**5. Conclusion**

*Bacterial Biofilms*

Other species producing biosurfactants and condition for their detection are able in research papers for example: *Lactobacillus paracasei* produced biosurfactants with anti-adhesive properties [56]. *Streptococcus mitis* biosurfactants plays a protective role in the oral cavity and protects against colonization of saliva-coated surfaces by cariogenic *Streptococcus mutans* [48]. Based on *Bacillus subtilis* SPB1 lipopeptides production researcher predict their possibility used in toothpaste formulation [53]. Biosurfactants

Lactic acid bacteria are the most frequently mentioned in studies of exopolysaccharides (EPS) in oral microbiota [57]. Except for lactobacilli, which are participated in the later stages of dental biofilm formation, streptococci are one of the first bacteria capable of producing EPS. Streptococci are able to assert themselves and adhere to the hard tissues of the oral cavity immediately after washing the teeth. This property of adherence is predetermined and is encoded in genes that are also responsible for the production of glucosyltransferases. Glucosyltransferases (Gtfs) are produced by several types of lactic acid bacteria [58]. Gtfs are generally characterized as Gtf-S (glucosyltransferase-soluble) or Gtf-I (glucosyltransferaseinsoluble) enzymes, depending on whether the glucan they produce is water soluble or insoluble [59]. For detection of exopolysaccharides production in oral lactic acid bacterial members is useful PCR with help of specific primers see in **Table 3**.

Testing of bacterial isolates as potential beneficial candidates or their products is necessary step in new discoveries. We are able declarate production of bioactive substance by very easy PCR reactions, as mentioned above in part 3. Activity of these substances is easy to declare by simply *in vitro* tests. At first for activity it is possible to use spot on or disc diffusion test. Same mechanism of declaration is for

If we found bacteria with interesting effect in spot or disc diffusion test it predict selection criteria of former characterized bacteria for next research.

**4.1 The disc diffusion method for** *Lactobacillus reuteri* **for testing of growth** 

We recommend the disc diffusion test for the detection of the inhibitory properties of beneficial microorganisms. Selected lactobacilli strains were grown on MRS agar (CONDA S.A, Madrid Spain) for 48 hours. anaerobically (Gas Pak Plus, BBL, Microbiology Systems, Cockeysville, USA) at 37°C. Then, a standardized suspension with an optical density of 1 McFarland by dissolving several solitary colonies in 5 ml of physiological saline was prepared. Sterile clean discs (6 mm diameter, BBL, Cockeysville, USA) were placed on Petri dishes (Ø 90 mm) with 20 ml of PYG agar (HiMedia Laboratories GmbH Einhausen, Germany). The sterile paper discs were

As a negative control, one Petri dish with PYG agar is served with a clean paper

The plates with discs were incubated for 48 hours. anaerobically (Gas Pak Plus, BBL, Microbiology Systems, Cockeysville, USA) at 37°C. The discs were removed with a sterile syringe needle or tweezer after incubation. Subsequently, 3 ml of 0.7% PYG agar was inoculated with 0.3 ml of the indicator pathogenic strain and put into

are promising bioactive molecules for oral-related health applications [47].

**4. Testing of growth inhibition activity against pathogens**

live bacteria isolates as for isolated bioactive substances.

inoculated with 5 μl of standardized suspensions of lactobacilli.

**inhibition activity against pathogens**

discs soaked with sterile MRS broth.

**3.4 Exopolysaccharides and methods for their detection**

**188**

It is necessary to know the composition of the dental biofilm of healthy individuals and the bacterial composition in pathological conditions to identify species responsible for disease initiation and progression. Identification of species and their characterization is essential for the selection of pathogenic, potentially pathogenic and potentially probiotic species. Blast n analysis of 16S RNA or MALDI-TOF mass spectrometry identification is perfect tools for identification of bacterial species. The ability to modulate the microbiocenosis of the dental biofilm by bacteria living together in the biofilm should be studied. The some bacteria are capable of producing bioactive substances whose presence we can quickly and easily declare with help of PCR. Sequencing and comparing of genes coding bioactive substances can uncover differences between tested bacteria isolates. Presence of these genes and prove the ability to inhibit the growth of other bacterial species are important steps in selection of potentially probiotic candidates. These bacteria are of great interest for further study and may be useful in the development of new antibacterial agents. Bioactive substances can be extracted by physical methods (centrifugation, separation and fractionation), by chemical methods (purification) and detected by modern analytical method (HPLC) or proteomic methods (MALDI-TOF MS). Next important step is declaration of activity pure extracted substance. Bioactive substances of bacterial origin can be used in dental preparations and serve as prevention or supplementary therapy of periodontal diseases. During recent years there has occurred a shift towards ecological and microbial community based approach to the therapy of oral cavity diseases. With the increasing resistance to antibiotics, the use of probiotics appears as a prospective alternative treatment or preventative measure in the control of periodontal diseases. From the clinical point

of view, it is not yet possible to give direct recommendations for the use of probiotics. However, the available scientific evidence indicates that probiotic therapy is a promising approach also in the field of stomatology. The potential beneficial strains of *Streptococcus salivarius* or *Lactobacillus reuteri* and others bacterial strains isolated from many oral biofilms can be selected for next research based on their production of bioactive substances and on growth inhibition level against oral pathogenic bacteria not only in human but also in social animals like dogs and cats.
