Biodiversity from Traditional Bulgarian and French Foods: Microbial and Plant Resources for Innovative Fermentation Processes

1. Introduction

Fermentation is one of the oldest biotechnological practices used by humans to transform and/or preserve food. In modern science, fermented foods are defined as foods and beverages obtained through the controlled growth of selected microorganisms – bacteria, yeasts, fungi, which often form complex and dynamic microbial communities. Their metabolic activity determines the acidification, the formation of aromatic compounds, proteolysis, lipolysis, the change of texture and the microbiological stability of the final product [1,2].

Traditional fermented foods represent a particularly valuable source of microbial biodiversity. In contrast to standardized industrial production, which usually uses a limited number of selected starter cultures, artisanal and home technologies are often based on spontaneous fermentation, back sloping or the use of natural starter cultures. As a result, locally adapted microbial populations accumulate in these foods, thus making them a valuable source of biodiversity. These microorganisms may possess technologically relevant and/or probiotic properties, such as resistance to low pH, salt, ethanol and temperature stress, production of organic acids, exopolysaccharides, aromatic substances, enzymes and antimicrobial compounds [2,3].

Bulgaria has a significant variety of traditional fermented products, including yogurt, white brine cheese, yellow cheese, katak, kefir-like products, boza, fermented vegetables, meat products and breads obtained with natural starter cultures. Bulgarian yogurt is obtained as a result of the symbiotic interaction between Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, but in home and artisanal production, additional representatives of the genera Lactobacillus sensu lato, Lactococcus, Leuconostoc and other microorganisms may be present [4]. Studies on traditional Bulgarian dairy products have shown that the isolated strains exhibit significant phenotypic and functional diversity, including different proteolytic activity, ability to form antimicrobial metabolites, resistance to gastrointestinal conditions and other potential probiotic properties [4,5,6].

France also has a deep-rooted tradition in the production of fermented foods, including raw milk cheeses, sourdough bread, wine, cider, fermented meat products and vegetables. Particularly characteristic of French products is the relationship between microbial biodiversity, geographical origin and the concept of terroir. In raw milk cheeses, the microbiota is formed under the influence of the animal species, the animal diet, the composition of the pasture vegetation, milking practices, the production environment and the ripening conditions. This complex microbiota actively participates in the formation of the typical taste, aroma and textural characteristics of traditional cheeses [7]. A large study of French milks and cheeses with a protected designation of origin shows that geographical and production factors shape specific bacterial and fungal profiles, with a significant part of the fungal diversity of the cheese being inherited from the milk used [3,8,9].

Aromatic and medicinal plants are an essential part of the traditional food systems of Bulgaria and France. They are used not only to shape the characteristic taste and aroma of foods, but also to improve their stability during storage. Before the introduction of modern preservation methods, the addition of thyme, savory, oregano, rosemary, sage, bay leaf, tarragon and other plants to meat, dairy, vegetable and grain products provided additional protection against oxidative and microbiological processes. Today, this traditional practice represents a scientific basis for the development of natural preservatives and “clean label” technologies. The technological value of herbs and spices is determined by their complex physicochemical composition. Aromatic plants of the Lamiaceae family, which includes thyme, oregano, savory, rosemary, sage, mint and marjoram, contain volatile and non-volatile bioactive substances that can modify the food matrix and give it certain functional properties. The ratio between the individual components depends on the plant species, genotype and chemotype, geographical origin, climatic and soil conditions, developmental stage, plant part used, drying method and extraction method. Therefore, plants of the same species can show significant differences in both chemical profile and antimicrobial activity. This variability is particularly important in the development of natural preservatives, since the use of the common botanical name is not sufficient to guarantee a consistent technological effect [10,11,12,13,14].

The aim of this study is to investigate the microbial diversity of microorganisms isolated from traditional French and Bulgarian fermented foods and beverages, as well as to select suitable plant raw materials (herbs, aromatic and medicinal plants) that can serve as a source of innovation in the development of so-called tailor-made foods. Other potential benefits of the study are the possibility of applying the concept of biopreservation in the development of new foods and beverages.

2. Bulgarian and French Traditional Fermented Foods as Sources of Microbial Biodiversity

2.1. Traditional French Fermented Products

France has a long-standing tradition of fermented foods and beverages, deeply rooted in regional agricultural systems, artisanal practices, and the concept of terroir. These products—particularly cheeses, sourdough breads, and fermented drinks—are characterized by complex microbial systems dominated by lactic acid bacteria (LAB), yeasts, and molds, which collectively determine their sensory, nutritional, and functional properties.

2.1. Traditional French Cheeses

French cheeses represent one of the most diverse fermented food categories globally, with over 1,000 varieties, many protected under Protected Designation of Origin (PDO) schemes. These cheeses are produced from cow, goat, or sheep milk and involve intricate fermentation and ripening processes driven by complex microbial consortia. LAB, including Lactococcus, Lactobacillus, and Leuconostoc species, play a primary role in early acidification, while non-starter LAB (NSLAB), yeasts, and surface-ripening bacteria contribute to maturation and flavor development [14].

Recent large-scale studies of French PDO cheeses have demonstrated that microbial composition is strongly influenced by geographical origin, milk type, and cheesemaking practices, supporting the concept of microbial terroir. These microbial communities are highly dynamic and include both dominant and subdominant taxa, with the latter contributing significantly to metabolic interactions and flavor complexity [8].

During cheese ripening, LAB and associated microorganisms drive proteolysis, lipolysis, and amino acid catabolism, leading to the formation of characteristic flavor compounds such as aldehydes, sulfur compounds, and short-chain fatty acids. This biochemical diversity gives rise to the wide range of textures and flavors found in French cheeses such as Camembert (soft, surface-ripened), Comté (hard, cooked-curd), and Roquefort (blue-veined) [15].

2.1.2. Sourdoughs and Fermented Cereal Products

Sourdough bread is another key component of French culinary heritage, particularly in artisanal bakeries. Sourdough fermentation involves the spontaneous or controlled growth of LAB and yeasts in a flour–water matrix, forming a stable symbiotic ecosystem. LAB species such as Fructilactobacillus sanfranciscensis and Lactiplantibacillus plantarum are frequently dominant, contributing to acidification, flavor, and shelf-life improvement [16].

The microbial diversity of sourdoughs is influenced by environmental conditions, flour type, and bakery-specific microbiota, resulting in unique, location-specific fermentations even when similar raw materials are used. LAB-mediated fermentation enhances the nutritional quality of bread by reducing phytate content, improving mineral bioavailability, and generating bioactive peptides and organic acids. In France, traditional sourdough breads such as pain au levain are valued for their sensory qualities (acidic flavor, complex aroma) and extended shelf life compared to yeast-leavened bread. These characteristics are primarily attributed to LAB metabolic activity, including the production of lactic and acetic acids and volatile compounds [16,17].

2.1.3. Fermented Beverages

French fermentation traditions also encompass a wide array of beverages, including wine, cider, and certain artisanal products like kefir and kombucha (more recently adopted). Among these, wine is the most emblematic, involving both alcoholic fermentation by yeasts (Saccharomyces cerevisiae) and malolactic fermentation (MLF) carried out by LAB, particularly Oenococcus oeni. Malolactic fermentation is a crucial step in winemaking, converting malic acid into lactic acid and carbon dioxide, thereby reducing acidity, stabilizing the product, and enhancing sensory complexity through the formation of flavor compounds such as diacetyl. The microbial composition of wines is shaped by vineyard microbiota, grape variety, and cellar practices, further reinforcing the concept of terroir in French fermented beverages. Cider production in regions such as Normandy and Brittany similarly relies on spontaneous fermentation, involving mixed populations of yeasts and LAB. These microorganisms contribute to acidification, flavor development, and product stability [18].

2.1.4. Microbial Ecology and Functional Relevance

Across French fermented foods, a common feature is the reliance on complex microbial ecosystems rather than single-strain fermentations. These ecosystems involve cooperative and competitive interactions among microorganisms, shaping the structure and function of the fermentation process. LAB play a central role by producing organic acids, antimicrobial compounds, and metabolites that enhance safety, shelf life, and nutritional quality [18,19,20,21].

2.2. Traditional Bulgarian Fermented Products

Traditional Bulgarian fermented foods represent complex microbial ecosystems, formed over generations under the influence of local raw materials, climatic conditions and specific home and artisanal production practices. Bulgarian fermented milk products, pickles, cereal drinks and sourdoughs can be considered as natural reservoirs of strains for the development of starter and protective cultures, functional foods and new fermentation technologies [1,2].

2.2.1. Dairy Products

The most recognizable Bulgarian fermented product is yogurt, whose classic microbial association includes Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. A protocooperative interaction occurs between the two species: S. thermophilus supports the initial acidification and forms metabolites that stimulate lactobacilli, while the proteolytic activity of L. delbrueckii subsp. bulgaricus releases peptides and amino acids necessary for the growth and development of streptococci. This interaction determines the rate of fermentation, the consistency and the characteristic aroma of the product [4,22]. In homemade yogurt and other traditional dairy products, the microbial composition is often more diverse than the regulatory starter combination. In white brine cheese, yellow cheese, katyk, kefir, cottage cheese and Rhodope skimmed milk, representatives of the genera Lactobacillus sensu lato, Lactococcus, Leuconostoc, Enterococcus, Streptococcus and Pediococcus have been identified, as well as various yeasts. The specific composition depends on the type of milk, the starter culture used, the salt concentration, the fermentation conditions, the temperature and the duration of maturation [1].

2.2.2. Pickles and Other Fermented Vegetables

Traditional Bulgarian pickles include sauerkraut, pickled cucumbers, fermented green tomatoes, peppers, cauliflower and mixed vegetable products. In most cases, fermentation occurs spontaneously in the presence of a saline solution and is initiated by microorganisms found on the surface of the vegetables, in the water used, the containers and the production environment. Under the influence of salt, anaerobic conditions and the gradual decrease in pH, microbial succession occurs, in which lactic acid bacteria gradually dominate over the unwanted microbiota. Among the bacteria characteristic of plant fermentations are Leuconostoc mesenteroides, Lactiplantibacillus plantarum, Levilactobacillus brevis, Pediococcus pentosaceus, Limosilactobacillus fermentum and other species adapted to acidic and salty environments. Heterofermentative bacteria usually participate in the early stages and form lactic and acetic acid, ethanol, carbon dioxide and aromatic substances. As the process progresses, the more acid-resistant lactobacilli become a dominant part of the community and contribute to the final stabilization of the product. When studying sauerkraut, fermented green tomatoes and pickled cucumbers, prepared according to traditional Bulgarian recipes, various lactic acid bacteria with different abilities for autoaggregation, coaggregation, biofilm formation and antagonistic activity against indicator pathogenic microorganisms were isolated. However, the microbial composition of pickles is highly dependent on salt concentration, temperature, vegetable to brine ratio, and the use of aged fermentation broth. It is this variability that is valuable from a biodiversity perspective, but also requires a thorough assessment of the safety, technological stability, and behavior of individual isolates through the conduction of controlled fermentations [2].

2.2.3. Traditional Fermented Beverages

The most characteristic Bulgarian fermented grain beverage is boza. It is produced from thermally processed grain raw materials, most often wheat, rye, millet or their mixtures, followed by combined lactic acid and alcoholic fermentation. The microbial ecosystem of boza includes lactic acid bacteria and yeasts, whose interactions determine acidification, the formation of carbon dioxide, small amounts of ethanol and the characteristic aromatic profile. When studying unpasteurized Bulgarian boza using cultural and molecular methods, Limosilactobacillus fermentum, Lentilactobacillus parabuchneri, Lentilactobacillus buchneri, Loigolactobacillus coryniformis, Pediococcus parvulus and representatives of the Lacticaseibacillus casei group were identified. Not only species, but also high intraspecific genetic diversity was established, which confirms boza as a rich source of potentially applicable cultures. Of particular interest are amylolytic lactic acid bacteria, capable of degrading starch and directly utilizing the resulting sugars. Strains with significant amylolytic activity have been isolated from Bulgarian boza, which creates opportunities for conducting fermentations of grain substrates with a reduced need for preliminary enzymatic hydrolysis [6].

The specific composition of the microbiota in boza can be divided into the following key groups:

• LAB: These microorganisms are dominant (usually reaching between 10⁵ and 10⁹ CFU/ml) and are responsible for fermentation. They regulate the acidity of the beverage (lowering the pH to around 3.5) and attribute beneficial probiotic properties [23,24,25,26,27]
• Yeast: These microorganisms aid in the fermentation process, impart the characteristic flavor of boza, and produce small amounts of ethanol (usually between 0.01% and 0.8%) and carbon dioxide, which account for the beverage's slight carbonation [23]

This symbiosis makes boza a functional food, as the live cultures in it support the intestinal microflora and the absorption of nutrients. Because these microorganisms are alive and active, unpasteurized boza has a short shelf life and must be stored in a refrigerator [23,24,25].

Boza was studied as a natural source of lactic acid bacteria and yeasts suitable for the development of symbiotic starter cultures [28,29]. Among the isolated microorganisms, the LAB strains Lactiplantibacillus plantarum BG24 and Lactiplantibacillus plantarum BG25 were selected, as well as the yeast strains Saccharomyces cerevisiae var. diastaticus 25-G and Saccharomyces cerevisiae 36-6G. L. plantarum BG24 was selected due to its established probiotic properties, its resistance under model conditions of the gastrointestinal tract and its amylolytic and proteolytic activity. The yeast strain Saccharomyces cerevisiae var. diastaticus 25-G was selected for its ability to participate in the fermentation of grain substrates. With the participation of these two strains, fermented functional drinks based on millet were developed. The resulting drinks combined the lactic acid fermentation carried out by the probiotic bacterial strain with the fermentation and aroma-forming activity of the yeast strain. Thus, the possibility of using the autochthonous microflora of boza to produce new non-dairy functional beverages from grain raw materials has been demonstrated [28,29]

2.2.4. Sourdough

A systematic approach was applied to develop starter cultures for controlled fermentation of sourdoughs, based on the selection of lactic acid bacteria isolated from spontaneously fermented grain substrates [30]. The isolated strains were comprehensively evaluated in terms of their fermentation activity, acidification rate, enzyme profile, antimicrobial activity, adaptability to different flour matrices and their compatibility in mixed microbial associations [31,32]. The strains Lactiplantibacillus plantarum Ph2, Levilactobacillus brevis X4, Lacticaseibacillus rhamnosus LBRC11, Limosilactobacillus fermentum LBRH10 and Fructilactobacillus sanfranciscensis R were selected as promising technological cultures. The strain Propionibacterium freudenreichii subsp. shermanii NBIMCC 327 was also used in the development of the symbiotic starter cultures. A basis multistrain starter containing L. plantarum Ph2, L. brevis X4, L. rhamnosus LBRC11 and L. fermentum LBRH10 in a ratio of 2:1:1:1, was developed. This combination has been upgraded by including F. sanfranciscensis R, and in the most complex version – also P. freudenreichii subsp. shermanii NBIMCC 327, in order to form a sustainable symbiotic microbial system with complementary metabolic and technological properties. The developed starter cultures were applied to produce sourdoughs from wheat, rye, spelled and spelt flour, and the influence of the flour type, the fermentation duration and the amount of sourdough added on the bread quality was studied. It was found that the addition of usually 7–10% 96-hour sourdough improved the volume, shape stability, structure and aroma profile of wheat, wheat-rye, spelled and spelt sourdough breads compared to the respective controls. The results obtained show that the selected autochthonous lactic acid bacteria can be used not only for standardization and reproducibility of fermentation, but also for the development of functional bakery products with improved sensory characteristics, increased microbiological stability and potentially extended shelf life without the use of chemical preservatives [33,34].

3. Lactic Acid Bacteria and Yeast Diversity

3.1. Lactic Acid Bacteria

Lactic acid bacteria (LAB) constitute a phylogenetically diverse group of Gram-positive, low-GC bacteria that include genera such as Lactobacillus sensu lato, Lactococcus, Streptococcus, Leuconostoc, Pediococcus, and Enterococcus. These taxa are central to spontaneous and controlled fermentations of dairy, cereal, vegetable, and meat products in both Bulgarian and French tradition [35,36].

Traditional fermented foods represent complex microbial ecosystems shaped by raw materials, geography, and artisanal practices. In Bulgarian products such as yogurt, white-brined cheese, kefir, and sourdough, LAB biodiversity is especially rich due to the preservation of autochthonous microflora through artisanal production processes. Studies employing 16S rRNA gene sequencing and culture-dependent methods have identified dominant species such as Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus helveticus, Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Levilactobacillus brevis, Lentilactobacillus hilgardii, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides, Pediococcus pentosaceus, Pediococcus acidilactici, Enterococcus faecium, and Enterococcus durans. Bulgarian fermented vegetables and dairy products have additionally yielded less commonly reported taxa, including Levilactobacillus koreensis and Levilactobacillus yonginensis. In traditional Bulgarian sourdoughs, Lactiplantibacillus plantarum, Levilactobacillus brevis, Pediococcus pentosaceus, and Pediococcus acidilactici have been reported among the most prevalent culturable LAB, whereas metagenomic studies have demonstrated an even broader diversity of lactobacilli and related genera [4,37].

Similarly, French traditional foods—including raw milk cheeses (e.g., Comté, Roquefort), sourdough breads, and fermented vegetables—harbor complex LAB consortia. Although industrialization has standardized starter cultures, artisanal production still favors strain-level diversity, often revealed through molecular approaches such as metagenomics, multilocus sequence typing (MLST), and whole-genome sequencing (WGS). These tools enable the identification of strain-specific functional genes involved in carbohydrate metabolism, proteolysis, and stress adaptation, providing a link between genetic diversity and technological performance [38].

Advances in omics technologies have significantly enhanced the resolution of LAB diversity analysis. Comparative genomics has uncovered horizontal gene transfer events and niche adaptation mechanisms, including genes encoding carbohydrate-active enzymes and stress-response proteins. These findings underscore the importance of preserving traditional microbial biodiversity as a reservoir for innovation in functional fermented foods.

LAB contribute to food fermentation through a broad spectrum of metabolic activities that directly affect product quality, safety, and functionality. The core metabolic trait is the conversion of carbohydrates into lactic acid via homo- or heterofermentative pathways, leading to pH reduction and inhibition of spoilage and pathogenic microorganisms [35].

Beyond acidification, LAB possesses diverse enzymatic systems that generate key organoleptic compounds. Proteolytic and lipolytic activities release peptides, amino acids, and fatty acids, which serve as precursors for volatile flavor compounds such as aldehydes, esters, ketones, and sulfur-containing molecules. These biochemical transformations are essential in traditional cheeses and fermented cereals, contributing to terroir-specific sensory profiles in Bulgarian and French products [39].

Another critical functional trait is the production of exopolysaccharides (EPS), which enhance texture, viscosity, and mouthfeel in fermented dairy products while also contributing to prebiotic effects. Furthermore, bacteriocin synthesis (e.g., nisin, plantaricin) provides a natural biopreservation mechanism by inhibiting closely related or pathogenic bacteria, thereby extending shelf life and improving food safety [40].

LAB from traditional Bulgarian foods have demonstrated strong probiotic and bioprotective potential. Depending on the strain, reported and documented properties include survival under simulated gastro-intestinal conditions, auto-aggregation and co-aggregation, adhesion to intestinal epithelial cell lines or mucus, and the production of antimicrobial metabolites. Their proven antagonistic activity has been associated with the production of a number of metabolites such as lactic and acetic acids, hydrogen peroxide, bacteriocins and bacteriocin-like peptides, as well as with competition for nutrients and adhesion sites. Bulgarian food-derived LAB have shown inhibitory activity against many important foodborne and opportunistic pathogens, including Escherichia coli, Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Helicobacter pylori. Antagonistic effects against spoilage microorganisms and fungal contaminants, including Candida albicans, Aspergillus sp., and Penicillium sp., have also been reported for selected LAB isolates. Together with their potential antioxidant, immunomodulatory, and barrier-supporting activities, these characteristics make autochthonous LAB promising candidates for the development of functional and naturally biopreserved foods that are also intended to support gastro-intestinal health. It should be stressed that these properties are highly strain-dependent and should be confirmed through comprehensive safety assessment, genomic characterisation, and appropriately designed in vivo and clinical studies before specific health benefits are claimed [37,41].

In the context of process innovation, the exploitation of strain-specific functional traits—such as stress tolerance, rapid acidification, or flavor-forming capacity—enables the design of tailored starter or adjunct cultures. This is particularly relevant for valorizing underutilized raw materials (e.g., cereals, vegetables) in sustainable fermentation processes, aligning with the growing demand for clean-label and functional foods.

The use of LAB in food fermentation is generally recognized as safe due to their long history of consumption. However, safety assessment remains a critical step, especially when introducing new strains isolated from traditional foods into industrial applications. Key safety criteria include the absence of virulence factors, hemolytic activity, and transferable antibiotic resistance genes. Phenotypic and genotypic analyses are routinely employed to ensure compliance with safety standards [37].

In the European context, regulatory frameworks such as the European Food Safety Authority (EFSA) Qualified Presumption of Safety (QPS) list provide guidelines for the safe use of microbial strains in food and feed. Most LAB species commonly used in fermentation (e.g., Lactococcus lactis, Lactiplantibacillus plantarum) are included in the QPS list, facilitating their approval for industrial use. However, strain-level evaluation is still required for novel isolates, particularly those derived from raw or minimally processed materials.

Another important consideration is the control of biogenic amine production (e.g., histamine, tyramine), which can occur in certain LAB strains and pose health risks. Screening for decarboxylase genes and monitoring metabolite production are essential to mitigate these risks.

Last but not least, the increasing interest in functional fermented foods necessitates compliance with health claim regulations. In the European Union, any probiotic or functional claim must be supported by robust clinical evidence demonstrating specific health benefits. This presents both a challenge and an opportunity for leveraging the biodiversity of LAB from traditional Bulgarian and French foods to develop scientifically validated, innovative fermented products.

3.2. Yeasts

Bulgarian and French traditional fermented matrices harbor a wide diversity of yeast species. Among the most frequently reported species are the genera Saccharomyces, Kluyveromyces, Debaryomyces, Candida, Pichia, Hanseniaspora, and Torulaspora [42,43,44,45]. In dairy fermentations such as traditional Bulgarian fermented milks or French raw milk cheeses, yeasts such as Kluyveromyces marxianus, Pichia fermentans, Debaryomyces hansenii, and Candida kefyr are commonly detected [46]. These species are able to metabolize lactose or lactate, contributing to microbial succession and stabilization of the fermentation ecosystem [47]. In cereal-based fermentations, including French sourdough, yeasts such as S. cerevisiae and Kazachstania humilis play key roles in carbohydrate metabolism and carbon dioxide production, which contributes to dough leavening and structure formation [48]. The French bakers’ sourdough contains yeast strains that have never been found elsewhere, such as K. bozae, K. australis, and K. saulgeensis. In fermented beverages such as wines or traditional fruit-based drinks, S. cerevisiae remains the dominant fermentative yeast due to its high ethanol tolerance and efficient sugar metabolism. However, non-Saccharomyces yeasts often dominate the early stages of fermentation and contribute to flavor complexity.

Beyond their role in fermentation kinetics, yeasts significantly contribute to the technological and sensory characteristics of fermented foods. S. cerevisiae, Candida and Geotrichum present in Bulgarian cereal-based beverage boza and other cereal-based beverages contribute to mild alcoholic notes, effervescence, and matrix acidification in mixed fermentation [44]. In cheese ripening, D. hansenii and G. candidum participate in surface microbiota development, influencing deacidification, proteolysis, and lipolysis processes [49]. These biochemical transformations contribute to the production of volatile compounds involved in the typical flavors of cheese. Moreover, interactions between yeasts and LAB may enhance fermentation efficiency, as yeasts can release amino acids, vitamins, and other growth factors that stimulate bacterial growth.

Recent research has highlighted the potential of non-conventional yeasts as drivers of innovation in food fermentation. Species such as Torulaspora delbrueckii, Metschnikowia pulcherrima, and Lachancea thermotolerans exhibit unique metabolic pathways that can enhance aroma complexity, modulate acidity, or reduce ethanol production in fermented beverages [50]. In addition, some yeast strains produce antimicrobial compounds or killer toxins, which may contribute to natural biopreservation strategies in fermented foods [51].

Representative yeast species and their technological roles in Bulgarian and French fermented foods are summarized in Table 1. The exploration of yeast biodiversity from traditional Bulgarian and French foods therefore represents a promising strategy for identifying novel starter cultures with desirable technological and functional properties.

4. Traditional Bulgarian and French Herbs and Spices as a Source of Natural Preservatives

4.1. Bulgarian Herbs and Spices

Among the most characteristic spices in the Bulgarian food tradition is the garden savory – Satureja hortensis L. It is used in the production and preparation of meat products, legumes and vegetable foods, cheeses, snacks and traditional mixed spices. The savory essential oil is usually rich in carvacrol or thymol, and γ-terpinene and p-cymene may be present in significant quantities. In the oils studied, carvacrol reaches approximately 67%, followed by γ-terpinene and p-cymene, while in other chemotypes, thymol is the main phenolic component. This indicates significant intraspecific chemical variability, which should be taken into account when selecting raw materials for food applications. The essential oil of S. hortensis exhibits activity against a number of gram-positive and gram-negative bacteria, yeasts and filamentous fungi. Among the sensitive microorganisms are representatives of the genera Staphylococcus, Bacillus, Listeria, Escherichia, Salmonella, Pseudomonas, Enterococcus and Candida [55,56].

Oregano – Origanum vulgare L. – is also widely distributed in Bulgaria and is used in meat, vegetable, legume and pasta foods. Its essential oil contains carvacrol, thymol, sabinene hydrate or other chemotype. In addition to the volatile fraction, oregano contains rosmarinic and caffeic acids, flavonoids and tannins, which contribute to its antioxidant properties [12,57]. Oregano oil inhibits the growth of food pathogens and spoilage agents, including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella sp., Bacillus cereus, yeasts and fungi. Carvacrol and thymol may act together with their biosynthetic precursors p-cymene and γ-terpinene. p-cymene itself usually exhibits weaker antimicrobial activity, but can facilitate the penetration of phenolic components by expanding and destabilizing the cell membrane [10,11].

Thyme, represented in Bulgaria by Thymus vulgaris L. and various wild species of the genus Thymus, is traditionally used to flavor meat, soups, bean and vegetable dishes, as well as for the preparation of herbal drinks. The main components of the essential oil can be thymol, carvacrol, linalool, geraniol, terpinen-4-ol or 1,8-cineole depending on the chemotype. Rosmarinic and caffeic acids, flavonoids and other polyphenolic compounds are found in non-volatile extracts. Thyme is a promising raw material for the preservation of meat and meat products, cheeses, sauces, bakery products and fresh fruits and vegetables. Its extracts can simultaneously limit microbial growth and slow down lipid oxidation. This dual effect is particularly valuable in products with a high content of unsaturated lipids [13].

Other plants typical for Bulgaria are spearmint and mint, belonging to the genus Mentha. Their essential oils contain different ratios of menthol, menthone, carvone, pulegone, 1,8-cineole and other terpenoids. Spearmint is traditionally used in bean, rice and meat foods, while mint is more often used in beverages, confectionery and herbal mixtures. Mentha oils and extracts exhibit antibacterial and antifungal activity, but are usually weaker than those rich in carvacrol and thymol. Due to their strong aroma and more moderate activity, their application as stand-alone preservatives may be limited, but they are suitable for inclusion in combined systems and for products in which the mint aroma is technologically and sensorially acceptable [11,12].

Dill – Anethum graveolens L. – is a traditional spice for Bulgarian fermented milk, vegetable, fish and pickled products. The fruits and aerial parts differ in composition. The oil from the fruits is usually rich in carvone and limonene, while the oil from the leaves may be dominated by α-phellandrene, limonene and ethers, characteristic of the fresh aroma. Dill exhibits moderate antimicrobial activity and can contribute to the stability of marinades, sauces and fermented vegetables, especially when combined with organic acids, salt or other plant components [11,12].

4.2. French Herbs and Spices

The French culinary tradition, especially that of Provence and Mediterranean France, makes extensive use of thyme, rosemary, savory, oregano, marjoram, sage, lavender, tarragon and bay leaf. Some of these plants are combined in traditional mixtures such as herbes de Provence and bouquet garni. Their role is mainly aromatic, but their physicochemical composition also suggests the possibility of limiting microbiological and oxidative changes in foods.

Rosemary – Salvia rosmarinus Spenn., synonym Rosmarinus officinalis L. – is one of the best-studied Mediterranean raw materials for natural preservation. Its volatile fraction usually contains 1,8-cineole, α-pinene, camphor, borneol, verbenone and camphene. However, a significant part of its technological value is due to non-volatile phenolic compounds, primarily carnosic acid, carnosol and rosmarinic acid. Rosemary extracts are particularly suitable for stabilizing meat, sausages, fats, oils and emulsion products. In addition to limiting lipid oxidation, they can inhibit some gram-positive bacteria, including Listeria monocytogenes, Staphylococcus aureus and members of the genus Bacillus [58,59].

Sage – Salvia officinalis L. – is traditionally used in France in meat products, poultry products, sauces, cheeses and dishes with high fat content. The main components of its essential oil are α- and β-thujone, camphor, 1,8-cineole, borneol, camphene and α-pinene, the ratios between which vary significantly according to the origin and chemotype [60]. Sage essential oil and extracts show activity against a number of bacteria and fungi, including S. aureus, L. monocytogenes, B. cereus, E. coli, Salmonella sp. and Candida albicans [60,61].

French tarragon – Artemisia dracunculus L. – is a characteristic spice for sauces, vinegars, marinades, fish, poultry and dairy products. Its specific aroma is mainly due to estragole, also called methylchavicol, as well as ocimene, sabinene, limonene and other volatile compounds. Depending on the chemotype and geographical origin, the composition can vary significantly. Extracts and essential oil of tarragon exhibit antibacterial and antifungal activity, including against S. aureus, E. coli and some fungi [62,63].

Bay leaf – Laurus nobilis L. – is used in both French and Bulgarian cuisine in meat and vegetable products, sauces, soups and marinades. Its essential oil contains mainly 1,8-cineole, sabinene, α-pinene, β-pinene, linalool, terpinen-4-ol and in some cases eugenol and methyleugenol. Bay leaf oil exhibits activity against bacteria, yeasts, and molds, but the strength of the effect depends on the chemotype and concentration. Due to its moderate activity and characteristic aroma, bay leaf is more suitable for combination with organic acids, heat treatment, cooling, or other plant extracts [10,11,63].

4.3. Mechanisms of Antimicrobial Action and Potential for Biological Preservation

The antimicrobial action of herbs and spices is not due to a single universal mechanism. The hydrophobic components of essential oils accumulate in cell membranes and can change their structure. The consequences include increased permeability, loss of potassium ions, nucleotides and other cellular components, imbalance of intracellular pH and disruption of membrane-bound enzyme systems. Phenolic components such as thymol and carvacrol can act as proton carriers and cause the collapse of the proton motive force. Other substances interact with cellular proteins, enzymes, nucleic acids or cell communication and biofilm formation systems [10,11].

However, the results obtained in vitro cannot be directly transferred to real foods. Fats and proteins can bind hydrophobic components and reduce their free concentration, and carbohydrates, salt, water activity and pH also modify their effectiveness. Therefore, the concentrations required to inhibit microorganisms in food are often higher than the minimum inhibitory concentrations determined in laboratory culture media [10,11,53].

To overcome these limitations, plant extracts and essential oils can be used as part of a combined preservation system. Promising combinations include reduced pH, organic acids, moderate heat treatment, refrigeration, modified atmosphere, bacterial protective cultures, bacteriocins and lowering of water activity. Combining different oils or individual components can also lead to additive or synergistic effects and allow the use of lower concentrations.

The use of traditional mixtures, for example combinations of savory, oregano and thyme or of rosemary, sage and thyme, is particularly promising. These mixtures contain phenolic monoterpenes, terpenoid oxides, alcohols and phenolic acids with different mechanisms of action. It is possible that the less active compounds increase membrane permeability and facilitate the action of carvacrol and thymol, while the phenolic diterpenes and acids provide additional antioxidant protection. This approach is in line with the traditional culinary use of herbal mixtures and could provide a more balanced aroma compared to a high dose of a single essential oil.

In the context of Bulgarian-French plant biodiversity, savory, oregano and thyme can be considered as major sources of carvacrol and thymol, while rosemary and sage are valuable primarily for their combination of volatile terpenoids and non-volatile phenolic antioxidants. Tarragon, lavender, mint, fennel and bay leaf offer different aromatic and antimicrobial profiles that can be used in specific food matrices. Comparative study of plants from both countries would allow the identification of geographical and chemotypic differences, selection of the most active populations, and development of standardized extracts or herbal combinations.

5. Process Innovation and Development of Functional Fermented Foods Based of Biodiversity

The EU’s Food 2030 initiative sets out the main guidelines for the current programming period, emphasizing the transformation of food systems into sustainable, healthy and inclusive ones. The main priorities set out in Food 2030 are: sustainable and healthy nutrition (improvement of nutrition; improvement of authenticity and safety systems); development of smart food systems; ensuring sustainable food systems that support biodiversity; application of the principles of the circular economy and minimal food processing; promotion of the application of innovative ecosystems and others [64]. As can be seen, ensuring biodiversity, for example by using new species and strains of microorganisms or including plant raw materials unique to a given country in food production, falls within the framework of the EU’s main priorities. The second main element of the Food 2030 initiative is the implementation of the principles of sustainability in the production of innovative, functional and healthy foods, oriented through tailor-made principles to different consumer groups. The sustainability of food systems, including these tailor-made foods, is promoted in a number of documents. Green Deal – EU’s Circular Economy Action Plan promotes the development of sustainable food systems, including through the use of waste raw materials [65]. The Farm to Fork Strategy relies on sustainable practices in the production of raw materials and food [66]. Horizon Europe which is the key EU research and innovation funding program, allocates significant resources to projects that develop sustainable, functional and healthy foods, funding research on alternative proteins, new technologies for food processing and fortification of foods with essential nutrients [67].

The production of fermented foods is in compliance with the principles of sustainability, while at the same time allowing to obtain products with increased nutritional value, improving the bioavailability of nutrients, providing accessible probiotic microorganisms. From an economic point of view, fermentation is a cost-effective method of preservation. Traditionally produced fermented foods worldwide are still the largest source of biodiversity, especially in terms of the unique species and strains of microorganisms isolated from them. The principles of sustainability can be effectively applied to the isolation, characterization and selection of microorganisms from traditional foods. The use of traditional fermented foods as a source of new species and strains of microorganisms continues to be the basis for the development of tailor-made foods, while also supporting the conservation of biodiversity and the preservation of cultural values [68,69,70,71].

The growing trend towards the development of new food products that do not use chemical preservatives, imposed by the legislation on the labelling of organic products (EC n°834/2007 in EU) or the market trends for the so-called “clean label”, has stimulated researchers to look for and develop new production and preservation methods. The consumption of ready-to-eat foods that are fresh and minimally processed leads to the emergence of new ecological pathways for application of selected microbial growth. This is necessitated from the fact that consumers demand the development of new, healthy, environmentally friendly production concepts that are based on natural components. Biodiversity in the plant world has always been a source of valuable components in food production. Plant extracts and/or essential oils are a valuable source of both biological value (in the form of providing antioxidants) and antimicrobial components that can replace traditional chemical preservatives. The successful combination of plant extracts and/or essential oils with microbial fermentation is a valuable strategy in the production of new tailor-made foods and beverages [71,72,73,74,75,76,77].

In the context of the Bulgarian-French partnership, the project “The synergistic action of essential oils and microorganisms in the development of functional fermented foods (SEO-MICRO-FOODS)” is being developed. It is focused on conducting scientific research on the synergistic action of plant extracts and/or essential oils and newly isolated and selected microorganisms (bacteria and yeasts) in food matrices, typical for Bulgaria and France, guaranteeing sustainability and diversity. The project will combine the efforts of teams from Bulgaria and France working in the fields of minimally processed food production, based on the use of fermented foods traditional for Bulgaria and France (yogurt, bread, boza, fermented beverages, meat products, etc.) and plant raw materials typical for both countries, which provide biological activity through specially prepared extracts and/or essential oils.

5.1. Microbial Fermentation as a Method for Providing Biological Value to Foods

Microbial fermentation is one of the main methods for both preserving foods and providing their biological value. Microbial fermentation often serves to improve the bioavailability of nutrients in the food matrix, increasing the protein and amino acid content of the matrix. Fermentation extends the shelf life of foods due to the produced and accumulated organic acids (such as lactic acid) and alcohol, which inhibit the growth of pathogenic and saprophytic microorganisms. Many fermented foods contain live beneficial bacteria (probiotics) that can colonize the intestines and restore and maintain the balance of the gastro-intestinal microflora [77,78,79,80,81]. This aids digestion, improves nutrient absorption and may improve the functioning of the immune system. Fermented foods can also be a source of prebiotics [78].

5.2. The Synergistic Effect of Plant Extracts and Essential Oils to Increase the Biological Value of Foods

The technological value of plant extracts is mainly associated with their complex composition of phenolic compounds, flavonoids, tannins, terpenoids, saponins, and alkaloids, which may exhibit antioxidant, antibacterial, and antifungal activity [81,82,83,84,85,86,87,88]. Essential oils additionally contain bioactive volatile compounds such as limonene, β-caryophyllene, α- and β-pinene, α- and γ-terpinene, sabinene, β-myrcene, cinnamyl alcohol, δ-3-carene, p-cymene, thymol, and carvacrol, many of which contribute to their antimicrobial effects [81,82,83,92,93,94,95,96,97,98]. Their application is particularly relevant in view of concerns associated with the excessive use of some conventional chemical preservatives [81,89,90,91,92].

The practical use of essential oils as effective antimicrobial agents in the food sector is still questionable due to their volatile components, hydrophobicity and low stability. Currently, methods for their encapsulation are being sought in practice in order to increase their stability and reduce harmful effects on the organoleptic profile of foods [81,100]. Another effective method for reducing the impact on the organoleptic properties of foods is their combination with another type of preservative agent/process – for example, microbial fermentation. As a result of such a combination, a significant synergistic effect can be achieved in food preservation and the growth of pathogenic and saprophytic microorganisms can be inhibited [79,80,81,100,101].

The incorporation of plant extracts and essential oils into fermented and probiotic foods represents a promising strategy for simultaneously improving product functionality, microbiological stability, and shelf life. In combination with selected probiotic cultures, these natural ingredients may contribute to biopreservation by inhibiting undesirable microorganisms, thereby reducing the need for synthetic preservatives [80]. Such functional foods can be developed through targeted fermentation, the addition of bioactive ingredients in forms compatible with the food matrix, and the selection of lactic acid bacteria capable of maintaining or enhancing the biological activity of plant-derived compounds during processing [80].

The combined use of plant-derived bioactive compounds and beneficial microorganisms may provide complementary or synergistic effects. While plant extracts can directly inhibit pathogens and spoilage microorganisms, fermentation may modify their composition, release bound phenolic compounds, or increase the bioavailability of selected constituents. At the same time, interactions with the food matrix may protect both microbial cells and plant bioactives during processing and storage. In addition to their preservative and functional roles, appropriately selected extracts may improve flavour, aroma, and overall sensory quality [55]. However, their use must be carefully optimized because high concentrations may adversely affect probiotic viability or produce excessive bitterness, pungency, or herbal notes. The practical application of these ingredients is facilitated by the fact that many plant extracts and essential oils, or their individual constituents, are permitted for food use and some have GRAS status [98].

Providing a synergistic effect in the application of plant extracts/essential oils and microbial fermentation with lactic acid bacteria is a relatively new strategy for the preservation of different types of food matrices. In fact, the combination of essential oils and lactic acid bacteria can reduce the concentration of oils required to achieve a sufficient inhibitory effect on the growth of pathogens and saprophytes, and both bioagents can have a synergistic effect [82,102,103,104,105]. The creation of an appropriate combination of lactic acid bacteria/essential oil should be sought not only in terms of the achieved biopreservative effect, but also in terms of preserving and improving the organoleptic qualities of the finished product.

5.3. “Non-Conventional” Yeasts as an Opportunity to Obtain New Types of Foods and Beverages with Increased Biological Potential

The fundamental role of yeasts in the production of a number of fermented foods and beverages is well known. The most significant share is still held by Saccharomyces cerevisiae, but increasing attention has recently been directed toward non-conventional yeasts as a source of new technological and functional properties. This group includes yeast species that are not traditionally used as standard starter cultures but possess distinctive metabolic capabilities that may be exploited in food biotechnology. The significant interest in unconventional yeasts is due to the more diverse genome, leading to different metabolic pathways and therefore the production of foods and beverages with a richer metabolic profile [105]. Their broader range of metabolic pathways may enable the utilization of alternative substrates and the production of organic acids, higher alcohols, esters, vitamins, polysaccharides, pigments, extracellular enzymes, and other biologically active metabolites. Consequently, their application may contribute to the development of fermented foods and beverages with more complex aroma profiles, modified nutritional composition, enhanced antioxidant activity, reduced alcohol content, and improved functional properties [104].

S. cerevisiae, lactic acid bacteria, or other suitable microorganisms in mixed or sequential fermentations. This makes it possible to combine the metabolic properties of different strains and, to some extent, to control the composition and characteristics of the final product. The outcome depends strongly on both the strain and the food matrix, but such fermentations can lead to a more complex flavour profile, support the release or transformation of phenolic compounds, increase the formation of certain bioactive metabolites, and improve microbial stability.

Cereal-based substrates are especially suitable for this type of application. They are rich in nutrients, easy to adapt technologically, and already form the basis of many traditional fermented foods and beverages in Europe. Brewing wort is a particularly useful model matrix because it contains fermentable sugars, amino acids, minerals, vitamins, and phenolic compounds that support the growth of different yeast species. By selecting appropriate non-conventional strains, it is possible to use wort for the production of low- and non-alcoholic beverages, functional cereal-based drinks, and beer-like products with new sensory characteristics and potentially higher biological value.

At the same time, the use of non-conventional yeasts requires careful evaluation. Their fermentation behaviour, safety, genetic stability, sensory impact, and ability to grow in the selected matrix should all be assessed at strain level. It is also important to monitor the possible formation of undesirable metabolites and to consider the relevant food safety and regulatory requirements. With proper strain selection and process control, non-conventional yeasts can provide a useful basis for developing a wider range of fermented foods and beverages with improved nutritional, functional, and sensory properties [105].

5.4. Contribution to Sustainable and Circular Food Systems

5.4.1. Valorization of Local Raw Materials

The development of functional fermented foods plays a pivotal role in advancing sustainable and circular food systems by enabling the valorization of local raw materials while simultaneously reducing waste and improving resource efficiency. Fermentation, as a low-input biotechnological process, aligns closely with circular bioeconomy principles, transforming underutilized biomass into value-added products and minimizing environmental impacts across the agri-food chain [105,106].

The use of locally available agricultural raw materials—including cereals, legumes, fruits, and traditional crops—represents a cornerstone of sustainable fermented food production. Fermentation enhances the nutritional, safety, and sensory properties of these substrates, allowing for their effective utilization even when they are otherwise underexploited or unsuitable for direct consumption [107]. This is particularly evident in traditional fermentation systems, where indigenous microorganisms convert region-specific raw materials into culturally important and nutritionally improved foods.

Local sourcing of substrates reduces dependency on global supply chains, thereby decreasing transportation-related emissions and fostering regional food resilience. Moreover, strengthening local food systems has been identified as a key pathway toward sustainability, improving economic viability for producers and supporting environmentally responsible practices [108]. Fermentation technologies further enhance this approach by enabling the conversion of seasonal or surplus local produce into stable, functional foods with extended shelf life and added health benefits.

Importantly, microbial fermentation enhances the bioavailability of nutrients and the production of bioactive compounds, including peptides, vitamins, and polyphenols, from local raw materials. This contributes to the development of functional fermented foods that meet both nutritional and health-promoting criteria while maintaining a low environmental footprint [109].

A defining contribution of fermented food processing to circular food systems lies in its capacity to valorize food waste and by-products. Significant volumes of residues generated along the food supply chain—including peels, pomace, spent grains, and whey—are rich in nutrients and represent a largely untapped resource [110]. Fermentation processes can convert these residues into high-value ingredients such as organic acids, enzymes, microbial proteins, and functional metabolites, effectively transforming “waste into wealth.” Recent reviews highlight fermentation as a key mechanism for integrating waste streams into circular bioeconomy models, where resource recycling and waste minimization are central objectives [105]. For instance, fruit and vegetable by-products, dairy residues, and cereal-processing wastes have been successfully fermented to yield nutraceutical compounds and bioactive ingredients with applications in functional foods and dietary supplements [111].

In addition, fermentation significantly reduces the environmental burden associated with waste disposal. Conventional disposal methods, such as landfilling, contribute to greenhouse gas emissions and environmental pollution, whereas biotechnological conversion via fermentation provides a sustainable alternative for waste management [112]. By diverting organic residues from waste streams and reintegrating them into the food system, fermentation contributes directly to zero-waste strategies and sustainable resource use [106].

5.4.2. Resource Efficiency and Circular Bioprocessing

Fermentation-based technologies enhance resource efficiency by optimizing the use of raw materials, water, and energy. Compared to many conventional food processing methods, fermentation is typically a low-energy process that relies on microbial metabolism rather than intensive physical or chemical treatments [107]. This intrinsic efficiency makes it particularly suitable for sustainable food production systems. Emerging technologies such as solid-state fermentation (SSF) further exemplify resource-efficient processing. SSF utilizes low-moisture substrates, often derived from agro-industrial residues, resulting in significantly reduced water consumption and improved process yields. It has been reported that SSF can reduce water usage by up to 70–90% compared to submerged fermentation while achieving high volumetric productivity [113]. Furthermore, SSF facilitates the direct conversion of solid wastes into value-added compounds, minimizing preprocessing requirements and associated energy inputs. The integration of fermentation into biorefinery frameworks also enhances overall system efficiency. Agro-food by-products can be sequentially processed to recover multiple valuable components, thereby maximizing the utility of each resource stream and reducing losses [106]. Such integrated approaches contribute to improved material circularity and economic viability within the agri-food sector.

5.4.3. Implications for Circular Functional Food Development

The convergence of fermentation technology with circular economy principles opens new pathways for the development of functional foods. By utilizing local raw materials and valorizing food processing residues, producers can create innovative products enriched with bioactive compounds while minimizing environmental impacts. These strategies also support the diversification of ingredients and the development of novel functional formulations based on non-conventional substrates. Moreover, fermentation contributes to sustainable food system transformation by enabling the production of functional ingredients without reliance on synthetic additives or resource-intensive inputs [109]. This aligns with consumer demand for natural, eco-friendly, and health-promoting foods.

6. Conclusions

Traditional Bulgarian and French fermented foods are valuable reservoirs of microbial biodiversity, while local herbs and spices provide a rich source of antimicrobial and antioxidant compounds. The biodiversity preserved in traditional foods should be viewed not only as part of cultural heritage, but also as an important resource for future food innovation. Together, these resources offer strong potential for the development of new fermented foods with improved safety, sensory quality, and biological value.

The most promising approach is the targeted combination of selected lactic acid bacteria, yeasts, plant extracts, and essential oils. Such systems may support biopreservation, improve fermentation performance, and reduce the need for synthetic additives and preservatives. Non-conventional yeasts are particularly relevant because of their ability to diversify aroma, modify product composition, and contribute to the development of low-alcohol and functional beverages.

These opportunities are at the core of the Bulgarian–French project SEO-MICRO-FOODS, which focuses on identifying compatible combinations of autochtonous bioprotective microorganisms and plant-derived extracts rich in bioactive compounds in real food matrices. By linking traditional biodiversity with modern fermentation technologies, the project may provide practical solutions for producing safer, more natural, and more sustainable foods and beverages.