**3. Probiotic application in poultry industry**

Although probiotics are considered potential alternatives to antibiotic use in poultry because they leave no residues in the meats and eggs given their modes of action, the variety of microorganisms in terms of species and even between strains of the same species, as well as their variation in metabolic activity, could affect their effectiveness. Furthermore, other factors that influence the effectiveness of probiotics in poultry are the species of origin, the probiotic preparation method, the survival of colonizing microorganisms in the gastrointestinal tract conditions, the environment where the birds are raised, the application time and administration route of probiotics, the immunologic state, the lineage of poultry, as well as age and concomitant use of antibiotics [45, 46]. Below are some of the applications of probiotics in poultry.

#### **3.1 Effects of lactic acid bacteria against** *pathogens* **of importance in poultry**

Several articles published by our laboratory have shown that the use of probiotics as a replacement of antibiotics in poultry production has had positive effects by reducing the growth of pathogens in *in vitro* models that simulate or not the three main compartments in birds (crop, proventriculus, and intestine) [47, 48], as well as the colonization of pathogens through the gastrointestinal tract in both turkeys and broiler chickens [26, 27, 49–51]. Although the results obtained have been promising, it is a fact that the isolated probiotics were characterized biochemically and by 16S rRNA sequence analyses (Microbial ID Inc., Newark, DE 19713, USA), subsequently, they were evaluated using *in vitro* models to determine their activity against pathogens, and, finally, the candidates were tested in *in vivo* models with the purpose of obtaining a well-characterized functional product.

Extensive research conducted by our laboratory determined the antimicrobial capability of several lactic acid bacteria (LAB) isolates mainly against *Salmonella* in *in vitro* models. However, only 11 were selected to produce a product called FloraMax®-B11 given their effect against *Salmonella*. Subsequently, these LAB were characterized by 16S rRNA sequence analyses (**Table 1**) [52].

However, since these LAB were grown together in a culture, the only LAB that remained viable were *Lactobacillus salivarius* and *Pediococcus parvulus*, two strains of poultry gastrointestinal origin. Despite this, *in vitro* studies showed that FloraMax®-B11 presented antimicrobial activity against *Salmonella* enteritidis, *Escherichia coli* (O157:H7), and *Campylobacter jejuni* [47] (**Table 2**). The antimicrobial activity of this probiotic culture could be due to the accumulation of primary metabolites such as lactic acid, ethanol, and carbon dioxide and to the production of other antimicrobial compounds such as bacteriocins [53]. Furthermore, the probiotic culture was capable of maintaining its viability under acidic conditions (pH = 3) for 4 h, which agrees with other studies where *Lactobacillus* spp. isolates were resistant to low pH, with high survival rates at

**221**

**Table 1.**

**Table 2.**

*Symbols: +, inhibition.*

reducing its toxic effect [59, 60].

*FloraMax®-B11 against enteropathogenic bacteria.*

In neonatal broilers, the administration of 1 × 106

and *Salmonella* typhimurium (ST) (1 × 104

*The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins*

**LAB identification 16S rRNA sequence analyses (Microbial ID Inc.)**

pH 3.0 for 1 h [54]. Although probiotic bacteria need to survive passage through the stomach (pH 1.5–2.0) [55], and maintain their viability for 4 h or more [56] before reaching the intestine, the feed passage rate for birds is faster; therefore, bacterial acid tolerance is not as critical in chickens as it is in other animals [57]. Additionally, this probiotic culture grew at low and high temperatures for 4 h of incubation. However, the ability to grow at high temperatures is an important advantage since the production of lactic acid increases, and, therefore, the bacterial load decreases [58]. The probiotic culture was also able to tolerate high osmotic concentrations of NaCl, but it is extremely important since it has been reported that a high salt concentration could affect the physiology of probiotics, as well as their enzymatic activity, water activity, and metabolism [58]. Finally, this probiotic culture has its ability to tolerate bile salt concentrations of 0.4, 0.5, and 0.6% for 2, 4, and 24 h of incubation. Bile resistance of probiotics is related to their enzyme activity of bile salt hydrolase that helps to hydrolyze conjugated bile,

*In vitro assessment of antimicrobial activity of Lactobacillus salivarius and Pediococcus parvulus present in* 

*Lactobacillus salivarius* + + + *Pediococcus parvulus* + + +

*Escherichia coli* **(O157:H7)**

*Campylobacter jejuni*

Furthermore, the effect of this commercial product (FloraMax®-B11) has been evaluated in different models of infection both in broiler chickens and turkeys.

oral gavage 1 h after the chicks were challenged with *Salmonella* enteritidis (SE)

SE and ST, as well as the SE counts by >2.9 log, 24 h post-LAB administration [61] (**Table 3**). In contrast, there were no significant differences at 6- and 12-h post-LAB administration, but a slight reduction was observed at 12-h post-LAB

cfu/bird FloraMax®-B11 by

cfu/bird) reduced the incidence of

*DOI: http://dx.doi.org/10.5772/intechopen.88817*

 *Pediococcus parvulus Weissella confusa Weissella confusa Pediococcus parvulus Lactobacillus salivarius* 37B *Weissella confusa Weissella confusa*

 *Weissella paramesenteroides Lactobacillus salivarius Lactobacillus salivarius Pediococcus parvulus*

*Identifications of FloraMax®-B11 (FM-B11) lactic acid bacteria (LAB).*

*Salmonella* **enteritidis**

*The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins DOI: http://dx.doi.org/10.5772/intechopen.88817*


#### **Table 1.**

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

**3. Probiotic application in poultry industry**

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

Although probiotics are considered potential alternatives to antibiotic use in poultry because they leave no residues in the meats and eggs given their modes of action, the variety of microorganisms in terms of species and even between strains of the same species, as well as their variation in metabolic activity, could affect their effectiveness. Furthermore, other factors that influence the effectiveness of probiotics in poultry are the species of origin, the probiotic preparation method, the survival of colonizing microorganisms in the gastrointestinal tract conditions, the environment where the birds are raised, the application time and administration route of probiotics, the immunologic state, the lineage of poultry, as well as age and concomitant use of antibiotics [45, 46]. Below are some of the applications of

**3.1 Effects of lactic acid bacteria against** *pathogens* **of importance in poultry**

Several articles published by our laboratory have shown that the use of probiotics as a replacement of antibiotics in poultry production has had positive effects by reducing the growth of pathogens in *in vitro* models that simulate or not the three main compartments in birds (crop, proventriculus, and intestine) [47, 48], as well as the colonization of pathogens through the gastrointestinal tract in both turkeys and broiler chickens [26, 27, 49–51]. Although the results obtained have been promising, it is a fact that the isolated probiotics were characterized biochemically and by 16S rRNA sequence analyses (Microbial ID Inc., Newark, DE 19713, USA), subsequently, they were evaluated using *in vitro* models to determine their activity against pathogens, and, finally, the candidates were tested in *in vivo* models with the purpose of obtaining a well-characterized

Extensive research conducted by our laboratory determined the antimicrobial capability of several lactic acid bacteria (LAB) isolates mainly against *Salmonella* in *in vitro* models. However, only 11 were selected to produce a product called FloraMax®-B11 given their effect against *Salmonella*. Subsequently, these LAB were

However, since these LAB were grown together in a culture, the only LAB that remained viable were *Lactobacillus salivarius* and *Pediococcus parvulus*, two strains of poultry gastrointestinal origin. Despite this, *in vitro* studies showed that FloraMax®-B11 presented antimicrobial activity against *Salmonella* enteritidis, *Escherichia coli* (O157:H7), and *Campylobacter jejuni* [47] (**Table 2**). The antimicrobial activity of this probiotic culture could be due to the accumulation of primary metabolites such as lactic acid, ethanol, and carbon dioxide and to the production of other antimicrobial compounds such as bacteriocins [53]. Furthermore, the probiotic culture was capable of maintaining its viability under acidic conditions (pH = 3) for 4 h, which agrees with other studies where *Lactobacillus* spp. isolates were resistant to low pH, with high survival rates at

characterized by 16S rRNA sequence analyses (**Table 1**) [52].

*2.1.2 Aflatoxins*

walls of probiotic strains [44].

probiotics in poultry.

functional product.

**220**

*Identifications of FloraMax®-B11 (FM-B11) lactic acid bacteria (LAB).*


#### **Table 2.**

*In vitro assessment of antimicrobial activity of Lactobacillus salivarius and Pediococcus parvulus present in FloraMax®-B11 against enteropathogenic bacteria.*

pH 3.0 for 1 h [54]. Although probiotic bacteria need to survive passage through the stomach (pH 1.5–2.0) [55], and maintain their viability for 4 h or more [56] before reaching the intestine, the feed passage rate for birds is faster; therefore, bacterial acid tolerance is not as critical in chickens as it is in other animals [57]. Additionally, this probiotic culture grew at low and high temperatures for 4 h of incubation. However, the ability to grow at high temperatures is an important advantage since the production of lactic acid increases, and, therefore, the bacterial load decreases [58]. The probiotic culture was also able to tolerate high osmotic concentrations of NaCl, but it is extremely important since it has been reported that a high salt concentration could affect the physiology of probiotics, as well as their enzymatic activity, water activity, and metabolism [58]. Finally, this probiotic culture has its ability to tolerate bile salt concentrations of 0.4, 0.5, and 0.6% for 2, 4, and 24 h of incubation. Bile resistance of probiotics is related to their enzyme activity of bile salt hydrolase that helps to hydrolyze conjugated bile, reducing its toxic effect [59, 60].

Furthermore, the effect of this commercial product (FloraMax®-B11) has been evaluated in different models of infection both in broiler chickens and turkeys. In neonatal broilers, the administration of 1 × 106 cfu/bird FloraMax®-B11 by oral gavage 1 h after the chicks were challenged with *Salmonella* enteritidis (SE) and *Salmonella* typhimurium (ST) (1 × 104 cfu/bird) reduced the incidence of SE and ST, as well as the SE counts by >2.9 log, 24 h post-LAB administration [61] (**Table 3**). In contrast, there were no significant differences at 6- and 12-h post-LAB administration, but a slight reduction was observed at 12-h post-LAB


*\* A significant (p* ≤ *0.05) difference was observed between control and treated within a single experiment in each column.*

#### **Table 3.**

*Effect of lactic acid bacteria (LAB) on Salmonella typhimurium (ST) or Salmonella enteritidis (SE) recovered from cecal tonsils or ceca of broiler chicks 24-h post-LAB administration.*

administration. These data suggest that the mechanism to reduce *Salmonella* was initiated within the first 12 h after treatment. Probably the reduction of *Salmonella* is due to the set of mechanisms of action of probiotics: bacterial interactions (competitive exclusion) or stimulation of a host innate immune response. The competitive exclusion could have included competition for receptor sites, production of volatile fatty acids that are inhibitors of certain enteric pathogens, production of bacteriocins, or competition with pathogens and native flora for limiting nutrients [62]. Furthermore, since the *Salmonella* recovery was performed in the early stages of infection, the innate immune response could be responsible for the reduction of *Salmonella*.

In our other studies, the administration of FloraMax®-B11 in drinking water (106 cfu/mL) for 3 days post-SE challenge (104 cfu/bird) using two presentations, liquid and lyophilized significantly reduced the incidence of *Salmonella* [63], which agrees with other studies [64]. Furthermore, the administration of FloraMax®-B11 at the same concentration as the previous study after 1-h post-*Salmonella* Heidelberg (SH) challenge practically eliminated the concentration of SH, as well as its incidence, since only one sample was positive. However, in turkey poults under the same experimental conditions (**Table 4**), although similar significant results were observed at day 3 post-FloraMax®-B11 administration, it is clear that poults were more susceptible to SH colonization than chicks [51].

Finally, trying to find FloraMax®-B11 applications in poultry, we opted for spray application since it could be more efficient and has lower cost than its application in


**223**

**Table 5.**

*\**

*and treatment regime in each column.*

*The Use of Probiotics in Poultry Production for the Control of Bacterial Infections and Aflatoxins*

drinking water since it is important to take into account water quality and medicator/proportioner function [65]. The results obtained were promising since when the probiotic was applied by spray and in drinking water, there was a reduction in the recovery of SE (55 and 50%, respectively; controls 85%) when chicks were held for 8 h prior to SE challenge and placement. In the same way, when probiotic was applied by spray or in drinking water and SE challenge occurred simultaneously, with placement 8 h after treatment, a marked and significant reduction of SE recovery was noted after 5d (10 and 40%, respectively; controls 55%). Furthermore, when the probiotic was sprayed and chickens were SE challenged simultaneously, with placement 8 h after treatment, a significant reduction of SE recovery was again noted in both the spray and DW application (80% controls, 15% spray, 15% drinking water) (**Table 5**). These results suggest that the spray application of this probiotic can be effective in protecting chicks against *Salmonella* infection. Furthermore, hatchery administration could prove to be a more effective way to administer probiotics because the chicks will be receiving the beneficial bacteria at the earliest

In this regard, an *in ovo* study was performed to know the effectiveness of FloraMax®-B11 [66]. For this, 18-day-old embryos were candled and inocu-

amnion. On day 21, chicks were pulled from hatchers to measure hatchability. Subsequently, all chickens were then orally gavaged with SE on the day of hatch

 cfu/chick) and maintained for 7 days. *Salmonella* recovery was done 24-h post-SE challenge. Body weight (BW) was determined at days 1, 3, and 7. In this experiment, a significant increase in BW was observed. Furthermore, chickens that received the probiotic culture showed a significant reduction in the incidence and counts of SE in cecal tonsils when compared with saline control chickens

These results agree with another study where the *in ovo* colonization with a probiotic could become an important method to reduce *Salmonella* and other intestinal bacterial infections in poultry [67]. Regarding the increase of BW in the group treated with the probiotic, this could be due to the significant morphometric

**Treatment regimen Group Cecal tonsils**

Treat-challenge-place immediately Control 95% (19/20) 95% (19/20)

Treat-hold 8 h-challenge-place Control 85% (17/20) 70% (14/20)

Treat-challenge-hold 8 h-place Control 55% (11/20) 80% (16/20)

*Indicates significant (p < 0.05) differences were observed between control and treated within a single experiment* 

*\*\*Significantly (p < 0.01) different than all groups within a single experiment and treatment regime in each column.*

*Salmonella enteritidis recovery from cecal tonsils of broiler chicks 5-day post-challenge.*

changes in the duodenum and ileum observed at day 1 of age.

cfu FloraMax®-B11 via *in ovo* injection into the

**Exp. 1 Exp. 2**

Probiotic (drinking water) 75% (15/20) 25% (5/25)\*\* Probiotic spray 90% (18/20) 80% (16/20)

Probiotic (drinking water) 50% (10/20)\* 70% (14/20) Probiotic spray 55% (11/20)\* 80% (16/20)

Probiotic (drinking water) 44% (7/20)\* 15% (2/20)\* Probiotic spray 20% (2/20)\*\* 15% (2/20)\*

*DOI: http://dx.doi.org/10.5772/intechopen.88817*

possible time, in the absence of *in ovo* administration.

lated with either saline or 104

(~104

(**Table 6**).

*a,bDifferent superscripts within columns indicate significant differences (p < 0.05).*

*1 Data expressed as positive/total poults (%).*

*2 Data expressed as mean ± SE.*

*\* p < 0.001.*

#### **Table 4.**

*In vivo evaluation of FloraMax-B11 against Salmonella Heidelberg (SH) at 24 and 72 h in poults.*
