*3.3.13.1 Isolation of genomic DNA*

In 20 ml of autoclaved LB broth, isolated colonies of chosen bacteria were inoculated. For 18 h, these falcons were kept at 37°C in an incubator that was constantly

shaken. After incubating for 18 h, the broth culture was put into an Eppendorf tube. That Eppendorf was centrifuged for 5 min at 40°C and 6500 rpm. The particle was kept, but the supernatant was discarded. Pellet was thoroughly washed with 200 PI of TEN buffer and mixed with a vortex.

This combination has been centrifuged for 5 min at 40°C and 6500 rpm. The supernatant was discarded once more. The pellets were preserved for future use. Following that, 100,111 of SET buffer was put into an Eppendorf and the pellet was well mixed using a vortex. The Eppendorf was then filled with 100 gl lysozyme and placed in the incubator at 37°C for 30 min.

Following that, 5 PI of 25% SDS solution and 100 PI of TEN buffers were added. The Eppendorf tube was gently inverted many times until lysis occurred, and then incubated at 600°C for 15 min. After withdrawing the Eppendorf from the incubator, it was allowed to cool at ambient temperature before being filled with 5 gl of 5 M NaCl. The mixture was treated with an equal proportion of chloroform and buffered phenol (l:l). Eppendorf was centrifuged at 40°C for 1 min at a speed of 6500 rpm. Supernatant was removed once more and transferred to a fresh Eppendorf tube. The DNA was then precipitated by adding twice the volume of absolute ethanol (100%) that had to be ice cold. Overnight, the Eppendorf was chilled.

The next day, Eppendorf was centrifuged for 5 min at 6500 rpm (40°C). The supernatant had been decanted. The pellet was rinsed with 70% ethanol. The Eppendorf was properly air dried. TE buffer (50 microliter) was added, and the DNA was kept at 20°C for future use.

#### *3.3.13.2 Gel electrophoresis*

Gel electrophoresis was used to determine if the treated materials contained isolated genomic DNA or not. 1% agarose gel was made for this purpose by dissolving 1 g of powdered agarose in 2% 50× TAE buffer. 2 ml of 50× TAE buffer was dissolved in 98 ml of autoclaved distilled water to get the 2% solution. Unless the agarose and buffer solution was correctly mixed, it was heated in the microwave for over 2 min. After cooling to room temperature, ethidium bromide 2 PI was added. When the temperature was reduced to 600°C, the gel was gently poured into the gel casting tray. Before pouring the gel, the comb was placed in the gel casting tray. The gel plate was put on a level surface to make the gel smooth and consistent. A 20 PI sample was obtained in an Eppendorf tube and 5 gl of 1× loading dye was applied to it. The sample was stored on ice during processing. After solidification, the comb was carefully removed from the tray, and the test sample was placed in each well. For over 40 min, electrophoresis was performed at 80 volts. A DNA ladder (10 Kb) was put into one well.

#### *3.3.13.3 Polymerase chain reaction*

Ferment was used as a PCR reagent in the PCR of 16S rDNA. The samples were put into a Thermo cycler (Progene, Techne) that was set for initial denaturation at 940°C for 20 min, melting at 940°C and annealing at 5200, primer extension at 720°C for 60 s each, for a total of 35 PCR cycles (**Figure 5**).

The last extension at 720°C lasted 10 min, and the ultimate storage temperature was set to 40°C for the maximum term.

*Probiotics in Processed Dairy Products and Their Role in Gut Microbiota Health DOI: http://dx.doi.org/10.5772/intechopen.104482*

**Figure 5.** *PCR steps.*

#### *3.3.13.4 Gel electrophoresis of amplified product*

1% agarose gel was created for this purpose by combining 1 g of powdered agarose with 2% of 50× TAE buffer. Amplified items were placed in the appropriate wells. It was ran at 80 volts for 40 min. The gel was then examined for the presence of amplified products using a UV trans-illuminator.

### *3.3.13.5 Purification of PCR product from agarose gel*

After amplification, the required DNA band was sliced, and the net weight of the agarose gel containing the DNA band was calculated. After that, an equivalent volume of binding buffer was added and the gel was incubated at 50°C until fully melted. The sample was then transferred to a column. The column was centrifuged at 10,000 rpm for 1 min, then washed with 750 gl of wash buffer and centrifuged again for 1 min. The flow through was removed, and the column was washed one more with 750 gl of was buffer. For 1 min, the column was centrifuged at 10,000 rpm. Flow through was discarded once again, the column was moved to a fresh micro centrifuge tube, and 30–50 PI elution buffer was added. It is then permissible to centrifuge for 2 min at 10,000 rpm for 30 s and store at −200°C for future use.

#### *3.3.13.6 Sequencing*

Following purification of the PCR products with the-Invitrogen gene clean, the samples were forwarded to the laboratory for 16S rRNA sequencing.

#### **3.4 Identification**

The initial stage in the identification of potential probiotics is to identify microorganisms inside the GIT or in dietary sources. Only a tiny proportion of microorganisms can be cultivated in various habitats in culture [106]. The taxonomic categorization characterized as the process of cataloging that was based on a polyphasic approach [107]. Phenotypical techniques used to identify microorganisms have historically been employed. For many decades, the taxonomy depends on significantly on the kind of sugar fermentation and fermentation products. The probiotics were therefore

categorized largely as LAB. The method of choice today is 16S rRNA gene analysis. Microbiologists have employed this conserved region for phylogenetic categorization for the past two decades, and the relatedness of species is inferred by comparing their sequences in publicly available databases. To detect bacterial communities from gutorecological sources, 16S rRNA gene analysis was coupled with other techniques.

The amplified 16S rDNA may relate to PAGE by means of the hybridization using fluorescent oligonucleotide probes (fluorescence in situ hybridization) or chemical denaturation using restricting enzymes (T-RFLP) with a specific 16S. However, in comparison with the bacterial Genome having base pair of 30,000–40,000, the 16S rDNA segment is exceedingly tiny (1500 bp).

#### **3.5 Characterization**

The two most significant probiotic-taxa are *Lactobacillus* and *Bifidobacterium* species in processed dairy products. When eaten, sufficient metabolically active bacteria are required to penetrate the GIT barrier and have transitory impact in GIT. This is essential since some writers have demonstrated the positive benefits of dead probiotics [108]. GIT has the challenge to survive on GIT with the potential to withstand with extremely low pH of about 1.5, availability of gastric enzymes, bile salts and other bowel enzymes [109]. Different in vitro tests to imitate these stress conditions have been devised.

#### **3.6 Identification and characterization of probiotics**

Isolate were identified by gram staining, endospore stain, catalase testing, and carbohydrate fermentation test. The growth and survivability of the stomach and small intestines is part of this. The stability of these properties following ingestion must thus be tested to verify that they are maintained in the host. Therefore, tests of acid and bile tolerance should include early screening and selection of probiotic strains.

Classical physiological and biochemical assays are not efficient for analyzing and quickly identifying microbial communities, as the bacterial population typically has comparable nutrient requirements and develops under similar environmental circumstances. Thus, it may often be difficult to clearly identify the species using simple phenotypic criteria. New possibilities for defining strains of fermented milk items have been developed using molecular methods. The 16S rDNA assays are fast, and cheap to detect the microbial species of yeast, acetic acid, and of some Gram-positive bacteria. Among PCR-based techniques is easy and cost effective. The chosen strain for diverse probiotic characteristics was characterized. These comprise the susceptibility analysis of antibiotics, the capacity to create bioactive metabolites and acid sensitivity tests for their antibiotically resistance potential.

#### *3.6.1 Antibiotic susceptibility test*

Several antibiotics were employed at different concentrations, ranging from 25 μg, 50 μg, 100 μg, 200 μg, 250 μg and 500 μg/ml agar medium, including the penicillin G, tetracyclines, gentamycin, vancomycin, and streptomycin. Lactobacillus has been streaked across an agar plate over the overnight culture. It was aerobically incubated at period of 48 h for 37°C, controlled and checked for lack of growth.
