## 4 Discussion  
The microbiota composition of smear-ripened cheeses from six commercial producers from the Czech Republic, Germany, and France was investigated in detail. 16S rRNA gene sequencing results indicated that each cheese exhibited a unique microbiota composition. Notably, only two bacterial species, likely originating from the starter culture, were shared across all six producers. A similar observation was reported by [PERSON] et al. [6], who analysed the microbial composition of surface microbiota cultivated from four Irish farmhouse smear-ripened cheeses. Despite analysing the same types of cheese, only two bacterial species--_C. casei_ and _G. anilaitensis_--were consistently found across all samples. The presence of various starter cultures was confirmed, with individual producers showing a distinct preference for specific strains. To illustrate, mesophilic LABs were predominantly used by producers A, C, E and F and thermophilic LABs were predominantly employed by producers B and D. Mesophilic SC are used to obtain soft or semi-hard smear-ripened cheese varieties, which are characterised by high moisture content and a soft or semi-hard texture. In contrast, thermophilic SCs are employed for harder smear-ripened cheese varieties, which also require heating the milk to higher temperatures and pressing the curd at higher pressure after syneresis, which results in a lower moisture content and  
Figure 4: A phylogenetic tree of 37 isolates obtained by aerobic culture from smear-ripened cheese. Bacterial isolates are clustered based on their full-length sequence of the 16S rRNA gene. Isolates of the phyla Firmicutes (green), Actinobacteria (yellow), Pseudomonas (pink), and Proteobacteria (blue) and their detection in cheeses from different producers are shown. a firmer texture [1; 2]. Although we did not measure the moisture content, cheese B is classified as a harder smear-ripened cheese variety. The term smear-ripened cheese refers to the layer of smear that develops on the cheese surface due to the activity of smear-associated bacteria when the young cheese is exposed to air at high relative humidity and suitable temperatures [6]. Nowadays, commercially available smear culture _B. aurantiacum_ is the most widely employed [3; 4; 15]. The relative abundance of _B. aurantiacum_ varied significantly across the cheeses from the six producers. This finding may suggest differences in production practices, for example, removing the smear or the possibility that individual strains of deliberately added bacteria may not always successfully colonise the cheese surface during the ripening process. Several findings were reported for _B. linens_ on French soft red-smear cheeses and _B. aurantiacum_ strains on Epoisses cheeses, where they were rather outcompeted by adventitious strains [15; 16], similarly as we observed with the cheeses A, C and F.  
As part of the adjunct cultures, yeasts are applied to the surface of the smear-ripened cheese to ensure deamination and production of growth factors such as vitamins, pantothenic acid, niacin, or riboflavin to promote smear-associated bacteria [17; 18; 19]. The results of ITS sequencing indicated that _D. hansenii_ was the most frequently used yeast culture in the production of smear-ripened cheese by all six producers. In cheese B, thermotolerant _K. marxianus_ was further employed. _Deharyomyces hansenii_ was described as being associated with the production of alcohol and carboxylic acids [20], while _K. marxianus_ was linked to the production of fruity ester flavours [3], thus influencing the cheese flavour. In addition to the defined starter and adjunct microbial cultures, microorganisms derived from the production environment can also make a substantial part of cheese microbiota and thus contribute to the organoleptic characteristics of products [2; 8; 11; 21]. These bacteria naturally appear during the ripening and are derived from the brine, as well as from the wooden shelves, the air, and the environment of the ripening cellers [6; 7; 8].