An Overview of the Use and Applications of Limosilactobacillus fermentum in Broiler Chickens

The implementation of government regulations on antibiotic use, along with the public’s concern for drug resistance, has strengthened interest in developing alternatives not only aimed at preserving animal production but also at reducing the effects of pathogenic infections. Probiotics, in particular, are considered microorganisms that induce health benefits in the host after consumption of adequate amounts; they have been established as a potential strategy for improving growth, especially by stimulating intestinal homeostasis. Probiotics are commonly associated with lactic acid bacteria, and Limosilactobacillus fermentum is a well-studied species recognized for its favorable characteristics, including adhesion to epithelial cells, production of antimicrobial compounds, and activation of receptors that prompt the transcription of immune-associated genes. Recently, this species has been used in animal production. Different studies have shown that the application of L. fermentum strains not only improves the intestinal ecosystem but also reduces the effects caused by potentially pathogenic microorganisms. These studies have also revealed key insights into the mechanisms behind the actions exerted by this probiotic. In this manuscript, we aim to provide a concise overview of the effects of L. fermentum administration on broiler chicken health and performance.


Introduction
In animal farming, antibiotics have been utilized not only for prophylaxis purposes but also for growth promotion, notwithstanding the forthcoming health threat associated with resistance [1][2][3]. The use of antibiotics as growth promoters has been forbidden in the U.S. and European Union [4,5], although this practice is still common in other regions, principally in rural areas that lack efficient administrative systems and legislative measures to curb drug misuse [6,7]. As a result, considerable attention has been drawn to the investigation of alternatives (e.g., probiotics) to replace the use of antibiotics for feed enrichment in animal production [8][9][10].
Probiotics have been defined by the Food and Agriculture Organization (FAO) and World Health Organization (WHO) as "live microorganisms that, when consumed in adequate amounts, confer a health effect on the host" [11]. However, an expert panel later reworked the definition to be utilized as follows: "products that deliver live microorganisms with a suitable viable count of well-defined strains with a reasonable expectation of delivering benefits for the wellbeing of the host" [12]. The most common probiotic microorganisms are bifidobacteria and lactic acid bacteria, although others are commonly recognized, including Enterococcus, Lactococcus, Streptococcus, Propionibacterium, and the

Properties of Limosilactobacillus fermentum
Limosilactobacillus fermentum was formerly known as Lactobacillus fermentum, and the taxonomy of Lactobacillaceae was revisited based on different approaches, including genomics and proteomics [50][51][52]. The genus classification refers to the synthesis of exopolysaccharides (limosus-slimy) [50]. The rod-shaped L. fermentum is recognized as a gram-positive, non-sporulating, catalase-negative, gas-producing facultatively anaerobic bacterium that is heterofermentative and capable of utilizing several carbohydrates, including arabinose, cellobiose, galactose, and maltose, among others [50,[52][53][54]. Strains of L. fermentum are acknowledged as nomadic or free-living and occur spontaneously in different environments. They have been isolated not only from fermenting plant materials and fermented cereals but also from dairy products, sewage, manure, and the gastrointestinal tract and feces of birds, pigs, and humans [50,[55][56][57][58]. Indeed, L. fermentum, as well as other lactobacilli, remain physiologically active in the gastrointestinal tract, with the potential to influence host physiology [55]. L. fermentum strains are known for exerting beneficial effects on human health [59][60][61][62][63]. This species is recognized as safe and is included in the official lists of European, American, and Chinese food safety authorities [64][65][66]. It has also been used for developing commercially available dietary supplements [61,67]. Selected strains have demonstrated particular probiotic characteristics that render them beneficial for the host (Table 1).
Once inside the host, probiotic bacteria are exposed to different types of stress, including low pH and elevated concentrations of bile salts. L. fermentum strains have evidenced high viability when encountering such conditions [57,68,69]; additionally, L. fermentum not only exhibits strong surface hydrophobicity but also high autoaggregation capacity; these characteristics have been associated with a facilitated interaction between bacterial and intestinal epithelial cells [53,70]. In general, lactobacilli are capable of adhering to intestinal mucosa [53,71,72]; this process is mainly mediated by adhesion proteins (e.g., binding proteins, sortases), but other molecules are also involved (e.g., LTA, LPS, PG) [53,73]. Particularly in L. fermentum, mucin-and fibronectin-binding proteins (Mub and Fbp, respectively), along with sortases, have been determined, with upregulation of mub, fbp, and sor observed in the presence of mucin, bile, and pancreatin [71,72]. Lipoteichoic acids have also been held responsible for the adhesion capabilities of some strains, along with other factors, including electrostatic interactions or passive forces [74,75]. Adherence of these molecules has proved beneficial for maintaining the integrity of the gut barrier; for instance, the LPS of L. fermentum CECT5716 increased the production of mucins in model intestinal cells [76]. This interaction permits the competitive exclusion of potential pathogens such as Helicobacter pylori, Campylobacter jejuni, and Staphylococcus aureus [77][78][79]. Pathogen clearance is enhanced by the capacity of L. fermentum strains to produce a variety of antimicrobial compounds, commonly known as bacteriocins. These ribosomally synthesized peptides are capable of disturbing the membrane or inducing cell wall degradation, although the mode of action of certain peptides remains unknown [79][80][81]. Various strains have been linked to these antimicrobial compounds (e.g., fermencin SD11, LF-BZ532, LBM97-1, LBM97-4, and LBM97-5), which have shown activity against gram-positive and gram-negative bacteria such as pathogenic E. coli, Salmonella spp., S. aureus, or Listeria spp. [82][83][84][85]. Also, other secondary metabolites (e.g., lactic and organic acids, hydrogen peroxide) contribute to the overall antibacterial activity of L. fermentum [78,86,87]. Bacterial infections can influence the concentration of reactive oxygen species/reactive nitrogen species (ROS/RNS) with the potential to induce pathological effects [88,89]. Some L. fermentum strains possess the entire glutathione-associated complex, which has made them attractive as potential modulators of oxidative stress [90][91][92]. This active redox tripeptide can reduce oxidative agents directly or indirectly as a cofactor of a group of enzymes involved in eliminating electrophilic compounds [93,94]. Moreover, the presence of L. fermentum is known to activate receptors that ultimately favor the transcription of antioxidant genes, which lessens oxidative stress [95]. L. fermentum PC-10 Poultry gut Inhibition of S. Gallinarum growth [56] L. fermentum PG1 Poultry digesta Adhesion to the epithelial cells; survival at low pH; tolerance to bile salts; antibacterial activity [57] L. fermentum Y57 Artisanal yogurt Reduction of hypercholesterolemia in rats [62] L. fermentum GR-3 Fermented food Ameliorates human hyperuricemia via degrading and promoting excretion of uric acid [63] L. fermentum MBD93 -Adhesion to gastrointestinal mucin; exclusion of enteropathogenic bacteria [71] L. fermentum 10 Human feces Strong adhesion to * HT29 epithelial cells; high tolerance to bile salt; autoaggregation activity; reduction of E. coli adhesion; antibacterial and antioxidant activity [75] L. fermentum J23 Cheese Antimicrobial activity of bacteriocin-containing fractions; growth inhibition of E. coli, S. aureus, L. innocua, and S. Typhimurium [82] L. fermentum SD11 Human oral cavity Production of fermencin SD11; antibacterial activity against oral pathogens [83] L. fermentum BZ532 Cereal beverage Production of bacteriocin LF-BZ532 with a broad antimicrobial spectrum, including anti-listerial and anti-pseudomonas activity [84] L. fermentum LBM97 Fermented vegetable Production of bacteriocins LBM97-4 and LBM97-5 with antibacterial activity against S.
L. fermentum interacts with intestinal epithelial cells (IECs), macrophages, dendritic cells, and immune cells; this induces the expression of different cytokines that modulate T cell polarization [65]. Such interactions are, on the one hand, associated with LTA, LPS, or PG of bacteria and, on the other hand, with Toll-like receptors (TLR2 and TLR4) and nucleotide-binding oligomerization domain-containing proteins (NOD2) of the host. This triggers the recruitment of adaptor proteins (MyD88, NF-κB) that transduce the signal to the nucleus and modulate the expression of response genes (e.g., cytokines) [102]. In intestinal cells, L. fermentum UCO-979C decreased expression of TNF-α, IL-1β, IL-6, and MCP-1 in H. pylori-challenged cells, although a slight increase was observed when compared to control conditions [96]. Exposure to L. fermentum CECT5716 also modulated the expression of TNF-α, IL-1β, and IL-6 in CMT-93 cells, which are used as a model cell line of the intestine [76]. Furthermore, L. fermentum DLBSA204 did not only activate macrophages and induce the synthesis of nitric oxide linked to bacterial clearance, virus inactivation, and tumor cytotoxicity but also reduced the expression of IL-6 and IL-1β [97]. Other strains (UCO-979C, IM12) have also demonstrated the ability to alter the expression of cytokines and other signaling molecules in macrophages [96,98]. In dendritic cells, L. fermentum AGR1487 modulated transcription of IL-6, TNFα, IL-10, and IL-12, whereas L. fermentum CECT5716 could induce the expression of MHC class II and other costimulatory molecules (e.g., CD40, CD80) [99,100]. The latter strain, when incubated with peripheral blood mononuclear cells (PBMCs), induced the activation of NK and Treg cells along with the production of cytokines including IL-1β, IL-18, TNF-α, and IFN-γ. PBMCs are constituted of lymphocytes and monocytes and are utilized for screening molecules with immunomodulatory properties [101]. The use of these cells has also demonstrated that exposure to L. fermentum B633 suppressed the production of IL-13 while prompting the synthesis of IL-12 and IFN-γ [103].

Applications of L. fermentum in Broiler Chickens
Broiler chickens have been bred exclusively for meat consumption, and the efficiency of the industry has been linked to innovations in management practices, breeding, nutrition, and disease control. However, complications from intestinal infectious diseases have negatively influenced production parameters, so antibiotics along with vaccines have extensively contributed to the efficiency of large-scale commercialization [104,105]. As the industry is detaching from the use of antibiotics for prophylaxis and performance, novel schemes have emerged for pathogen control and body weight enhancement, including probiotics, prebiotics, plants and algae, organic acids, bacteriophages, and essential oils [32-36,106]. Probiotics, in general, modulate key physiological characteristics that ultimately ameliorate animal development [48,49]. Strains of L. fermentum, in particular, have proven convenient for augmenting growth parameters, which has been related to their abilities to improve gut health by regulating architecture, epithelial integrity, microbial diversity, and inflammation. Moreover, these strains have been employed to antagonize the effects of potentially harmful bacteria such as Campylobacter, Salmonella, Clostridium, and Pasteurella (Table 2). ↑ VH and ↑ VH:CD ratio in the small intestine; ↑ GC count in the duodenum and jejunum; positive correlation between gut architecture and BW in early stages ↑ mRNA expression of IL-4, IL-18, IL-13; ↓ mRNA expression of IL-15, IL-16, IL-17RA, IL-9, IL-6RA and CXCL-12; ↑ percentages of IgM and CD8 cells in the cecum of young chickens Antagonistic effects against C. jejuni, C. coli, and S. Infantis; attenuation of intestinal impairments and regulation of cecal inflammatory response [107][108][109][110]

Gut Health, Microbiota, and Homeostasis
The gut ecosystem is acknowledged as a complex environment involving different constituents. The gut epithelium not only acts as a barrier against invading microorganisms and their toxins but also plays a fundamental role in host immunity and nutrient acquisition [125,126]. Intestinal epithelial as well as immune-associated cells are of prime importance; the metabolism of these cells could be modulated by various factors including age, housing, gender, or diet [127,128]. Furthermore, the development of a stable microbiota is known to stimulate the immune system and prevent enteric diseases [129][130][131]. A suspension of L. fermentum Biocenol CCM 7514 (1 × 10 9 CFU/0.2 mL), administered orally during the first week of growth, augmented villus height in the small intestine in 8-day-old and 11-day-old chicks. The probiotic ultimately improved the villus-height-to-crypt-depth (VH:CD) ratio in the duodenum and ileum; a positive correlation between such conditions and the animal body weight was also determined [107]. This strain has also improved the aforementioned parameters in duodenal and jejunal sections of 15-day-old chicks; however, in this case, the number of goblet cells was determined and proved to be higher in animals exposed to the probiotic than in untreated ones, although no differences were observed regarding the expression of muc2 [108]. On the contrary, in jejunal and ileal sections of 21-day-old chicks inoculated with L. fermentum 1.2029 (1 × 10 8 CFU/0.5 mL), expression of this gene was higher than that of untreated birds. Nonetheless, an overall increment of goblet cell density was only evidenced in the jejunum [111].
Dietary supplementation of L. fermentum KGL4 (1 × 10 8 CFU/mL) during the starter phase did not alter intestinal architecture; although a decrease in coliform and enterococci counts was reported, this was accompanied by a proliferation of lactobacilli. An overall increase in animal body weight was observed in probiotic-treated animals [114]. Likewise, dietary administration of L. fermentum NKN51 (1 × 10 7 CFU/gM) for a period of 28 days reduced the total count of cecal E. coli while augmenting those of lactobacilli. In jejunal sections, this strain improved villus height, villus width, VH:CD ratio, and surface area; feed conversion ratio and body weight were also ameliorated [115]. Moreover, birds fed a diet containing L. fermentum 1.2133 (2.5 × 10 8 CFU) showed larger numbers of lactic acid bacteria than control animals in the ileum and cecum; in the latter, a reduction in Salmonella counts was also registered [116]. Finally, L. fermentum has been used to develop multi-strain probiotics with potential applications in broilers. For instance, this species, along with L. plantarum, Pediococcus acidilactici, Enterococcus faecium, and Saccharomyces cerevisiae, has been mixed at equal ratios and added to the diet at a dose of 1 × 10 8 CFU/kG between the third and 21st days. Incorporation of this mixture into the diet did not only reduce enterobacteria counts but also augmented the number of lactobacilli in both the ileal and cecal contents of 28-day-old chicks. Exposure to the probiotic also improved body weight and the feed conversion ratio [117]. Furthermore, a rapeseed meal fermented with a mixture of probiotics, including L. fermentum CICC 20176 and L. fermentum CGMCC 0843, improved the VH:CD ratio in the jejunum and ileum of 21-and 42-day-old chicks; no differences were found regarding animal performance [118,119].
Nutrition is crucial not only for sustaining the prooxidant-antioxidant balance but also for regulating fat metabolic function [132,133]. Reactive oxygen or nitrogen species can modulate primary immune defense, albeit prolonged exposure leads to a disruption of the oxidant/antioxidant network; this imbalance ultimately results in an acceleration of pathological inflammation [134,135]. The inclusion of L. fermentum CCM 7158 (1 × 10 9 CFU) in drinking water reduced the total antioxidant status in 42-day-old broiler chickens, although it influenced neither bilirubin nor albumin levels. Its administration, however, reduced the content of serum triglycerides. This has also been observed in chickens (42 days old) fed a diet enriched with L. fermentum KGL4 (1 × 10 8 CFU/mL); furthermore, the probiotic reduced LDL content while augmenting levels of HDL. In both cases, an increment in body weight was observed in probiotic-treated animals [114,120]. Similarly, L. fermentum CIP 102980 (1 × 10 7 CFU/mL) improved growth performance and feed conversion ratio in 36-day-old birds [121].

Modulation of Immune Reaction
Strains of L. fermentum are recognized for their immunomodulatory properties, as they are able to interact with immune cells and either suppress or stimulate the production of various inflammatory cytokines [136][137][138]. Oral administration of L. fermentum Biocenol CCM 7514 (1 × 10 9 CFU/0.2 mL) during the first week of growth did not only induce expression of anti-inflammatory cytokines (IL-13, IL-4), but also reduced transcription of pro-inflammatory factors in the cecum of one-week-old chickens, including IL-15, IL-16, IL-17RA, LIF, IL-6RA, and CXCL-12 [107,109,110]. This treatment also increased the percentages of lamina propria IgM plasma cells and intraepithelial CD8 cells [109]. The latter were also augmented in the jejunum of 21-and 42-day-old chickens when a probiotic product was added to the basal diet; this product contained 1× 10 7 CFU/g of L. fermentum JS and 2 × 10 6 CFU/g of S. cerevisiae. The percentages of intraepithelial CD4 and CD3 cells were also enhanced, and overexpression of TLR2 and TLR4 was registered [122]. Additionally, a mixture of probiotics, containing approximately 5 log CFU/mL of L. fermentum CICC 20176 and Bacillus subtilis (1:1), was used to ferment a meal based on rapeseed; dietary administration of this mixture improved the concentration of serum IgG and IgM in 21-day-old chickens [118].

Antagonism against Potentially Harmful Bacteria
The ability of L. fermentum to antagonize a variety of dangerous bacteria is not only associated with competitive exclusion but also with the secretion of bacteriocins and secondary metabolites that contribute to the overall antimicrobial activity [77,78,[82][83][84][85]. Moreover, stimulation of the immune system by L. fermentum could prime the host's response to potential infections [96,98]. For example, the use of L. fermentum Biocenol CCM 7514 could prime the immune response during Campylobacter spp. infections. Campylobacter has been traditionally regarded as commensal in birds, although it has been reported that its presence induces the expression of pro-inflammatory cytokines, which may lead to intestinal damage and ultimately to weight loss [139,140]. Inoculation with the probiotic (1 × 10 9 CFU/0.2 mL) during the first week of growth enhanced the immune response in 8-day-old challenged chicks. In cecal sections, the percentage of CD8 and IgA plasma cells in the epithelium and lamina propria was augmented compared to C. coli-infected animals; furthermore, a downregulation of inflammatory cytokines (e.g., IL-15 and IL-16) was also observed [109]. A similar cecal response has been registered in the context of a C. jejuni infection; early treatment with the aforementioned strain (1 × 10 9 CFU/0.2 mL) modulated the expression of inflammatory cytokines, including IL-1β, IL-17, and IL-15, in 8-day-old challenged chicks. Moreover, in these animals, C. jejuni invasion reduced the height of villi in the duodenum, jejunum, and ileum; in the latter section, crypt depth was also affected. Application of L. fermentum Biocenol CCM 7514 did not only prevent these effects but actually ameliorated intestinal architecture, even when compared to untreated animals [107,110].
Different serovars of Salmonella are capable of eliciting intestinal mucosal damage in broiler chickens [141,142]. The beneficial effects exerted by L. fermentum Biocenol CCM 7514 regarding gut health have also been evidenced in chickens challenged with S. Infantis. Infection with this serovar reduced the VH:CD ratio in the small intestine of 15-day-old birds. Early probiotic treatment (1 × 10 9 CFU/0.2 mL) did not only relieve the observed impairments but also improved the calculated ratios when compared to basal levels. In animals previously exposed to the probiotic, the presence of S. Infantis increased the surface of villi and augmented the number of goblet cells in the small intestine compared to control conditions. Finally, higher IgM serum levels were also reported in the co-exposure group than in untreated birds [108]. Infection with S. Pullorum also affected intestinal homeostasis in 15-day-old chicks. First, the pathogen decreased total anaerobic bacteria while increasing the number of total aerobic bacteria in the ileum and cecum; these outcomes were relieved by animal exposure to L. fermentum 1.2133 (2.5 × 10 8 CFU). In particular, probiotic administration reduced the presence of Salmonella in challenged animals. Second, S. Pullorum infection triggered lesions in duodenal villi, evidencing accumulation of erythrocytes and autolysis; the latter was also observed in ileal goblet cells. Previous inoculation with the probiotic relieved these conditions, as few erythrocytes were found in villi and injuries were local and fewer in number [116]. Similarly, S. Enteritidis negatively affected intestinal homeostasis, as it elicited hemorrhagic lesions and the expression of inflammatory cytokines (IL-1β and LITAF) in the cecal tonsils of 11-day-old chickens. These effects were lessened by oral inoculation of a mixture called Lactobacilli-based probiotic, containing L. acidophilus, L. reuteri, L. salivarius, and L. fermentum (1 × 10 5 CFU). Ingestion of the mixture proved to increase the percentage of macrophages and CD4 T cells, which was not observed when birds were only infected with S. Enteritidis [123].
C. perfringens is associated with intestinal barrier damage, unstable intestinal microbiota, and reduced immunity in birds [143,144]. L. fermentum strains have also been shown to be beneficial in diminishing pathogenic outcomes induced by these bacteria, regardless if the probiotic was supplemented orally or in the diet. First, oral administration of L. fermentum 1.2029 (1 × 10 8 CFU/mL) demonstrated protection against the negative effects caused by C. perfringens in the ileum of 28-day-old animals. Infection prompted the upregulation of inflammatory factors, such as IFN-γ and TLR2, and the downregulation of IL-10. The latter was upregulated in the presence of the probiotic, whereas the former two were downregulated. In addition, the pathogen induced-hyperplasia of the lamina propria, along with lymphocyte infiltration and crypt structure deterioration. Again, the lesions derived from infection were not detected in birds previously exposed to the probiotic strain [112]. Second, the incorporation of L. fermentum (1 × 10 9 CFU/g) into the basal diet relieved the intestinal damage elicited by C. perfringens in 13-day-old chickens, which involved a decrease in VH:CD ratio in the duodenum, jejunum, and ileum as well as a downregulation of key factors including ZO-1, Mucin-2, and Occludin in the jejunum. Previous exposure of infected animals to probiotic treatment induced even better conditions than those registered in untreated birds [124]. Likewise, C. perfringens inoculation stimulated the expression of the pleiotropic and potentially inflammatory cytokine TGF-β4 in the jejunum; such expression levels were reduced by dietary administration of L. fermentum 1.2029 (1 × 10 9 CFU/kG) in 21-day-old animals. However, treatment with the probiotic also increased transcription of cytokines such as IL-1β, IFN-γ, IL-17, and TGF-β4 in older chicks (28-day-old); this has been linked to the inhibitory and stimulatory effects of the probiotic in both the acute and recovery phases of infection [113]. Finally, P. multocida causes the contagious disease known as "avian cholera", which is linked to high morbidity and mortality [145]. Infection by P. multocida did not only alter the ileal and cecal microbiota but also reduced body weight and increased mortality rates in 28-day-old chickens. A mix of probiotics, including L. fermentum, was supplemented in the feed (1 × 10 8 CFU/kG). Challenged animals exposed to the enriched diet showed no evidence of P. multocida effects on the intestine; body weight loss and mortality rates were also attenuated. In general, previous exposure to the probiotic reduced intestinal enterobacteria counts while augmenting the total number of lactic acid bacteria. Furthermore, the probiotic mixture reduced cholesterol and glucose while eliciting the production of lymphocytes and upregulating the expression of anti-inflammatory genes in the cecal mucosa [117].
Results from animal trials involving L. fermentum strains have demonstrated the beneficial effects of this probiotic on intestinal health and growth performance. These outcomes have also evidenced the protective effects of L. fermentum against potential pathological conditions induced by other bacteria, as it can adhere to the epithelium and secrete antimicrobial compounds. Moreover, treatment with these lactic acid bacteria improves intestinal health, namely gut architecture as well as the immune response (Figure 1). Despite the relevance of current research, further studies must be conducted to ensure the safety and efficiency of these strains, especially regarding possible side effects.

Conclusions
The in vivo studies summarized here exhibit the beneficial effects of L. fermentum administration on broiler chicken physiology and growth, especially with regards to gut health, nutrition, and modulation of the immune response. Furthermore, this species has demonstrated the potential for antagonizing the negative effects exerted by potentially pathogenic bacteria. In particular, strains of L. fermentum have proven beneficial for ameliorating conditions in the small intestine, including VH:CD ratio, microbial composition, integrity of the epithelium, and inflammation. Broiler chickens are bred for meat, and the productivity of the industry has been associated with management, breeding, and disease control practices that normally employ antibiotics for both prophylaxis and performance. However, due to the public concerns raised by the use of antibiotics in animal husbandry, many countries have banned their use as growth promoters. Thus, alternatives must be designed not only to maintain production performance but also to curb the effects of infectious diseases. Probiotics have been established as a potential strategy for preventing the disruption of the gut microbiota and preserving intestinal homeostasis. They represent a possible feed additive that may, or may not, have an influence on profitability; however, in the absence of antibiotics, these species definitely represent an important option for supporting animal growth and providing protection against invading pathogens. A variety of L. fermentum strains, administered orally, dietary, or in drinking water, have proved advantageous for improving such conditions in broiler chickens. Further research, however, should not only focus on determining the effects of probiotics on animal physiological conditions but also on deciphering the mechanisms behind their action, which might lead to the discovery of novel potential therapeutic targets. Undoubtedly, the evidence gathered so far demonstrates that L. fermentum should be considered as a potential ingredient when developing nutritional supplements aimed not only at improving growth conditions but also at preventing and treating infectious diseases.