The first step in selecting for psychobiotic strains is to conduct a wide-scale in vitro assessment of the general probiotic properties of bacteria. Bacterial strains should be selected based on their ability to withstand the lethally acidic environment of the stomach and the bile salts in the duodenum. At this stage, the strain’s ability to adhere to intestinal epithelial cells and to suppress the growth of pathogenic bacteria should be investigated as well. Then, preliminary safety studies are needed to confirm the absence of mobile genes, accounting for transferable pathogenicity and antibiotic resistance. The next step is to look for other properties, such as the production of active compounds—namely, bacteriocins, organic acids and exopolysaccharides—and the conversion of carbohydrates, proteins and other food components into short-chain fatty acids and other molecules. Expanding our knowledge of the technical characteristics of potential psychobiotics is crucial for production. The viability of the strains in the end product depends on their ability to withstand the freeze-drying process. Just as important is to test the viability of the strains during long storage. There are tests specifically designed to check the activity of the strain at all stages of the manufacturing process. After all these steps are completed, the strain’s specific activity, whether antidepressant, anti-inflammatory, anticarcinogenic or immunomodulatory, should be studied in both in vitro and in vivo systems. Finally, perhaps the most necessary and revealing part of this protocol is to validate the strain’s effects in human volunteers [17].

The antidepressant potential of bacteria has been attested to by hundreds of studies conducted on laboratory animals—mainly rats and mice. The bacterial strains tested in these studies belonged predominantly to the genera Lactobacillus and Bifidobacterium [18]. These studies also emphasized the strain-specific rather than species-specific nature of these properties [19]. Most experiments are designed similarly, and they involve the administration of a single or a mixture of strains of bacteria to a control group made up of conventional or gnotobiotic animals as opposed to a control group receiving a placebo. At the same time, the animals are exposed to a stressor for the duration of the experiment and tested afterwards in relevant behavioral tests, such as sucrose preference, forced swimming test, elevated plus maze, open field and others. The biochemical results show that specific probiotic strains can impact the HPA (hypothalamic–pituitary–adrenal) axis by lowering the corticosterone and ACTH (adrenocorticotropic hormone) blood levels. The levels of dopamine, serotonin, tryptophan and other neurotransmitters are also normalized in various regions of the brain and the blood serum. Other effects involve the regulation of the activity of gamma-aminobutyric acid (GABA) receptors in various regions of the brain and the level of the brain-derived neurotrophic factor in the hippocampus and blood serum. Most importantly, some probiotic bacteria can reduce inflammation by inhibiting the release of a C-reactive protein and other proinflammatory cytokines (IL-6, IFN-γ, TNF-α and IL-1β) and upregulating anti-inflammatory cytokines such as IL-10 in the blood plasma and the spleen. A recent in vitro study conducted by Dyakov et al. demonstrated that the protein FN3 produced by bifidobacteria possesses TNF-α-binding properties. Bifidobacterium longum contains the gene cluster PFNA, which is presumably involved in species-specific communication between the bacteria and their hosts. The gene cluster PFNA consists of five genes, including fn3, which codes for a protein containing two fibronectin type III domains. E. coli BL21 (DE3), containing the recombinant plasmid pET16b:fn3, was genetically engineered to produce a recombinant FN3 protein [20]. Probiotic bacteria are also known for their ability to activate many antioxidant enzymes in the brain and blood serum—namely, superoxide dismutase and glutathione peroxidase. The relationship between the gut microbiome-derived enzymes and the human organism remains largely uncharted territory. Enzyme preparations, for instance, work differently in the human body. The most important question is how enzymes/metabiotics penetrate human cells. These could be type III secretion systems, bacteriophage infections, Gram-positive bacterial extracellular vesicles, ergothioneine and microbe-derived metabolites such as L. reuteri [21,22,23,24]. Unfortunately, none of the abovementioned mechanisms were described in other species. Probiotics can interact with the cell components; it is also plausible that probiotics interact with the metabolites of human cells, thus giving rise to new compounds. However, these are often contradictive and speculative pathways that will not be addressed in this study. Probiotics can achieve this in many ways, including by the direct production of antioxidant enzymes or by reduction of the lipid peroxidation levels in the blood [12,25]. Aside from that, the role of probiotic bacteria is central to sustaining the integrity of the gut–brain barrier and the balance of the gut microbiota composition [18].

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