can decrease the concentration of flavor compounds, while the rapid acetification method must be carefully managed to avoid excessive oxygen levels that could alter the vinegar’s taste [12].

Various by-products and compounds are formed and significantly contribute to the unique characteristics of Port wine vinegar, impacting its sensory profile [7]. In the initial phase of wine vinegar production, the by-products produced are directly influenced by the raw material used. Since different types of wines result in vinegar, the final product, while belonging to the same category, differs in its chemical and sensory characteristics. Additionally, ethanol oxidation during the oxidative fermentation phase forms secondary compounds [12], further contributing to the complexity and unique profile of the Port wine vinegar.

Table 2 and Table 4 show that some compounds appear in the alcoholic and oxidative fermentation lists, such as acetic acid, ethanol, ethyl acetate, diacetyl, minerals, and vitamins. This overlap occurs because these substances can be generated through different metabolic pathways or stages of fermentation, contributing distinct characteristics to the final Port wine vinegar.

Ribeiro et al. (2023) [93] conducted a comprehensive study on the volatile composition of fortified grape spirit and Port wine, detailing the various methodologies used to characterize them. The authors report that grape spirit contains 2 alcohols, 9 aldehydes, 8 esters, one phenol, and 3 terpenic compounds. In contrast, Port wine comprises 8 acids, 18 alcohols, 29 aldehydes, 11 dioxanes and dioxalanes, 76 esters, 9 furaldehydes and lactones, 21 ketones, 6 norisoprenoids, 5 phenols, 15 sulfur compounds, 5 terpenic compounds, and 5 other compounds. All 22 volatile compounds in grape spirit are present in Port wine, except formaldehyde. Among these 22 compounds, several can reach concentrations greater than 1000 µg/L in the grape spirit, including 2-phenyl ethanol, acetaldehyde, 2-isopropanol, 2-methyl butanal, 3-methyl butanal, diethyl succinate, ethyl octanoate, and ethyl 3-phenylpropionate. Consequently, these compounds are highly present in Port wine and, by extension, in its vinegar. These compounds contribute distinct aromas: 2-phenyl ethanol provides rose and honey notes; acetaldehyde imparts an overripe apple scent; 2-oxopropanal gives an intense and stinging aroma; 2-methyl-butanal is sharp and pungent; 3-methyl-butanal is powerful and choking; diethyl succinate offers fruity, melon, and yeasty notes; and both ethyl octanoate and ethyl 3-phenylpropanoate contribute sweet, floral, and fruity aromas.

Although Port wine vinegar is a relatively recent product on the market and remains underexplored [7], it possesses a distinctive production process and chemical profile [12]. Despite its uniqueness, this vinegar shares similarities with other vinegar types, especially red wine vinegar. Figure 3 provides a comparative case study of Port wine vinegar, balsamic vinegar, cider vinegar, and Sherry vinegar, which are globally renowned and highly valued in European gastronomy.

4. Sensory Characteristics and Consumer Perception

The hidden world of microbes is pivotal in defining the sensory experiences of various foods and beverages. Whether cooperative or competitive, the interactions among microbes profoundly influence these products’ taste, aroma, texture, and appearance. For producers, a deep understanding of these microbial dynamics is essential to achieve consistent and desirable sensory qualities.

4.1. Influence of Microbial Interactions on Sensory Properties and Chemical-Sensory Correlations

The microbial interaction resulting from the metabolic activity of yeasts and AAB leads to the formation of various compounds that enhance the complexity and depth of sensory attributes in Port wine vinegar [12,23]. These organic compounds contribute to the aroma profile of vinegars, often imparting fruity and floral notes and occasionally buttery aromas.

Esters like ethyl acetate are particularly influential in creating pleasant fruity aromas reminiscent of apples or bananas [38,96]. Higher alcohols, including fuel oils, add depth to the aromatic profile with slightly oily or spicy notes [38,97]. Aldehydes, like acetaldehyde, can provide a fresh, green, and somewhat intense character to the vinegar [98].

The complex Port wine vinegar’s aroma results from the intricate balance between these compounds. Yeasts’ initial alcoholic fermentation produces a variety of primary aromas [99], while AAB’s subsequent oxidative fermentation enhances these with additional layers of aroma. This sequential fermentation process ensures a rich and multi-dimensional aromatic profile.

Furthermore, the specific strains of yeasts and AAB used and the conditions under which fermentation occurs can significantly influence the types and concentrations of aromatic compounds produced. Variations in temperature, oxygen availability, and nutrient levels can lead to different aromatic outcomes, making the control of these parameters vital for achieving desired sensory qualities [12,100].

Aroma is crucial in accepting oenological products such as wine and vinegar and their quality [101]. Consumers often rely on the aromatic profile as a primary indicator of quality and authenticity. Understanding and controlling these microbial processes are essential for vinegar producers aiming to achieve consistent and desirable aromatic attributes. Factors such as the specific strains of yeasts and AAB used, fermentation temperatures, oxygen exposure, and nutrient availability all influence the types and concentrations of aromatic compounds produced. For instance, higher fermentation temperatures may favor the formation of certain esters and alcohols, while oxygen exposure during the oxidative phase enhances the development of complex aromas [2,12,102].

According to Xie et al. (2022) [103], fruit vinegars can contain approximately 160 volatile organic compounds (VOCs), including 25 acids, 22 alcohols, 17 aldehydes, 53 esters, 20 ketones, 10 lactones, 11 phenols, one pyrazine, and one furan. Despite the study focusing on a single wine vinegar, the Sherry vinegar, these findings highlight the complex chemical profile characteristic of fruit vinegars.

To assess the aromatic profile of Port wine vinegar, producers rely on various analytical and sensory evaluation methods [7,104]. Sensory analysis remains a fundamental approach, where trained panels evaluate vinegar samples for aroma intensity, quality, and complexity. These sensory assessments provide valuable qualitative insights into the overall aromatic profile, guiding producers in refining their fermentation processes to achieve specific flavor profiles. The quality of the samples is then tested by a panel of trained tasters [105]. The samples can be tasted directly in a tasting glass or indirectly in a salad (lettuce only) [2]. Various methodologies can be employed; however, the descriptive quantitative analysis test is efficient. This method allows the creation of a sensory profile (aromatic, flavor, and color profile) for the samples under study using a scale of intensities. This comprehensive approach ensures that the sensory characteristics of Port wine vinegar are meticulously evaluated and documented, helping producers enhance their products’ appeal and consistency [105].

Gas Chromatography–Mass Spectrometry (GC-MS) is employed for a more detailed and quantitative analysis. This analytical technique identifies and quantifies individual volatile compounds in vinegar samples, offering a precise chemical profile of their aroma constituents [41]. GC-MS enables producers to monitor the concentration of essential aroma compounds such as ethyl acetate, fuel oils, and various aldehydes [106], which directly influence the sensory perception of the vinegar.

In recent years, electronic nose (e-nose) technology has emerged as a non-invasive and rapid method for assessing aroma profiles. E-noses consist of sensor arrays that mimic human olfaction, detecting and quantifying specific odorants based on their electronic responses. This technology provides a complementary approach to traditional sensory and analytical methods, offering producers additional tools for quality control and flavor optimization [107,108].

Recently, Liu and colleagues [109] studied the aromatic properties of European vinegars using an ultra-fast gas chromatographic electronic nose. This advanced technology combines the principles of GC and an e-nose to analyze volatile compounds quickly. This device is typically used for high-speed detection and identification of complex aromatic profiles. Separation occurs in a small column with faster temperature programming, reducing analysis time while maintaining resolution and sensitivity. Through the e-nose, sensors identify compounds that emit a unique signal for each compound. These signals are translated into advanced algorithms that allow for the identification and quantification of compounds based on their characteristic patterns. This study detected 83 volatile organic compounds (VOCs) in 11 vinegar samples, including one malt vinegar, one lemon vinegar, four wine vinegars, three Italian balsamic vinegars, and two apple vinegars. The detected compounds included 9 alcohols, 32 esters, 12 aldehydes, 5 ketones, and 7 acids. Additionally, the sensory analysis revealed that a rich, decadent aroma characterized wine and cider vinegars, while only the vinegar made from non-fruit raw materials exhibited a more pronounced sourness.

Solid-phase microextraction (SPME) is another technique commonly used with GC-MS. SPME allows for the extraction and concentration of volatile compounds directly from the headspace of vinegar samples, providing insights into the volatile composition and aroma release characteristics [41,110].

Perestrelo et al. (2018) [111] investigated the aromatic profiles of wine-based aromatic vinegars that underwent maceration with banana, passion fruit, apple, and pennyroyal. Using the HS-SPME/GC-MS methodology, they analyzed volatile organic compounds (VOCs) responsible for the distinct aroma of these products compared to a control sample of wine vinegar. The study identified 103 VOCs across the vinegar samples, with 34 compounds detected exclusively in the flavored vinegar and not in control. These included ethyl esters (e.g., ethyl 3-hexanoate) and terpenoids (e.g., limonene oxide, bornyl acetate, menthol), characteristic of the maceration process. This research highlights how adding fruits and herbs during maceration can significantly alter the aromatic profile of wine-based vinegars, introducing new aroma compounds that enhance their sensory appeal and complexity.

In 2019, Ríos-Reina and colleagues [112] conducted a comparative study of the volatile profile of 50 samples of Spanish wine vinegar from three regions with protected designation of origin. The analysis was performed using headspace stir bar sorptive extraction. This method involves the sorption of analytes onto a polydimethylsiloxane (PDMS) film coated onto the magnet of a stir bar named Twister, which is placed in the headspace of a glass vial. This technique offers several advantages, including a low risk of contamination, a high analyte recovery rate, and an increased stir bar and fiber lifetime. The study found that acetates were the predominant group of compounds and identified seven new compounds in this type of vinegar: benzoic acid, methyl benzeneacetate, 6-methyl-5-hepten-2-one, and p-cresol. Between 150 and 160 VOCs were detected across the three samples.

Another crucial parameter assessed in Port wine vinegar sensorial analysis is taste. Taste encompasses the physical sensations in the mouth produced by food or drink, including texture, astringency, viscosity, and overall tactile sensation. Mouthfeel plays a significant role in a product’s overall sensory experience and acceptance [113]. In the case of this agricultural product, the mouthfeel is directly influenced by the acetic acid concentration since higher levels of this acid impact a sharp, pungent sensation, while lower levels contribute to a smoother mouthfeel; the presence of other organic acids, like lactic acid and citric acid, which can influence the overall acidity and tactile sensation [114]; residual sugars, which can enhance viscosity and add a sense of body [115] to the vinegar; and alcohol content, often adding a slight warming effect. Advanced technologies such as the electronic tongue (e-tongue) and high-performance liquid chromatography (HPLC) can be employed to assess and analyze mouthfeel.

The electronic tongue is an innovative device designed to mimic human taste perception, providing objective and reproducible measurements of taste and mouthfeel. It employs an array of sensors that detect various taste attributes, including sourness, bitterness, sweetness, and umami, as well as textural properties [116,117,118].

Besides that, HPLC is an analytical technique used to separate, identify, and quantify individual components in complex mixtures. It is widely used in the food and beverage industry to analyze the chemical composition of products, including Port wine vinegar [41]. A liquid sample is injected into a column packed with a stationary phase. As the sample passes through the column, its components interact with the stationary phase to varying degrees, causing them to elute at different times. These eluted compounds are then detected and quantified by a detector, typically a UV or mass spectrometer [41,119]. Fluorescence spectroscopy is a technique that measures light emission by fluorescent compounds in vinegar. It helps dictate target compounds and provide a more complete sensory profile [41,120].

Finally, flavor is also a crucial parameter assessed in food and beverages on a sensory level. Flavor represents a complex amalgamation of taste, aroma, and mouthfeel experienced during consumption. It encompasses the fundamental tastes perceived by the taste buds (sweet, sour, salty, bitter, and umami) and the intricate interplay of volatile compounds detected by the olfactory system [121,122].

4.2. Consumer Preferences and Acceptance

Consumer preferences are shaped by sensory characteristics, product quality, culinary versatility, origin, production methods, nutritional content, health considerations, and packaging. The integration of sensory analysis and consumer acceptance studies provides invaluable insights into these preferences, guiding producers in developing products that meet market demands and exceed consumer expectations [123,124,125].

In today’s digital age, the success and commercialization of food and beverage products heavily rely on their visibility across digital platforms [126,127]. Social media channels such as TikTok, Instagram, and Spotify, along with advertising and podcasts, play crucial roles in influencing consumer perceptions and purchasing decisions. The impact of influencers and online content creators further amplifies product visibility and consumer engagement.

Moreover, a discernible societal shift towards nutraceutical and probiotic products reflects a growing preference for health-conscious and balanced dietary choices [128,129]. As consumers become more selective, they seek products that fulfill their nutritional needs and resonate with their values [130]. In response to these evolving consumer preferences, producers are encouraged to innovate and adapt their offerings to cater to a more informed and conscientious consumer base [131].

Understanding consumer preferences and acceptance of Port wine vinegar entails considering its distinctive sensory profile, quality standards, and production methods. Derived from fortified wine, Port wine vinegar is prized for its rich and complex aromas, balanced acidity, and distinctive flavor notes that reflect its heritage and aging in oak barrels [7,12]. Consumers who appreciate Port wine typically value its traditional craftsmanship and the assurance provided by adherence to geographical indications or protected designations of origin, which enhances their trust in these products [132]. Furthermore, the rising popularity of artisanal and premium food products aligns with the growing consumer demand for authentic [133], high-quality vinegars like Port wine vinegar.

Wang et al. (2019) [134] conducted a comprehensive review of how both intrinsic sensory factors (such as a product’s color, aroma, texture, and viscosity) and extrinsic sensory factors (including temperature, packaging, and ambient light) influence the perception of sweetness in food and drink. Their framework suggests integrating multisensory cues from within and outside the body to design effective sugar-reduced products while maintaining consumer satisfaction.

Recently, Ker et al. (2023) [135] conducted a comprehensive study comparing the sensory impact of black and balsamic vinegar in traditional Taiwanese-style thick soup. The study involved 189 participants, mostly aged between 20 and 29 years. By using survey questionnaires and experimental methods with purposive and snowball sampling, the study aimed to understand consumer culinary preferences. The findings revealed significant variations in sensory evaluations, encompassing visual, olfactory, and overall satisfaction, between the two types of vinegar. Notably, the study suggests that participants’ habitual taste memories from previous experiences with the traditional soup influenced their preferences for its specific flavor profile. Additionally, the findings indicate that combining different vinegars can influence consumers’ liking levels, as individuals tend to use their preferred condiments to enhance food taste, aligning with their sensory expectations.

These studies show that sensory expectation and memory are pivotal in shaping consumer preferences for specific tastes and food products. Sensory expectation refers to individuals’ anticipatory beliefs or assumptions about how a food or drink will taste based on previous experiences, advertising, or cultural influences. These expectations can significantly influence a product, as can perception and enjoyment [136,137,138]. On the other hand, sensory memory involves recalling past sensory experiences associated with certain foods, which can evoke positive or negative emotions and influence current preferences [139,140]. These factors shape consumer choices and perceptions of food and beverage consumption.

5. Challenges, Future Directions, and Applications

5.1. Current Challenges and Research Opportunities in Port Wine Vinegar Production

Port wine vinegar production faces intricate challenges requiring continuous research and innovation to enhance its quality, sustainability, and market competitiveness. One of the primary challenges lies in optimizing ethanol tolerance within AAB [7]. Since Port wine typically contains around 19% alcohol by volume [12], higher ethanol concentrations can inhibit AAB growth and acetic acid production. This phenomenon poses a risk of functional loss for certain AAB strains essential for vinegar fermentation. Moreover, residual sugars from the initial alcoholic fermentation stage can exacerbate challenges by promoting the overoxidation of ethanol [7,141]. This process increases acetic acid levels and triggers a physiological response in AAB, potentially leading to the degradation of acetic acid into water and carbon dioxide, thereby compromising vinegar quality.

Future research should prioritize investigating genetic and metabolic mechanisms that influence ethanol tolerance in AAB. Understanding these mechanisms could develop more robust AAB strains that thrive in high-alcohol environments without compromising vinegar flavor and stability. Techniques such as genetic engineering or adaptive evolution could be explored to enhance ethanol tolerance and improve the resilience of AAB strains.

Sustainability considerations are increasingly integral to vinegar production practices. This includes exploring eco-friendly vineyard management techniques to minimize chemical inputs and reduce carbon footprints [142,143]. Sustainable fermentation practices, such as energy-efficient processing methods and waste management strategies, aim to mitigate environmental impacts while maintaining product quality and economic viability [144]. Innovations in water conservation, renewable energy utilization, and adopting circular economy principles [145] could further enhance sustainability in vinegar production. Maintaining adherence to stringent quality standards and geographical indications is paramount in preserving the authenticity and market competitiveness [146] of Port wine vinegar. Rigorous quality assurance protocols encompass sensory evaluations, chemical analyses, and microbiological assessments to ensure consistency and excellence in product attributes. Advancements in rapid testing technologies and digital tools for quality monitoring could streamline quality assurance processes and facilitate real-time decision-making in vinegar production.

Looking ahead, the future of Port wine vinegar production will likely be shaped by emerging trends in consumer preferences, technological innovations, and regulatory developments. Collaborative efforts between researchers, producers, and stakeholders will drive innovation and sustainability in the vinegar industry. Interdisciplinary approaches that integrate biotechnological advancements, sustainable practices, and consumer-driven insights will pave the way for enhanced vinegar quality, optimized production efficiency, and expanded applications across culinary, health, and industrial sectors worldwide.

5.2. Pratical Applications

Port wine vinegar, with its rich heritage and distinctive flavor profile [12], finds diverse practical applications across various culinary, health, and industrial sectors. The unique characteristics derived from its fermentation process make it a versatile ingredient prized for its acidity, flavor complexity, and culinary enhancement properties.

In culinary applications, Port wine vinegar is a critical ingredient in vinaigrettes, marinades, sauces, and dressings, where its robust acidity and nuanced flavors complement a wide range of dishes [7]. Its use extends beyond traditional salad dressings to include meat marinades, pickling solutions for vegetables, and reductions in gourmet cuisine. The vinegar’s ability to balance flavors and add depth to dishes makes it a preferred choice among chefs and home cooks seeking to elevate culinary creations with sophistication and depth.

Health-conscious consumers increasingly appreciate Port wine vinegar for its potential health benefits and functional properties. As a fermented product, it contains bioactive compounds such as polyphenols and antioxidants derived from its raw material, Port wine. These compounds contribute to its antioxidant activity and potential health-promoting effects, including anti-inflammatory properties and support for digestive health [5,11]. Incorporating Port wine vinegar into dietary regimens or functional foods aligns with modern dietary trends emphasizing natural, minimally processed ingredients with potential health benefits.

Industrial applications of Port wine vinegar encompass diverse sectors, including food processing, pharmaceuticals, and cosmetics. Its antimicrobial properties, derived from acetic acid and other organic acids produced during fermentation, make it a natural preservative in food products [1,7]. In pharmaceuticals, vinegar extracts may find applications in traditional medicine or as functional ingredients in nutraceuticals targeting metabolic health [147,148,149,150]. Cosmetically, vinegar-based products are used in skincare formulations for their astringent properties and potential benefits in skin pH balance and acne management [151,152].

Moreover, Port wine vinegar production contributes to sustainable practices within the food industry. Producers minimize waste and reduce environmental impact by utilizing winery by-products and adopting eco-friendly fermentation processes. Sustainable vineyard management practices further support biodiversity conservation and soil health [153], aligning with global sustainability goals. Figure 4 illustrates the diverse applications of Port wine vinegar.

6. Final Remarks

Port wine vinegar represents a harmonious blend of tradition and innovation, offering a product that excels in sensory richness and versatility. During fermentation, the intricate microbial interactions between yeast and AAB are crucial in developing its distinctive flavor and aromatic profile. Advanced analytical techniques and sensory evaluations are essential for understanding these properties and meeting the high expectations of both consumers and chefs.

The growing health consciousness and preference for authentic, high-quality products enhance Port wine vinegar’s market appeal. Its unique sensory characteristics and potential health benefits position it favorably in the culinary and functional food sectors. Nonetheless, producers encounter significant challenges, such as optimizing AAB’s ethanol tolerance and adopting sustainable production practices. Addressing these issues through targeted research and technological advancements will be vital for enhancing production efficiency and product quality.

Port wine vinegar’s applications extend across culinary, health, and industrial fields, adding value to gourmet dishes, functional foods, and cosmetic products. The industry’s future will benefit from collaborative efforts among researchers, producers, and stakeholders to drive innovation and sustainability. By focusing on these areas, Port wine vinegar can maintain its status as a premium product with broad global appeal.