**2.1 The selection of experimental indicator pathogen**

The choice has relied on the target disease that the probiotic is thought to treat. Thus, for vulvovaginal candidiasis *C. albicans,* the predominant pathogen has been chosen [24, 25], even though *Candida glabrata* has also been screened [26, 27]. Enterotoxigenic *E. coli* and *Salmonella typhimurium* is the choice for studying gastrointestinal infection [28]. However, while screening new probiotic microorganisms for general antimicrobial activity, major classes of pathogens of medical importance should be representatively tested [29]. For example, studies on fungal pathogens should include at least a dermatophyte, non-dermatophyte, and yeast. Antibacterial should consist of a Gram-positive and a Gram-negative bacterial pathogen. Furthermore, clinical, typed microorganism and drug-resistant strains should be included due to emerging resistance [10].

#### **2.2 The choice of probiotic microorganism and postbiotics**

The WHO/FAO has listed the criteria for evaluation of probiotic microorganism, which include; the ability of the microorganism to adhere to epithelial cells, bile salts, resist stomach acid and enzymes, persistence within the system, produce antimicrobial compounds, antibiotic resistance profile inability to confer resistance or genome stability and ability to stabilize the normal microbiota among others [7, 15, 30]. Probiotic antimicrobial activity is strain-specific; therefore, the species level and strain of the selected probiotic should be identified.

#### **2.3 Inoculum size**

The actual number of viable indicator pathogens in the inoculum size directly influences the outcome. Too little may lead to false-positive results, while too heavy inoculum may give a false negative result [29, 31]. A foundation for the inoculum size can be suggested by CLSI [32]. Researchers have used different inoculum size, incubation temperature, and time for both probiotic and indicator pathogen in *in vitro*, *in vivo* and clinical studies [9]. We propose that the viability and dose of probiotic microorganisms used (also in the production of postbiotics) be established by dose-dependent experiments. This should be indicated in experimental reports.

#### **2.4 The experimental conditions and incubation time**

Lactic acid bacteria and Bifidobacteria are fastidious; subsequently, the media chosen should have a specific nutrient requirement, for example, growth factors. MRS, which is an appropriate media for the growth of probiotic microorganisms, is widely used. MRS is both a selective and an enriched media for the growth and isolation of only lactic acid bacteria and other bacteria. Therefore, if this medium cannot support the indicator pathogen, for example, dermatophytes (J. [33]), probiotic growth factors can be incorporated in any media of choice such as potato dextrose agar (PDA), sabouraud dextrose agar (SDA) and nutrient agar (NA) to favor the growth of both the indicator pathogen and the probiotic microorganism. Proper choice of media and specific modifications is key to a successful experiment [33, 34]. Therefore, media supplemented with growth factors should be screened for the ability to grow both probiotic microorganisms and indicator pathogen. We propose that the specific incubation conditions such as time and oxygen requirements for both probiotic microorganisms and indicator pathogen be optimized before the experiment and confirmed by the growth curve of individual microorganisms (**Figure 2**). Furthermore, fresh media should always be prepared and used for reproductive results, especially in the case of disc diffusion results.

#### **2.5 The technique of production of postbiotics**

The postbiotics is also referred to as cell-free supernatant (CFS) or biogenics or spent media. The preparation of postbiotics is varied and attests to the need to harmonize the methods. The process entails the following steps; the probiotic microorganism is inoculated in broth media and incubated in an incushaker [35]. Cells are then removed by centrifuging to obtain the CFS ([36]; J. [33, 35, 37]). The supernatant obtained can then be screened for antimicrobial activity [35], or the supernatant is further filter-sterilized [35, 36]. The CFS is then used to screen for the microbial activity or concentrated to obtain concentrated CFS (cCFS) [36, 38] or freeze-dried [36].

The advantage of using postbiotics is that the properties of the active component can be deduced. To ascertain if the active ingredient is proteinaceous, heat treatment and enzymes are used. If the activity is reduced or is lost compared

*Probiotics and Postbiotics from Food to Health: Antimicrobial Experimental Confirmation DOI: http://dx.doi.org/10.5772/intechopen.99675*

to non-treated postbiotic, it infers that the antimicrobial agent is proteinaceous [33, 38]. To ascertain if the antimicrobial activity is pH-related, the postbiotic is neutralized and buffered [30].

#### **Figure 2.**

*Detailed proposed method for conclusively screening probiotic antimicrobial activity. Step 1 entails choice of optimal media and growth conditions, step 2 is the preliminary screening on agar and step 3 is the confirmation of probiotic antimicrobial activity in liquid cocultures.*
