Introduction
The Greek feta cheese is a traditional semi-soft white pickled cheese produced from 0-30% goat milk and 70-100% sheep milk or exclusively from sheep milk (1). Since 2002, it has been recognised as a protected designation of origin (PDO) product, in accordance with Commission Regulation (EC) No 1829/2002 (2,3). PDO cheeses ensure the authenticity of tte product linked to its geographical indication where it is produced, processed and developed with recognised expertise characterizing traditional and craft production. Greek feta PDO is a regional product of particular areas of Greece (Macedonia, Epirus, Thessaly and mainland Greece) and is matured in tins or wooden barrels for at least 60 days (4). Although there is a variety of >600 different oak species globally (5), only four have been reported to be suitable for feta PDO cheese production (6). These are Quercus frainetto, Quercus pubescens, Quercus petraea and Quercus robur and in Greece, these are found in Western Macedonia, Thrace, Eurytania and Pindus National Park (7-10). These species are endemic to specific Greek regions and have traditionally been used in the construction of wooden barrels for the maturation of feta PDO cheese. Alongside oak, beech wood is also considered appropriate.
Wood in contact with raw or pasteurised milk and cheese is rapidly covered by a microbial biofilm (11,12). In previous studies, researchers have shown a high biodiversity of microorganisms that are present in biofilms, not only between the different types of cheeses studied, but also within the same cheese variety, between different wooden vats (tina or gerle) used in different cheese plants (13,14). The biodiversity confirmed and demonstrated the strong characterisation of the territorial origin of the products (15-17). In cheese, the correct maintenance of wooden vats promotes the selection of microbial flora, able to play an active role in the achievement of food safety through the biocompetitive activity of lactic acid bacteria (LAB) and their inhibitory activity against pathogenic bacteria, particularly Listeria monocytogenes (13,18-20). Greek dairies have traditionally produced feta PDO cheese, using equipment consisting of wooden utensils and wooden barrels of a capacity of up to 60 kg (6) (as illustrated in Fig. 1).
According to the guidelines of the Hellenic Food Safety Authority (EFET) (21), and Hazard Analysis and Critical Control Point (HACCP) principles for the traditional small dairy production units in Greece (22), the wooden barrel can be used among other wooden utensils, to mature traditional feta PDO and white cheese, such as barrel-aged goat cheese. Their porous structure supports beneficial microflora, while their use enhances the texture and flavour through the natural maturation process (23-25). On the other hand, the use of traditional and reusable equipment, particularly in small and medium-sized enterprises producing barrel-aged feta PDO (2), requires special attention concerning the implementation of critical control points (CCPs), that are designed to reduce microbiological (M), chemical (C) and physical (P) risks in line with HACCP guidelines. These CCPs and the related Operational Prerequisite Programs (OPRPs) are outlined throughout the production routine, as illustrated in the flowchart of the feta PDO cheese production (Fig. 2) (26). Some studies have demonstrated that the wooden vats used for raw milk cheese production allow for the development of a biofilm on the inner surface, which actively participates in the production process by adding unique sensory characteristics to the traditional products, such as aroma, appearance, taste and texture, which is associated with the total viable count of microorganisms, mesophilic LAB, lactic cocci/streptococci and yeast/moulds (11,12,27,28).
The present study aimed to monitor the microbiological parameters during all the stages of the ripening period of the Fagus sylvatica (beech) barrel-aged feta PDO cheese (2), to enhance the established control according to the methodology that complies with food safety in the final product.
Discussion
Greek feta PDO cheese is a national treasure, representing 90% of the total cheese production of the country (44). The essential ingredients for its production include milk, salt, rennet and microorganisms (46). The matrix of dairy products facilitates microorganism proliferation, such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Brucella abortus, Campylobacter jejuni, Salmonella, Yersinia enterocolitica, and several others, compromising the public health status (26,47).
Traceability plays a key role in food chain management, particularly in the case of rapidly deteriorating products, such as milk and cheese (48). In this context, traceability has become a requirement for a better understanding of the life cycle of products and conscientious delight. Thus, the major concern of the dairy companies is the respect of the traditional practices through a certified quality control system according to ISO 22000:2005 and implementing a HACCP daily workflow, illustrated by Casino et al (49), and in the workflow of Fig. 2, to guarantee excellent quality.
Food safety is an increasingly critical public health concern, and the proposed traceability procedure enhances the improved process control for managing any future product recalls and/or product quality. Microbial food contamination in dairy plants is of utmost significance for the monitoring of food quality and safety (6,50). For instance, it is known that Listeria monocytogenes has the potential to survive at low temperatures (0˚C), low pH values (pH 4.4) and in salt media with 10-20% (w/v) of NaCl (28), conditions that are close to the physicochemical characteristics of feta cheese. For the purpose of the present study, further investigated the food safety of beech barrel-aged Feta PDO, 2.5% (w/w) NaCl, following the monitoring of pH and through the in-depth microbiological analysis across the time-points of the production workflow (Fig. 2).
Previous studies describe that the microbial ecology found in cheese-making establishments exhibits a great differentiation in composition, regarding taxonomy classification, due to the processing procedures (51-53). The conditions that favour microbiome settlement are several and are associated with the age, conservation, quality and nature of the wooden vats. Additionally, the presence of microbial ecosystems and environmental conditioning (54), such as temperature, humidity, or pH, markedly affects the selective survival of the microbiome (55). Humidity, cleaning protocols, nutrient availability and composition also have a regulatory impact on the ability of microbes to develop biofilms. The availability of nutrients in wooden barrels can promote biofilm development. However, this effect is modulated by key environmental factors, such as salinity, moisture and pH, which suppress the the proliferation of pathogenic microorganisms even in nutrient-rich conditions. This specific microenvironment favours the development of stable, functional biofilms that enhance the microbial stability and hygiene of the final product (56). In particular, the limited moisture, regulated by the high salinity of the brine, combined with the acidic pH (~4.4), creates a selective ecosystem. Within this environment, the growth of pathogens, such as Listeria monocytogenes, Staphylococcus aureus and Salmonella spp. is inhibited, while microorganisms naturally adapted to high-salt and low-pH conditions are favoured. These salt- and acid-tolerant microbiota include lactic acid bacteria such as Lactobacillus spp., as well as probiotic strains such as Lactobacillus paracasei and Bifidobacterium spp., which form resilient, protective biofilms (17). In the present study, the aggregation of microorganisms referred to as ‘biofilm’ formed in the wooden barrels used in the production of feta PDO cheese is characterised by minimal variation in TMC, with a quantitative change of <5% following 150 days of ripening. Additionally, the structural and chemical properties of beech wood exert further selective pressure. Cleaning protocols effectively eliminate pathogens while preserving these non-pathogenic, functionally advantageous biofilms. Nutrient limitation during advanced ripening likely restricts microbial overgrowth, supporting a balanced beneficial microbiota and ensuring the microbiological safety and quality of the final product (57-59). However, in the case of feta PDO production, which has always been an artisanal process, the improvements through empirical trials have emerged long before the current scientific knowledge of these fermentations.
The establishment of the LAB protects against spoilage bacteria through differential colonisation capability (60), which creates food bio-protection conditions, providing antifungal and antimicrobial activity. This could be explained through mechanisms of competition for nutrients (61). In the case of the traditional art that characterises feta PDO aged in a wooden barrel, surface spoilage is subject to the irregularity of the surface material, which represents a cardinal issue for compliance with standard hygiene regulations in food processing plants (50). In the present study, it was demonstrated that the traditional practice for the production of feta PDO cheese using wooden-beech barrels for cheese ageing met the standards of Good Manufacturing Practice (GΜP) (62), including the appropriate practices of sanitation to ensure food safety.
Wood, due to its organic properties, particularly its porosity and hygroscopic nature, is a renewable natural resource that allows a progressive inlet of oxygen into the barrel and attributes a sharp flavour to the cheese, but also a slightly harder texture (23,54,60). Previous studies have demonstrated that some commonly used wood species have antimicrobial activities and are suitable for food contact surfaces (63,64). Wooden barrels remain an indispensable requirement characterising the traditional practice of barrel-aged feta PDO. This peculiar maturing process could significantly influence the future and the perspective of this iconic Greek product.
Specifically, the use of beech barrels (Fagus sylvatica) allows for controlled micro-oxygenation, as wood enables oxygen to reach the inside of feta cheese as it matures. This oxygen interaction enhances the flavour, contributes to the development of a sharp, piquant and distinct sensory profile, but also defines the texture of the cheese. These organoleptic characteristics are associated to the presence of wood-derived compounds, such as phenolics, for example, syringyl derivatives, and mild, insignificant tree lactones (65-67), as well as tannins, that exert antimicrobial efficacy and structural resilience in brine (68). This maturation technique highlights the artisanal and terroir-specific identity of feta, affirming its value as a PDO product.
Additionally, wooden barrels used in the production of feta PDO support sustainability goals, as they are biodegradable and eco-friendly. Their local sourcing supports circular production and promotes the cultural legacy of cheese, which in turn fosters the rural economy and helps prevent land abandonment. Furthermore, the implementation of forest certification protocols could help prevent and mitigate the decline of tree species commonly used for timber of beech (Fagus spp.) populations in Mediterranean regions, while contributing to improved stand structure by protecting mature trees and promoting natural regeneration (69).
Consequently, the long-term commitment to using beech wooden barrels in the production of feta PDO cheese (8,70,71) ensures not only the preservation of the cultural and nutritional heritage, but also highlights the use of sustainable forest management (72,73). This contributes to establishing the continuity and the viability of agricultural livelihoods in Greece.
Despite its advantages, wooden barrel sanitisation poses challenges for product safety, as it carries the risk of microbial contamination due to the porous nature of the wood (74). Nevertheless, forward-looking strategies could further develop Greek feta PDO cheese, ensuring product safety and enhancing its nutritional profile, with the potential to qualify as a functional food through targeted fortification and the integration of adjunct co-culture strains exhibiting probiotic and technological potential (28,75-76). These strategies may not only satisfy consumer expectations for authenticity and safety, but may also boost the market prospects of traditional cheeses, particularly feta PDO cheese.
Although the specifications for Greek feta PDO clearly define the origin of the milk, they do not impose specific requirements on the type of wood used for barrels, since the regulation does not include this parameter (2). For example, chestnut (Castanea sativa) has been recommended for fresh cheeses due to its antioxidant properties and its capacity to enhance product stability as well as the shelf life in other agro-industrial by-products (56,77). Likewise, sweet cherry (Prunus avium L.) is known for its high phenolic content, contributing to organoleptic, antioxidant, and anti-hyperglycemic benefits (78,79). At the same time, walnut (Juglans regia L.) has been incorporated into functional cheese fortification, as seen in the case of Beyaz cheese (78,79). Despite these favourable attributes, none of these woods aligns with the cultural and historical framework that governs the production of feta PDO.
Although oak is beneficial for cheese ageing, the porosity of the wood, creates an organic wicking effect that favours the conditions of drying and ageing of cheese, because it sustains bacterial growth and the development of biofilms. This is beneficial, as the prevalence of desirable organisms that are immobilised within the thick layer of polysaccharides, promotes the contribution of flavour and positive organoleptic characteristics to the Feta PDO cheese. Specifically, lactococci and lactobacilli are critical as they contribute to the cheese acidification and flavour formation of the feta cheese (23).
To better illustrate how exogenous or endogenous environmental factors impact microbial populations during feta PDO cheese ripening, Table IV summarises the selective pressure acting on several bacterial species, deduced from both our observations and the literature evidence. The perspective is that characterisation and mapping of the microbiome of feta PDO cheese may become a valuable tool for the monitoring of environmental contamination, to improve the quality control in the dairy plants, particularly under routine and experimental conditions such as salinity, moisture, temperature and pH adjustments. Therefore, understanding the interactions between feta cheese and selected bacterial flora could be meaningful for feta PDO cheese manufacturing of a standard quality (50) and variability of organoleptic features. Moreover, the growth of pathogens and the risk of their survival is a fundamental issue and needs to be addressed by validated and frequently monitored good sanitation practices.
In conclusion, the present study provides a comprehensive assessment of the microbiological safety of handcrafted feta PDO cheese aged in wooden barrels, in accordance with current food safety regulations in Greece. The results confirm that the traditional maturation process using beech barrels, when accompanied by proper cleaning protocols inhibit the presence of key foodborne pathogens, including Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus. In particular, to the best of our knowledge, the present study is the first to formally document and evaluate under microbiological criteria the traditional cleaning method of hot whey rinsing during the ripening of Greek artisanal feta PDO cheesemaking in Fagus sylvatica barrels, demonstrating that it contributes to the effective hygienic maintenance of wooden barrels used in the maturation process.
The observed TMC in biofilm samples revealed minimal fluctuations (≤5%) over a 150-day ripening period, suggesting a stable microbial ecosystem. The dominance of beneficial LAB, fostered by selective environmental pressures such as low pH and high salinity, supports the formation of functional, protective biofilms that contribute to both microbial safety and flavour development. While the complexity and variability of the cheese microbiota pose challenges for standardisation, they also highlight the unique sensory identity of feta PDO. In particular, the presence of beneficial microbial communities within the beech wood-associated biofilm contributes to both microbial stability and the organoleptic profile of the cheese during ripening. Future studies utilizing advanced molecular approaches, such as next generation sequencing (NGS) and metagenomic analysis, will be essential for elucidating the role of the microbiome in ripening dynamics, biofilm formation, and sensory development.
Overall, the present study reinforces the compatibility of traditional wooden-barrel aging practices with modern food safety requirements and highlights the potential of microbial mapping as a future tool for quality control and process optimisation in artisanal dairy production. Furthermore, the presence of beneficial microbial populations, particularly probiotic strains, provides a promising basis for the development of functional feta cheese with potential health-promoting properties.