**3.10 Probiotic potential of LAB isolates**

This study examined the propensity to promote the activity of β-galactosidase, CSH, generation of hydrogen peroxide, antibiotic sensitivity, and pathogenic microbes, in vitro, of eight strains of lactobacillus isolated from Iranian conventional cheese, "koozeh," The results demonstrate that all lactic strains are powerful probiotics in developing novel formulations for the design of health-promoting functional food items. Analysis showed that the best of test probiotics among the tested are *L. fermentum* named MT. ZH893 and MT. ZH993 and MT. ZH593 *L. plantarum. Lactobacillus acidophilus* is an initially isolated bacterial bacterium economically important and was named *Bacillus acidophilus* from human gastrointestinal tract in 1900. During the development of technologies for the identification of bacteria *L acidophilus* is the typical species of a very varied and heterogeneous Lactobacillus group that has undergone several taxonomical revisions. The characterization of

*L. acidophilus* has suffered with misrepresentation because of the difficulties of distinguishing phenotypically identical species by morphologic and different biochemical techniques. In comparison, *L. acidophilus* sensu stricto is currently one of the most characterized species of *Lactobacillus* and it is used as a supplement for probiotic that is present in functional foods. The source of *L. Acidophilus* strains is established, *L. acidophilus* historically and now misidentified, and the probiotic has genomic, and physiological features.

Another study conducted by Maged in [118] identified 93 lactic acid bacteria (LAB) from the local and fermented milk samples of 13 in number that were gathered, including fresh raw milk, crude frozen milk, and different types of cheese like (fresh, salty, cooked) yogurt stirred, (shrimp milk) yogurt, and butter. The LAB counts were greater under circumstances of microaerobic incubation than in conditions of aerobics. On the MRS solid medium surface, the colony morphologies of the isolates were seen; the colors of colonies observed were white to pale creamy with circular shape and the width that ranged from a diameter of 0.5–4 mm. About 92.47% strains were gram-positive. Catalase and cytochrome oxidase activities varied in isolated isolates, as seen that 71% were unfavorable for the activity of catalase and cytochrome oxidase production were negligible about 72%. Also, 96% of isolates were nonmotile and the motility of isolated strains varied. All the isolated strains were amplified with the 16S rDNA gene. Then by the usage of amplicon it was being sequenced and purified. The 46 distinct genera's nucleotide sequence, i.e., *Enterococcus, Lactococcus, Lactobacillus, Streptococcus* and *Weissella*, are in line with 16S rDNA sequences from 14 species. The nine strains of the genus *L. acidophilus, L. casei, L. paracasei, L. plantaarum* and *L. futsaii* were analyzed to indicate that there could be two strains named (i.e., Hadhramaut4 and Musallam2), four strains named (i.e. MSJ 1, BgShn3, MasaLam7, and Dwan5), one strain namely (i.e. NMBM1), and another strain, (i.e., EMBM2), respectively.

Ten condensed yogurts imported from China were studied by Qian [119]. In the MRS medium, the strains of *Lactobacillus* grew at a temperature of 37°C at 16–24 h. Brain Heart Infusion Gardnerella vaginalis (ATCC49145) with yeast extract supplement (1%), maltose (0.1%), glucose (0.1%), and horse serum (10%) (BHIS) at 37°C for 24 h under anaerobic circumstances. It was cultured on Luria- Bertani Medium (LB) for 12 h at a temperature of 37°C (ATCC 25922). Streaking is done by MRS agar and broth and enriching them for the strains of *Lactobacillus*. Gram-positive colonization's were chosen and inoculated in a broth of MRS with characteristic *Lactobacillus* shape, white in color and fruity fragrance. Then genetic investigation with the use of PCR and 16S rDNA sequencing has validated and identified isolates. A bacterial DNA isolation kit was used to extract the genomic DNA from the strains of *Lactobacillus*. The 16S rDNA genes were amplified using the universal PCR primers 27F (AGAGTTGATCGGCTCAG) and 1492R (TACGGC TACCTTGTAGACTT).

#### **3.11 Study of phylogenetic analysis of probiotics**

Hajigholizadeh et al. [120] isolating LAB from traditional cheeses and characterizing them. In 225 ml the quantity of peptone water is about 0.1% w/v was added and then mix the 25 g of each sample of cheese. Then dilute the sample of cheese in a suspension containing 2% of the sodium citrate and then grown on MRS agars and incubated for 1–2 days at a temperature of 37°C under anaerobic and aerobic conditions. From each plate of cultivation, the 3–4 distinct colonies were picked randomly. Gram staining microscopically inspected, and the catalase analysis was carried out.
