**2. Microbiota of the unique Bulgarian dairy products**

The diversity of milk, selected starter and nonstarter cultures, and technological treatments give exclusively heterogeneous products with various organoleptic, texture, and nutritional qualities [16]. The uniqueness of presented traditional dairy products is a complex result of the climate conditions, the composition, and activities of milk microbiota [17]. With an understanding of its important role, several studies aimed to characterize LAB diversity, not only in Bulgaria. Discovery of rod- and globular-shaped bacteria (cocci), named initially *Thermobacterium bulgaricum* and *Streptococcus thermophilus,* respectively, started in 1905 with the work of Bulgarian scientist Dr. Stamen Grigorov. The cooperation between the two microorganisms was considered to be one of the most important characteristics of the typical Bulgarian yoghurt. In 1938 Dr. K. Popdimitrov considered that using only the two bacterial species is proper for the production of Bulgarian yoghurt and classified all other microorganisms as undesirable. According to the national standard, the starter cultures for Bulgarian yoghurt include strains of Bulgarian origin from species *Lactobacillus delbrueckii* subsp. *bulgaricus* and *Streptococcus salivarius* subsp. *thermophilus*. At least 107 CFU/g in a ratio of 1:2–1:5 is needed as a yoghurt starter, according to the definition of FAO/WHO (2003) [1]. LAB reach 107 –108 CFU/g for *L. bulgaricus* and 108 –109 CFU/g for *Str. thermophilus* in the final product. However, homemade and artisanal yoghurts contain different non-starter species that add to the different sensory qualities of the final products. The biodiversity of autochthonous microbiota and its use as starter cultures make Bulgarian dairy products exceptional. Species from the family *Lactobacillaceae* and genera *Lactococcus, Leuconostoc, Streptococcus, Enterococcus*, *Pediococcus*, and especially the new genera of previously determined genus *Lactobacillus* have been isolated throughout the years from different homemade and artisanal dairy products [18–21]. The autochthonous microbiota of "*brano mliako*" is comprised of yeast species as well, including *Kl. lactis* and *Saccharomyces cerevisiae* [2]. Our research proved that such products contain more diverse microbiota (**Figure 3**).

A special protocol with an overnight enrichment step, however, was needed. More than 90 artisanal samples from yoghurt, white-brined cheese, and "kashkaval", made at home or small farms in different rural regions of Bulgaria, have been collected. The combination of classical phenotypic and microbiological with molecular and typing methods, according to the polyphasic taxonomic approach applied, allowed us to establish the biodiversity of their LAB microbiota (**Figure 3**). The most persistent was the species *Lactiplantibacillus plantarum.*

Multiplex PCR analyses (according to Torriani et al. [22]) allowed to identify closely related *Lactiplantibacillus paraplantarum*, *Lactiplantibacillus pentosus*, and *L. plantarum* and revealed their presence in homemade yoghurt and white-brined cheese [23, 24]. By the gold standard, 16S rDNA sequencing, *L. bulgaricus* was identified in yoghurt samples. In addition, *L. plantarum* presence in yoghurt with a dominance in ripened samples of cheese was confirmed. In the early stage, however, significant number of enterococci and lactococci have to be pointed for white-brined cheese samples. Originally, *lactobacilli* have a low density in cheese (<50 CFU/g). During the ripening period, they significantly increase and become the dominating microbiota in the final product (107 –108 CFU/g). Therefore, an enrichment step with pre-culturing of collected samples allowed us to establish LAB diversity.

Nemska et al. [20] isolated 74 pure cultures from different dairy products, made by traditional recipes without the addition of industrial starters. Using classical phenotypic tests, 45 out of them were identified as lactobacilli [20]. The group of lactobacilli from white-brine cheese was the most numerous (23 strains from cow, sheep, buffalo, goat, and a mixture of cow and buffalo milk), followed by yoghurt isolates (14 strains from cow, sheep, and buffalo milk), 1 strain from curd (from goat

#### **Figure 3.**

*Biodiversity of LAB microbiota in artisanal Bulgarian dairy products, studied during a period of 10 years.*

milk), and 4 strains from yellow cheese (from goat and cow milk) [20]. Twenty-two strains were identified as *L. plantarum*. A predominance of *L. plantarum* in the nonstarter microflora of Bulgarian white-brined cheese has been reported [23, 25]. This species is also reported to be present in high density in many different types of cheese [26–28]. *Ligilactobacillus acidipiscis* was also identified in a homemade ripened sample of cheese, made from raw ewe milk, in the region of nuclear power plant (Kozloduy, Bulgaria) (Danova, 2015, unpublished data). The tendency of rich LAB microbiota in samples made from non-pasteurized milk was proven in several samples from sheep, cow, and mixed milk [19, 20]. Most traditional cheese achieve their specific taste and aroma due to the natural microflora of raw milk. The raw milk that is used impacts the unique organoleptic and sensory qualities of the final product. Different cheese products can be made with rennet and natural microflora, with or without starter cultures.

Today, the wide variety of cheese with specific organoleptic properties is due to the different combinations of LAB species. It is important to maintain an optimal strain balance in the starter because of the complex microbial interactions. Even small differences in the microbial composition can lead to surprising or unwanted effects on the quality of the cheese product.

According to Bulgarian National Standard for white-brined cheese, a mesophilic and/or thermophilic starter is used in a ratio of 2:1. The mesophilic starter is predominant and contributes to the ripening processes and developing the taste and aroma of the final product. The mesophilic LAB for white-brined cheese and "*Kashkaval*" are represented by *Lactococcus lactis* subsp. *lactis* and *Lacticaseibacillus casei*. The thermophilic starter includes *L. bulgaricus* and *Str. thermophilus.* For yellow cheese "*Kashkaval*", in addition to *L. bulgaricus* and *S. thermophilus*, the thermophilic *Lactobacillus helveticus* is also present. For all Bulgarian dairy products, the strains in the starters must be isolates of Bulgarian origin.
