*3.11.1 PCR and RFLPs*

Koohestani [121] study based upon the spot-on-the-lawn approach was performed to assess the performance regarding antibacterial activity of 8 LAB isolates with various patterns of RFLP. For all 70 bacterial isolates, a DNA fragment contains the size of 1.540 bp has been enlarged as shown in **Figure 6**. Three distinct digestive patterns were shown in the RFLP PCR product analysis (patterns I–III) shown in **Figure 6**. Out of 70 isolates, RFLP pattern I was shown in amplified fragments from 16 rRNA from

63 (90%) isolates. The RFLP patterns I and III were all RFLP patterns discovered with the HinfI named endonuclease enzyme, and each with two and five isolates.

LAB isolates were identified based on the generation of the phylogenetic trees from the sample of cheese named *Enterococcus* subsp*., Lb. lactis*, *Lb. farciminis*, and *Lb. paracasei Enterococcus* subspecies was made up of majority of the LAB (90%) in that RFLPs isolates named [2, 97, 122, 123] isolate it. RFLP pattern II was classified jointly in Isolates namely 14 and 32. The RFLP pattern III was identical to two isolates 22 and 44; nevertheless, these two isolates were grouped into two different clusters based on a phylogenetic tree. The most common LAB in traditional cheeses in this research were *enterococcus* subsp.

#### **3.12** *Enterococcus* **safety evaluation and probiotic potential by genomic analysis**

Enterococci are the ordinary people of the human and animal gastrointestinal system. Recently, the selection of helpful microorganisms by bacteriocins was a novel probiotic characteristic [124]. The 1st OB14 strain and 2nd OB15 strain of lactic acid were isolated and identified as *E. faecalis* from Testori and Rigouta typical Tunisian fermented milk products. These novel isolates have been examined for the character of the gastrointestinal tract and proven tolerant to severe circumstances. They were moderate biofilm makers that increase the trans epithelial resistance and they can attach to the cells of intestines to reinforce the barrier. Different antibiotics susceptibility is seen includes Ampicillin, vancomycin, gentamicin, and erythromycin and the evidence shows the susceptibility of *E. faecalis* OB14 and for OB15 to essential ampicillin and vancomycin clinical drugs. The tetracycline resistance existence and the presence of cytolysin genes in *E. faecalis* OB14 1st strain, nevertheless, was found in the Whole Genome Sequencing (WGS). Hierarchical cluster analysis reveals the tight connection between *E. faecalis* OB15 and *E. faecalis* Symbioflor 1 against OB14. *E. faecalis* OB15 looks therefore trustworthy as a probiotic food or feed business for future growth.

#### **3.13 Study of strain of probiotic potential by isolation and identification**

Samples of Ezine type of cheese have been mixed and attenuated into the solution of Ringer and plated with aerobic incubation at 37°C at a species of kanamycin aesculin azide agar for 48 h. After incubation, colonies exhibiting Enterococcus-typical shape were randomly chosen and spotted using sterile toothpicks on plates containing agar. A total of 114 colonies with characteristic Enterococcal shape were transferred to BHI Agar in KAA agar. It was observed that 84 colony areas greater than 10 mm versus indicator strains displayed inhibitory zones (results not shown). PMD74 was identified as the strongest antibacterial activity for additional tests. Among these colonies. The distinctive characteristics of the strain are compatible with general characteristics. The isolate has been identified as an Enterococcus lactis strain, according to the sequence of 16S rRNA, undertaken to ensure molecular identification.

The latest investigation showed that Ezine (PDO), which consists of nonstarter Turkish white long-ripened cheese, serves as an isolation source for new enterococcal strains. This is the first analysis on *E. lactis* isolation in Turkey, to the best of our understanding. *E. Lactis* is a probiotic candidate because of the results such as strong strain tolerance to GI tract virtual circumstances, other physiological features, and remarkable antibacterial activity to both near relatives and dietaryborne pathogens.

### **3.14 Commercial interest in probiotics and sensory evaluation of food matrix**

Commercial interests also exist for the idea of probiotic food, as is shown in the range of probiotic products accessible in supermarkets and specialized stores, that make up a major portion of the functional food market. Singh (2011) observed that various author has demonstrated that frequent ingestion of live probiotic microbes might be useful to improve lactose tolerability, reduce cholesterol levels. It was observed that probiotics may directly or indirectly affect the intestines by modifying the physiology of Endogenous Microflora or Immune System, as colonization of some strains can decrease the severity of acute diarrhea in children. Probiotics have a positive impact on the intestinal microbiology, including antagonistic effects, competition for immunological effects and improved infections resistance.

The usage of bacteria at the expense of potentially dangerous bacterial proliferation therefore encourages the proliferation of beneficial bacteria which enhances the natural defensive systems of the host. In fermented milk products, the application of probiotic bacteria has been extensively explored due to problems in maintaining the vitality of these organisms during cooling storage. The survival of probiotic bacteria in fermented dairy product may be influenced by factors such as acidity and dissolved oxygen and species interactions, inoculation techniques and stock conditions.

The quantity of viable bacteria in the intestines and the level of pH that is low in stomach leads to the limit the survival of probiotics. Furthermore, there are still numerous difficulties with the poor viability of probiotic bacteria in milk meals. In fermented dairy products there are several variables that impact the survivability of probiotics: acidity, pH and hydrogen peroxide, dissolved concentrations, stocktemperature, interaction in products with other microorganisms, lactic and acetic acid concentration, and protein concentration [125].
