From Raw to Fermented: Uncovering the Microbial Wealth of Dairy

Abstract

Dairy products harbor complex and dynamic microbial communities that contribute to their sensory properties, safety, and cultural distinctiveness. Raw milk contains a diverse microbiota shaped by seasonality, storage conditions, lactation stage, animal health, farm management, and genetics, serving as a variable starting point for further processing. Fermentation, whether spontaneous or starter driven, selects for subsets of lactic acid bacteria (LAB), yeasts, and molds, resulting in microbial succession that underpins both artisanal and industrial products such as kefir and cheese. Kefir represents a balanced LAB–yeast symbiosis, with species composition influenced by grain origin, milk type, and processing parameters, whereas the cheese microbiota reflects the interplay of starter and non-starter LAB, coagulants, ripening conditions, and “house microbiota”. Methodological factors—including DNA extraction, sequencing platform, and bioinformatic pipelines—further impact the reported microbial profiles, highlighting the need for standardization across studies. This review synthesizes current knowledge on raw milk, kefir, and cheese microbiomes, emphasizing the biological, technological, environmental, and methodological factors shaping microbial diversity. A holistic understanding of these drivers is essential to preserve product authenticity, ensure safety, and harness microbial resources for innovation in dairy biotechnology.

1. Introduction

Microbial communities are integral to the quality, safety, and cultural heritage of dairy products. From raw milk to complex fermented foods such as kefir and cheese, microorganisms play a decisive role in shaping sensory attributes, extending shelf life, and contributing to product identity. Advances in culture-independent methods have revealed that the microbial ecosystems of dairy are far more diverse than previously appreciated, uncovering both abundant taxa and rare organisms with potential technological or probiotic significance.

Raw milk constitutes the initial microbial reservoir, reflecting animal health, farm management practices, environmental exposures, and handling conditions. Its microbiota is highly dynamic, influenced by seasonality, storage conditions, and lactation stage, with psychrotrophic bacteria often dominating under cold storage, while LAB and aroma-associated species prevail in warmer conditions. Once milk enters fermentation, microbial succession reduces initial diversity but enriches for acid-tolerant LAB and yeasts that drive acidification, flavor formation, and textural changes.

Kefir exemplifies a natural symbiotic ecosystem, where kefir grains composed of LAB, yeasts, and exopolysaccharide matrices maintain structural stability and microbial homeostasis. Microbial composition differs between grains and the beverage, and it varies across regions, milk types, and processing strategies. Cheese microbiota, by contrast, is one of the most complex among fermented foods, shaped by multiple interacting parameters including milk treatment, coagulant type, ripening environment, rind management, and local “house microbiota”.

Despite these biological and technological influences, methodological variation remains a major confounding factor in dairy microbiome research. DNA extraction techniques, target gene selection, sequencing platforms, and data analysis approaches can all alter observed community profiles, complicating cross-study comparisons. Recognizing these limitations is essential for accurate interpretation of microbial diversity and for developing standardized frameworks that link microbial ecology to sensory quality, safety, and geographical authenticity.

This review offers a novel perspective by bringing together three cornerstone dairy products, raw milk, kefir, and cheese, within a single comparative framework. While previous reviews have tended to focus on one product category in isolation, here, we systematically analyze these products side by side, allowing common drivers and distinct features of their microbiota to be identified. In addition, we emphasize methodological aspects, highlighting how differences in culture-based, molecular, and multi-omics approaches affect reported microbial diversity. By explicitly linking biological and technological factors with methodological biases, this review provides an integrative framework that not only refines our understanding of dairy microbial ecology but also offers practical implications for product quality, safety, and industrial applications.

2. Microbial Composition of Raw Milk

Raw milk from healthy animals contains a complex microbial community originating from the animal, its environment, and processing practices. While culture-based techniques remain the traditional standard for analyzing the microbiology of milk and dairy products, rapid progress in omics-driven methodologies, including metagenomics, transcriptomics, and proteomics, have expanded the use of sequencing approaches for exploring the microbiota of raw milk and milk products. Such strategies reduce biases inherent in traditional methods and provide deeper insights into microbial diversity, functional potential, and ecological dynamics over time. Recent advances in these technologies have shifted research from solely profiling microbial community structures to revealing how individual strains influence the sensory and technological properties of milk and dairy products and to identifying rare taxa that may play site-specific roles in production systems [1]. For instance, Debaryomyces hansenii has been shown to produce branched-chain aldehydes such as 2-methylpropanal and 3-methylbutanal on cheese surfaces, generating nutty and malty flavor notes that directly influence sensory quality [2]. Likewise, Kluyveromyces marxianus contributes to fruity aroma development through the production of esters, highlighting its technological importance in fermented dairy products [3].

A review of the literature on raw milk microbiota reveals substantial differences in the diversity and dominant species reported across studies. This variation can primarily be attributed to numerous environmental and biological factors that shape the microbiota, including seasonal changes, storage conditions, stage of lactation, animal health, farm management, and geographical differences. Seasonality has been identified as a key determinant of microbial diversity in many studies. For example, a study conducted in Italy reported that bioprotective species such as Lactobacillus and Lactococcus were predominant during the summer months, whereas psychrotrophic bacteria (e.g., Pseudomonas, Enterobacteriaceae) dominated in winter [4]. Similarly, long-term observations in Poland indicated that microbial diversity increased in summer and autumn but declined in winter and spring [5]. Extensive field studies in Ireland further demonstrated a direct correlation between climatic parameters (temperature, rainfall, sunlight duration) and microbial diversity [6].

Storage temperature and duration represent critical factors influencing raw milk microbiota. For instance, psychrotrophic bacteria often dominate under prolonged refrigeration, while different bacterial groups emerge depending on storage time and handling conditions. In a German study, psychrotrophic bacteria such as Pseudomonas and Acinetobacter became dominant after three to four days of storage, contributing to spoilage through proteolytic and lipolytic activities [7]. Similarly, research conducted in China showed that Pseudomonas species predominated with extended storage, followed by the emergence of Carnobacterium, Serratia, and Lactococcus [8,9]. A Spanish study also reported increased dominance of Pseudomonas between days 11 and 21, while LAB species became prevalent during longer storage periods [10]. Furthermore, a study from Korea revealed that storage temperature played a critical role, with psychrotrophic bacteria dominating at 4 °C, whereas LAB species prevailed at 25 °C [11]. On the other hand, research from Portugal demonstrated that hyperbaric storage significantly extended milk shelf life by suppressing microbial diversity [12]. The stage of lactation is also a biological factor that shapes microbial diversity.

In a study conducted in the United States on goat milk, members of Proteobacteria were reported to be predominant during early and mid-lactation, whereas Actinobacteria and species such as Micrococcus became more dominant in late lactation [13]. Similarly, research carried out in Ireland found that Bacteroides and Faecalibacterium were enriched in mid-lactation, while members of Actinobacteria prevailed in late lactation [14]. Another study from China demonstrated that differences across lactation stages were shaped through the connections between rumen microbiota and milk metabolites [15]. Moreover, distinct microbiota profiles were observed in transgenic goats producing lysozyme, highlighting the influence of host genetics [13].

Animal health is also among the factors influencing microbiota diversity. A study from Italy reported that cases of subclinical mastitis caused by Streptococcus agalactiae or Prototheca infections led to a marked microbial shift and reduced diversity compared to healthy milk samples [16]. Likewise, in goat milk, samples obtained from mastitic animals were dominated by Staphylococcus, Streptococcus, and Trueperella species, whereas a higher microbial diversity was observed in healthy animals [17].

Farm management practices, sanitation standards, and milking methods collectively determine the microbial composition of raw milk. Factors such as housing conditions, equipment cleaning, teat preparation, and overall hygiene operate in combination, shaping microbial diversity at both the farm and processing levels. A large-scale study on bulk tank milk in the United States revealed that microbial profiles shifted rapidly during transfer to the processing facility, with Acinetobacter in particular becoming dominant [18]. Research conducted in Norway reported substantial differences at the farm level, noting that even samples collected on the same day could exhibit distinct microbiota compositions [19]. A Canada-based review further demonstrated that milking hygiene, housing conditions, and tank sanitation directly shape the microbiota [20]. In addition, a study carried out in Italy suggested that each farm harbors a unique “microbial barcode”, which could be used for traceability within geographical indication systems [21]. Practical application of a microbial barcode concept could involve using product-specific microbial signatures as authenticity and traceability markers. For example, artisanal cheeses often harbor facility-specific ‘house microbiota’ that can be profiled and stored as a microbial fingerprint. Such barcodes could then be used to verify product origin, detect adulteration, and support quality control systems, in parallel with existing chemical and molecular traceability methods.

Host genetics also contributes to shaping raw milk microbiota. Comparative studies have shown that dairy cow breeds differ in microbial composition, partly due to genetic predisposition for traits such as feed efficiency and resistance to mastitis. For example, in Holstein cows with high vs. low genetic resilience to mastitis, significant differences in alpha and beta diversity have been reported, along with taxa such as Corynebacterium and Psychrobacter differing in abundance [22]. Another study among Girolando, Gyr, Guzera, and Holstein breeds indicated that “breed” accounted for measurable variation in milk bacterial community structure even when controlling for udder health status [23]. A study on sheep milk in Türkiye reported variations in microbial diversity and volatile organic compounds among Merino, Lacaune, and Assaf breeds, with correlations particularly observed between Lactobacillus populations and ketone/hydrocarbon profiles [24]. Milking method and equipment sanitation are also critical. Hand milking versus pipeline milking, or conventional parlors versus automated robotic systems, create different contamination routes [25]. A study comparing milking systems found that the housing and milking system type significantly altered the raw milk microbiota at the genus level [26]. For instance, farms using automated milking systems (AMS) tended to have milk with greater proteolytic activity (from microbial enzymes) and differences in genera like Pseudomonas, Acinetobacter, and Lactococcus relative to conventional milking. Inadequate cleaning of milking robots can allow biofilms to develop on equipment surfaces, harboring bacteria such as Bacillus, Acinetobacter, Chryseobacterium, and Pseudomonas. These biofilm-producing microbes can continuously seed milk if not properly removed [27]. Effective teat cleaning prior to milking is equally important—the microbiota of the cow’s teat skin (e.g., Staphylococcus or environmental bacteria) can directly transfer into milk if hygiene is poor [28]. Seasonal and farm-to-farm variations further contribute to raw milk’s microbial composition. In general, higher temperatures in summer favor fast-growing mesophiles and spore-formers (e.g., Bacillus spp.), while psychrotrophic bacteria (such as Pseudomonas) often proliferate during cold storage [4]. Overall, maintaining good farm hygiene, proper milking procedures, and quick cooling of milk are vital to keep undesirable microbes low while still preserving the natural microbiota that can positively influence product quality. The microbial composition of raw milk is thus a variable starting point—one that both reflects its production conditions and profoundly affects the character and safety of any dairy product made from it.

Although several studies consistently report the dominance of psychrotrophic bacteria during cold storage, the relative role of initial farm hygiene versus storage duration remains unclear. Conflicting results across European and Asian studies may partly reflect methodological biases (16S vs. shotgun) rather than true ecological variation. Future studies should integrate multi-omics approaches to disentangle biological drivers from analytical artifacts.

Taken together, these findings indicate that the variations in raw milk microbiota cannot be attributed to a single factor; rather, they result from the combined influence of seasonality, storage conditions, lactation stage, animal health, farm management, geographical differences, and genetic factors in shaping microbial communities. Factors affecting raw milk microbiota are summarized in Figure 1.

3. Evolution of Microbial Diversity During Fermentation

When milk undergoes fermentation, its microbial community shifts dramatically. The process of fermentation—whether spontaneous or using added cultures—typically selects for a subset of microbes (particularly LAB and sometimes yeasts) that can thrive in milk and produce acid, while many other initial organisms decline [29]. In spontaneous fermentations (relying on milk’s own microbiota or environmental inocula), the microbial diversity can be initially high, comprising various LAB species, yeasts, and others co-fermenting. Over time, as fermentation proceeds and pH drops, the community tends to converge to acid-tolerant, fermentation-specialist microbes [30]. In general, spontaneously fermented dairy products exhibit an abundance of microbial resources initially, but the active fermentation environment favors LAB (and sometimes yeasts or molds), leading to a community skewed toward those groups by the end of fermentation [29]. The coexistence of LAB and yeasts in fermented dairy systems is supported by mutualistic metabolic cross-feeding, ecological niche partitioning, and environmental modulation. For example, in traditional kefir, LAB lowers pH and produces lactic acid, creating conditions under which acid-tolerant yeasts such as Kluyveromyces marxianus and Saccharomyces cerevisiae can grow; yeasts in turn contribute essential metabolites such as vitamins, amino acids, and growth factors [31]. In model and natural cheese rind communities, yeasts such as Geotrichum candidum and Debaryomyces hansenii degrade lipids and proteins, releasing substrates usable by bacteria, while also consuming oxygen and producing CO₂, which help LAB survive micro-aerobic niches [32]. These interactions help stabilize microbial communities and enhance flavor complexity in fermented dairy products.

3.1. Natural (Spontaneous) vs. Starter Culture Fermentation

Traditionally, many dairy fermentations were spontaneous or employed ‘backslopping’ (inoculating fresh milk with a portion of a previous batch), resulting in a rich consortium of microbes. This co-fermentation of multiple LAB and yeasts produces a complex flavor profile and often includes wild strains with probiotic potential [33]. In contrast, modern industrial dairy fermentations typically use defined starter cultures, one or a few standardized strains added to pasteurized milk. While starter cultures offer consistency and control, they dramatically simplify the microbiota. A single dominant strain will usually drive the fermentation, suppressing other microbes and thereby reducing diversity of the final product. For instance, in the case of yogurt production, the use of just Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus as starters leads to a fermented product where those two species vastly outnumber any other microbes. Similarly, a comparison of artisanal vs. industrial cheeses found that the industrial versions (made with commercial starter cultures) had a much less complex microbiota, whereas artisanal cheeses contained a broader array of bacteria unique to each farmhouse or region. The artisanal cheeses’ higher microbial diversity was associated with more distinctive flavors and aromas, while the industrial cheese, though safer and more uniform, was microbiologically and sensorially ‘flatter’. Recent studies provide concrete comparative evidence for the distinct microbial and sensory profiles of industrial vs. traditional dairy products. For example, Papadimitriou et al. [34] showed that artisanal homemade feta cheeses possessed significantly higher microbial alpha-diversity compared to industrial feta; artisanal samples were dominated by Lactococcus lactis, whereas industrial samples favored Str. thermophilus and Lb. delbrueckii species. Similarly, Nelli et al. [35] found that Gidotyri cheeses made using artisanal methods (with commercial or in-house starters) exhibited a more complex bacterial community, including prominent non-starter lactic acid bacteria (NSLAB) families, whereas industrial Gidotyri were overwhelmingly dominated by Streptococcaceae. In addition, a comparison of Manouri cheeses revealed that artisanal Manouri had distinct volatile compound profiles and sensory attribute differences (e.g., aroma, texture, flavor intensity) compared to industrial Manouri, suggesting that differences in manufacturing scale and microbial community contribute directly to sensory divergence [36].

Notably, industrial dairy processing can lead to a loss of microbial diversity compared to traditional methods. Pasteurization or sterilization of milk before fermentation eliminates the native microbiota, meaning only the introduced starter strains (and perhaps a few contaminants) will be present. This ensures safety and repeatability but foregoes the “microbial terroir” that raw or spontaneously fermented dairy can have [37]. For example, artisanal kefir made with traditional kefir grains contains dozens of bacterial and fungal species interacting in a symbiotic matrix, whereas commercial “kefir” is often produced by adding a couple of freeze-dried LAB strains and possibly a yeast to milk. The latter approach yields health benefits but does not replicate the full microbial diversity of true grain-fermented kefir [38,39]. As another example, many small-scale dairies across Eurasia maintain their own heirloom yogurt or cheese cultures (in-house starters), which are mixtures of microorganisms accumulated over years—these mixed starters preserve more diversity than highly purified commercial cultures [40]. Overall, the evolution of microbial diversity during fermentation is characterized by a winnowing process: from a diverse starting community (in raw milk or traditional inoculum) down to a selected few dominant microbes that carry out the fermentation. Whether this results in a biodiverse fermented food or a monoculture depends on the method. Contemporary trends show a renewed appreciation for microbial diversity—artisanal and spontaneous fermentations are being valued for their flavor complexity—but they must be balanced with safety considerations. Industrial dairy fermentations, in striving for safety and consistency, often sacrifice microbial richness. Recognizing this trade-off, some producers are exploring ways to retain more of the natural microbiota (for instance, through raw milk cheeses or by adding adjunct cultures) to combine the best of both worlds: a safe product with a rich flavor from a diverse microbiome.

3.2. Traditional Fermented Dairy Products: Hidden Microbial Treasures

Traditional fermented dairy products around the world are treasure troves of microbial diversity, each developed through local practices and harboring unique communities of beneficial microbes. Fermented dairy traditions from diverse geographies provide more directly relevant examples than non-dairy fermentations. In Zambia, mabisi is produced via the spontaneous fermentation of raw cow’s milk using a variety of local methods such as back-slopping and fermentation in calabash gourds or plastic containers; factors like container type and fermentation duration strongly shape its microbial composition and sensory characteristics [41]. Similarly, koumiss, a traditional fermented mare’s milk from Central Asia, harbors complex bacterial and fungal consortia, with Lactobacillus, Saccharomyces, and Kluyveromyces species playing central roles. Comparative analyses show that industrial koumiss has reduced microbial richness and less diverse flavor compounds than traditional versions [42]. Notably, the map underscores the richness of dairy fermentation heritage—from the cheese and yogurt belt of Europe and the Middle East to fermented mare’s milk in Central Asia—illustrating that every region has its own microbial riches in these foods [41].

3.2.1. Kefir

The microbial differences reported in milk kefir research can be primarily explained along three main axes: (i) the origin of the grains and their “domestication” history, (ii) the milk matrix and processing parameters, and (iii) the choice of analytical method. Without considering these factors collectively, it remains debatable to what extent the observed diversity across studies reflects biological reality versus methodological artifacts.

Although kefir microbiota is often discussed in terms of diversity, recent studies increasingly demonstrate that commercial and retail kefir products share a stable microbial core. For example, a global pan-metagenome analysis of kefir from 25 countries observed that species such as Lc. lactis (or Lc. cremoris) and Lb. helveticus or Lb. kefiranofaciens are consistently present across most samples, regardless of geography, indicating a core microbiota in milk kefir beverages [43]. Similarly, longitudinal monitoring of kefir grains and the corresponding fermented kefir showed that, while minor bacterial and yeast taxa fluctuate over time, dominant taxa such as Lb. kefiranofaciens and Lc. lactis remain relatively stable in kefir grains [44]. This suggests that retail kefir, though varying in minor components, tends to maintain a predictable microbial backbone. Culture-dependent methods and MALDI-TOF analyses have consistently reported Lb. kefiranofaciens as the dominant species across grains of different origins, followed by Lentilactobacillus kefiri, with smaller proportions of Len. parakefiri, Leu. mesenteroides, Lc. lactis, and the yeast Kazachstania exigua [45]. Similarly, high-throughput 16S rRNA sequencing of Greek kefir grains showed that over 99% of the bacterial community belonged to Firmicutes, with Lactobacillaceae dominating at the family level (98%) and Lb. kefiranofaciens accounting for 95–96% at the species level [46]. A multi-method study conducted in Italy, employing Scanning Electron Microscopy (SEM), culture, PCR-DGGE, and pyrosequencing, likewise identified Lb. kefiranofaciens as the principal bacterium, while Dekkera anomalus was reported as the dominant yeast; in addition, Str. thermophilus, Lc. lactis, and Acetobacter species were detected as accompanying microorganisms [47].

Kefir beverages produced from grains exhibit a distinct microbial community structure compared to the grains themselves. Time-resolved studies have shown that, while the grains remain dominated by Lb. kefiranofaciens, the relative abundance of Lc. lactis and Leuconostoc increases in the beverage, accompanied by shifts in the yeast community. Multidimensional scaling analyses clearly demonstrate the divergence between grain and beverage microbiotas [48]. This pattern can be explained by the advantage Lb. kefiranofaciens gains within the grains through EPS matrix production, whereas, in the liquid phase, it is outcompeted by the faster-growing Lc. lactis and Leuconostoc.

Retail products further broaden this diversity. In a study conducted in Ireland on 28 retail kefir products (21 milk kefirs, 3 kefir yogurts, and 4 water kefirs), both bacterial and fungal components were analyzed using full-length 16S and ITS sequencing. The dominant bacteria in these products were Lc. cremoris and Str. thermophilus/suis, while the dominant fungi were Zygotorulaspora florentina, Brettanomyces anomalus, and Kazachstania unispora. It was reported that the overall diversity was lower compared to traditional grain-fermented kefirs, with starter cultures being more predominant [49].

Milk type and grain origin significantly affect kefir microbiota. Studies have examined kefirs produced from different milk sources (e.g., cow, goat), and whether UHT, pasteurized, or raw differ in terms of LAB and yeast abundance, as well as in pH dynamics across storage [50]. Likewise, a comparative analysis demonstrated that kefirs made from goat, sheep, buffalo, and cow milk present distinct microbial and biochemical profiles, with goat milk kefir generally harboring lower Lactobacillus counts and slower acidification than cow milk kefir [51]. These findings underscore that both milk matrix and the geographical origin of grains shape kefir microbial community structures and sensory properties. In a study where Chinese, German, and United States kefir grains were combined in varying proportions and fermented in goat milk, the microbiota largely exhibited “homeostasis,” meaning that it preserved its initial community structure. However, clear microbiota–aroma relationships were identified: positive correlations were observed between Lb. helveticus and ethyl/amyl acetate and ethanol, as well as between Kazachstania unispora and octyl formate, heptanol, and hexanoic acid [52].

Comparisons between raw milk and heat-treated milk also reveal differences in microbiota. In raw milk kefir, in addition to starter-derived species (e.g., Lc. lactis, Str. thermophilus, Leuconostoc, Debaryomyces), Pichia and Galactomyces species were reported exclusively in raw milk kefir. Moreover, only raw milk kefir was found to suppress allergic skin responses, indicating that the microbiota of the raw material plays a decisive role in functional outcomes [53].

Production strategy further influences diversity. When starter-based and backslopping methods were compared in raw milk kefir, both groups were dominated by Lactobacillus and Lactococcus; however, lactobacilli were found at higher proportions in the backslopping group, and peptide profiles were shown to differ between the two methods [54].

Methodological factors are also critically important in the interpretation of results. Long-read sequencing platforms (e.g., Nanopore) enhance species-level resolution, while shotgun metagenomics has revealed not only the taxonomic composition but also the functional gene profiles of the microbiota. In contrast, amplicon-based 16S approaches may underreport certain species due to primer biases. Culture conditions can likewise introduce variability; for instance, some subspecies of Lb. kefiranofaciens do not grow on standard MRS medium and therefore cannot be detected through culture-dependent methods. In a study by Kesmen and Kaçmaz [55], classical culture techniques identified Lc. lactis, Leu. mesenteroides, and Lb. kefiri as dominant in kefir, whereas PCR-DGGE analysis of the same samples revealed Lb. kefiranofaciens as the predominant species. Reports on the dominance of Lb. kefiranofaciens versus Lb. helveticus remain inconsistent. While some differences clearly originate from grain origin and milk type, others may result from culture-dependent versus culture-independent methods. Such discrepancies highlight the need for standardized protocols to accurately compare kefir microbiota across regions.

3.2.2. Cheese

Studies conducted across different geographies and production systems demonstrate marked diversity within cheese microbiota. These variations are influenced not only by the type of milk and starter cultures used but also by the type of coagulant, ripening conditions, rind treatments, geographical environment, hygiene practices, and analytical methods. Cheeses produced from raw milk generally exhibit higher NSLAB diversity. For instance, Enterococcus, Lactobacillus, and Leuconostoc species were found to dominate Divle Obruğu and Edirne White cheeses in Türkiye [56,57], whereas, in cheeses produced from pasteurized milk such as Fontina Protected Designation of Origin (PDO) or Caciotta, starter-derived species remained predominant [58,59]. This phenomenon is attributed to heat treatment reducing natural microbial diversity, thereby enhancing the competitive advantage of starter cultures.

The choice of coagulant also shapes microbial structure. While typical LAB communities dominate in cheeses made with animal rennet, different species such as Leu. mesenteroides have been reported in Spanish cheeses produced with vegetable rennet (Cynara cardunculus) [60]. Polyphenols and protease activities present in plant rennet influence microbiota through pH and peptide profiles. Similarly, the use of starter cultures is decisive for microbial diversity: in PDO cheeses such as Fontina [58] and Comté [61], starters remained dominant throughout ripening, whereas, in traditional cheeses without starters (e.g., Turkish otlu cheese, Malta Ġbejna, Brazil Canastra), environmental flora and NSLAB contributed to broader microbial diversity [62,63,64].

Ripening time has a pronounced impact on cheese microbiota, not merely on pathogen survival but also on community succession, peptide formation, texture, and overall sensory profile. For example, in Idiazabal cheese, SLAB dominatse in the initial 30-60 days, but NSLAB increases later, changing the genus-level composition significantly as ripening progresses [65]. In goat milk cheese ripened for 90 days, Lactococcus remains dominant but total viable counts, titratable acidity and organoleptic traits continue evolving through the final stages [66]. Moreover, in handmade cheeses from China, Streptococcus abundance decreases while Lactococcus increases with ripening from 1 to 120 days; yeast and mold taxa also emerge as texture and biochemical parameters change over time [67]. These examples together show that ripening time is not just a passive waiting period: it dynamically reshapes cheese microbiota, flavor, and texture. In Pecorino and Caciocavallo cheeses ripened on wooden shelves, rind communities were dominated by Corynebacterium, Brevibacterium, and Staphylococcus species [59,68], while, in Edirne White cheese ripened in brine-filled tins, Enterococcus faecium and Lc. lactis were predominant [57]. In Divle cheese, ripened in cave environments, molds such as Penicillium species and yeasts including Debaryomyces hansenii defined its characteristic color and aromatic properties [56].

Rind treatments further contribute to microbial diversity. In Belgium’s washed-rind Herve cheese, Corynebacterium casei and Psychrobacter spp. are found among the dominant bacterial taxa on the rind, and high rind diversity is reported using metagenomic profiling [69]. In Poland’s smoked cheeses made from native cow breeds in the Low Beskids, while the overall microbiota composition remains similar, the volatile compound profile is significantly altered by the smoking process and storage, with notable shifts in ketones, aldehydes, and carboxylic acids [70]. These examples illustrate how rind management practices such as washing or smoking shape both microbial and sensory trajectories in PDO or traditional cheeses. In Turkish otlu cheese, the addition of herbs was associated with unique LAB species (e.g., Companilactobacillus ginsenosidimutans, Weissella jogaejeotgali), although the impact of plant diversity on the overall microbiota was found to be limited [62].

PDO cheeses represent a particularly rich source of information on how traditional practices influence microbial biodiversity. For example, Comté and Parmigiano Reggiano, both aged on untreated wooden boards, develop complex surface communities dominated by yeasts (Debaryomyces hansenii, Geotrichum candidum) and coryneform bacteria (Brevibacterium linens, Corynebacterium casei), which contribute to rind deacidification and flavor development [71]. Similarly, Fontina and Pecorino cheeses ripened on wood show that the board surface serves as a reservoir of LAB, yeasts, and molds, enhancing microbial succession and stability during maturation [72]. These studies highlight that wooden boards are not only passive supports but active ecological niches, transferring beneficial microbes that shape the rind microbiota and contribute to the distinctive sensory identity of PDO cheeses.

In addition, seasonal and geographical differences substantially influence cheese microbiota. In Norwegian goat cheeses, distinct LAB profiles were observed in cheeses made from spring versus summer milk [73], while, in Malta’s Ġbejna cheese, bacterial diversity increased in summer samples whereas fungal diversity remained stable [63]. Moreover, as demonstrated in Italian PDO cheeses, microbiota differences are often attributed less to cheese type and more to the “house microbiota” specific to each production facility [59,74].

Finally, the analytical method employed is a key factor in shaping perceptions of diversity. Culture-dependent approaches only capture species that can grow under laboratory conditions, whereas culture-independent methods (16S rRNA, ITS, shotgun metagenomics) have revealed a much broader diversity. For example, PacBio analyses clearly demonstrated the dominance of Lc. lactis [75], while shotgun metagenomic studies provided more detailed insights into NSLAB and fungal diversity [74,76].

In conclusion, cheese microbiota is too multilayered to be explained by a single parameter. Factors such as the type and processing of milk, choice of coagulant, use of starter cultures, ripening duration and environment, rind treatments, geographical differences, and the analytical methods employed all influence microbial composition. These multidimensional interactions play a fundamental role in shaping the sensory properties, food safety, and geographical distinctiveness of cheeses.

3.2.3. Comparison of Raw Milk, Kefir, and Cheese Microbiota

A review of studies on raw milk, kefir, and cheese microbiota indicates that the reported differences in diversity cannot be attributed solely to product types; rather, they arise from the combined influence of biological, technological, environmental, and methodological factors. The microbiota of raw milk is particularly shaped by conditions within the storage chain. During cold storage, psychrotrophic bacteria such as Pseudomonas, Acinetobacter, and Psychrobacter become dominant, while LAB remain limited. This demonstrates that raw milk is directly influenced not only by animal health and milking practices but also by logistics and cold chain conditions [4,6,18]. In addition, seasonal variations diversify the milk microbiota: LAB and aroma-associated species have been reported as dominant in summer, whereas psychrotrophic bacteria prevail in winter. These findings highlight the high sensitivity of raw milk microbiota to both seasonal and storage conditions.

Compared to milk, kefir microbiota represents a more complex and balanced symbiotic ecosystem. During fermentation, the metabolic cooperation established between LAB and yeasts shapes the characteristic microbial composition of the product. In Turkish kefirs, LAB species such as Lb. kefiranofaciens, Lb. kefiri, Lc. lactis, and Leu. mesenteroides are predominant, while the yeast community is dominated by Kluyveromyces marxianus, Saccharomyces cerevisiae, and Kazachstania unispora [77,78,79]. However, kefir microbiota composition is influenced not only by grain origin but also by production practices, geography, and the type of milk used. For instance, S. cerevisiae and Lb. helveticus have been reported as dominant in Brazilian kefirs [80]. Methodological differences further affect perceptions of diversity: culture-dependent methods emphasize a limited range of species, while shotgun metagenomic approaches have identified Bifidobacterium, Acetobacter, and rare yeasts. Thus, the microbial differences reported in kefir are shaped not only by biological factors but also by the resolution of the analytical methods employed.

Cheese microbiota represents one of the most complex ecosystems, shaped by a wider range of interacting factors compared to raw milk and kefir. Milk treatment plays a decisive role: cheeses produced from raw milk generally harbor greater NSLAB diversity, including Enterococcus, Lactobacillus, and Leuconostoc species, whereas pasteurized-milk cheeses tend to remain dominated by starter-derived LAB such as Str. thermophilus and Lb. delbrueckii [56,57,58,59]. The type of coagulant also affects microbial structure. In Spanish cheeses produced with vegetable rennet, species such as Leu. mesenteroides become prevalent, while animal rennets favor more typical LAB communities [60]. Ripening duration and environment drive microbial succession, with short-ripened cheeses retaining mainly starter LAB, while long-ripened cheeses such as Pecorino and Caciocavallo develop rich rind communities dominated by Corynebacterium, Brevibacterium, and Staphylococcus [59,68]. In cave-ripened varieties such as Divle, molds (Penicillium) and yeasts (Debaryomyces hansenii) contribute to characteristic aroma and texture [56], whereas in brined cheeses including Edirne White, E. faecium and Lc. lactis predominate [57]. Rind treatments and additives also shape diversity: washed-rind Herve cheese is enriched in Corynebacterium casei and Psychrobacter [69], while smoked goat cheeses show significant alterations in volatile profiles [70]. In Turkish otlu cheese, the addition of herbs has been linked with the presence of unique LAB such as Companilactobacillus ginsenosidimutans and Weissella jogaejeotgali, though the overall effect of herbal diversity is limited [62]. Seasonal and geographical factors further influence microbiota, as seen in Norwegian goat cheeses and Malta’s Ġbejna, while “house microbiota” has been identified as a decisive determinant in Italian PDO cheeses [63,73,74].

Methodological aspects remain a critical source of variation in cheese microbiome studies. DNA extraction protocols, particularly those lacking mechanical disruption, tend to underrepresent Gram-positive taxa while overrepresenting Gram-negative bacteria [81,82]. Marker selection (16S rRNA V1–V3 vs. V3–V4), sequencing platform (PacBio vs. Illumina), and analytical approach (OTU vs. ASV) all significantly influence reported outcomes. For instance, full-length 16S sequencing with PacBio revealed the dominance of Lc. lactis [75], while shotgun metagenomics uncovered both bacterial and fungal taxa, including the roles of molds such as Geotrichum and Penicillium in cheese rinds [64,76]. Reuben et al. [83] showed that methodological choices alone accounted for more than half of the observed variability across studies. Thus, comparisons must be made cautiously, with careful consideration of methodological parameters to avoid misinterpretation of ecological differences.

In conclusion, research on the microbiota of raw milk, kefir, and cheese demonstrates that differences in reported diversity reflect both biological realities and methodological artifacts. Raw milk tends to shift toward psychrotrophic dominance during storage, kefir achieves stabilization through LAB–yeast symbiosis, and cheese is shaped by the complex interplay of milk treatment, starters, NSLAB, coagulants, ripening conditions, and facility-specific microbiota. However, only by accounting for methodological variation can these ecological differences be reliably linked to sensory quality, safety, and geographical distinctiveness.

4. Techniques for Identifying Microbial Diversity in Dairy

Understanding the complex microbiomes of dairy products has been greatly advanced by modern identification techniques. Historically, microbiologists relied on culture-dependent methods—sampling milk or cheese, plating on selective media, incubating, then identifying isolates via biochemical tests or microscopy. While foundational, traditional culturing has clear limitations: it recovers only the fraction of microbes that can grow under specific laboratory conditions [84]. In dairy microbiology, a substantial proportion of microorganisms are either viable but non-culturable (VBNC) or fail to compete effectively on standard culture media, which likely led to significant underestimations of microbial diversity in earlier studies [85,86]. For example, fastidious anaerobes or subdominant rind-associated organisms may never be detected if they do not thrive in Petri dishes. Nonetheless, culture-based approaches remain crucial for obtaining pure strains (e.g., isolating Lactobacillus or Bifidobacterium from yogurt) and for phenotypic characterization such as acid or flavor production. Some specialized techniques—cultivation on milk agar, gradient plates for yeast, or anaerobic culturing—help broaden the diversity of culturable dairy microbes, but consensus now is that culture-dependent surveys capture only a small slice of the true microbial population. Metagenomic and culture-independent methods routinely detect around 100 times more microbial taxa than culturing alone [87].

The revolution in microbial ecology has come from culture-independent, DNA-based methods. In particular, high-throughput sequencing (HTS) technologies—often referred to as next-generation sequencing—allow us to profile entire communities directly from a sample’s DNA (metagenomics) or specific genetic markers (amplicon sequencing) [85]. Two cross-kingdom-specific genetic markers commonly used in amplicon sequencing approaches are the 16S rRNA gene for bacteria and archaea and the internal transcribed spacer (ITS) region for fungi. These methods allow for taxonomic profiling and relative abundance analyses of organisms in a sample by amplifying DNA barcodes [88]. Using such methods, researchers have dramatically expanded our view of dairy microbiomes: numerous previously undetected or “unculturable” taxa have been identified in milks and fermented products [18]. For example, metagenomic sequencing of traditional cheese rinds has revealed dozens of bacterial genera, including some novel ones, whereas conventional culturing would have recovered only a few [32]. In one study, advanced sequencing of kefir detected not only dominant LAB and yeast but also minor bacterial populations that were not previously associated with kefir [31]. These low-abundance organisms, though minor, can influence sensory traits and bioactive potential and were essentially invisible to older methods.

Beyond DNA-based methods, several complementary approaches have been developed for the identification and monitoring of dairy microbiota. Immunological assays, particularly the enzyme-linked immunosorbent assay (ELISA), have been validated for the detection of Listeria monocytogenes and other pathogens in dairy matrices, offering rapid and reliable results compared to culture-based techniques [89]. Spectroscopic techniques, such as Fourier-transform infrared (FT-IR) and Raman spectroscopy, provide metabolic ‘fingerprints’ without the need for microbial isolation. FT-IR combined with chemometrics has been successfully applied to detect microbial spoilage in pasteurized milk by correlating spectral patterns with bacterial counts [90], while portable Raman spectroscopy has been used to screen liquid milk for adulterants with high sensitivity and reproducibility [91]. Sensor-based platforms, including electronic noses (e-nose) and electronic tongues (e-tongue), have also shown potential for real-time quality monitoring; for example, e-nose systems have been applied to classify cheeses based on aroma signatures and to detect early spoilage in raw milk [92]. These immunological, spectroscopic, and sensor-based methods complement DNA-based techniques by offering rapid, cost-effective, and functional insights into dairy microbial dynamics.

Metagenomics refers broadly to sequencing all genetic material in a sample. Shotgun metagenomic sequencing can identify microbes down to the species or even strain level and simultaneously reveal functional gene content (e.g., enzymes for flavor production or antibiotic resistance) [93]. This approach has been successfully applied to fermented dairy: for example, shotgun metagenomics of mixed milk cultures has enabled assembly of metagenome-assembled genomes (MAGs) for novel species not previously isolated, providing insights into their metabolic capabilities [94]. Some recent studies have integrated amplicon sequencing, shotgun metagenomics, and metatranscriptomics to generate a comprehensive view of dairy fermentations—from taxonomy to gene expression. Another powerful culture-independent tool is MALDI-TOF mass spectrometry, which identifies bacteria based on their protein spectral fingerprints. It is used to characterize isolates from fermented foods quickly, though it still requires culturing organisms first [95,96]. Earlier molecular fingerprinting methods such as DGGE (denaturing gradient gel electrophoresis) produced DNA banding patterns that offered early evidence of high microbial diversity in traditional fermented milks—less detailed than sequencing but foundational to the shift in microbial ecology.

In the last five years, the adoption of metagenomics and high-throughput sequencing in dairy research has exploded, leading to comprehensive microbial inventories for many traditional products—sometimes revealing hundreds of species—and to the discovery of new bacterial species in fermented milks and cheeses [97]. Sequencing-based methods have unveiled “minority” taxa and functional genes that older methods would miss—such as low-abundance ripening bacteria producing flavor enzymes or antibiotic resistance determinants [98]. These approaches are also vital for safety assessment, detecting pathogens or spoilage organisms present at very low levels. However, each method has limitations: DNA-based detection does not distinguish between live and dead cells, and it may overestimate diversity if DNA from non-viable organisms is present [99]. Therefore, many recent studies pair sequencing with viability qPCR—often using propidium monoazide (PMA or PMAxx) to exclude dead cell DNA—or with culture-based confirmation to verify that key organisms are viable and functional [100,101]. Microbial identification techniques are summarized in Figure 2. For studies on microbiota of raw milk and fermented dairy products, methods and key findings are summarized in

Table 1. Studies on microbiota of raw milk and fermented dairy products: Methods and key findings.

Product/Matrix	Species	Method(s)	Key Findings	References
Raw milk (multi-species)	Cow, sheep, goat, donkey, horse, camel, yak	16S rRNA amplicon (V3–V4)	Clear inter-species differences: horse milk enriched in Bacteroidetes; sheep milk enriched in Gammaproteobacteria	[102]
Raw milk	Cow (bulk tank)	16S rRNA amplicon	Pararhizobium and Agrobacterium abundances were found to be related to spring and Pseudomonas abundance was related to winter and spring seasons; core raw milk microbiota identified	[6]
Dairy chain (farm bulk tank → milk powder)	Cow	16S rRNA + shotgun metagenomics	Microbiota tracked along the processing chain; day-to-day variability noted	[103]
Retail raw milk	Cow	Shotgun metagenomics	Dominated by Pseudomonadaceae (mainly Pseudomonas spp.); LAB at very low levels. At 4 °C microbiota remained stable; at room temperature rapid fermentation and bacterial growth occurred Very low LAB; retail raw milk is a reservoir of ARGs	[104]
Raw milk	Holstein; monthly samples for 12 months	16S rRNA gene amplicon sequencing (high-throughput, Illumina)	Dominant genera: Pseudomonas, Lactococcus, Acinetobacter; also Firmicutes, Proteobacteria, Actinobacteria at the phylum level	[105]
Raw milk	Cow (bulk tank milk)	Culture-dependent isolation (>500 colonies) + 16S rRNA amplicon sequencing (Illumina MiSeq)	Culture revealed ~70–110 bacterial species; Gram(+) dominant genera: Staphylococcus, Corynebacterium, Streptococcus, Janibacter; Gram(−): Chryseobacterium, Acinetobacter. 16S sequencing detected anaerobes and hard-to-culture species, increasing diversity.	[106]
Raw ewe milk	Sheep (Assaf)	16S rRNA amplicon	Core genera: Staphylococcus, Lactobacillus, Corynebacterium, Streptococcus, Escherichia/Shigella	[107]
Raw ewe milk	Sheep (Assaf, Lacaune, Merino)	16S rRNA amplicon (V3–V4)	Lactobacillus, Jeotgalicoccus, Enterococcus, Corynebacterium	[24]
Raw goat milk	Saanen vs. Guanzhong breeds	16S rRNA gene amplicon sequencing (Illumina, V3–V4)	Proteobacteria dominant phylum (~71%); Enterobacter (~25%) most abundant genus. Saanen goat milk had higher levels of lactose-fermenting LAB (Lactococcus, Lactobacillus, Bifidobacterium, Streptococcus) compared to Guanzhong.	[108]
Milk kefir and kefir grains	Cow milk kefir	16S rRNA (V1–V3 region) pyrosequencing (bacteria) + 26S rDNA pyrosequencing (yeasts)	16S: ~20 major bacterial species detected; dominant: Lb. kefiranofaciens, Lc. lactis ssp. cremoris, Gluconobacter frateurii, Lb. kefiri, Acetobacter orientalis, A. lovaniensis. 3 main yeasts in grains: Naumovozyma spp., Kluyveromyces marxianus, Kazachastania khefir.	[109]
Kefir (home-made vs. industrial)	Cow milk kefir	Culture + qPCR + 16S/ITS amplicon	Grains: Lb. kefiranofaciens, Lb. kefiri; Beverage: Streptococcus, Lactobacillus, Lactococcus; yeasts: Kluyveromyces, Debaryomyces	[46]
Kefir (home-made vs. industrial)	Cow milk kefir	16S rRNA amplicon (V3–V4)	Industrial: Lactococcus and Streptococcus. Home-made: Lactobacillus	[98]
Kefir (global collection)	Cow milk kefir	Shotgun metagenomics (pan-metagenome)	Global kefir community types; recurrent LAB (Lb. kefiranofaciens, Lb. kefiri) and yeasts (Kazachstania)	[43]
Kefir (Brazil)	Cow milk kefir	Shotgun metagenomics + peptidomics	Species-level LAB profiling; bioactive peptide repertoire described	[110]
Yogurt (back-slopped, Turkey)	Cow	16S rRNA amplicon	Firmicutes-dominated; potential next-generation probiotics highlighted	[111]
Cheese	Raw milk cheese production chain (cow teat skin → milk → cheese)	16S rRNA gene amplicon sequencing (Illumina)	Teat skin microbiota is an important source for milk and cheese microbiota. 85% of raw milk and 27% of ripened cheeses contained teat skin-derived OTUs. Shared taxa included Micrococcales, Staphylococcaceae, and LAB important for cheese flavor. Grazing influenced subdominant LAB levels in cheese.	[112]
Cheese plants and cheeses	Cow (industrial plants)	Shotgun metagenomics	Dairy plant microbiomes harbor flavor- and probiotic-related genes; microbial transfer networks from environment to product	[113]

.

In summary, to identify and catalog microbial diversity in dairy products, researchers now employ a comprehensive toolbox of methods:

Culture-dependent techniques: These include improved culturing strategies—such as anaerobic cultivation, extended incubation periods, and high-throughput culturing (e.g., culturomics)—which aim to isolate as many microbial species as possible [114].

Sequencing-based techniques: Standard tools include 16S rRNA/ITS amplicon sequencing for community profiling; shotgun metagenomics for deeper taxonomic and functional gene insight; and long-read sequencing (PacBio HiFi or Oxford Nanopore) to assemble near-complete genomes from complex communities.

Other modern methods: Modern approaches extend to metatranscriptomics, to identify which microbial genes are actively expressed, and metabolomics, to map the chemical metabolites of microbial activity. When combined with classical microbiology, these methods give a far more complete and dynamic view of dairy microbiomes [115].

Crucially, these advances have shown that dairy microbial communities are far more complex than previously appreciated, with interactions among many strains. This insight has enabled purposeful management of fermentation microbiota—such as reintegrating wild strains into industrial starters to enhance flavor—or implementing DNA-based quality control to detect spoilage organisms at early stages. The continuous evolution and cost reduction of sequencing technologies ensures that even the most “hidden” microorganisms in dairy ecosystems can now be uncovered and studied comprehensively. Comparative overview of microbiota detection methods summarized in

Table 2. Comparative overview of microbiota detection methods.

Method	Description	Advantages	Disadvantages
Culture-dependent methods (classical microbiology)	Isolation of microorganisms on selective media, colony morphology, biochemical tests.	Allows isolation and characterization of live strains; enables testing of probiotic potential; antimicrobial susceptibility testing possible	Detects only cultivable organisms (many species cannot be cultured); underestimates diversity; time-consuming
Molecular PCR-based techniques (16S rRNA PCR, qPCR, RT-PCR)	Amplification and analysis of target genes (mainly 16S rRNA).	Rapid and sensitive; can detect low-abundance organisms; culture-independent detection possible	Not always species-level resolution; sensitive to contamination; limited to known taxa with available primers
DGGE/TGGE (Denaturing/Temperature Gradient Gel Electrophoresis)	Separation of PCR products on gradient gels to profile communities.	Provides an overview of community structure and diversity; multiple taxa detectable simultaneously	Low resolution; rare species often missed; time consuming
T-RFLP (Terminal Restriction Fragment Length Polymorphism)	Fluorescent-labeled PCR fragments digested with restriction enzymes and analyzed.	Rapid overview of microbial community; provides semi-quantitative diversity information	Limited species-level resolution; band interpretation difficult in complex samples
Microarray (PhyloChip, LAB-Chip)	Hybridization of target DNA fragments to taxon-specific probes.	Simultaneous analysis of many taxa; high-throughput and relatively fast	Detects only known species with probes; cannot discover novel taxa
NGS (Next-Generation Sequencing—16S rRNA amplicon sequencing)	High-throughput sequencing of specific 16S rRNA regions (e.g., V3–V4).	Culture-independent, comprehensive profiling; detects rare taxa; enables calculation of diversity indices	Species/subspecies resolution sometimes limited; requires bioinformatics; relatively costly
Shotgun metagenomics	Sequencing of all genomic DNA from a sample.	Species- and even strain-level resolution; functional gene and metabolic pathway analysis; detects bacteria, fungi, and viruses simultaneously	High costs; computationally demanding; requires expertise in bioinformatics
Metatranscriptomics	Sequencing of total microbial RNA, focusing on expressed genes.	Reveals active functional genes; provides insight into metabolic activity	RNA degrades easily; technically challenging; high costs
Metaproteomics and Metabolomics	Profiling of microbial proteins and metabolites using MS.	Direct functional readout; links to fermentation quality and aroma compounds	Complex sample preparation; expensive; lack of standardization

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5. Effects of the Microbiome on Flavor and Texture

The microbial inhabitants of fermented dairy products have profound effects on the organoleptic properties (flavor, aroma, texture) of the food, as well as on its nutritional profile and potential health benefits. These effects can be placed into several categories.

5.1. Flavor and Aroma Development

During fermentation and ripening, microbes generate a multitude of flavor compounds that define a product’s character. LAB ferment lactose into lactic acid, imparting the characteristic tartness of yogurt, kefir, and sour cream, and helping preserve the product by lowering pH and inhibiting spoilage organisms [116]. Beyond lactic acid, certain LAB including Leuconostoc and some Lactococcus strains metabolize citrate present in milk into diacetyl, which produces a buttery aroma crucial for the flavor of cultured butter, buttermilk, and select cheeses [117,118]. In Swiss-type cheeses, Propionibacterium freudenreichii ferments lactate to propionic acid, acetic acid, and CO₂; these products impart nutty-sweet notes and create the characteristic “eyes” (holes) in cheeses such as Emmental [119,120]. Proteolytic ripening bacteria, such as non-starter lactobacilli or Brevibacterium species on cheese rinds, break down milk proteins into peptides and amino acids, which are then converted into savory flavor molecules including amines, sulfur compounds, and short-chain fatty acids, contributing umami depth to aged cheeses. Likewise, lipolytic microbes, including certain molds or Micrococcus on rinds, liberate fatty acids from milk fat, such as butyric and caproic acids, which impart pungent notes characteristic of blue cheeses [116].

Yeasts and molds in fermented dairy contribute an additional layer of complexity to flavor. In kefir, yeasts produce ethanol and carbon dioxide: the ethanol provides a mild fruity or yeasty aroma, while CO₂ adds gentle carbonation that enhances aroma perception [121]. In blue cheeses, molds such as Penicillium roqueforti generate methyl ketones—particularly 2-heptanone and 2-nonanone, which are primarily responsible for the piquant, earthy flavor characteristic of Roquefort, Stilton, and similar cheeses [122,123]. On the rinds of soft cheeses including Camembert, the yeast-like fungus Geotrichum candidum produces sulfur compounds—such as dimethyl sulfide, dimethyl disulfide, and other thiols—that contribute to the distinctive ripe, creamy, and buttery aroma of these cheeses [124,125]. Together, these microbial actions form a “flavor-generating orchestra”, transforming plain raw milk into a symphony of aromas and taste. Studies show that many volatile compounds—such as benzaldehyde, buttery diacetyl, or malty 3-methylbutanol—are absent or minimal in pasteurized milk but rise markedly in fermented products as microbes metabolize lactose, fat, or amino acids over time [126].

Notably, microbiome diversity often correlates with flavor complexity: artisanal cheeses hosting many microbial species tend to exhibit richer and more distinctive flavor profiles than industrial cheeses dominated by just one or two strains [34,127,128]. When a product’s microbial community is simplified, such as by using only Str. thermophilus and Lb. bulgaricus, some flavor nuances may be missing. For example, ordinary yogurt made solely with these strains has a clean acidic note with mild yogurt aroma, but, if wild Leuconostoc or Lc. lactis diacetylactis are introduced (via traditional backslopping), buttery and yeasty notes may also emerge [129]. Similarly, fermented milks like kefir or koumiss, which naturally rely on bacteria–yeast synergy, provide a lactic tang, mild effervescence, and subtle ester aromas reminiscent of mild beer or cider—flavor signatures of yeast metabolism working alongside LAB [30,130,131,132,133].

5.2. Texture and Rheology

Microbial activity also influences the texture and body of dairy products. A prime example is the production of exopolysaccharides (EPS) by certain LAB. Strains of Lc. lactis or Str. thermophilus that are “ropy” secrete polysaccharides (e.g., kefiran in kefir or dextrans), which thicken the milk gel and enhance viscosity in products such as yogurt and drinking yogurt [134]. Some traditional fermented milks inherently select for EPS-producing cultures. For example, Scandinavian ropy milks including viili exhibit a characteristic stringy, custard-like consistency due to EPS from wild Lc. lactis subspecies [135]. In kefir, the polysaccharide kefiran—produced by Lb. kefiranofaciens in the grain matrix—not only helps maintain the structural integrity of grains but also partially dissolves into the beverage, imparting a silky mouthfeel and functioning as a dietary prebiotic [131,136,137].

Microbial gas production significantly influences the texture of dairy products. In kefir, CO₂ produced by yeasts (e.g., Kluyveromyces or Saccharomyces) contributes a subtle effervescence, adding lightness to the mouthfeel and enhancing flavor perception [138]. Similarly, in spontaneously fermented ayran, native yeasts produce CO₂, yielding tiny bubbles that create a slight fizz on the tongue and modify how creaminess and flavor are perceived [139]. In Swiss-type and mold-ripened cheeses, microbial respiration leads to open textures with gas holes. Propionibacterium freudenreichii ferments lactate into propionate, acetate, and CO₂; the CO₂ accumulates in the curd to form the characteristic eyes and affects texture by increasing internal pressure and elasticity [119,140].

Microbes can also stabilize or weaken the protein network in fermented milk. Initially, the lactic acid produced by starter cultures lowers pH and precipitates casein, causing coagulation to form yogurt or cheese curds—a significant textural shift from liquid milk [141]. Following coagulation, microbial proteases play critical roles. For instance, Lb. delbrueckii proteases break some casein chains during yogurt fermentation, resulting in a smoother, less elastic gel and improved sensory properties [132,142]. In cheese, both starter (SLAB) and NSLAB produce proteolytic enzymes during ripening, softening texture over time—from crumbly to creamy or pliable—such as in Brie or aged Gouda, where NSLAB reduce bitterness and degrade casein intactness [143]. Moreover, some microbes produce compounds that enhance water retention and reduce syneresis (wheying off) in yogurt—improving stability and water-holding capacity [141].

In summary, the microbiome is a key texturizer: whether it is through EPS thickening (improving viscosity and creaminess), gas and acid production (creating curd structure and lightness), or enzymatic proteolysis (modifying firmness), the presence and metabolic traits of specific microbes dictate the final mouthfeel of fermented dairy.

6. Conclusions and Recommendations

This review highlights that the microbial diversity of raw milk, kefir, and cheese arises not from product type alone, but from the combined effects of biological, technological, environmental, and methodological factors. Raw milk microbiota is highly sensitive to season, storage, and farm practices, while kefir represents a stable LAB–yeast consortium shaped by milk type and grain origin. Cheese microbiota is the most complex, integrating milk treatment, starter and non-starter LAB, ripening duration, rind ecology, and facility-specific “house microbiota”.

Beyond biological determinants, methodological choices such as DNA extraction protocols, marker regions, sequencing platforms, and bioinformatics pipelines can significantly influence reported outcomes, underscoring the need for transparency and standardization. Recognizing these methodological biases is crucial for ensuring comparability across studies and for accurately linking microbial data to sensory quality, safety, and geographical distinctiveness.

From an applied perspective, understanding dairy microbial ecosystems has direct relevance for improving product quality, authenticity, and safety management. The concept of microbial barcodes, for instance, may support traceability systems, while knowledge of starter–non-starter dynamics can guide industrial fermentation strategies. Looking ahead, the integration of metagenomics, metatranscriptomics, and metabolomics with culture-based approaches will provide deeper functional insights into microbial interactions. Such advances will not only enhance our scientific understanding but also enable innovation in artisanal and industrial dairy production, bridging tradition with modern biotechnology.