**2.1 Mechanisms of action probiotics**

### *2.1.1 Pathogenic bacteria*

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

Nevertheless, in the last decades, these practices have changed considerably due to the concern of the increase of bacteria resistant to antibiotics, since they can be transmitted zoonotically from animals to humans, causing serious problems in public health and even death because of the failure of the antibiotic at therapeutic doses [10]. Furthermore, another problem for human health is the presence of antibiotic residues in animal-derived food, by the use of antibiotics for long periods of time, since it is associated in some cases with allergic reactions, imbalance of the intestinal microbiota, and especially, the development of antibacterial resistance [11]. Consequently, one of the measures taken in the face of the problems of bacterial resistance was the restriction of antibiotics at subtherapeutic doses in the EU in 2006 [12] and the USA in 2017 [13], and although in countries as Mexico they have not been officially banned, the Ministry of Agriculture and Rural Development (SADER), through its decentralized administrative body, the National Health Service, Food Safety and Food Quality (SENASICA), has promoted initiatives to prevent their use since 2012 [14–17]. However, as a consequence of this measure, the incidence of enteric diseases in animals has increased significantly [18], as well as the use of antibiotics, but at therapeutic doses for the purpose of controlling and preventing diseases, which could lead to a worse scenario of bacterial resistance [2, 19–21]. In this context, the European One Health Action Plan against antimicrobial resistance calls for the phasing out of routine prophylactic (Prevent) and metaphylactic (Control) antimicrobial use in animal production and investment in the research of new alternatives [22], since they could be regulated in the coming years. Therefore, the poultry industry has been under pressure to seek and investigate new alternatives to reduce the problems of bacterial resistance, prevent and control diseases, reduce the mortality rate, and finally promote the growth of animals. Among these alternatives, the most popular are probiotics (yeasts or bacteria) since it has been reported that they can improve the performance [23, 24], as well as prevent and control enteric pathogens in poultry [25–27]. Furthermore, it has been reported that probiotics could be an interesting alternative to prevent and control the toxic effects of aflatoxins. For these reasons, the probiotic market has expanded rapidly and is expected to grow to around 7% in 2020. However, this market is led mainly by Asia and Europe given the growing demand for dietary supplements [18].

Probiotics are defined as "live strains of strictly selected microorganisms which, when administered in adequate amounts, confer a health benefit on the host" [28]. The most common microorganisms used as probiotics in livestock production are lactic acid bacteria (LAB) from the genus *Lactobacillus*, *Pediococcus*, *Lactococcus*, *Enterococcus*, *Streptococcus*, and *Leuconostoc*. Nevertheless, only the genera

*Lactobacillus*, *Streptococcus*, *Pediococcus*, *Enterococcus*, and *Weissella* are the most frequently used in poultry production [29]. Although the efficacy of probiotics reducing enteric pathogens is evident, one of the disadvantages is that they require refrigeration or lyophilization to survive for long storage periods or can be encapsulated to increase their stability/viability when included in the feed, which would increase the cost of production at the industrial level, making it unprofitable [30]. Unlike LAB, direct-fed microbials (DFM) as *Bacillus* spores, other types of probiotics, have several potential applications since they can be included as feed additives in poultry diets, due to their remarkable heat stability and long shelf life [31, 32]. Bacteria of the genus *Bacillus* are Gram-positive, frequently found in the soil. However, several studies have shown that *Bacillus* spores can also be present, germinate, and survive

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**2. Probiotics**

Although a large number of studies have shown the possible mechanisms by which probiotics have a beneficial action in inhibiting of pathogens, more studies are needed to elucidate them.

The possible modes of action of probiotics for the inhibition of pathogens include two basic mechanisms [29, 36, 37]: competitive exclusion and modulation of the host immune system (**Figure 1**). Competitive exclusion involves mechanisms such as (1) production of inhibitory compounds, that is, hydrogen peroxide, bacteriocins, and defensins [38, 39], (2) prevention of the pathogen adhesion [38], (3) competition for nutrients [40], and (4) reduction of toxin bioavailability [36]. Meanwhile, in the modulation of the host immune system, both innate and adaptive immune responses are involved [29]. The adaptive immune response depends on B and T lymphocytes to induce an antigen-specific response and produce antibodies [29, 41]. In contrast, physical and chemical barriers (innate immunity), such as intestinal epithelial cells (IEC), are the first line of defense to prevent the spread of pathogens and subsequent infections. Furthermore, IEC are the target cells for probiotics, which can improve the function of the intestinal barrier by stimulating the production of mucus and antimicrobial peptides such as defensins [42, 43].

**Figure 1.** *Mechanism of action of probiotics.*

## *2.1.2 Aflatoxins*

Similar as for pathogenic bacteria, probiotics can (1) compete for space and nutrients with aflatoxigenic mold strains, (2) degrade aflatoxins by the production of enzymes, or (3) avoid the intestinal absorption of AFB1 by its binding to the cell walls of probiotic strains [44].
