*2.10.5 The probiotics as anti-cancerous*

On the World Health Organization's cancer data page, there were around 14 million new cancer diagnoses and approximately 8.2 million cancer-related deaths in 2012 alone. Asia, Africa, and the Americas account for more than 70% of cancer fatalities worldwide [98]. The attention has shifted to natural sources that impart anti-cancer benefits, such as probiotics, in recent years [99]. They are interested in working together to bring the illness down as well as produce a treatment with minimal or no adverse effects [100].

#### **2.11** *In vitro* **studies**

The probiotic strains, the *Lactobacillus fermentum* NCIMB-5221, and -8829, have been shown to be extremely strong for the suppression and development of normal colonic cell epithelial growth by producing SCFAs in vitro studies (ferulic acid). Kahouli [101], in 2015 compared *L. acidophilus* ATCC 314 and *L. rhamnosus* ATCC 51303, that were characterized by tumorigenic activity. Probiotic strains of *L. acidophilus* LA102 and *L. casei* LC232 have shown the cytotoxic activities with two colorectal cell lines (Caco-2 and HRT-18) being in vitro anti-proliferative [102]. Although probiotics may play an important role in cancer neutralization, only *in-vitro* tests are confined to research. Therefore in vivo models and animals' clinical trials must prove the potential of anti-cancer probiotics.

#### **2.12 Efficacy of probiotics in COVID 19**

The recent Xu et al. [103] trials indicated the ability of probiotics in the avoidance of secondary infections in those afflicted by COVID 19. Some COVID-19 individuals suffered from microbial intestinal dysbiosis. All patients need to examine their dietary and gastrointestinal functioning. The regulation of the stability of gut microbiota and the decreasing probability of secondarily infected bacterial translocation should be supplemented with nutrition and application of probiotics.

#### *2.12.1 Probiotics for COVID prevention*

Probiotic medicines against viruses which lead to respiratory tract infections are proposed in the last two decades as antimicrobial agents. There are numerous conceivable action mechanisms to increase breath-probiotic activity; the modulation of the innate immune system and better immune response are nevertheless most probable. According to earlier research of certain viral illnesses, preventing infectious diseases can be achieved by increasing and activating human immunological activity by healthy, equilibrated meals and administrative complements such as vitamins, minerals, fiber, and probiotics [104].

Live microorganisms which provide a sufficient intake of health advantages, including an increase in immune activity and removal of respiratory tract diseases, are probiotics. Probiotics can obviously lower the prevalence and severity of illnesses, showing their promise to cure or prevent COVID-19**.** To manage viral infection, it is important to understand the immune cell activation, cytokine profile and immunological regulation. The preservation of the human GI or lung microbiota might help prevent COVID-19, as dysbiosis plays an essential role in people's vulnerability to infectious illnesses The potential preventative and therapeutic impact of probiotics against SARS-CoV-2 infection should be examined in in vitro and clinical investigations.

### **3. Isolation of probiotic microorganism**

The initial process that was carried out for the isolation of probiotic bacteria is to keep the sample selectively before the step of incubation in suitable conditions. Many probiotics are anaerobic; hence, the samples then placed into anaerobic conditions immediately (within 3 h). The samples should be immediately homogenized, diluted, and cultivated in selective media Several mediums were developed to isolate *Probiotics in Processed Dairy Products and Their Role in Gut Microbiota Health DOI: http://dx.doi.org/10.5772/intechopen.104482*

bifidobacteria and lactobacilli either electively or selectively. As a source of isolation for probiotic bacteria were utilized milk fermented products (curd, buttermilk, cheese) and vegetable pickles. Direct plating and enrichment methods were used for the isolation of MRS agar and MRS broth, respectively. These samples were diluted in 9 ml of saline water serially up to 10-4 (0.89% NaCl) with a spread of semi-solid MRS on Petri flat plates. The inoculates were then incubated at room temperature for 2 days to examine and control microbial colonies that grown on medium (without inoculation).

## **3.1 Spreading**

Appropriate sample dilutions were made, and a concentration of just 50 PI-J was dispersed on MRS-agar plates. For 48 h, each plate was kept at 37°C in a static incubator.

#### **3.2 Streaking**

A single bacterial colony was plucked with a sanitized loop and streaked over plates to get isolated colonies. The plates were then put in a static incubator at 37°C for 48 h.

#### **3.3 Characterization of bacterial isolates**

#### *3.3.1 Morphological tests*

These morphological tests were performed to identify bacterium isolates. These tests are as follows:

#### *3.3.2 Gram's staining*

This staining was used to discriminate between gram-positive and gram-negative microorganisms. Gram staining may be used to distinguish the morphology of bacteria, such as bacillus or coccus.

A neat and clean glass slide was prepared initially for gram's staining, and a thin smear of a single colony was created. The slide was then air dried. After that, the slip was fixed by running it through the flame 5–6 times. The prepared smear was then coated with crystal violet for 30–60 s. To remove any remaining discoloration from the slide, distilled water was utilized. For 30–60 s, Gram's iodine solution was applied to the smear. Following that, the initial stain was removed with alcohol for 30 s. The slide was drained with tap or distilled water before applying the secondary stain safranin for 1 min. Rinse the slide once again with distilled water. To dry the slide, blotting paper was employed. The slide was then examined under low and high magnification towards the end. Microscope power, i.e., at IOOX in oil immersion [105].

#### *3.3.3 Endospore staining*


The malachite green stain was then put on blotting paper over steam for 15–20 min. After the slide had cooled to room temperature, the blotting paper was removed, and the slide was washed with distilled water for 30 s. The slide was then treated with safranin for about 2 min before being rinsed with deionized water. To dry the slide, blotting paper was used. Finally, the slide was examined under a microscope with low and high magnification powers, i.e., at 100× with oil immersion [105].

#### *3.3.4 Motility test*

A motility test is a test that is used to determine if bacteria are motile or not. Semi-solid medium was necessary for this purpose. Tryptone log, 5 g yeast extract, 13 g agar, and NaCl 5(g) were used to make the medium, which was then diluted in 1000 ml distilled water.

After that, 10 ml of it was poured into a variety of test tubes, cotton plugs were used to seal the test tubes' mouths, and it was sterilized in an autoclave at 121°C for 15 min. Finally, the medium was allowed to harden vertically. The medium was infected using a red-hot inoculating needle and then incubated at 37°C for 24 h [105].

#### *3.3.5 Biochemical characterization*

#### *3.3.6 Catalase test*

This test was carried out to determine the capacity of microorganisms to digest hydrogen peroxide (1-1202). A nice and clean glass slide was used to perform the catalase test. In the center of the slide, one drop of water was placed. Using an inoculating loop, I took some isolate culture and mixed it with water. I applied 2 drops of hydrogen peroxide to it and witnessed the results [105].

#### *3.3.7 Casein hydrolysis test*

Skim milk agar medium was made for the casein test by combining 2 g tryptone, 1 g yeast extract, 6 g agar, 4 g glucose, and 4 g skim milk in 400 ml of distilled water. The media was steam sterilized in an autoclave for about 15 min at 121°C, and the petri plates were poured under sterilized conditions. The isolated colony of each bacterium was then streaked in the middle of the petri dish under sterilized conditions. Each plate was placed in the incubator at 37°C for 24 h before the change was detected in each petri dish [105].

#### *3.3.8 Carbohydrate fermentation test*

The goal of this test is to confirm the microorganisms' ability to ferment carbohydrates via gas and acid [105].

#### *3.3.9 Glucose fermentation*

The medium for this experiment was phenol red broth. 5 g NaCl, 0.018 g phenol red, 10 g peptone, and 5 g glucose were dissolved in 1 liter of distilled water to make phenol red broth. The medium was then autoclaved for 15 min. The bacterial culture was then injected in the medium. For 24 h, test tubes were put in an incubator set to 37°C. If the color goes from red to yellow, the result is good. To observe gas generation, a Durham tube was inserted in each test tube [105].

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

#### *3.3.10 Lactose fermentation*

The medium for the lactose fermentation test was made by combining 0.018 g phenol red, 5 g sodium chloride, and 10 g peptone 5-gram lactose in 1000 milliliter deionized water. The medium was then autoclaved sterilized. The medium was then injected with a bacterial culture. After that, each test tube was placed in the static incubator overnight at 37°C. The presence of yellow suggests a favorable outcome. Durham's tube was used to monitor gas output.
