**Probiotic Applications in Autoimmune Diseases**

**Probiotic Applications in Autoimmune Diseases**

DOI: 10.5772/intechopen.73064

Gislane L.V. de Oliveira Additional information is available at the end of the chapter

Gislane L.V. de Oliveira

[39] Moroti C. Souza Margi Loyanne Francine, Costa Marcela de Rezende, Cavallini D. Sivieri K. Effects of the consumption of a new symbiotic shake on glycemia and cholesterol levels in elderly people w/type 2 diabetes mellitus. Lipids in Health and Disease. 2012;**11**(29)

[40] Jones M, Martoni C, Parent M, Prakash S. Cholesterol-lowering efficacy of a microencapsulated bile salt hydrolase-active Lactobacillus reuteri NCIMB 30242 yoghurt formulation in hypercholesterolaemic adults. British Journal of Nutrition. 2011;**107**(10):1505-1513

[41] Alajeeli, Fakhri, Flayyih, May, Alrubaie, Abdulhadi, Ak Khazaal, Faris. Effect of Probiotic Consumption in the Level of Pep- tide yy, Ghrelin Hormone and Body Weight in Iraqi Obese Female. World Journal of Pharmacy and Pharmaceutical Sciences. 2016;**5**:242-248

[42] Kiessling G, Schneider J, Jahreis G. Long-term consumption of fermented diary produces over 6 months increases HDL cholesterol. European Journal of Clinical Nutrition.

2002;**56**(9):843-849

68 Probiotics - Current Knowledge and Future Prospects

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73064

**Abstract**

Evidences from animal models and humans have implied the involvement of alterations in the gut microbiota in development of some autoimmune diseases. Dysbiosis observed in autoimmune diseases is associated with decreased bacteria function and diversity, impaired epithelial barrier function, inflammation, and decreased regulatory T cells in the gut mucosa. Studies suggest that probiotics influence systemic immune responses, ensure the homeostasis of the healthy microbiota in the intestinal mucosa, and could, therefore, be used as adjuvant therapy to treat immune-mediated diseases. The mechanisms proposed to achieve this include mucus secretion; antimicrobial peptide production; the maintenance of the function of the gastrointestinal-epithelial barrier, ensuring adequate interactions between the gut microbiota and the mucosal immune cells; and, finally, helping the activation of host immune system in response to pathobionts. Here, we described several reports concerning probiotic applications in several animal models of autoimmune diseases and data of the main clinical trials concerning the applicability of probiotics in type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

**Keywords:** dysbiosis, barrier disruption, inflammation, autoimmunity, probiotics

#### **1. Introduction**

Thousands of years ago, Hippocrates, father of medicine, coined the concept that food would serve as medicine and postulated, "Let food be thy medicine, and let medicine be thy food." Nowadays, the concept of food as a medicine appeared as functional foods, referring to any foods or ingredients with nutritional value and that promote a health benefit to the host [1]. Probiotics, prebiotics, and synbiotics are the most popular ingredients used as functional foods and dietary supplements [2].

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

According to the World Health Organization (2002) and the International Scientific Association for Probiotics and Prebiotics (2013), probiotics is defined as "a live organism, which provides a benefit to the host when provided in adequate quantities" [2–4]. Most commonly used probiotic includes lactic acid-producing bacteria, such as *Lactobacillus*, *Bifidobacterium*, and *Streptococcus* species. Non-lactic acid-producing bacteria, such as *Bacillus* and *Propionibacterium*, species and nonpathogenic yeasts, including *Saccharomyces boulfecesardii*, non-spore-forming and nonflagellated rod or coccobacilli, and some helminths, such as *Trichuris suis ova*, could also been used as probiotics [5, 6]. Some of these strains were chosen based on origin, in vitro adherence to intestinal cells, and survival during passage through the gastrointestinal tract [5].

to control blood glucose levels [11]. The etiopathogenesis may involve the interaction of predisposing human leucocyte antigens (HLA) alleles and environmental factors, such as viral infections, vitamin deficiencies, and disruption of the gut microbiota [12]. According to the International Diabetes Federation, more than 96,000 children and adolescents under 15 years will be diagnosed with T1D annually worldwide, and this number is estimated to be more

Probiotic Applications in Autoimmune Diseases http://dx.doi.org/10.5772/intechopen.73064 71

The role of the gut microbiota in T1D etiology has been the subject of research over the last decade to clarify its role in disease development and determine preventive approaches, such as diet manipulation and probiotic administration [12]. Several researches have been carried out to verify whether the administration of probiotics may improve the prognosis of diabetes through modulation of gut microbiota. Probiotics have been identified as effective adjuvants in insulin resistance therapies [14–16]. This health claims apparently stem from the ability of probiotics to secrete antimicrobial substances, competing with other pathogens, strengthen-

The intestinal microbiota might modulate the autoimmune T1D pathogenesis via two mechanisms, recently proposed by Knip and Honkanen [18], from the University of Helsinki, in Finland. In the first phase, an impaired tolerance process in infancy leads to a susceptibility to develop autoimmune diseases, such as T1D, and may result in appearance of autoreactive T cells and autoantibodies. At the second phase, the intestinal dysbiosis predisposes children with genetic susceptibility and positive autoantibodies to develop clinical disease [18].

The inflammasome signaling components are innate immune sensors that are highly influenced by the gut environment and play pivotal roles in maintaining intestinal immune homeostasis [19]. Previous studies suggested the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of

The earliest study to evaluate the efficacy of probiotics in T1D was published in 2005. The study performed by Calcinaro and colleagues, in the University of Perugia, in Italy, investigated the effects of oral administration of the probiotic VSL#3 in nonobese diabetic (NOD) mice development. VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a reduced β-cell destruction. Prevention was associated with an increased production of interleukin (IL)-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive isletinfiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. Oral VSL#3 administration prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis. These data provide a sound rationale for future clinical trials of the

Eleven years later, Kim and colleagues evaluated the effects of *Bifidobacterium lactis* HY8101 on insulin resistance induced by tumor necrosis factor-alpha (TNF-α) in the skeletal muscle

than 132,600 when the age range extends to 20 years [13].

ing the intestinal barrier, and modulating the immune system [17].

probiotics can modulate local and systemic immune responses [20].

primary prevention of T1D by oral VSL#3 administration [21].

*3.1.1. Probiotics in animal models of autoimmune diabetes*

#### **2. Intestinal dysbiosis in autoimmune diseases**

Evidence from animal models has implied the involvement of intestinal dysbiosis in development of some autoimmune diseases [24–26]. Dysbiosis observed in autoimmune diseases is associated with decreased bacteria function and diversity, impaired epithelial barrier function, inflammation, and decreased regulatory T cells (Treg cells) in the gut mucosa [7, 8]. The hypotheses proposed to link dysbiosis with autoimmune diseases include molecular mimicry, bystander T cell activation, and the amplification of autoimmunity by pro-inflammatory cytokines, which is elicited by dysbiotic gut microbiota [9]. In 2016, Lerner and colleagues, from Institute Wendelsheim, in Germany, proposed the posttranslational modification of luminal proteins, promoted by enzymes from altered microbiota, which modify substrates in a different way than performed under homeostatic conditions. The defective posttranslational modification of luminal proteins could generate neo-epitopes that may become immunogenic and induce systemic autoimmunity and trigger autoimmune diseases [9].

Here, we described several reports concerning probiotic applications in several animal models of autoimmune diseases and data of the main clinical trials concerning the applicability of probiotics in type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA), and systemic lupus erythematosus.

#### **3. Probiotics in autoimmune diseases**

Studies suggest that probiotics influence systemic immune responses, ensure the homeostasis of the healthy microbiota in the intestinal mucosa, and could, therefore, be used as adjuvant therapy to treat immune-mediated diseases [4]. The mechanisms proposed to achieve this include mucus secretion, antimicrobial peptide production, the maintenance of the function of the gastrointestinal-epithelial barrier, decreasing oxidative stress, ensuring adequate interactions between the gut microbiota and the mucosal immune cells, and, finally, helping the activation of host immune system in response to pathobionts [10].

#### **3.1. Type 1 diabetes**

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by autoimmune reactions against the insulin-secreting pancreatic β-cells, resulting in exogenous insulin dependence to control blood glucose levels [11]. The etiopathogenesis may involve the interaction of predisposing human leucocyte antigens (HLA) alleles and environmental factors, such as viral infections, vitamin deficiencies, and disruption of the gut microbiota [12]. According to the International Diabetes Federation, more than 96,000 children and adolescents under 15 years will be diagnosed with T1D annually worldwide, and this number is estimated to be more than 132,600 when the age range extends to 20 years [13].

The role of the gut microbiota in T1D etiology has been the subject of research over the last decade to clarify its role in disease development and determine preventive approaches, such as diet manipulation and probiotic administration [12]. Several researches have been carried out to verify whether the administration of probiotics may improve the prognosis of diabetes through modulation of gut microbiota. Probiotics have been identified as effective adjuvants in insulin resistance therapies [14–16]. This health claims apparently stem from the ability of probiotics to secrete antimicrobial substances, competing with other pathogens, strengthening the intestinal barrier, and modulating the immune system [17].

#### *3.1.1. Probiotics in animal models of autoimmune diabetes*

According to the World Health Organization (2002) and the International Scientific Association for Probiotics and Prebiotics (2013), probiotics is defined as "a live organism, which provides a benefit to the host when provided in adequate quantities" [2–4]. Most commonly used probiotic includes lactic acid-producing bacteria, such as *Lactobacillus*, *Bifidobacterium*, and *Streptococcus* species. Non-lactic acid-producing bacteria, such as *Bacillus* and *Propionibacterium*, species and nonpathogenic yeasts, including *Saccharomyces boulfecesardii*, non-spore-forming and nonflagellated rod or coccobacilli, and some helminths, such as *Trichuris suis ova*, could also been used as probiotics [5, 6]. Some of these strains were chosen based on origin, in vitro adherence

to intestinal cells, and survival during passage through the gastrointestinal tract [5].

and induce systemic autoimmunity and trigger autoimmune diseases [9].

activation of host immune system in response to pathobionts [10].

systemic lupus erythematosus.

**3.1. Type 1 diabetes**

**3. Probiotics in autoimmune diseases**

Evidence from animal models has implied the involvement of intestinal dysbiosis in development of some autoimmune diseases [24–26]. Dysbiosis observed in autoimmune diseases is associated with decreased bacteria function and diversity, impaired epithelial barrier function, inflammation, and decreased regulatory T cells (Treg cells) in the gut mucosa [7, 8]. The hypotheses proposed to link dysbiosis with autoimmune diseases include molecular mimicry, bystander T cell activation, and the amplification of autoimmunity by pro-inflammatory cytokines, which is elicited by dysbiotic gut microbiota [9]. In 2016, Lerner and colleagues, from Institute Wendelsheim, in Germany, proposed the posttranslational modification of luminal proteins, promoted by enzymes from altered microbiota, which modify substrates in a different way than performed under homeostatic conditions. The defective posttranslational modification of luminal proteins could generate neo-epitopes that may become immunogenic

Here, we described several reports concerning probiotic applications in several animal models of autoimmune diseases and data of the main clinical trials concerning the applicability of probiotics in type 1 diabetes (T1D), multiple sclerosis (MS), rheumatoid arthritis (RA), and

Studies suggest that probiotics influence systemic immune responses, ensure the homeostasis of the healthy microbiota in the intestinal mucosa, and could, therefore, be used as adjuvant therapy to treat immune-mediated diseases [4]. The mechanisms proposed to achieve this include mucus secretion, antimicrobial peptide production, the maintenance of the function of the gastrointestinal-epithelial barrier, decreasing oxidative stress, ensuring adequate interactions between the gut microbiota and the mucosal immune cells, and, finally, helping the

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by autoimmune reactions against the insulin-secreting pancreatic β-cells, resulting in exogenous insulin dependence

**2. Intestinal dysbiosis in autoimmune diseases**

70 Probiotics - Current Knowledge and Future Prospects

The intestinal microbiota might modulate the autoimmune T1D pathogenesis via two mechanisms, recently proposed by Knip and Honkanen [18], from the University of Helsinki, in Finland. In the first phase, an impaired tolerance process in infancy leads to a susceptibility to develop autoimmune diseases, such as T1D, and may result in appearance of autoreactive T cells and autoantibodies. At the second phase, the intestinal dysbiosis predisposes children with genetic susceptibility and positive autoantibodies to develop clinical disease [18].

The inflammasome signaling components are innate immune sensors that are highly influenced by the gut environment and play pivotal roles in maintaining intestinal immune homeostasis [19]. Previous studies suggested the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotics can modulate local and systemic immune responses [20].

The earliest study to evaluate the efficacy of probiotics in T1D was published in 2005. The study performed by Calcinaro and colleagues, in the University of Perugia, in Italy, investigated the effects of oral administration of the probiotic VSL#3 in nonobese diabetic (NOD) mice development. VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a reduced β-cell destruction. Prevention was associated with an increased production of interleukin (IL)-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive isletinfiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. Oral VSL#3 administration prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis. These data provide a sound rationale for future clinical trials of the primary prevention of T1D by oral VSL#3 administration [21].

Eleven years later, Kim and colleagues evaluated the effects of *Bifidobacterium lactis* HY8101 on insulin resistance induced by tumor necrosis factor-alpha (TNF-α) in the skeletal muscle cell from L6 rat. The treatment using HY8101 improved the insulin-stimulated glucose uptake and translocation of GLUT4 via the insulin signaling pathways AKT and IRS-1(Tyr) in TNFtreated L6 cells. HY8101 increased the mRNA levels of GLUT4 and several insulin sensitivity-related genes in TNF-α-treated L6 cells. HY8101 improved diabetes-induced plasma total cholesterol and triglyceride levels and increased the muscle glycogen content. *Bifidobacterium lactis* HY8101 can be used to moderate glucose metabolism, lipid metabolism, and insulin sensitivity in mice and in cells. *Bifidobacterium lactis* HY8101 might have potential as a probiotic candidate for alleviating metabolic syndromes such as diabetes [22].

microbiota on T1D onset, scientists manipulated gut microbes by fecal transplantation between NOD and resistant NOD mice (NOR) and by oral antibiotic and probiotic treatment of NOD mice. The intestinal microbiota from NOD mice harbored more pathobionts and fewer beneficial microbes in comparison with NOR mice. Fecal transplantation of NOD microbes induced insulitis in NOR hosts, suggesting that the NOD microbiome is diabetogenic. Moreover, antibiotic exposure accelerated diabetes onset in NOD mice accompanied by increased Th1 and Th17 cells in the mucosal-associated lymphoid tissues. The diabetogenic microbiome was characterized by a metagenome altered in several metabolic gene clusters. Furthermore, diabetes susceptibility correlated with reduced fecal short chain fatty acids. In an attempt to correct the diabetogenic microbiome, researchers administered VLS#3 probiotic to NOD mice and found that VSL#3 colonized the intestine poorly and did not delay diabetes onset. Authors concluded that NOD mice harbor gut microbes that induce diabetes and that their diabetogenic microbiome can be amplified early in life through antibiotic exposure. Protective microbes like VSL#3

Probiotic Applications in Autoimmune Diseases http://dx.doi.org/10.5772/intechopen.73064 73

Another recent work, performed in Jiangnan University, in China, Jia and colleagues [28], investigated whether administration of probiotic *Clostridium butyricum* CGMCC0313.1 (CB0313.1) could induce Treg cells in pancreas, and consequently inhibit the diabetes onset in NOD mice. CB0313.1 supplementation was delivered daily to female NOD mice from 3 to 45 weeks of age. Researchers observed that probiotic administration suppressed the insulitis, delayed the disease onset, and improved the glucose metabolism. These beneficial effects could involve the migration of intestinal Treg cells to the pancreatic lymph nodes and changes in the Th1/Th2/Th17 balance, favoring an anti-inflammatory milieu in the gut and pancreas. Additionally, probiotic supplementation increased the Firmicutes/*Bacteroidetes* ratio,

Probiotic supplementation has been hypothesized to affect innate and adaptive immune responses to environmental antigens by supporting healthy gut microbiota and could therefore be used to prevent the onset of T1D-associated islet autoimmunity and treat the stab-

In humans, a TEDDY study group, published in JAMA Pediatrics in 2016, evaluated the association between probiotic supplementation and islet autoimmunity in children with genetic risk for T1D, during their first year of life. This multicenter prospective cohort study (United States, Finland, Germany, and Sweden) investigated 7473 children ranging from 4 to 10 years old. Early probiotic administration (0–27 days of life) was correlated with a decreased risk of islet autoimmunity when compared with the group that received probiotics after 27 days of life or no supplementation. This study concludes that early probiotic supplementation could decrease the risk of islet autoimmune reactions in children with high-genetic-risk alleles for T1D [30].

A current clinical trial, performed by Medical University of Warsaw, in Poland, involves the evaluation of the effect of *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* BB12 on β-cell function in children with newly diagnosed T1D. The double-blind, randomized, placebocontrolled clinical trial included 96 children aged 8 to 17 years old. During 1 year, patients

are insufficient to overcome the effects of a diabetogenic microbiome [27].

*Clostridium* species, and butyrate-producing bacteria in the gut [28].

*3.1.2. Probiotic applications in T1D patients*

lished disease [29].

Another work, performed in Yale University, by Peng and colleagues, in 2014, demonstrated that the protection from T1D development observed in MyD88-deficient NOD mice (MyD88−/− NOD) could be transferred to wild-type NOD mice [23, 24]. The gut bacteria isolated from MyD88−/−NOD mice, administered over a 3-week period, altered the family composition of the gut microbiome, mainly increasing the *Lachnospiraceae* and Clostridiaceae members and decreasing *Lactobacillaceae* family members. The gut microbiota-transferred mice had a higher concentration of IgA and transforming growth factor-beta (TGF-β) in the lumen that was accompanied by an increase in CD8+CD103+ and CD8αβ T cells in the lamina propria of the large intestine. The data obtained in this study suggest that gut bacterial composition can be altered after the neonatal period, affects the mucosal immune system, and might delay the onset of autoimmune diabetes. These results have important implications for the development of probiotic adjuvant treatment for T1D [24].

In 2015, Le and colleagues, from the National Institute for Food Control, by using C57BL/6 J mice with streptozotocin-induced diabetes, evaluated whether *Bifidobacterium* species induce the expression of proteins of the insulin signaling pathway and enhance adipocytokine gene expression. Oral administration of *Bifidobacterium* species significantly reduced blood glucose levels and increased the protein expressions of insulin receptor beta, insulin receptor substrate 1, protein kinase B (Akt/PKB), IκB kinase alpha (IKKα), and nuclear factor-kappaB inhibitor alpha (IκBα). *Bifidobacterium* species also induce the adiponectin gene expression and decrease in macrophage chemoattractant protein-1 (MCP-1) and IL-6 expression. In conclusion, the results from this work suggest that *Bifidobacterium* species may be the promising bacteria for treat diabetes [25].

A study performed in Diabetes Research Institute, in Milan, Italy, by Dolpady and colleagues, in 2016, reported that the oral administration of a *Lactobacillaceae*-enriched probiotics VSL#3, alone or in combination with retinoic acid, protects NOD mice from diabetes by suppressing inflammasome activation and IL-1β expression and by inducing the immunomodulatory indoleamine 2,3-dioxygenase (IDO) and IL-33 secretion. In addition, VSL#3-treated NOD mice showed modulation of the gut immunity by promoting differentiation of CD103+ tolerogenic dendritic cells and suppressing the differentiation of inflammatory Th1 and Th17 subsets in the gut mucosa [26].

Accumulating evidence supports that the intestinal microbiome is involved in T1D pathogenesis through the gut-pancreas axis. A recent study, performed in the University of British Columbia, in Canada, Brown and colleagues [27], aimed to determine whether the gut microbiota in the NOD mice played a role in T1D through the gut mucosa. To examine the effect of the intestinal microbiota on T1D onset, scientists manipulated gut microbes by fecal transplantation between NOD and resistant NOD mice (NOR) and by oral antibiotic and probiotic treatment of NOD mice. The intestinal microbiota from NOD mice harbored more pathobionts and fewer beneficial microbes in comparison with NOR mice. Fecal transplantation of NOD microbes induced insulitis in NOR hosts, suggesting that the NOD microbiome is diabetogenic. Moreover, antibiotic exposure accelerated diabetes onset in NOD mice accompanied by increased Th1 and Th17 cells in the mucosal-associated lymphoid tissues. The diabetogenic microbiome was characterized by a metagenome altered in several metabolic gene clusters. Furthermore, diabetes susceptibility correlated with reduced fecal short chain fatty acids. In an attempt to correct the diabetogenic microbiome, researchers administered VLS#3 probiotic to NOD mice and found that VSL#3 colonized the intestine poorly and did not delay diabetes onset. Authors concluded that NOD mice harbor gut microbes that induce diabetes and that their diabetogenic microbiome can be amplified early in life through antibiotic exposure. Protective microbes like VSL#3 are insufficient to overcome the effects of a diabetogenic microbiome [27].

Another recent work, performed in Jiangnan University, in China, Jia and colleagues [28], investigated whether administration of probiotic *Clostridium butyricum* CGMCC0313.1 (CB0313.1) could induce Treg cells in pancreas, and consequently inhibit the diabetes onset in NOD mice. CB0313.1 supplementation was delivered daily to female NOD mice from 3 to 45 weeks of age. Researchers observed that probiotic administration suppressed the insulitis, delayed the disease onset, and improved the glucose metabolism. These beneficial effects could involve the migration of intestinal Treg cells to the pancreatic lymph nodes and changes in the Th1/Th2/Th17 balance, favoring an anti-inflammatory milieu in the gut and pancreas. Additionally, probiotic supplementation increased the Firmicutes/*Bacteroidetes* ratio, *Clostridium* species, and butyrate-producing bacteria in the gut [28].

#### *3.1.2. Probiotic applications in T1D patients*

cell from L6 rat. The treatment using HY8101 improved the insulin-stimulated glucose uptake and translocation of GLUT4 via the insulin signaling pathways AKT and IRS-1(Tyr) in TNFtreated L6 cells. HY8101 increased the mRNA levels of GLUT4 and several insulin sensitivity-related genes in TNF-α-treated L6 cells. HY8101 improved diabetes-induced plasma total cholesterol and triglyceride levels and increased the muscle glycogen content. *Bifidobacterium lactis* HY8101 can be used to moderate glucose metabolism, lipid metabolism, and insulin sensitivity in mice and in cells. *Bifidobacterium lactis* HY8101 might have potential as a probiotic

Another work, performed in Yale University, by Peng and colleagues, in 2014, demonstrated that the protection from T1D development observed in MyD88-deficient NOD mice (MyD88−/− NOD) could be transferred to wild-type NOD mice [23, 24]. The gut bacteria isolated from MyD88−/−NOD mice, administered over a 3-week period, altered the family composition of the gut microbiome, mainly increasing the *Lachnospiraceae* and Clostridiaceae members and decreasing *Lactobacillaceae* family members. The gut microbiota-transferred mice had a higher concentration of IgA and transforming growth factor-beta (TGF-β) in the lumen that was accompanied by an increase in CD8+CD103+ and CD8αβ T cells in the lamina propria of the large intestine. The data obtained in this study suggest that gut bacterial composition can be altered after the neonatal period, affects the mucosal immune system, and might delay the onset of autoimmune diabetes. These results have important implications for the develop-

In 2015, Le and colleagues, from the National Institute for Food Control, by using C57BL/6 J mice with streptozotocin-induced diabetes, evaluated whether *Bifidobacterium* species induce the expression of proteins of the insulin signaling pathway and enhance adipocytokine gene expression. Oral administration of *Bifidobacterium* species significantly reduced blood glucose levels and increased the protein expressions of insulin receptor beta, insulin receptor substrate 1, protein kinase B (Akt/PKB), IκB kinase alpha (IKKα), and nuclear factor-kappaB inhibitor alpha (IκBα). *Bifidobacterium* species also induce the adiponectin gene expression and decrease in macrophage chemoattractant protein-1 (MCP-1) and IL-6 expression. In conclusion, the results from this work suggest that *Bifidobacterium* species may be the promising

A study performed in Diabetes Research Institute, in Milan, Italy, by Dolpady and colleagues, in 2016, reported that the oral administration of a *Lactobacillaceae*-enriched probiotics VSL#3, alone or in combination with retinoic acid, protects NOD mice from diabetes by suppressing inflammasome activation and IL-1β expression and by inducing the immunomodulatory indoleamine 2,3-dioxygenase (IDO) and IL-33 secretion. In addition, VSL#3-treated NOD mice showed modulation of the gut immunity by promoting differentiation of CD103+ tolerogenic dendritic cells and suppressing the differentiation of inflammatory Th1 and Th17 sub-

Accumulating evidence supports that the intestinal microbiome is involved in T1D pathogenesis through the gut-pancreas axis. A recent study, performed in the University of British Columbia, in Canada, Brown and colleagues [27], aimed to determine whether the gut microbiota in the NOD mice played a role in T1D through the gut mucosa. To examine the effect of the intestinal

candidate for alleviating metabolic syndromes such as diabetes [22].

ment of probiotic adjuvant treatment for T1D [24].

72 Probiotics - Current Knowledge and Future Prospects

bacteria for treat diabetes [25].

sets in the gut mucosa [26].

Probiotic supplementation has been hypothesized to affect innate and adaptive immune responses to environmental antigens by supporting healthy gut microbiota and could therefore be used to prevent the onset of T1D-associated islet autoimmunity and treat the stablished disease [29].

In humans, a TEDDY study group, published in JAMA Pediatrics in 2016, evaluated the association between probiotic supplementation and islet autoimmunity in children with genetic risk for T1D, during their first year of life. This multicenter prospective cohort study (United States, Finland, Germany, and Sweden) investigated 7473 children ranging from 4 to 10 years old. Early probiotic administration (0–27 days of life) was correlated with a decreased risk of islet autoimmunity when compared with the group that received probiotics after 27 days of life or no supplementation. This study concludes that early probiotic supplementation could decrease the risk of islet autoimmune reactions in children with high-genetic-risk alleles for T1D [30].

A current clinical trial, performed by Medical University of Warsaw, in Poland, involves the evaluation of the effect of *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* BB12 on β-cell function in children with newly diagnosed T1D. The double-blind, randomized, placebocontrolled clinical trial included 96 children aged 8 to 17 years old. During 1 year, patients received *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* BB12 at a dose of 10<sup>9</sup> colonyforming units or an identically appearing placebo, orally, daily, for 6 months. The follow-up will be for 12 months. The primary outcome measures will be the area under the curve of the C-peptide levels during 2 h response to a mixed meal [31].

reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group [41]. In the same year, Maassen and colleagues presented data showing that strain-specific differences on the effect of commercially available probiotic depend on physiological use (normal route, dose, growth phase, specific strain, or substrain/species) and overwhelm (high dose) or cir-

Probiotic Applications in Autoimmune Diseases http://dx.doi.org/10.5772/intechopen.73064 75

Two years later, Lavasani and colleagues, from Lund University, in Sweden, evaluated the effect of five daily-administered *Lactobacillus* strains in inhibiting disease onset in EAE mice. The *Lactobacillus paracasei* DSM 13434 and *Lactobacillus plantarum* DSM 15312 and DSM 15313 diminished autoreactive T cell responses and inflammation in the CNS*. Lactobacillus paracasei* and *Lactobacillus plantarum* DSM 15312 induce Treg cells in mesenteric lymph nodes and TGF-β secretion. *Lactobacillus plantarum* DSM 15313 induces increase in the IL-27 serum concentrations. The isolated *Lactobacillus* strains failed to be therapeutic in EAE mice. On the other hand, the combination of three strains inhibited the disease progression and reversed the clinical and histological signs of EAE, probably by suppressing inflammatory Th1 and

In 2010, Kobayashi and colleagues, from Yakult Central Institute for Microbiological Research, in Japan, evaluated the safety of two probiotic bacterial strains, *Lactobacillus casei* strain Shirota (LcS) and *Bifidobacterium breve* strain Yakult (BbY), that were orally administered to EAE Lewis rats. EAE was induced with a homogenate of guinea pig spinal cord as the sensitizing antigen, and LcS was orally administered from 1 week before this sensitization until the end of the experiment. The oral administration of LcS tended to suppress the development of neurological symptoms. Differences in neurological symptoms between the control group and the administration groups did not reach statistical significance and support the notion

Two years later, Kobayashi and colleagues investigated the safety use of *Lactobacillus casei* strain Shirota (LcS) in prevention of EAE in a relapse and remission models. LcS was administered 1 week prior antigen sensitization until the end of the experiments. Probiotics did not exacerbate neurological symptoms or histopathological changes of the spinal cord in either model. LcS administration transiently induces IL-17 production by antigen-stimulated lymphocytes 7 days after sensitization. Increased production of IL-10 and an increase in the percentages of CD4+CD25+ Treg cells were observed. Strong expression of IL-17 mRNA was detected in the spinal cord of mice that displayed severe neurological symptoms on day 12,

In 2013, Kwon and colleagues, from School of Life Sciences and Immune Synapse Research Center, in Republic of Korea, evaluated the prophylactic and therapeutic actions of a mixture of five probiotics (IRT5) in EAE mice. IRT5 includes *Lactobacillus casei*, *Lactobacillus acidophilus*, *Lactobacillus reuteri*, *Bifidobacterium bifidum*, and *Streptococcus thermophilus*. IRT5 prior treatment, before EAE induction, abrogated the disease development and delayed the EAE onset. Furthermore, the inflammatory subset Th1 and Th17 polarization was suppressed by the administration of IRT5 probiotic. These actions were due probably by induction of CD4+Foxp3+ Treg cells and IL-10 secretion at sites of inflammation and peripheral

cumvent natural immune processing [42].

Th17 pathways and inducing regulatory mechanisms [43].

that neither LcS nor BbY exacerbates EAE [44].

lymph nodes [46].

but this expression was not enhanced by LcS administration [45].

The *Lactobacillus* and *Bifidobacterium* are the major bacteria genera that make up the colon microbiota in humans and help in the intestinal microbial homeostasis, inhibit growth of pathobionts, improve the gut mucosal barrier, and modulate local and systemic immune responses. Intestinal dysbiosis may influence the immune system by increasing gut permeability, intestinal inflammation, and impaired oral tolerance in T1D patients. Beneficial effect of *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* BB12 on β-cell function would create a rationale for its routine use in patients with newly diagnosed T1D [31]. Taken together, the studies imply that bacteriotherapy may potentially be used as a tool to modulate the immune system for preventing islet autoimmunity [31, 32].

#### **3.2. Multiple sclerosis**

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease that affects the central nervous system (CNS) and is characterized by immune reactions against myelin proteins and gangliosides. Susceptible HLA alleles and environmental factors, such as virus infection, a hypercaloric diet, vitamin D deficiency, and intestinal dysbiosis, have been implicated in triggering MS [33]. MS promotes disability in young adults and affects twice more women than men. According to the Multiple Sclerosis International Federation and World Health Organization, the prevalence of MS increased from 2.1 million in 2008 to 2.3 million in 2013 [34].

Studies have shown that gut microbiota can affect the development of MS, and these works implicated intestinal dysbiosis as one of the possible causes of extraintestinal disease development [35]. The colonization of germ-free mice with segmented filamentous bacteria promotes an increase in the number of Th17 cells in the lamina propria and CNS, worsening disease severity in experimental autoimmune encephalomyelitis (EAE), a MS animal model [36]. Likewise, the colonization of the same mice with *Bacteroides fragilis* and polysaccharide A (PSA), which induces Foxp3+ Treg cell differentiation, decreases symptoms in EAE mice [37].

#### *3.2.1. Probiotics in experimental autoimmune encephalomyelitis*

Several studies in experimental autoimmune encephalomyelitis (EAE) mice reported the immunomodulatory functions of probiotic administration. Treatment with *Lactobacillus* species, *Pediococcus acidolactici*, *Bifidobacterium bifidum*, *Bifidobacterium animalis*, and *Bacteroides fragilis* decreased CNS inflammation through the induction of Treg cells in the gastrointestinal mucosa, IL-10 and TGF-β secretion, and decreased expansion of Th1 and Th17 inflammatory subsets [37–40].

In previous studies, performed in National Institute for Public Health and the Environment, in the Netherlands, in 2008, Ezendam and colleagues evaluated the effect of the probiotic *Bifidobacterium animalis* on Th1- and Th2-mediated immune responses, including a rat EAE model. *Bifidobacterium animalis* administration started when the rats were 2 weeks old and EAE were induced when the animals were 6–7 weeks old. *Bifidobacterium animalis* significantly reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group [41]. In the same year, Maassen and colleagues presented data showing that strain-specific differences on the effect of commercially available probiotic depend on physiological use (normal route, dose, growth phase, specific strain, or substrain/species) and overwhelm (high dose) or circumvent natural immune processing [42].

received *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* BB12 at a dose of 10<sup>9</sup>

C-peptide levels during 2 h response to a mixed meal [31].

74 Probiotics - Current Knowledge and Future Prospects

system for preventing islet autoimmunity [31, 32].

*3.2.1. Probiotics in experimental autoimmune encephalomyelitis*

**3.2. Multiple sclerosis**

subsets [37–40].

forming units or an identically appearing placebo, orally, daily, for 6 months. The follow-up will be for 12 months. The primary outcome measures will be the area under the curve of the

The *Lactobacillus* and *Bifidobacterium* are the major bacteria genera that make up the colon microbiota in humans and help in the intestinal microbial homeostasis, inhibit growth of pathobionts, improve the gut mucosal barrier, and modulate local and systemic immune responses. Intestinal dysbiosis may influence the immune system by increasing gut permeability, intestinal inflammation, and impaired oral tolerance in T1D patients. Beneficial effect of *Lactobacillus rhamnosus* GG and *Bifidobacterium lactis* BB12 on β-cell function would create a rationale for its routine use in patients with newly diagnosed T1D [31]. Taken together, the studies imply that bacteriotherapy may potentially be used as a tool to modulate the immune

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease that affects the central nervous system (CNS) and is characterized by immune reactions against myelin proteins and gangliosides. Susceptible HLA alleles and environmental factors, such as virus infection, a hypercaloric diet, vitamin D deficiency, and intestinal dysbiosis, have been implicated in triggering MS [33]. MS promotes disability in young adults and affects twice more women than men. According to the Multiple Sclerosis International Federation and World Health Organization, the prevalence of MS increased from 2.1 million in 2008 to 2.3 million in 2013 [34]. Studies have shown that gut microbiota can affect the development of MS, and these works implicated intestinal dysbiosis as one of the possible causes of extraintestinal disease development [35]. The colonization of germ-free mice with segmented filamentous bacteria promotes an increase in the number of Th17 cells in the lamina propria and CNS, worsening disease severity in experimental autoimmune encephalomyelitis (EAE), a MS animal model [36]. Likewise, the colonization of the same mice with *Bacteroides fragilis* and polysaccharide A (PSA), which induces Foxp3+ Treg cell differentiation, decreases symptoms in EAE mice [37].

Several studies in experimental autoimmune encephalomyelitis (EAE) mice reported the immunomodulatory functions of probiotic administration. Treatment with *Lactobacillus* species, *Pediococcus acidolactici*, *Bifidobacterium bifidum*, *Bifidobacterium animalis*, and *Bacteroides fragilis* decreased CNS inflammation through the induction of Treg cells in the gastrointestinal mucosa, IL-10 and TGF-β secretion, and decreased expansion of Th1 and Th17 inflammatory

In previous studies, performed in National Institute for Public Health and the Environment, in the Netherlands, in 2008, Ezendam and colleagues evaluated the effect of the probiotic *Bifidobacterium animalis* on Th1- and Th2-mediated immune responses, including a rat EAE model. *Bifidobacterium animalis* administration started when the rats were 2 weeks old and EAE were induced when the animals were 6–7 weeks old. *Bifidobacterium animalis* significantly

colony-

Two years later, Lavasani and colleagues, from Lund University, in Sweden, evaluated the effect of five daily-administered *Lactobacillus* strains in inhibiting disease onset in EAE mice. The *Lactobacillus paracasei* DSM 13434 and *Lactobacillus plantarum* DSM 15312 and DSM 15313 diminished autoreactive T cell responses and inflammation in the CNS*. Lactobacillus paracasei* and *Lactobacillus plantarum* DSM 15312 induce Treg cells in mesenteric lymph nodes and TGF-β secretion. *Lactobacillus plantarum* DSM 15313 induces increase in the IL-27 serum concentrations. The isolated *Lactobacillus* strains failed to be therapeutic in EAE mice. On the other hand, the combination of three strains inhibited the disease progression and reversed the clinical and histological signs of EAE, probably by suppressing inflammatory Th1 and Th17 pathways and inducing regulatory mechanisms [43].

In 2010, Kobayashi and colleagues, from Yakult Central Institute for Microbiological Research, in Japan, evaluated the safety of two probiotic bacterial strains, *Lactobacillus casei* strain Shirota (LcS) and *Bifidobacterium breve* strain Yakult (BbY), that were orally administered to EAE Lewis rats. EAE was induced with a homogenate of guinea pig spinal cord as the sensitizing antigen, and LcS was orally administered from 1 week before this sensitization until the end of the experiment. The oral administration of LcS tended to suppress the development of neurological symptoms. Differences in neurological symptoms between the control group and the administration groups did not reach statistical significance and support the notion that neither LcS nor BbY exacerbates EAE [44].

Two years later, Kobayashi and colleagues investigated the safety use of *Lactobacillus casei* strain Shirota (LcS) in prevention of EAE in a relapse and remission models. LcS was administered 1 week prior antigen sensitization until the end of the experiments. Probiotics did not exacerbate neurological symptoms or histopathological changes of the spinal cord in either model. LcS administration transiently induces IL-17 production by antigen-stimulated lymphocytes 7 days after sensitization. Increased production of IL-10 and an increase in the percentages of CD4+CD25+ Treg cells were observed. Strong expression of IL-17 mRNA was detected in the spinal cord of mice that displayed severe neurological symptoms on day 12, but this expression was not enhanced by LcS administration [45].

In 2013, Kwon and colleagues, from School of Life Sciences and Immune Synapse Research Center, in Republic of Korea, evaluated the prophylactic and therapeutic actions of a mixture of five probiotics (IRT5) in EAE mice. IRT5 includes *Lactobacillus casei*, *Lactobacillus acidophilus*, *Lactobacillus reuteri*, *Bifidobacterium bifidum*, and *Streptococcus thermophilus*. IRT5 prior treatment, before EAE induction, abrogated the disease development and delayed the EAE onset. Furthermore, the inflammatory subset Th1 and Th17 polarization was suppressed by the administration of IRT5 probiotic. These actions were due probably by induction of CD4+Foxp3+ Treg cells and IL-10 secretion at sites of inflammation and peripheral lymph nodes [46].

Three years later, Abdurasulova and coworkers, from Institute of Experimental Medicine, in St. Petersburg, Russian Federation, evaluated the effect of probiotic *Enterococcus faecium* strain L-3 that was studied in EAE rats. Glatiramer acetate (GA) was used as control drug. *Enterococcus faecium* strain L-3 and GA were able to reduce the severity of EAE. Both approaches prolonged the inductive phase of EAE and reduced the disease duration. Study of the phenotypes of immune cells in the blood revealed the differences in immunoregulatory pathways that mediate the protective action of probiotic or GA treatment of EAE. The presence of pronounced protective and immunomodulating effects of the probiotic *Enterococcus faecium* strain L-3 opens an opportunity of its application for the adjuvant treatment of MS [47].

*3.2.2. Probiotic applications in MS patients*

Probiotic applications based on the hygiene hypothesis, such as administration of the eggs from nonpathogenic helminth *Trichuris suis ova* (TSO), have proven safe and effective in autoimmune inflammatory bowel disease. Based on this, Fleming and colleagues [6], from the University of Wisconsin, in the United States, evaluated the safety and effects of TSO administration in newly diagnosed, non-treated relapsing-remitting MS patients. Researchers conducted the phase 1 helminth-induced immunomodulatory therapy (HINT 1) study by enrollment of five MS patients that took orally 2500 TSO, every 2 weeks, for 3 months. The preliminary outcomes showed increase in the serum levels of IL-4 and IL-10 cytokines and decreased in the mean number of new gadolinium-enhancing magnetic resonance imaging (MRI) lesions. TSO was well tolerated in this first human study of the probiotic application in relapsing-remitting MS, and favorable trends were observed in exploratory MRI and immunological parameters [6].

Probiotic Applications in Autoimmune Diseases http://dx.doi.org/10.5772/intechopen.73064 77

Two years later, Rosche and colleagues, from the Department of Neurology and Experimental Neurology, in Berlin, Germany, evaluated the administration of 2500 *Trichuris suis ova* eggs orally, every 2 weeks, for 12 months, in relapsing-remitting MS patients. Fifty patients with relapsingremitting MS with clinical activity, not undergoing any standard therapies, were enrolled. The safety, tolerability, and effect on disease activity and in vivo mechanisms of action of TSO in MS will be assessed by neurological, laboratory, and immunological exams and MRI throughout the 12-month treatment period and over a follow-up period of 6 months. No adverse effects were observed, and the *Trichuris suis ova* group was more effective than the placebo in preventing new T2 and gadolinium-positive lesions, quantified by MRI. Authors also expect the Th1 and Th17

pro-inflammatory responses polarize toward the anti-inflammatory Th2 response [51].

check index and HDL-cholesterol levels compared with the placebo [52].

*Lactobacillus casei*, *Bifidobacterium bifidum*, and *Lactobacillus fermentum* (2 × 10<sup>9</sup>

In a recent study, Kouchaki and colleagues [52], from School of Medicine from Kashan, in Islamic Republic of Iran, reported improved Expanded Disability Status Score (EDSS), insulin resistance, and a decrease in inflammatory markers in MS patients treated with probiotic supplementation containing *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus fermentum*, and *Bifidobacterium bifidum*. This randomized double-blind, placebo-controlled clinical trial analyzed probiotic intake for 12 weeks in 60 MS patients. Compared with the placebo group, probiotic administration improved EDSS, beck depression inventory, general health questionnaire, and depression anxiety and stress scale. Furthermore, changes in high-sensitivity C-reactive protein, plasma nitric oxide metabolites, and malondialdehyde in the probiotic group were significantly different from the changes in these parameters in the control group. In addition, the probiotic intake significantly decreased insulin levels and total high-density lipoprotein (HDL) cholesterol and significantly increased quantitative insulin sensitivity

Another recent randomized, double-blind, placebo-controlled clinical trial, performed in Islamic Republic of Iran, by Tamtaji and colleagues [53], evaluated the role of probiotic administration on gene expression associated to inflammatory, glucose, and lipid signaling pathways in MS patients. The study included 40 patients with MS. Participants were randomly assigned into two groups to receive either a probiotic capsule containing *Lactobacillus acidophilus*,

units/g each) or placebo, for 12 weeks. Researchers observed that probiotic administration

colony-forming

The Goudarzvand group [48], from School of Medicine, in Karaj, Iran, investigated the effect of *Lactobacillus plantarum* (LP) and *Bifidobacterium* B94 (BB94) on acquisition phase of spatial memory in the local demyelination of rats' hippocampus. Thirty-two male Wistar rats were divided into control, damage group and treatment group. After the induction of demyelination, probiotics were administered by gavage for 28 days. Findings demonstrated that probiotics have no significant effect on swimming speed compared with lesion and saline groups. According to some studies, probiotics have a positive impact on improving the performance of spatial memory and learning, although this current study could not indicate finality of this assumption [48].

A recent study, performed by Secher and colleagues [49], from the University of Toulouse, in France, evaluated the effects of the probiotic *Escherichia coli* strain Nissle 1917 (ECN) in EAE model. The daily oral administration of ECN significantly decreased the disease severity induced by myelin oligodendrocyte glycoprotein (MOG) peptide mice immunization. The therapeutic effects could be explained by the increase in the IL-10 anti-inflammatory cytokine and reduction in inflammatory cytokines in the CNS and in the periphery. They also observed a decreased frequency of MOG-specific CD4+ T cells in the CNS, suggesting that ECN modulate the T cell homing from the lymph nodes to the CNS by affecting their activation and differentiation. In this study, authors showed that EAE trigger is associated with increased gut permeability [49].

Another recent study, performed in Immunology Research Center, in Mashhad, Iran, Salehipour and colleagues [50], evaluated the therapeutic effect of probiotic strains, *Lactobacillus plantarum* A7, *Bifidobacterium animalis* PTCC 1631, or both. Probiotics were administered orally for 22 days starting at same time with the induction of EAE in female C57BL/6 mice. Results showed that treatment with both strains caused a more significant delay in the time of disease onset and clinical score compared with strains used alone. Mononuclear cell infiltration into the CNS was significantly inhibited by the combinational approach. The treatment with both strains enhanced the population of CD4+CD25+Foxp3+ Treg cells in the lymph nodes and spleen. Additionally, *Lactobacillus plantarum* A7 and *Bifidobacterium animalis* ameliorated EAE condition by inhibiting IL-6 production, decreasing the release of IFN-γ, a Th1-type cytokine, and IL-17, a Th17 pro-inflammatory molecule, and increasing the secretion of IL-4, a Th2-type cytokine, and IL-10 and TGF-β, anti-inflammatory cytokines, in the lymph nodes and spleen. The treatment with *Bifidobacterium animalis* induced a downregulation of transcription factors T-bet and ROR-γt that generate Th1 and Th17 inflammatory subsets, in the brain and spleen, and promoted an upregulation of GATA3 and Foxp3, which contributes for the Th2 and Treg cell differentiation [50].

#### *3.2.2. Probiotic applications in MS patients*

Three years later, Abdurasulova and coworkers, from Institute of Experimental Medicine, in St. Petersburg, Russian Federation, evaluated the effect of probiotic *Enterococcus faecium* strain L-3 that was studied in EAE rats. Glatiramer acetate (GA) was used as control drug. *Enterococcus faecium* strain L-3 and GA were able to reduce the severity of EAE. Both approaches prolonged the inductive phase of EAE and reduced the disease duration. Study of the phenotypes of immune cells in the blood revealed the differences in immunoregulatory pathways that mediate the protective action of probiotic or GA treatment of EAE. The presence of pronounced protective and immunomodulating effects of the probiotic *Enterococcus faecium* strain L-3

The Goudarzvand group [48], from School of Medicine, in Karaj, Iran, investigated the effect of *Lactobacillus plantarum* (LP) and *Bifidobacterium* B94 (BB94) on acquisition phase of spatial memory in the local demyelination of rats' hippocampus. Thirty-two male Wistar rats were divided into control, damage group and treatment group. After the induction of demyelination, probiotics were administered by gavage for 28 days. Findings demonstrated that probiotics have no significant effect on swimming speed compared with lesion and saline groups. According to some studies, probiotics have a positive impact on improving the performance of spatial memory and learning, although this current study could not indicate finality of this assumption [48]. A recent study, performed by Secher and colleagues [49], from the University of Toulouse, in France, evaluated the effects of the probiotic *Escherichia coli* strain Nissle 1917 (ECN) in EAE model. The daily oral administration of ECN significantly decreased the disease severity induced by myelin oligodendrocyte glycoprotein (MOG) peptide mice immunization. The therapeutic effects could be explained by the increase in the IL-10 anti-inflammatory cytokine and reduction in inflammatory cytokines in the CNS and in the periphery. They also observed a decreased frequency of MOG-specific CD4+ T cells in the CNS, suggesting that ECN modulate the T cell homing from the lymph nodes to the CNS by affecting their activation and differentiation. In this study, authors showed that EAE trigger is associated with increased gut

Another recent study, performed in Immunology Research Center, in Mashhad, Iran, Salehipour and colleagues [50], evaluated the therapeutic effect of probiotic strains, *Lactobacillus plantarum* A7, *Bifidobacterium animalis* PTCC 1631, or both. Probiotics were administered orally for 22 days starting at same time with the induction of EAE in female C57BL/6 mice. Results showed that treatment with both strains caused a more significant delay in the time of disease onset and clinical score compared with strains used alone. Mononuclear cell infiltration into the CNS was significantly inhibited by the combinational approach. The treatment with both strains enhanced the population of CD4+CD25+Foxp3+ Treg cells in the lymph nodes and spleen. Additionally, *Lactobacillus plantarum* A7 and *Bifidobacterium animalis* ameliorated EAE condition by inhibiting IL-6 production, decreasing the release of IFN-γ, a Th1-type cytokine, and IL-17, a Th17 pro-inflammatory molecule, and increasing the secretion of IL-4, a Th2-type cytokine, and IL-10 and TGF-β, anti-inflammatory cytokines, in the lymph nodes and spleen. The treatment with *Bifidobacterium animalis* induced a downregulation of transcription factors T-bet and ROR-γt that generate Th1 and Th17 inflammatory subsets, in the brain and spleen, and promoted an upregulation of GATA3 and Foxp3, which contributes for the Th2 and Treg

opens an opportunity of its application for the adjuvant treatment of MS [47].

76 Probiotics - Current Knowledge and Future Prospects

permeability [49].

cell differentiation [50].

Probiotic applications based on the hygiene hypothesis, such as administration of the eggs from nonpathogenic helminth *Trichuris suis ova* (TSO), have proven safe and effective in autoimmune inflammatory bowel disease. Based on this, Fleming and colleagues [6], from the University of Wisconsin, in the United States, evaluated the safety and effects of TSO administration in newly diagnosed, non-treated relapsing-remitting MS patients. Researchers conducted the phase 1 helminth-induced immunomodulatory therapy (HINT 1) study by enrollment of five MS patients that took orally 2500 TSO, every 2 weeks, for 3 months. The preliminary outcomes showed increase in the serum levels of IL-4 and IL-10 cytokines and decreased in the mean number of new gadolinium-enhancing magnetic resonance imaging (MRI) lesions. TSO was well tolerated in this first human study of the probiotic application in relapsing-remitting MS, and favorable trends were observed in exploratory MRI and immunological parameters [6].

Two years later, Rosche and colleagues, from the Department of Neurology and Experimental Neurology, in Berlin, Germany, evaluated the administration of 2500 *Trichuris suis ova* eggs orally, every 2 weeks, for 12 months, in relapsing-remitting MS patients. Fifty patients with relapsingremitting MS with clinical activity, not undergoing any standard therapies, were enrolled. The safety, tolerability, and effect on disease activity and in vivo mechanisms of action of TSO in MS will be assessed by neurological, laboratory, and immunological exams and MRI throughout the 12-month treatment period and over a follow-up period of 6 months. No adverse effects were observed, and the *Trichuris suis ova* group was more effective than the placebo in preventing new T2 and gadolinium-positive lesions, quantified by MRI. Authors also expect the Th1 and Th17 pro-inflammatory responses polarize toward the anti-inflammatory Th2 response [51].

In a recent study, Kouchaki and colleagues [52], from School of Medicine from Kashan, in Islamic Republic of Iran, reported improved Expanded Disability Status Score (EDSS), insulin resistance, and a decrease in inflammatory markers in MS patients treated with probiotic supplementation containing *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus fermentum*, and *Bifidobacterium bifidum*. This randomized double-blind, placebo-controlled clinical trial analyzed probiotic intake for 12 weeks in 60 MS patients. Compared with the placebo group, probiotic administration improved EDSS, beck depression inventory, general health questionnaire, and depression anxiety and stress scale. Furthermore, changes in high-sensitivity C-reactive protein, plasma nitric oxide metabolites, and malondialdehyde in the probiotic group were significantly different from the changes in these parameters in the control group. In addition, the probiotic intake significantly decreased insulin levels and total high-density lipoprotein (HDL) cholesterol and significantly increased quantitative insulin sensitivity check index and HDL-cholesterol levels compared with the placebo [52].

Another recent randomized, double-blind, placebo-controlled clinical trial, performed in Islamic Republic of Iran, by Tamtaji and colleagues [53], evaluated the role of probiotic administration on gene expression associated to inflammatory, glucose, and lipid signaling pathways in MS patients. The study included 40 patients with MS. Participants were randomly assigned into two groups to receive either a probiotic capsule containing *Lactobacillus acidophilus*, *Lactobacillus casei*, *Bifidobacterium bifidum*, and *Lactobacillus fermentum* (2 × 10<sup>9</sup> colony-forming units/g each) or placebo, for 12 weeks. Researchers observed that probiotic administration downregulated gene expression of IL-8 and TNF-α mRNA in peripheral blood mononuclear cells of MS patients. On the other hand, probiotics did not affect the gene expression of IL-1, peroxisome proliferator-activated receptor gamma (PPAR-γ), or oxidized low-density lipoprotein receptor (LDLR) in peripheral blood mononuclear cells of MS patients [53].

demonstrated the ability of *Lactobacillus helveticus* SBT2171 to downregulate the abundance of immune cells and the subsequent production of CII-specific antibodies and IL-6, thereby sup-

Probiotic Applications in Autoimmune Diseases http://dx.doi.org/10.5772/intechopen.73064 79

*Lactobacillus helveticus* SBT2171 (LH2171) is a lactic acid bacterium with high protease activity and used in starter cultures in the manufacture of cheese. Scientists have demonstrated that LH2171 inhibited the proliferation of lipopolysaccharide (LPS)-stimulated mouse T and B cells and the human lymphoma cell lines, Jurkat and BJAB. The findings of this study suggest that LH2171 inhibits the proliferation of lymphocytes through the suppression of the JNK signaling pathway and exerts an immunosuppressive effect in vivo, reinforcing their use in

Intestinal dysbiosis has been previously identified in patients with RA, and the administration of certain probiotics showed an improvement in RA. Study from Gohil and colleagues [62], from the Institute of Pharmaceutical Education and Research, in Gujarat, India, was designed to find out the antiarthritic activity of cell wall content of *Lactobacillus plantarum* in complete Freund's adjuvant (CFA)-induced arthritis in rats. The change in body weight, paw volume and arthritic index, joint stiffness, gait test, mobility test, erythrocyte sedimentation rate, serum C-reactive protein level, serum rheumatoid factor, and serum TNF-α was measured on day 21. Cell wall content of *Lactobacillus plantarum*-treated animals showed improvement in all the parameters as compared to that in CFA-treated animals and exert antiarthritic activity [62].

Some performed studies evaluating the effect of probiotics as an adjuvant therapy for RA treatment have shown no significant results, and some of these conducted studies have

The earliest study to evaluate the efficacy of probiotics in RA was performed in Rheumatism Foundation Hospital, in Finland, and was published in 2003. In a pilot study, Hatakka and colleagues evaluated 25 non-treated RA patients that were randomized to receive either two capsules of a *Lactobacillus rhamnosus* or placebo, twice daily for a year. Overall, no statistically significant differences were seen between the case and the placebo. Both groups had a decline in tender and swollen joints, and the physician global scores improved in the probiotic group. Mean erythrocyte sedimentation rates and C-reactive protein levels remained normal in both groups. The serum concentrations of IL-1β increase in patients treated with *Lactobacillus* species; however, this increase was not associated with any detectable change in disease status. Fecal sampling showed an increase in the presence of *Lactobacillus rhamnosus* in the probiotic group at 1 year. Based on these results, researchers concluded that *Lactobacillus rhamnosus* preparation did not alter RA activity. However, study cohort was small, and enrolled patients have low disease activity [65].

A double-blind, placebo-controlled clinical trial, performed in the University of Western Ontario, Canada, by Pineda and colleagues [63], evaluated the effect of the oral administration of *Lactobacillus rhamnosus* and *Lactobacillus reuteri* for 3 months to 29 RA patients. Fifteen patients were randomized to the probiotic group and 14 to placebo. Alterations in cytokines favored placebo over probiotic group. There was a significant improvement in the Health Assessment Questionnaire score in the probiotic group. Although researchers did not detect

smaller number of patients and a short period of evaluation [63, 64].

pressing the CIA symptoms, indicating its potential for use in the prevention of RA [60].

treatment of immune-mediated diseases [61].

*3.3.2. Probiotic applications in RA patients*

#### **3.3. Rheumatoid arthritis**

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic inflammation of multiple joints, bone erosion, and cartilage destruction. Moreover, RA can affect internal organs such as the lungs, heart, and kidneys. Anti-cyclic citrullinated peptide and rheumatoid factor are the most important autoantibodies in RA and can be found before disease onset [54]. The disease is three times more common in women, and according to the World Health Organization, the worldwide prevalence, which is between 0.3 and 1%, ranks the disease among the most common autoimmune disorders. The triggering of RA involves the interaction of HLA genes and environmental factors, such as smoking and infections [55]. Among environmental factors, dysbiosis has been identified as a possible trigger factor for autoimmunity and RA development [56].

#### *3.3.1. Probiotics in animal models of RA*

Experiments in animal models suggest that gut microbiota influences local and systemic immunity and might trigger joint inflammation [57]. Studies in collagen-induced arthritic (CIA) mice showed that the administration of antibiotics exacerbates the disease and increases the level of IL-6, IFN-γ, and IL-17 pro-inflammatory cytokines. Further study showed differences in the gut microbiota composition between CIA-susceptible and CIA-resistant mice, with a prevalence of *Desulfovibrio*, *Prevotella*, *Parabacteroides*, *Odoribacter*, *Acetatifactor*, *Blautia*, *Coprococcus*, and *Ruminococcus* genera in arthritic mice, in addition to increased levels of serum IL-17 and CD4 Th17 cells in the spleen [58].

The study performed by Abhari and colleagues [59], in Shiraz University, in Iran, investigated the possible role of probiotic *Bacillus coagulans* and prebiotic inulin on the downregulation of immune responses and the progression of RA, by using rat models of the disease. The sporeforming probiotic strain *Bacillus coagulans* has an anti-inflammatory and immunomodulatory effects in animals and humans. The treatment with the probiotic and prebiotic significantly inhibits serum amyloid A in arthritic rats, and a significant decrease in the secretion of the pro-inflammatory TNF-α was detected [59].

Another work, performed in the Department of Probiotics Immunology, Sapporo University, in Japan, Yamashita and colleagues [60], evaluated the effect of the oral administration of *Lactobacillus helveticus* SBT2171 on CIA development and on the regulation of antigen-specific antibody production and inflammatory immune cells, implicated in the RA development. Probiotic administration promotes decrease in joint swelling, body weight loss, and the serum level of bovine type II collagen (CII)-specific antibodies in the CIA mouse model. In addition, the intraperitoneal inoculation of *Lactobacillus helveticus* SBT2171 also decreased the arthritis incidence, joint damage, and serum concentrations of IL-6. Furthermore, the numbers of total immune cells, total B cells, germinal center B cells, and CD4+ T cells in the draining lymph nodes were decreased following intraperitoneal inoculation of *Lactobacillus helveticus* SBT2171. Findings of this study demonstrated the ability of *Lactobacillus helveticus* SBT2171 to downregulate the abundance of immune cells and the subsequent production of CII-specific antibodies and IL-6, thereby suppressing the CIA symptoms, indicating its potential for use in the prevention of RA [60].

*Lactobacillus helveticus* SBT2171 (LH2171) is a lactic acid bacterium with high protease activity and used in starter cultures in the manufacture of cheese. Scientists have demonstrated that LH2171 inhibited the proliferation of lipopolysaccharide (LPS)-stimulated mouse T and B cells and the human lymphoma cell lines, Jurkat and BJAB. The findings of this study suggest that LH2171 inhibits the proliferation of lymphocytes through the suppression of the JNK signaling pathway and exerts an immunosuppressive effect in vivo, reinforcing their use in treatment of immune-mediated diseases [61].

Intestinal dysbiosis has been previously identified in patients with RA, and the administration of certain probiotics showed an improvement in RA. Study from Gohil and colleagues [62], from the Institute of Pharmaceutical Education and Research, in Gujarat, India, was designed to find out the antiarthritic activity of cell wall content of *Lactobacillus plantarum* in complete Freund's adjuvant (CFA)-induced arthritis in rats. The change in body weight, paw volume and arthritic index, joint stiffness, gait test, mobility test, erythrocyte sedimentation rate, serum C-reactive protein level, serum rheumatoid factor, and serum TNF-α was measured on day 21. Cell wall content of *Lactobacillus plantarum*-treated animals showed improvement in all the parameters as compared to that in CFA-treated animals and exert antiarthritic activity [62].

#### *3.3.2. Probiotic applications in RA patients*

downregulated gene expression of IL-8 and TNF-α mRNA in peripheral blood mononuclear cells of MS patients. On the other hand, probiotics did not affect the gene expression of IL-1, peroxisome proliferator-activated receptor gamma (PPAR-γ), or oxidized low-density lipo-

Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by chronic inflammation of multiple joints, bone erosion, and cartilage destruction. Moreover, RA can affect internal organs such as the lungs, heart, and kidneys. Anti-cyclic citrullinated peptide and rheumatoid factor are the most important autoantibodies in RA and can be found before disease onset [54]. The disease is three times more common in women, and according to the World Health Organization, the worldwide prevalence, which is between 0.3 and 1%, ranks the disease among the most common autoimmune disorders. The triggering of RA involves the interaction of HLA genes and environmental factors, such as smoking and infections [55]. Among environmental factors, dysbiosis has been identified as a possible trigger factor for

Experiments in animal models suggest that gut microbiota influences local and systemic immunity and might trigger joint inflammation [57]. Studies in collagen-induced arthritic (CIA) mice showed that the administration of antibiotics exacerbates the disease and increases the level of IL-6, IFN-γ, and IL-17 pro-inflammatory cytokines. Further study showed differences in the gut microbiota composition between CIA-susceptible and CIA-resistant mice, with a prevalence of *Desulfovibrio*, *Prevotella*, *Parabacteroides*, *Odoribacter*, *Acetatifactor*, *Blautia*, *Coprococcus*, and *Ruminococcus* genera in arthritic mice, in addition to increased levels of

The study performed by Abhari and colleagues [59], in Shiraz University, in Iran, investigated the possible role of probiotic *Bacillus coagulans* and prebiotic inulin on the downregulation of immune responses and the progression of RA, by using rat models of the disease. The sporeforming probiotic strain *Bacillus coagulans* has an anti-inflammatory and immunomodulatory effects in animals and humans. The treatment with the probiotic and prebiotic significantly inhibits serum amyloid A in arthritic rats, and a significant decrease in the secretion of the

Another work, performed in the Department of Probiotics Immunology, Sapporo University, in Japan, Yamashita and colleagues [60], evaluated the effect of the oral administration of *Lactobacillus helveticus* SBT2171 on CIA development and on the regulation of antigen-specific antibody production and inflammatory immune cells, implicated in the RA development. Probiotic administration promotes decrease in joint swelling, body weight loss, and the serum level of bovine type II collagen (CII)-specific antibodies in the CIA mouse model. In addition, the intraperitoneal inoculation of *Lactobacillus helveticus* SBT2171 also decreased the arthritis incidence, joint damage, and serum concentrations of IL-6. Furthermore, the numbers of total immune cells, total B cells, germinal center B cells, and CD4+ T cells in the draining lymph nodes were decreased following intraperitoneal inoculation of *Lactobacillus helveticus* SBT2171. Findings of this study

protein receptor (LDLR) in peripheral blood mononuclear cells of MS patients [53].

**3.3. Rheumatoid arthritis**

78 Probiotics - Current Knowledge and Future Prospects

autoimmunity and RA development [56].

serum IL-17 and CD4 Th17 cells in the spleen [58].

pro-inflammatory TNF-α was detected [59].

*3.3.1. Probiotics in animal models of RA*

Some performed studies evaluating the effect of probiotics as an adjuvant therapy for RA treatment have shown no significant results, and some of these conducted studies have smaller number of patients and a short period of evaluation [63, 64].

The earliest study to evaluate the efficacy of probiotics in RA was performed in Rheumatism Foundation Hospital, in Finland, and was published in 2003. In a pilot study, Hatakka and colleagues evaluated 25 non-treated RA patients that were randomized to receive either two capsules of a *Lactobacillus rhamnosus* or placebo, twice daily for a year. Overall, no statistically significant differences were seen between the case and the placebo. Both groups had a decline in tender and swollen joints, and the physician global scores improved in the probiotic group. Mean erythrocyte sedimentation rates and C-reactive protein levels remained normal in both groups. The serum concentrations of IL-1β increase in patients treated with *Lactobacillus* species; however, this increase was not associated with any detectable change in disease status. Fecal sampling showed an increase in the presence of *Lactobacillus rhamnosus* in the probiotic group at 1 year. Based on these results, researchers concluded that *Lactobacillus rhamnosus* preparation did not alter RA activity. However, study cohort was small, and enrolled patients have low disease activity [65].

A double-blind, placebo-controlled clinical trial, performed in the University of Western Ontario, Canada, by Pineda and colleagues [63], evaluated the effect of the oral administration of *Lactobacillus rhamnosus* and *Lactobacillus reuteri* for 3 months to 29 RA patients. Fifteen patients were randomized to the probiotic group and 14 to placebo. Alterations in cytokines favored placebo over probiotic group. There was a significant improvement in the Health Assessment Questionnaire score in the probiotic group. Although researchers did not detect clinical improvement, measured by the American College of Rheumatology criteria, authors reported functional improvement within the probiotic supplementation group compared with the placebo [63].

inflammation in SLE patients [71]. Some *Lactobacillus* species have been demonstrated to have immunomodulatory properties in the host gut mucosa, such as inhibiting neutrophil extracellular trap formation, improving antioxidant status, and increasing the expression of adhesion

Probiotic Applications in Autoimmune Diseases http://dx.doi.org/10.5772/intechopen.73064 81

In a recent study, performed by Tzang and colleagues [75], in Chung Shan Medical University, in Taiwan, scientists investigated the effects of oral administration of *Lactobacillus paracasei* GMNL-32, *Lactobacillus reuteri* GMNL-89, and *Lactobacillus reuteri* GMNL-263 in NZB/W F1 mice. When researchers evaluated the administration of the three probiotic strains, they observed a significant decrease in IL-6 and TNF-α serum concentrations and increase in antioxidant activity in serum and liver samples (higher glutathione GSH and 1,1-diphenyl-2-picrylhydrazyl levels and lower malondialdehyde levels). Additionally, the supplementation with *Lactobacillus reuteri* GMNL-263 significantly increased the differentiation of CD4+CD25+FoxP3+ Treg cells in NZB/W F1 mice, suggesting that these strains could be used as adjuvant treatment of SLE patients [75]. Another investigation from the same group demonstrated that supplementation with these three probiotic strains ameliorates hepatic apoptosis, matrix metalloproteinase-9 activity, C-reactive protein, and inducible nitric oxide synthase expressions. In addition, probiotics decrease the gene expression of hepatic IL-1β, IL-6 and TNF-α proteins, by

suppressing the mitogen-activated protein kinase and NF-κB signaling pathways [76].

the role of probiotics as an adjuvant therapy in the treatment of SLE patients.

probiotic administration in patients with autoimmune diseases are needed.

Thanks for the School of Health Sciences Dr. Paulo Prata, Barretos, Sao Paulo, Brazil.

Although some studies in SLE animal models showed promising results using probiotic supplementation, currently, there are no clinical trials reported at clinicaltrials.gov investigating

Evidences associate intestinal dysbiosis with autoimmune disease pathogenesis. Impaired gut microbiota function and diversity could represent a trigger site of autoimmunity by neoantigen generation under dysbiotic conditions. Emerging findings point to the use of probiotics as a preventive functional food and as adjuvant treatment of autoimmune diseases. However, further clinical trials, with large cohorts, to evaluate the security and efficacy of the

molecules in the gut [73, 74].

**4. Conclusions**

**Acknowledgements**

**Conflict of interest**

The author reports no conflict of interest.

Another randomized, double-blind placebo-controlled trial, performed in Tabriz University of Medical Sciences, in Iran, by Vaghef-Mehrabany and colleagues [64], investigated the role of *Lactobacillus casei* 01 intake in 46 RA patients for 8 weeks. This clinical trial showed improvement in disease activity score, increased levels of serum IL-10, and decreased levels of pro-inflammatory TNF-α, IL-6, and IL-12 cytokines in treated patients. In this study, scientists concluded that supplementation improved the disease activity and inflammatory status in RA patients [64].

Another clinical trial, with the same study design, performed by Zamani and colleagues [66], in Kashan University of Medical Sciences, Iran, evaluated the effect of probiotic administration on clinical and metabolic parameters in RA patients. Sixty patients aged 25–70 years were enrolled into two groups to receive either probiotic or placebo. Probiotic group received a daily capsule containing three strains: *Lactobacillus acidophilus*, *Lactobacillus casei*, and *Bifidobacterium bifidum*, for 8 weeks. After intervention, probiotic administration improved Disease Activity Score of 28 joints (DAS-28). In addition, a significant decrease in serum insulin levels, homeostatic model assessment-B cell function (HOMA-B), and serum high-sensitivity C-reactive protein concentration was also observed in the probiotic group [66].

#### **3.4. Systemic lupus erythematosus**

Systemic lupus erythematosus (SLE) is an autoimmune and heterogeneous disease characterized by damage to the skin, kidneys, lungs, joints, heart, and brain [67]. The disease affects mainly females, and its worldwide prevalence varies from 30 to 60 per 100,000 in the United Kingdom and the United States [68]. SLE pathogenesis may involve genetic and environmental factors, such as viral infections, defective apoptosis, elevated oxidative stress, and solar exposure to ultraviolet-B waves. Regarding immune response, it is known that autoantibodies bind mainly with nuclear and cytoplasmic antigens [69]. Moreover, increased evidence has emerged in a recent year that suggests the role of intestinal dysbiosis in SLE development [70].

#### *3.4.1. Probiotics in animal models of SLE*

In female lupus-prone mice, Zhang and colleagues [71] reported a decrease in the relative abundance of *Lactobacillus* species and an increase in *Lachnospiraceae* members when compared with controls. Early disease onset and severe symptoms correlated with increased *Lachnospiraceae* reads in female lupus-prone mice. Additionally, the number of Clostridiaceae and *Lachnospiraceae* reads increased at specific time points during disease progression [71]. Another study reported that dietary intervention, such as caloric restriction, in NZB/WF1 mice promoted changes in the intestinal microbiota and delayed disease progression in this animal model [72].

In a lupus-like animal model, the administration of retinoic acid restored *Lactobacillus* species and improved lupus symptoms, suggesting the use of these species as a probiotic to diminish inflammation in SLE patients [71]. Some *Lactobacillus* species have been demonstrated to have immunomodulatory properties in the host gut mucosa, such as inhibiting neutrophil extracellular trap formation, improving antioxidant status, and increasing the expression of adhesion molecules in the gut [73, 74].

In a recent study, performed by Tzang and colleagues [75], in Chung Shan Medical University, in Taiwan, scientists investigated the effects of oral administration of *Lactobacillus paracasei* GMNL-32, *Lactobacillus reuteri* GMNL-89, and *Lactobacillus reuteri* GMNL-263 in NZB/W F1 mice. When researchers evaluated the administration of the three probiotic strains, they observed a significant decrease in IL-6 and TNF-α serum concentrations and increase in antioxidant activity in serum and liver samples (higher glutathione GSH and 1,1-diphenyl-2-picrylhydrazyl levels and lower malondialdehyde levels). Additionally, the supplementation with *Lactobacillus reuteri* GMNL-263 significantly increased the differentiation of CD4+CD25+FoxP3+ Treg cells in NZB/W F1 mice, suggesting that these strains could be used as adjuvant treatment of SLE patients [75]. Another investigation from the same group demonstrated that supplementation with these three probiotic strains ameliorates hepatic apoptosis, matrix metalloproteinase-9 activity, C-reactive protein, and inducible nitric oxide synthase expressions. In addition, probiotics decrease the gene expression of hepatic IL-1β, IL-6 and TNF-α proteins, by suppressing the mitogen-activated protein kinase and NF-κB signaling pathways [76].

Although some studies in SLE animal models showed promising results using probiotic supplementation, currently, there are no clinical trials reported at clinicaltrials.gov investigating the role of probiotics as an adjuvant therapy in the treatment of SLE patients.

#### **4. Conclusions**

clinical improvement, measured by the American College of Rheumatology criteria, authors reported functional improvement within the probiotic supplementation group compared

Another randomized, double-blind placebo-controlled trial, performed in Tabriz University of Medical Sciences, in Iran, by Vaghef-Mehrabany and colleagues [64], investigated the role of *Lactobacillus casei* 01 intake in 46 RA patients for 8 weeks. This clinical trial showed improvement in disease activity score, increased levels of serum IL-10, and decreased levels of pro-inflammatory TNF-α, IL-6, and IL-12 cytokines in treated patients. In this study, scientists concluded that supplementation improved the disease activity and inflammatory status

Another clinical trial, with the same study design, performed by Zamani and colleagues [66], in Kashan University of Medical Sciences, Iran, evaluated the effect of probiotic administration on clinical and metabolic parameters in RA patients. Sixty patients aged 25–70 years were enrolled into two groups to receive either probiotic or placebo. Probiotic group received a daily capsule containing three strains: *Lactobacillus acidophilus*, *Lactobacillus casei*, and *Bifidobacterium bifidum*, for 8 weeks. After intervention, probiotic administration improved Disease Activity Score of 28 joints (DAS-28). In addition, a significant decrease in serum insulin levels, homeostatic model assessment-B cell function (HOMA-B), and serum high-sensitivity C-reactive

Systemic lupus erythematosus (SLE) is an autoimmune and heterogeneous disease characterized by damage to the skin, kidneys, lungs, joints, heart, and brain [67]. The disease affects mainly females, and its worldwide prevalence varies from 30 to 60 per 100,000 in the United Kingdom and the United States [68]. SLE pathogenesis may involve genetic and environmental factors, such as viral infections, defective apoptosis, elevated oxidative stress, and solar exposure to ultraviolet-B waves. Regarding immune response, it is known that autoantibodies bind mainly with nuclear and cytoplasmic antigens [69]. Moreover, increased evidence has emerged in a recent year that suggests the role of intestinal dysbiosis in SLE development [70].

In female lupus-prone mice, Zhang and colleagues [71] reported a decrease in the relative abundance of *Lactobacillus* species and an increase in *Lachnospiraceae* members when compared with controls. Early disease onset and severe symptoms correlated with increased *Lachnospiraceae* reads in female lupus-prone mice. Additionally, the number of Clostridiaceae and *Lachnospiraceae* reads increased at specific time points during disease progression [71]. Another study reported that dietary intervention, such as caloric restriction, in NZB/WF1 mice promoted changes in the

In a lupus-like animal model, the administration of retinoic acid restored *Lactobacillus* species and improved lupus symptoms, suggesting the use of these species as a probiotic to diminish

intestinal microbiota and delayed disease progression in this animal model [72].

protein concentration was also observed in the probiotic group [66].

with the placebo [63].

80 Probiotics - Current Knowledge and Future Prospects

in RA patients [64].

**3.4. Systemic lupus erythematosus**

*3.4.1. Probiotics in animal models of SLE*

Evidences associate intestinal dysbiosis with autoimmune disease pathogenesis. Impaired gut microbiota function and diversity could represent a trigger site of autoimmunity by neoantigen generation under dysbiotic conditions. Emerging findings point to the use of probiotics as a preventive functional food and as adjuvant treatment of autoimmune diseases. However, further clinical trials, with large cohorts, to evaluate the security and efficacy of the probiotic administration in patients with autoimmune diseases are needed.

#### **Acknowledgements**

Thanks for the School of Health Sciences Dr. Paulo Prata, Barretos, Sao Paulo, Brazil.

#### **Conflict of interest**

The author reports no conflict of interest.

## **Appendices and nomenclature**


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#### **Author details**

Gislane L.V. de Oliveira Address all correspondence to: glelisvilela@gmail.com Microbiome Study Group, School of Health Sciences Dr. Paulo Prata, Barretos, Brazil

#### **References**

**Appendices and nomenclature**

82 Probiotics - Current Knowledge and Future Prospects

NOD mice Nonobese diabetic mice

TNF-α Tumor necrosis factor-alpha

IDO Indoleamine 2,3-dioxygenase

NOR mice Resistant NOD mice

MS Multiple sclerosis

PSA Polysaccharide A GA Glatiramer acetate

TSO *Trichuris suis ova*

CNS Central nervous system

TGF-β Transforming growth factor-beta

MCP-1 Macrophage chemoattractant protein-1

EAE Experimental autoimmune encephalomyelitis

MOG Myelin oligodendrocyte glycoprotein

MRI Magnetic resonance imaging

HDL High-density lipoproteins

RA Rheumatoid arthritis

LPS Lipopolysaccharide

**Author details**

Gislane L.V. de Oliveira

NZB New Zealand black mice

EDSS Expanded Disability Status Score

CIA mice Collagen-induced arthritic mice

CII Type II collagen-specific antibodies

Address all correspondence to: glelisvilela@gmail.com

Microbiome Study Group, School of Health Sciences Dr. Paulo Prata, Barretos, Brazil

TEDDY The Environmental Determinants of Diabetes in the Young

Treg T regulatory cells T1D Type 1 diabetes

IL Interleukin


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[42] Maassen CB, Claassen E. Strain-dependent effects of probiotic lactobacilli on EAE auto-

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[44] Kobayashi T, Kato I, Nanno M, Shida K, Shibuya K, Matsuoka Y, Onoue M. Oral administration of probiotic bacteria, *Lactobacillus casei* and *Bifidobacterium breve*, does not exacerbate neurological symptoms in experimental autoimmune encephalomyelitis. Immunopharmacology and Immunotoxicology. 2010;**32**:116-124. DOI:

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**Chapter 5**

**Provisional chapter**

**The Role of Probiotics in Acne and Rosacea**

**The Role of Probiotics in Acne and Rosacea**

DOI: 10.5772/intechopen.79044

Through basic science as well as animal and human clinical trials, the evidence is growing for the use of probiotics in the treatment of acne. Acne formation is dependent upon several processes, including follicular hyperkeratinization, excess sebum production, *Propionibacterium acnes* colonization and an inflammatory cascade. The antimicrobial properties of probiotics as well as the modification of the skin microbiome may decrease levels of *P. acnes* on the skin. Additionally, successful acne outcomes are influenced by compliance with topical regimens, which can commonly cause skin barrier disruption, leading to dryness and irritation. Consequently, calming inflammation as well as maintaining skin hydration and barrier repair is of primary importance when treating acne. In this chapter, we discuss how probiotics affect several factors in the pathophysiology of

Acne is an inflammatory disorder involving the pilosebaceous unit. A multifactorial cascade including excess sebum production, follicular hyperkeratinization, and bacterial overgrowth conspire to incite an inflammatory response. Acne therapies have focused on modulating this inflammatory response as well as targeting components of this cascade. Probiotics is an emerging area of research that continues to gain momentum for the treatment of acne. A probiotic is defined as a "live microorganism which, when administered in adequate amounts, confers a health benefit on the host" [1]. Both oral and topical preparations of probiotics have

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Caitlin F. Porubsky, Alexandria B. Glass, Victoria Comeau, Christopher Buckley,

Caitlin F. Porubsky, Alexandria B. Glass, Victoria Comeau, Christopher Buckley,

http://dx.doi.org/10.5772/intechopen.79044

**Abstract**

**1. Introduction**

shown promise in the treatment of acne.

Marcus B. Goodman and Mary-Margaret Kober

Marcus B. Goodman and Mary-Margaret Kober

acne development and can improve the treatment outcomes.

**Keywords:** acne, probiotics, pathogenesis, inflammation, therapy

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Chapter 5 Provisional chapter**

#### **The Role of Probiotics in Acne and Rosacea The Role of Probiotics in Acne and Rosacea**

DOI: 10.5772/intechopen.79044

Caitlin F. Porubsky, Alexandria B. Glass, Victoria Comeau, Christopher Buckley, Marcus B. Goodman and Mary-Margaret Kober Caitlin F. Porubsky, Alexandria B. Glass, Victoria Comeau, Christopher Buckley, Marcus B. Goodman and Mary-Margaret Kober

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79044

#### **Abstract**

Through basic science as well as animal and human clinical trials, the evidence is growing for the use of probiotics in the treatment of acne. Acne formation is dependent upon several processes, including follicular hyperkeratinization, excess sebum production, *Propionibacterium acnes* colonization and an inflammatory cascade. The antimicrobial properties of probiotics as well as the modification of the skin microbiome may decrease levels of *P. acnes* on the skin. Additionally, successful acne outcomes are influenced by compliance with topical regimens, which can commonly cause skin barrier disruption, leading to dryness and irritation. Consequently, calming inflammation as well as maintaining skin hydration and barrier repair is of primary importance when treating acne. In this chapter, we discuss how probiotics affect several factors in the pathophysiology of acne development and can improve the treatment outcomes.

**Keywords:** acne, probiotics, pathogenesis, inflammation, therapy

#### **1. Introduction**

Acne is an inflammatory disorder involving the pilosebaceous unit. A multifactorial cascade including excess sebum production, follicular hyperkeratinization, and bacterial overgrowth conspire to incite an inflammatory response. Acne therapies have focused on modulating this inflammatory response as well as targeting components of this cascade. Probiotics is an emerging area of research that continues to gain momentum for the treatment of acne. A probiotic is defined as a "live microorganism which, when administered in adequate amounts, confers a health benefit on the host" [1]. Both oral and topical preparations of probiotics have shown promise in the treatment of acne.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. The gut-brain-skin axis**

The field of dermatology continues to investigate the interconnected relationships between the skin and other systems of the body. The unifying theory of the gut-brain-skin axis outlines the relationship between the skin, gastrointestinal (GI) system, and mental health. Notably, the role of the gastrointestinal (GI) system is an area of particular interest as it relates to inflammatory skin conditions. The gut microbiome reacts to various stimuli resulting in systemic responses. By altering the GI microbiome, it is possible to decrease systemic inflammation, and these results can improve the severity of inflammatory skin diseases such as acne.

Hypochlorhydria and small intestinal bacterial overgrowth (SIBO) are two conditions that demonstrate an association with cutaneous pathology and mental health. Alterations in gastric acid secretion, such as hypochlorhydria, increase the risk for SIBO. While increased rates of SIBO have long been associated with psychological disorders of anxiety, depression, and fibromyalgia, more recent evidence has demonstrated increased rates of SIBO in rosacea patients, as well, with one study reporting an SIBO rate of 50% in rosacea patients. Those patients were then treated for SIBO with the antimicrobial rifaximin, and most had significant improvement or clearance of their rosacea [13, 14]. Additionally, an Australian study used the probiotic *Lactobacillus casei* to successfully reduce SIBO [15]. The authors not only noted improvement in

The Role of Probiotics in Acne and Rosacea http://dx.doi.org/10.5772/intechopen.79044 93

Intestinal permeability has also been linked to cutaneous pathology. As far back at 1916, acne patients have shown reactivity to stool-isolated bacteria using a serum complement test [4]. Later, Juhlin and Michaelsson tested *Escherichia coli* polysaccharide endotoxin in the blood samples of acne patients. The patients with severe acne had reactivity while the control had none [18]. A second study of human subjects with IBS identified increased levels of *E. coli* lipopolysaccharide (LPS)-induced cytokines in patients with IBS compared to controls [19]. Increased intestinal permeability and heightened immune responses has also been associated with chronic constipation [20]. Several studies have noted a higher prevalence of constipation in those with acne vulgaris [12, 21]. These correlations suggest increased intestinal permeability and consequently higher levels of circulating endotoxin may contribute to acne formation [21].

The gut microbiome is dynamic; it changes with the stress on its environment and reacts to feedback from other systems. As stated previously, changes in the microbiome may influence levels of systemic inflammation. Acne is an inflammatory disorder that has demonstrated its response to systemic inflammation and oxidative stress. A Russian study found that not only is the intestinal microflora of acne patients altered, but therapy for the GI disruption reduced

The gut's commensal bacteria can induce immune responses that ultimately reach T cells in the skin. Probiotics interact with the gastrointestinal mucosal immune system, altering levels of inflammatory cytokines in the blood [23]. For instance, the levels of gamma-aminobutyric acid (GABA) are modified by intestinal bacteria [24, 25]. Microbial-fermented food enriched with GABA has been shown to improve atopic dermatitis in mice through a Th-1 mediated-

Major histocompatibility cell (MHC) class II complexes are found on antigen presenting cells, such as dendritic cells and macrophages, and interact with immune-regulating T cells. Gastrointestinal bacteria, including strains of probiotics, have been shown to bind to the MHC II complex and modify their expressions [27]. A study that administered *Lactobacillus paracasei* NCC2461 (ST11) to mice found the probiotic to induce T regulatory cells and inhibited CD4+ T-cell proliferation, while increasing the secretions of anti-inflammatory cytokines, specifically IL-2, IL-10, and TGF-β [28]. Other strains of lactic acid bacteria continue to show that they induce T cell communication, decrease inflammatory response, and regulate antigen

the GI symptoms but also in cutaneous and psychological symptoms [13, 16, 17].

**2.3. Impact of the gut microbiome**

the duration of acne treatment [22].

immune response [26].

#### **2.1. History of the gut-brain-skin axis**

The gut-brain-skin axis originated in 1930 when John H. Stokes and Donald M. Pillsbury reported their clinical observations and colleagues' studies. They linked emotional states with gastrointestinal (GI) disorders through various mechanisms, including diet and neuronal responses. Stokes and Pillsbury reported cases of individuals with colitis who also suffered from urticaria and dermatographism. They purported that alterations of microflora increase gut permeability and lead to systemic inflammation. Higher levels of systemic inflammation result in altered cutaneous physiology. This association was further supported by the observation that hypochlorhydria is associated with multiple dermatologic conditions such as rosacea, eczema, pruritus, psoriasis, dermatitis herpetiformis, neurodermatitis, and acne [2].

Similarly, other case studies support the connection between gut physiology and cutaneous and psychological pathology. A psychopathic institute administered *Bacillus acidophilus* to their patients and recorded improvement in their mental distress, gastrointestinal disturbances, and skin eruptions [3]. A 1916 study showed patients with acne exhibiting alterations in their intestinal permeability [4]. In 1937, novel therapies for acne included acidophilus cultures, which acted as "intestinal flora changers" and improved pustular acne [5].

Stokes and Pillsbury's early concept of the gut being linked with the brain and skin started with anecdotal evidence and preliminary studies. They discovered that alteration of gastric acid levels and fluctuation of the gut microflora could have further effects beyond the GI system. From this evidence, they even proposed a treatment of Bacillus and cod liver oil, which is similar to present day probiotics and omega-3 fatty acids, to restore homeostasis in the gut. This early research has led to larger animal and human studies on the GI system, its microbiome, and its relation to the skin.

#### **2.2. Theory of the gut-brain-skin axis**

The negative impact of acne and other cutaneous diseases on the quality of life has been welldocumented [6–10], and can cause long-lasting personality changes [10]. It has been reported that 8.8% of patients with acne exhibit depression [11]. Apart from psychological distress, acne patients often suffer from gastrointestinal disturbances at higher rates compared to the normal population [12]. The gastrointestinal tract houses the largest population of commensal bacteria, and in this reservoir of bacteria lies the central theme to the gut-brain-skin axis.

Hypochlorhydria and small intestinal bacterial overgrowth (SIBO) are two conditions that demonstrate an association with cutaneous pathology and mental health. Alterations in gastric acid secretion, such as hypochlorhydria, increase the risk for SIBO. While increased rates of SIBO have long been associated with psychological disorders of anxiety, depression, and fibromyalgia, more recent evidence has demonstrated increased rates of SIBO in rosacea patients, as well, with one study reporting an SIBO rate of 50% in rosacea patients. Those patients were then treated for SIBO with the antimicrobial rifaximin, and most had significant improvement or clearance of their rosacea [13, 14]. Additionally, an Australian study used the probiotic *Lactobacillus casei* to successfully reduce SIBO [15]. The authors not only noted improvement in the GI symptoms but also in cutaneous and psychological symptoms [13, 16, 17].

Intestinal permeability has also been linked to cutaneous pathology. As far back at 1916, acne patients have shown reactivity to stool-isolated bacteria using a serum complement test [4]. Later, Juhlin and Michaelsson tested *Escherichia coli* polysaccharide endotoxin in the blood samples of acne patients. The patients with severe acne had reactivity while the control had none [18]. A second study of human subjects with IBS identified increased levels of *E. coli* lipopolysaccharide (LPS)-induced cytokines in patients with IBS compared to controls [19]. Increased intestinal permeability and heightened immune responses has also been associated with chronic constipation [20]. Several studies have noted a higher prevalence of constipation in those with acne vulgaris [12, 21]. These correlations suggest increased intestinal permeability and consequently higher levels of circulating endotoxin may contribute to acne formation [21].

#### **2.3. Impact of the gut microbiome**

**2. The gut-brain-skin axis**

92 Probiotics - Current Knowledge and Future Prospects

**2.1. History of the gut-brain-skin axis**

microbiome, and its relation to the skin.

**2.2. Theory of the gut-brain-skin axis**

The field of dermatology continues to investigate the interconnected relationships between the skin and other systems of the body. The unifying theory of the gut-brain-skin axis outlines the relationship between the skin, gastrointestinal (GI) system, and mental health. Notably, the role of the gastrointestinal (GI) system is an area of particular interest as it relates to inflammatory skin conditions. The gut microbiome reacts to various stimuli resulting in systemic responses. By altering the GI microbiome, it is possible to decrease systemic inflammation,

The gut-brain-skin axis originated in 1930 when John H. Stokes and Donald M. Pillsbury reported their clinical observations and colleagues' studies. They linked emotional states with gastrointestinal (GI) disorders through various mechanisms, including diet and neuronal responses. Stokes and Pillsbury reported cases of individuals with colitis who also suffered from urticaria and dermatographism. They purported that alterations of microflora increase gut permeability and lead to systemic inflammation. Higher levels of systemic inflammation result in altered cutaneous physiology. This association was further supported by the observation that hypochlorhydria is associated with multiple dermatologic conditions such as rosacea, eczema, pruritus, psoriasis, dermatitis herpetiformis, neurodermatitis, and acne [2]. Similarly, other case studies support the connection between gut physiology and cutaneous and psychological pathology. A psychopathic institute administered *Bacillus acidophilus* to their patients and recorded improvement in their mental distress, gastrointestinal disturbances, and skin eruptions [3]. A 1916 study showed patients with acne exhibiting alterations in their intestinal permeability [4]. In 1937, novel therapies for acne included acidophilus

and these results can improve the severity of inflammatory skin diseases such as acne.

cultures, which acted as "intestinal flora changers" and improved pustular acne [5].

Stokes and Pillsbury's early concept of the gut being linked with the brain and skin started with anecdotal evidence and preliminary studies. They discovered that alteration of gastric acid levels and fluctuation of the gut microflora could have further effects beyond the GI system. From this evidence, they even proposed a treatment of Bacillus and cod liver oil, which is similar to present day probiotics and omega-3 fatty acids, to restore homeostasis in the gut. This early research has led to larger animal and human studies on the GI system, its

The negative impact of acne and other cutaneous diseases on the quality of life has been welldocumented [6–10], and can cause long-lasting personality changes [10]. It has been reported that 8.8% of patients with acne exhibit depression [11]. Apart from psychological distress, acne patients often suffer from gastrointestinal disturbances at higher rates compared to the normal population [12]. The gastrointestinal tract houses the largest population of commensal bacteria, and in this reservoir of bacteria lies the central theme to the gut-brain-skin axis.

The gut microbiome is dynamic; it changes with the stress on its environment and reacts to feedback from other systems. As stated previously, changes in the microbiome may influence levels of systemic inflammation. Acne is an inflammatory disorder that has demonstrated its response to systemic inflammation and oxidative stress. A Russian study found that not only is the intestinal microflora of acne patients altered, but therapy for the GI disruption reduced the duration of acne treatment [22].

The gut's commensal bacteria can induce immune responses that ultimately reach T cells in the skin. Probiotics interact with the gastrointestinal mucosal immune system, altering levels of inflammatory cytokines in the blood [23]. For instance, the levels of gamma-aminobutyric acid (GABA) are modified by intestinal bacteria [24, 25]. Microbial-fermented food enriched with GABA has been shown to improve atopic dermatitis in mice through a Th-1 mediatedimmune response [26].

Major histocompatibility cell (MHC) class II complexes are found on antigen presenting cells, such as dendritic cells and macrophages, and interact with immune-regulating T cells. Gastrointestinal bacteria, including strains of probiotics, have been shown to bind to the MHC II complex and modify their expressions [27]. A study that administered *Lactobacillus paracasei* NCC2461 (ST11) to mice found the probiotic to induce T regulatory cells and inhibited CD4+ T-cell proliferation, while increasing the secretions of anti-inflammatory cytokines, specifically IL-2, IL-10, and TGF-β [28]. Other strains of lactic acid bacteria continue to show that they induce T cell communication, decrease inflammatory response, and regulate antigen presenting cells [29, 30]. The anti-inflammatory cytokines affect the differentiation of keratinocytes, while TGF-β has a considerable role in enhancement of the skin barrier [28, 31]. These findings were supported by a second study. Mice that were treated with *Lactobacillus casei* recruited T regulatory cells to inflamed skin and released higher levels of the anti-inflammatory cytokine IL-10 [32].

The concept of acne as a result of inflammation is based upon the understanding that the immune system is designed to defend the human body against actual threats. However, in the acne patient, we are recognizing a chronic, low level of inflammation in the absence of threat [40]. Ideally, probiotics would eliminate this chronic inflammatory state, and in turn, halt the

The Role of Probiotics in Acne and Rosacea http://dx.doi.org/10.5772/intechopen.79044 95

Subclinical microcomedones are established as the earliest lesions of acne, and even at this early stage, inflammatory cells have been observed to be already present in these primary lesions. A modern research study was performed comparing immunohistochemistry and immunofluorescence of early, inflamed papules less than 6 h old in acne patients to both uninvolved skin of acne patients and a nonacne control group. In papules less than 6 h old, a remarkable increase in K16 and K67 activity is observed [39]. Uninvolved skin in acne patients exhibited increased expression of CD4+ T cells, and an even more significant upregulation of CD4+ T cells was observed in papules less than 6 h old [39]. The presence of macrophages was found to be higher in both uninvolved skin of acne patients as well as papules less than 6 h

One of the strongest pieces of evidence supporting the theory of baseline inflammation in acne patients is the increased presence of interleukin-1-alpha (IL-1), a well-known proinflammatory cytokine. In the above study, an increased level of IL-1-alpha was observed in both early lesional skin and uninvolved skin of acne patients in comparison with the control group [39]. IL-1 has been proposed as the signal that triggers the entire inflammatory cascade in the setting of a wound. In response to endothelial injury, IL-1 is the first cytokine to be produced, attracting lymphocytes to the area as well as activating endothelial cells to produce a hyperproliferative state [41]. It is further proposed that increased expression of K6 and K16, TNF-alpha, and endothelial growth factors then occur as a result [41]. Considering the above information, it may be deduced that IL-1 is a powerful inflammatory cascade that trigger in

Collectively, several conclusions can be drawn from this information. These findings support the theory of acne as an inflammatory disease. Significant evidence reinforces the theory that inflammation precedes the overproduction of sebum, hyperproliferative state, and other physical manifestations of acneiform lesions. Taking into account the subtype of T cell activation observed, it is prudent to believe the inflammation is specific and antigenic in nature

Therefore, the anti-inflammatory actions of probiotics may be beneficial in the treatment of acne. Although the exact mechanism remains unclear, literature exists that suggests that *Lactobacilli* have been shown to modulate Th1/Th2 activity [42]. A separate study examined the Th1/Th2 inflammatory response of rats, when faced with an antigen challenge, in the setting of pretreatment with a combination of *Lactobacilli* and *Bifidobacterium* strains. It was found that the combination probiotic treatment did in fact alter both the Th1 and Th2 response [43]. As previously discussed in this section, a dysregulation of the T-cell response has been demonstrated in the skin of acne patients and it may be deduced that normalizing this response may be a critical step toward decreasing the baseline inflammatory state in this population.

development of acne lesions.

the setting of acne as well.

rather than an innate response [39].

old of acne patients compared to nonacne controls [39].

The composition of the gut microbiome can inhibit or promote the release of substance P in both the skin and intestinal tract [33, 34]. When a specific strain of *Lactobacillus paracasei* ST11 was orally administered, secretions of substance P decreased. Lower systemic levels of substance P enhanced skin barrier function and decreased local skin inflammation [35]. Inhibition of substance P directly affects acne pathogenesis, as substance P increases sebum production [36].

The interconnected relationship described by the gut-brain-skin axis illustrates the significant role of the gut microbiome, and its alteration, for instance by probiotics, may play in the development of acne. Modification of local and systemic inflammatory profiles by GI flora presents a target for potential therapy.

### **3. Pathophysiology of probiotics and acne**

It is quite evident that the gut-brain-skin axis plays a theoretically significant role in the formation of acne lesions. In the following sections, we will discuss the pathophysiology behind probiotics and their ensuing potential impact in the arena of acne treatment. As previously discussed, the early theories introduced by Stokes and Pillsbury conceptualized the functional interdependence of the gut-brain-skin axis. It was further proposed that alterations in the neural axis result in gastrointestinal dysfunction, thereby disrupting the local normal flora, and resulting in widespread inflammatory response [37]. As we will see, the concept of systemic inflammatory response as well as oxidative stress is at the core of the rationale behind probiotics and their role in acne treatment.

#### **3.1. Inflammation**

The initial research during the era of Stokes and Pillsbury began with the discovery of concomitant hypochlorhydria in a significant portion of acne patients [37]. Additionally, the expanded SIBO theory suggested that an increased pH in the stomach resulted in a migration of bacteria proximally, increased gut permeability, and significant resultant inflammation [37]. This inflammation is the key starting point for the inflammatory cascade ultimately resulting in acne lesions.

The inflammatory state associated with acne has received much attention from dermatologic research studies and literature in recent years. While it was previously thought that events such as follicular keratinization and bacterial colonization preceded inflammation [38], it is now known that inflammation is actually the herald event [39].

The concept of acne as a result of inflammation is based upon the understanding that the immune system is designed to defend the human body against actual threats. However, in the acne patient, we are recognizing a chronic, low level of inflammation in the absence of threat [40]. Ideally, probiotics would eliminate this chronic inflammatory state, and in turn, halt the development of acne lesions.

presenting cells [29, 30]. The anti-inflammatory cytokines affect the differentiation of keratinocytes, while TGF-β has a considerable role in enhancement of the skin barrier [28, 31]. These findings were supported by a second study. Mice that were treated with *Lactobacillus casei* recruited T regulatory cells to inflamed skin and released higher levels of the anti-inflamma-

The composition of the gut microbiome can inhibit or promote the release of substance P in both the skin and intestinal tract [33, 34]. When a specific strain of *Lactobacillus paracasei* ST11 was orally administered, secretions of substance P decreased. Lower systemic levels of substance P enhanced skin barrier function and decreased local skin inflammation [35]. Inhibition of substance P directly affects acne pathogenesis, as substance P increases sebum

The interconnected relationship described by the gut-brain-skin axis illustrates the significant role of the gut microbiome, and its alteration, for instance by probiotics, may play in the development of acne. Modification of local and systemic inflammatory profiles by GI flora

It is quite evident that the gut-brain-skin axis plays a theoretically significant role in the formation of acne lesions. In the following sections, we will discuss the pathophysiology behind probiotics and their ensuing potential impact in the arena of acne treatment. As previously discussed, the early theories introduced by Stokes and Pillsbury conceptualized the functional interdependence of the gut-brain-skin axis. It was further proposed that alterations in the neural axis result in gastrointestinal dysfunction, thereby disrupting the local normal flora, and resulting in widespread inflammatory response [37]. As we will see, the concept of systemic inflammatory response as well as oxidative stress is at the core of the rationale

The initial research during the era of Stokes and Pillsbury began with the discovery of concomitant hypochlorhydria in a significant portion of acne patients [37]. Additionally, the expanded SIBO theory suggested that an increased pH in the stomach resulted in a migration of bacteria proximally, increased gut permeability, and significant resultant inflammation [37]. This inflammation is the key starting point for the inflammatory cascade ultimately

The inflammatory state associated with acne has received much attention from dermatologic research studies and literature in recent years. While it was previously thought that events such as follicular keratinization and bacterial colonization preceded inflammation [38], it is

tory cytokine IL-10 [32].

94 Probiotics - Current Knowledge and Future Prospects

production [36].

**3.1. Inflammation**

resulting in acne lesions.

presents a target for potential therapy.

**3. Pathophysiology of probiotics and acne**

behind probiotics and their role in acne treatment.

now known that inflammation is actually the herald event [39].

Subclinical microcomedones are established as the earliest lesions of acne, and even at this early stage, inflammatory cells have been observed to be already present in these primary lesions. A modern research study was performed comparing immunohistochemistry and immunofluorescence of early, inflamed papules less than 6 h old in acne patients to both uninvolved skin of acne patients and a nonacne control group. In papules less than 6 h old, a remarkable increase in K16 and K67 activity is observed [39]. Uninvolved skin in acne patients exhibited increased expression of CD4+ T cells, and an even more significant upregulation of CD4+ T cells was observed in papules less than 6 h old [39]. The presence of macrophages was found to be higher in both uninvolved skin of acne patients as well as papules less than 6 h old of acne patients compared to nonacne controls [39].

One of the strongest pieces of evidence supporting the theory of baseline inflammation in acne patients is the increased presence of interleukin-1-alpha (IL-1), a well-known proinflammatory cytokine. In the above study, an increased level of IL-1-alpha was observed in both early lesional skin and uninvolved skin of acne patients in comparison with the control group [39]. IL-1 has been proposed as the signal that triggers the entire inflammatory cascade in the setting of a wound. In response to endothelial injury, IL-1 is the first cytokine to be produced, attracting lymphocytes to the area as well as activating endothelial cells to produce a hyperproliferative state [41]. It is further proposed that increased expression of K6 and K16, TNF-alpha, and endothelial growth factors then occur as a result [41]. Considering the above information, it may be deduced that IL-1 is a powerful inflammatory cascade that trigger in the setting of acne as well.

Collectively, several conclusions can be drawn from this information. These findings support the theory of acne as an inflammatory disease. Significant evidence reinforces the theory that inflammation precedes the overproduction of sebum, hyperproliferative state, and other physical manifestations of acneiform lesions. Taking into account the subtype of T cell activation observed, it is prudent to believe the inflammation is specific and antigenic in nature rather than an innate response [39].

Therefore, the anti-inflammatory actions of probiotics may be beneficial in the treatment of acne. Although the exact mechanism remains unclear, literature exists that suggests that *Lactobacilli* have been shown to modulate Th1/Th2 activity [42]. A separate study examined the Th1/Th2 inflammatory response of rats, when faced with an antigen challenge, in the setting of pretreatment with a combination of *Lactobacilli* and *Bifidobacterium* strains. It was found that the combination probiotic treatment did in fact alter both the Th1 and Th2 response [43]. As previously discussed in this section, a dysregulation of the T-cell response has been demonstrated in the skin of acne patients and it may be deduced that normalizing this response may be a critical step toward decreasing the baseline inflammatory state in this population.

Further solidifying this concept, in a study aimed at examining the immunomodulatory effects of probiotics in subjects with food allergies, it was determined that probiotics do in fact increase production of anti-inflammatory cytokines such as IL-10, TNF-α, and INF-γ [44]. This discovery may be re-enforced by looking back to the research involving rats and pretreatment with combined *Lactobacilli* and *Bifidobacterium*. In this study, significant reductions were also observed in the production of inflammatory cytokines, most notably IL-1α and IL-1β [43]. It should be noted that TNF-α production was decreased as well [43]. As previously discussed, the IL-1 cytokines play a key role upstream in the inflammatory cascade and altering the production of this cytokine via probiotics may prove advantageous in treating the acne patient.

**4. Probiotics used for the treatment of acne**

The idea of treating acne with probiotics dates back to the 1930s. During that time, *Lactobacillus acidophilus (*a common probiotic found in foods such as yogurt) was a popular diet supplement for the treatment of acne among the public [5]. Although this trend was widely accepted, formal research had not been carried out proving its effectiveness. It was not until 1961 that the first official clinical trial regarding probiotics and its relationship to acne was published. The trial was performed by a physician from the Union Memorial Hospital in Baltimore, Maryland named Robert H. Siver. Dr. Siver followed 300 patients who were taking a commercially available oral probiotic tablet called "Latinex" (combination of *L. acidophilus* and *L. bulgaricus*). Subjects ingested this supplement for eight consecutive days followed by a twoweek break and then repeated the process. Over time, he noticed that 80% of patients with acne experienced clearing of their skin, especially in those with inflammatory acne lesions. Despite this study lacking a placebo group to compare results and having an unconventional probiotic dosing regimen, the findings did suggest a promising linkage between the intestinal

The Role of Probiotics in Acne and Rosacea http://dx.doi.org/10.5772/intechopen.79044 97

After Dr. Siver's research was published, other researchers became interested in a correlation between oral probiotics and acne. Two studies, both published in a non-English language journal, continued to demonstrate a connection. In 1987, an Italian article was published by Marchetti et al., 20 of the 40 patients with acne were given 250 mg of freezedried *L. acidophilus* and *Bifidobacterium bifidum* in addition to standard acne treatment. Subjects in the study group exhibited better compliance with their antibiotic regimen in addition to seeing improved clinical results in their acne [51]. In 2001, a similar investigation was performed in Russia by Vokova et al. using 114 subjects with acne. He found that 61% of the subjects had impaired bacterial microflora, and after probiotic supplementation in addition to combined acne therapy, their duration of treatment was greatly reduced to

More recent studies have continued to confirm these results. In 2010, Kim et al. randomized 36 subjects with acne to receive either lactoferrin (a milk protein with anti-inflammatory, bactericidal, and fungicidal properties) added to fermented milk (experimental group) or fermented milk alone (control group). After 12 weeks, the experimental group experienced significant decreases in total lesion count (23.1%), inflammatory lesion count (38.6%), acne grade (20.3%), and sebum content (31.1%) compared to the control group. Although this study had the additional element of lactoferrin, both groups responded to the fermented milk and saw a reduction in total skin surface lipids. Furthermore, the addition of lactoferrin decreased a specific group of lipids called triacylglycerols, directly related to the decreased sebum content, acne

An interesting open-label study was published in 2013 by Jung et al. concerning probiotics versus antibiotics in 45 women between the ages of 18 to 35 years old. The females were randomized into one of three groups: probiotics only (a mixture of *L. acidophilus*, *L. delbrueckii*, and *B. bifidum*), oral minocycline only, or both probiotics and minocycline. After the first 4 weeks,

**4.1. Oral probiotics**

flora and acne [50].

that of subjects without dysbacteriosis [22].

lesion counts, and acne grade [52].

Given the aforementioned research, it is once again reasonable to conclude that the addition of probiotics in the acne-prone patient would positively affect the causatory state of inflammation. It is clear that further research is needed to solidify the definitive effects of probiotics on the low-level inflammatory state and subsequent inflammatory cascade.

#### **3.2. Oxidative stress**

An alternative theory proposed by Allan L. Lorincz suggested that oxidative breakdown of lipids and squalene was a cause of acne rather than a consequence. The theory then goes on to suggest that this oxidative process is a trigger for the inflammatory condition seen in acne patients [38]. Subsequent studies reinforced this theory. In 1975, A Tappel also supported the theory of inflammation stemming from the damaging effects of lipid peroxidation [45].

This is both important and relevant in the setting of acne as squalene, a key component in the formation of the comedone, is sensitive to oxidative stress. In an independent study, squalene, when exposed to UV radiation (a source leading to oxidative stress), became increasingly comedogenic [46].

It has thus been proposed that alongside inflammation, oxidative stress may play a significant role in the development of acne lesions. Reactive oxygen species (ROS) are produced by environmental factors as well as cellular metabolism byproducts. Higher levels of ROS encourage an environment that is more hospitable to bacteria such as *P. acnes* [38]. In a study examining the activity of antioxidants defense enzymes in leukocytes, acne patients were found to have low levels of both superoxide dismutase and glutathione peroxidase [47].

Faulty antioxidant response seen in acne patients provides yet another role for probiotics in the treatment of acne. Probiotics have been proven to assist in antioxidant activity. In a study performed on the probiotic, *Bacillus coagulans RK-02*, evidence came to light that the bacteria produced a potent extracellular polysaccharide with significant antioxidant activity as well as superoxide radical scavenging activity and hydroxyl radical scavenging activity, even when measured against classic antioxidants including vitamin C [48].

In a separate study, researchers combined various strains of *Lactobacillus* with a gene encoding for superoxide dismutase. The *Lactobacilli* were found not only to successfully express the gene, but were also found to provide measurable defense against hydrogen peroxide species [49].

Probiotics provide a mechanism to counter free radical damage and increase antioxidant activity, resulting in an environment that is less attractive for *P. acnes* colonization.

#### **4. Probiotics used for the treatment of acne**

#### **4.1. Oral probiotics**

Further solidifying this concept, in a study aimed at examining the immunomodulatory effects of probiotics in subjects with food allergies, it was determined that probiotics do in fact increase production of anti-inflammatory cytokines such as IL-10, TNF-α, and INF-γ [44]. This discovery may be re-enforced by looking back to the research involving rats and pretreatment with combined *Lactobacilli* and *Bifidobacterium*. In this study, significant reductions were also observed in the production of inflammatory cytokines, most notably IL-1α and IL-1β [43]. It should be noted that TNF-α production was decreased as well [43]. As previously discussed, the IL-1 cytokines play a key role upstream in the inflammatory cascade and altering the production of this cytokine via probiotics may prove advantageous in treating the acne patient.

Given the aforementioned research, it is once again reasonable to conclude that the addition of probiotics in the acne-prone patient would positively affect the causatory state of inflammation. It is clear that further research is needed to solidify the definitive effects of probiotics

An alternative theory proposed by Allan L. Lorincz suggested that oxidative breakdown of lipids and squalene was a cause of acne rather than a consequence. The theory then goes on to suggest that this oxidative process is a trigger for the inflammatory condition seen in acne patients [38]. Subsequent studies reinforced this theory. In 1975, A Tappel also supported the theory of inflammation stemming from the damaging effects of lipid peroxidation [45].

This is both important and relevant in the setting of acne as squalene, a key component in the formation of the comedone, is sensitive to oxidative stress. In an independent study, squalene, when exposed to UV radiation (a source leading to oxidative stress), became increasingly

It has thus been proposed that alongside inflammation, oxidative stress may play a significant role in the development of acne lesions. Reactive oxygen species (ROS) are produced by environmental factors as well as cellular metabolism byproducts. Higher levels of ROS encourage an environment that is more hospitable to bacteria such as *P. acnes* [38]. In a study examining the activity of antioxidants defense enzymes in leukocytes, acne patients were found to have

Faulty antioxidant response seen in acne patients provides yet another role for probiotics in the treatment of acne. Probiotics have been proven to assist in antioxidant activity. In a study performed on the probiotic, *Bacillus coagulans RK-02*, evidence came to light that the bacteria produced a potent extracellular polysaccharide with significant antioxidant activity as well as superoxide radical scavenging activity and hydroxyl radical scavenging activity, even when

In a separate study, researchers combined various strains of *Lactobacillus* with a gene encoding for superoxide dismutase. The *Lactobacilli* were found not only to successfully express the gene, but were also found to provide measurable defense against hydrogen peroxide species [49].

Probiotics provide a mechanism to counter free radical damage and increase antioxidant

activity, resulting in an environment that is less attractive for *P. acnes* colonization.

on the low-level inflammatory state and subsequent inflammatory cascade.

low levels of both superoxide dismutase and glutathione peroxidase [47].

measured against classic antioxidants including vitamin C [48].

**3.2. Oxidative stress**

96 Probiotics - Current Knowledge and Future Prospects

comedogenic [46].

The idea of treating acne with probiotics dates back to the 1930s. During that time, *Lactobacillus acidophilus (*a common probiotic found in foods such as yogurt) was a popular diet supplement for the treatment of acne among the public [5]. Although this trend was widely accepted, formal research had not been carried out proving its effectiveness. It was not until 1961 that the first official clinical trial regarding probiotics and its relationship to acne was published. The trial was performed by a physician from the Union Memorial Hospital in Baltimore, Maryland named Robert H. Siver. Dr. Siver followed 300 patients who were taking a commercially available oral probiotic tablet called "Latinex" (combination of *L. acidophilus* and *L. bulgaricus*). Subjects ingested this supplement for eight consecutive days followed by a twoweek break and then repeated the process. Over time, he noticed that 80% of patients with acne experienced clearing of their skin, especially in those with inflammatory acne lesions. Despite this study lacking a placebo group to compare results and having an unconventional probiotic dosing regimen, the findings did suggest a promising linkage between the intestinal flora and acne [50].

After Dr. Siver's research was published, other researchers became interested in a correlation between oral probiotics and acne. Two studies, both published in a non-English language journal, continued to demonstrate a connection. In 1987, an Italian article was published by Marchetti et al., 20 of the 40 patients with acne were given 250 mg of freezedried *L. acidophilus* and *Bifidobacterium bifidum* in addition to standard acne treatment. Subjects in the study group exhibited better compliance with their antibiotic regimen in addition to seeing improved clinical results in their acne [51]. In 2001, a similar investigation was performed in Russia by Vokova et al. using 114 subjects with acne. He found that 61% of the subjects had impaired bacterial microflora, and after probiotic supplementation in addition to combined acne therapy, their duration of treatment was greatly reduced to that of subjects without dysbacteriosis [22].

More recent studies have continued to confirm these results. In 2010, Kim et al. randomized 36 subjects with acne to receive either lactoferrin (a milk protein with anti-inflammatory, bactericidal, and fungicidal properties) added to fermented milk (experimental group) or fermented milk alone (control group). After 12 weeks, the experimental group experienced significant decreases in total lesion count (23.1%), inflammatory lesion count (38.6%), acne grade (20.3%), and sebum content (31.1%) compared to the control group. Although this study had the additional element of lactoferrin, both groups responded to the fermented milk and saw a reduction in total skin surface lipids. Furthermore, the addition of lactoferrin decreased a specific group of lipids called triacylglycerols, directly related to the decreased sebum content, acne lesion counts, and acne grade [52].

An interesting open-label study was published in 2013 by Jung et al. concerning probiotics versus antibiotics in 45 women between the ages of 18 to 35 years old. The females were randomized into one of three groups: probiotics only (a mixture of *L. acidophilus*, *L. delbrueckii*, and *B. bifidum*), oral minocycline only, or both probiotics and minocycline. After the first 4 weeks, all patients observed significant improvement in their total lesion count; however, after 8 and 12 weeks, the group using both probiotics and minocycline experienced a significant decrease in their total lesion count compared to the other two groups. In addition, two subjects in the minocycline-only group developed vaginal candidiasis, an adverse event not observed in the group taking both. This study demonstrated that not only can probiotics augment antibiotic therapy, but they may also alleviate particular side effects experienced with chronic antibiotic use by suppressing the growth of unwanted organisms [53].

additional four subjects were treated with sphingomyelinase purified from *Bacillus cereus* to ensure that the results produced were specific to the sphingomyelinase and not another component within the bacterium. After seven consecutive days of application, the probiotic formulation containing *S. thermophilus* caused an increase in the production of ceramides in the stratum corneum, which was comparable to the results seen using the sphingomyelinase extracted from *B. cereus.* These results demonstrated that the sphingomyelinase produced by

The Role of Probiotics in Acne and Rosacea http://dx.doi.org/10.5772/intechopen.79044 99

Ceramides not only have a role in water permeability, but they also play a part in the antimicrobial and anti-inflammatory properties of the skin. The exact antimicrobial mechanism of ceramides has not been confirmed; however, there are many theories: reduction of bacteria adherence to epithelial cells, inhibition of bacterial protein kinases, and/or damage to the cell wall of the bacteria [59, 60]. Aware of their antimicrobial properties, Pavicic et al. performed a study in 2007 to evaluate the role of ceramides in patients with acne. The study consisted of both an *in vitro* and *in vivo* phase. *In vitro*, he found that phytosphingosine (PS), one of the four types of sphingoid bases that make up ceramides, inhibited growth of *Propionibacterium acnes*, an important contributor to acne formation. From these findings, he performed a two-part *in vivo* pilot study testing a 0.2% PS formulation on subjects with acne. In the first part, 30 subjects with acne applied a topical medication containing PS with benzoyl peroxide (PS-BPO) to half of their face versus benzoyl peroxide (BPO) alone to the contralateral side of their face two times per day. After 2 months, comedones were reduced by 72% and inflammatory papules and pustules by 88% in the PS-BPO group versus 22 and 32%, respectively, in BPO only group. In another arm of the trial, 10 subjects applied PS alone to half of their face and a placebo cream alone to the other side of their face twice a day. After 2 months, the placebo increased comedones by 43% compared to only 6% in the PS group. More significant results were seen in inflammatory acne numbers with an 89% reduction observed in the PS group

Topical probiotics may also help with stress-induced acne. It is known that acne can be exacerbated due to stress, primarily due to a release of a chemical called substance P. Sebocytes stimulated by substance P show higher levels of proinflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6) tumor necrosis factor-alpha (TNF-alpha), and peroxisome proliferators activated receptors-gamma (PRAR-gamma), compared to a control [36]. Studies by Gueniche et al. in 2010, using both *in vitro* and clinical trials, have shown that two bacteria, *Lactobacillus paracasei* and *Bifidobacterium longum,* may improve inflammatory skin conditions by inhibiting substance P [33, 62]. As an adjunctive to current acne therapy, these two bacteria in topical formulations may provide relief for individuals suffering from inflammatory and/

Studies of topical and oral probiotics have demonstrated the beneficial anti-inflammatory and antibacterial properties of probiotics in the treatment of acne. Newer research is focusing on yet another treatment mechanism—the production of antimicrobial peptides (AMPs). AMPs are molecules produced by the innate immune system of a wide range of organisms, including

or stress-induced acne not responding to conventional treatment methods.

*S. thermophilus* may improve skin barrier function [58].

compared to no change in the placebo group [61].

**4.3. Future directions**

A 2016 clinical trial identified 57 patients with erythematous papulopustular facial rashes that were diagnosed as either acne, seborrheic dermatitis, or rosacea. The participants were started on a vegetarian diet and appropriate standard therapy for their disorder, including antibiotics, retinoids, and/or steroids. 37 patients of these patients were randomized to receive a daily oral probiotic supplement with *E. coli* Nissle. The group receiving probiotics showed an 89% improvement in their facial dermatoses compared to 56% improvement achieved with diet and standard therapy in the control group. In addition, white blood cell count via blood draw and immunoassays of IL-8, INF-α, and IgA levels were measured throughout the trial. After treatment, lymphocytosis disappeared by 78% in the probiotic group compared to 42% in the control group. Also, levels of INF-α, IL-8, and Ig-A normalized only in the probiotic group compared to no change seen in the control group [54].

#### **4.2. Topical probiotics**

With the growing body of evidence for the role of systemic probiotics in the treatment of acne, the efficacy of topical probiotics is also generating interest and investigation. Similar to oral probiotics, the use of topical probiotics dates back to the early 1900s [55]; however, proper clinical trials were not conducted until much later. In 1999, Di Marzio et al. completed the first clinical trial evaluating topical probiotics and their effects on ceramide production in the skin. Ceramides are waxy lipid molecules that comprise 50% of the lipid matrix within the intercellular spaces of the stratum corneum. Along with cholesterol and long-chain fatty acids, they are essential to maintaining the water permeability of the skin barrier. Ceramides have been found to be low in patients with aged skin, xerosis, atopic dermatitis, psoriasis, and even acne; therefore, increasing their production may significantly impact these disorders [56]. Initially, Di Marzio et al. conducted an *in vitro* study during which he added the bacterium *Streptococcus thermophilus* to human keratinocyte cell cultures and found an increase in the production of ceramides. He believed this was due to *S. thermophilus'* possession of sphingomyelinase, an enzyme that hydrolyzes sphingomyelin into ceramides. Many bacteria have been reported to produce extracellular sphingomyelinase including the genera *Bacillus, Listeria, Staphylococcus, Mycobacterium, Chlamydia, Pseudomonas, Leptospira,* and some species of *Helicobacter.* Although this enzyme primarily functions as a virulence factor for the bacteria, its ability to increase ceramide production may provide a benefit in treating skin diseases [57].

In the next phase of the study, Di Marzio tested this theory *in vivo* on 17 healthy subjects with normal skin. The subjects were instructed to apply 0.5 g of a topical probiotic formulation consisting of *Streptococcus thermophilus* twice a day to the volar surface of one of their forearms. They applied the vehicle alone to the contralateral forearm for comparison. An additional four subjects were treated with sphingomyelinase purified from *Bacillus cereus* to ensure that the results produced were specific to the sphingomyelinase and not another component within the bacterium. After seven consecutive days of application, the probiotic formulation containing *S. thermophilus* caused an increase in the production of ceramides in the stratum corneum, which was comparable to the results seen using the sphingomyelinase extracted from *B. cereus.* These results demonstrated that the sphingomyelinase produced by *S. thermophilus* may improve skin barrier function [58].

Ceramides not only have a role in water permeability, but they also play a part in the antimicrobial and anti-inflammatory properties of the skin. The exact antimicrobial mechanism of ceramides has not been confirmed; however, there are many theories: reduction of bacteria adherence to epithelial cells, inhibition of bacterial protein kinases, and/or damage to the cell wall of the bacteria [59, 60]. Aware of their antimicrobial properties, Pavicic et al. performed a study in 2007 to evaluate the role of ceramides in patients with acne. The study consisted of both an *in vitro* and *in vivo* phase. *In vitro*, he found that phytosphingosine (PS), one of the four types of sphingoid bases that make up ceramides, inhibited growth of *Propionibacterium acnes*, an important contributor to acne formation. From these findings, he performed a two-part *in vivo* pilot study testing a 0.2% PS formulation on subjects with acne. In the first part, 30 subjects with acne applied a topical medication containing PS with benzoyl peroxide (PS-BPO) to half of their face versus benzoyl peroxide (BPO) alone to the contralateral side of their face two times per day. After 2 months, comedones were reduced by 72% and inflammatory papules and pustules by 88% in the PS-BPO group versus 22 and 32%, respectively, in BPO only group. In another arm of the trial, 10 subjects applied PS alone to half of their face and a placebo cream alone to the other side of their face twice a day. After 2 months, the placebo increased comedones by 43% compared to only 6% in the PS group. More significant results were seen in inflammatory acne numbers with an 89% reduction observed in the PS group compared to no change in the placebo group [61].

Topical probiotics may also help with stress-induced acne. It is known that acne can be exacerbated due to stress, primarily due to a release of a chemical called substance P. Sebocytes stimulated by substance P show higher levels of proinflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6) tumor necrosis factor-alpha (TNF-alpha), and peroxisome proliferators activated receptors-gamma (PRAR-gamma), compared to a control [36]. Studies by Gueniche et al. in 2010, using both *in vitro* and clinical trials, have shown that two bacteria, *Lactobacillus paracasei* and *Bifidobacterium longum,* may improve inflammatory skin conditions by inhibiting substance P [33, 62]. As an adjunctive to current acne therapy, these two bacteria in topical formulations may provide relief for individuals suffering from inflammatory and/ or stress-induced acne not responding to conventional treatment methods.

#### **4.3. Future directions**

all patients observed significant improvement in their total lesion count; however, after 8 and 12 weeks, the group using both probiotics and minocycline experienced a significant decrease in their total lesion count compared to the other two groups. In addition, two subjects in the minocycline-only group developed vaginal candidiasis, an adverse event not observed in the group taking both. This study demonstrated that not only can probiotics augment antibiotic therapy, but they may also alleviate particular side effects experienced with chronic antibiotic

A 2016 clinical trial identified 57 patients with erythematous papulopustular facial rashes that were diagnosed as either acne, seborrheic dermatitis, or rosacea. The participants were started on a vegetarian diet and appropriate standard therapy for their disorder, including antibiotics, retinoids, and/or steroids. 37 patients of these patients were randomized to receive a daily oral probiotic supplement with *E. coli* Nissle. The group receiving probiotics showed an 89% improvement in their facial dermatoses compared to 56% improvement achieved with diet and standard therapy in the control group. In addition, white blood cell count via blood draw and immunoassays of IL-8, INF-α, and IgA levels were measured throughout the trial. After treatment, lymphocytosis disappeared by 78% in the probiotic group compared to 42% in the control group. Also, levels of INF-α, IL-8, and Ig-A normalized only in the probiotic group

With the growing body of evidence for the role of systemic probiotics in the treatment of acne, the efficacy of topical probiotics is also generating interest and investigation. Similar to oral probiotics, the use of topical probiotics dates back to the early 1900s [55]; however, proper clinical trials were not conducted until much later. In 1999, Di Marzio et al. completed the first clinical trial evaluating topical probiotics and their effects on ceramide production in the skin. Ceramides are waxy lipid molecules that comprise 50% of the lipid matrix within the intercellular spaces of the stratum corneum. Along with cholesterol and long-chain fatty acids, they are essential to maintaining the water permeability of the skin barrier. Ceramides have been found to be low in patients with aged skin, xerosis, atopic dermatitis, psoriasis, and even acne; therefore, increasing their production may significantly impact these disorders [56]. Initially, Di Marzio et al. conducted an *in vitro* study during which he added the bacterium *Streptococcus thermophilus* to human keratinocyte cell cultures and found an increase in the production of ceramides. He believed this was due to *S. thermophilus'* possession of sphingomyelinase, an enzyme that hydrolyzes sphingomyelin into ceramides. Many bacteria have been reported to produce extracellular sphingomyelinase including the genera *Bacillus, Listeria, Staphylococcus, Mycobacterium, Chlamydia, Pseudomonas, Leptospira,* and some species of *Helicobacter.* Although this enzyme primarily functions as a virulence factor for the bacteria, its ability to increase ceramide production may provide a benefit in treating skin diseases [57]. In the next phase of the study, Di Marzio tested this theory *in vivo* on 17 healthy subjects with normal skin. The subjects were instructed to apply 0.5 g of a topical probiotic formulation consisting of *Streptococcus thermophilus* twice a day to the volar surface of one of their forearms. They applied the vehicle alone to the contralateral forearm for comparison. An

use by suppressing the growth of unwanted organisms [53].

98 Probiotics - Current Knowledge and Future Prospects

compared to no change seen in the control group [54].

**4.2. Topical probiotics**

Studies of topical and oral probiotics have demonstrated the beneficial anti-inflammatory and antibacterial properties of probiotics in the treatment of acne. Newer research is focusing on yet another treatment mechanism—the production of antimicrobial peptides (AMPs). AMPs are molecules produced by the innate immune system of a wide range of organisms, including humans, plants, and insects, that act as a first line of defense against natural antimicrobial agents [63]. These peptides are extremely small, are anti-inflammatory, and have been shown to exhibit properties against bacteria, fungi, viruses, and tumors. They have even shown the ability to overcome bacterial resistance because it is difficult to develop complete resistance to AMPs, making them potential candidates for future therapeutic medications [64, 65]. Besides being produced by many eukaryotic organisms, numerous bacteria have been found to produce AMPs. These bacterial AMPs, called bacteriocidins, have been isolated from about 50 various bacterial species, especially lactic acid-producing bacteria [66–68]. Some researchers refer to bacteriocidins as only those produced by Gram-positive bacteria, which are further classified into two subgroups: lantibiotics (class I) and nonlantibiotics (class II). Many of the lactic acid bacteria produce AMPs in the nonlantibiotics group. AMPs produced by Gramnegative bacteria are sometimes referred to as microcins and classified further into two groups: class I and class II [68]. For simplicity, the general term "bacteriocidins" will be used here.

While these findings suggest that probiotics and the AMPs produced may benefit patients with acne, larger randomized controlled clinical trials are needed. Further studies will elucidate the most efficacious strains, preparations, and treatment regimens for the treatment of

In summary, oral and topical probiotics are emerging as an exciting treatment option or adjuvant treatment for acne. Although additional research needs to be performed, the clinical trials conducted so far have continued to provide evidence that probiotics can improve acne, along with multiple other inflammatory disorders, with very limited adverse effects. In the upcoming years, probiotic formulations have the potential to be a fundamental component of acne treatment and may augment the efficacy of current treatments today.

, Victoria Comeau2

\*

2 Department of Dermatology, Philadelphia College of Osteopathic Medicine, Roswell, GA,

[1] Morelli L, Capurso L. FAO/WHO guidelines on probiotics: 10 years later. Journal of

[2] Stokes JH, Pillsbury DM. The effect on the skin of emotional and nervous states: I II. Theoretical and practical consideration of a gastro-intestinal mechanism. Archives of

[3] Saunders A. The bacillus acidophilus treatment. Institution Q. 1924;**15**:85-88

, Christopher Buckley1,2,

The Role of Probiotics in Acne and Rosacea http://dx.doi.org/10.5772/intechopen.79044 101

acne and potential uses in other conditions.

No conflict of interest to be reported by the authors.

, Alexandria B. Glass2

\*Address all correspondence to: mmkober@gmail.com

Clinical Gastroenterology. 2012;**46**(Suppl):S1-S2

Dermatology and Syphilology. 1930;**22**:962-993

1 Riverchase Dermatology, Naples, FL, USA

and Mary-Margaret Kober1

**5. Conclusion**

**Conflict of interest**

**Author details**

Caitlin F. Porubsky<sup>2</sup>

USA

**References**

Marcus B. Goodman2

Compared to AMPs produced by eukaryotic organisms, bacteriocidins have a narrower spectrum of activity, only capable of targeting a few species but have the advantage of being more potent. Bacteriocidins are active at pico- to nanomolar concentrations compared to micromolar concentrations required when produced by eukaryotes. Bacteriocidins are bactericidal, causing pore formation in cell membranes [69].

There have been multiple studies performed observing the effects of AMPs on many disorders, including acne vulgaris. In 2006, Bowe et al. discovered that a normal oral flora bacterium, *Streptococcus salivarius,* was capable of inhibiting the growth of *P. acnes* by producing a bacteriocidin called bacteriocin-like inhibitory substances (BLIS). While BLIS is responsible for inhibiting group A streptococcus (GAS), a pathogenic bacterium responsible for causing many upper respiratory infections, its activity against *P. acnes* had not previously been evaluated. In this *in vitro* study, oral swabs were taken from 106 subjects and cultured for the growth of *S. salivarius.* Out of 106, 33 specimens yielded growth of *S. salivarius* and were available for assays of *P. acnes* and GAS. Results found 11 (33.3%) inhibited the growth of *P. acnes* and 13 (39.4%) inhibited the growth of GAS. Although these results focused only on *in vitro* activity, this study demonstrated the potential use of BLIS or BLIS-producing bacteria in future as acne topical treatment formulations [70].

A similar study in 2009 by Kang et al. demonstrated the effects of the bacterium, *Enterococcus faecalis* SL-5 (a very common inhabitant of the human gastrointestinal tract) and its effect on *P. acnes*. He conducted *in vitro* and *in vivo* studies. In the *in vitro* aspect of the study, *E. faecalis* proved to be bacteriocidal to *P. acnes* due to a bacteriocidin named ESL5. In the clinical trial, 70 subjects with mild-to-moderate acne were enrolled in an 8-week double-blind, randomized, placebo-controlled phase III study. Subjects were randomized into the probiotic or placebo group. Those in the experimental group applied a lotion containing ESL5 to the areas of the face involved with acne twice per day, and the control group applied a placebo lotion twice daily. After 8 weeks of application, a decrease in the number of comedones was seen in the probiotic group compared to the placebo group; however, these results were not statistically significant. In the inflammatory lesion counts, a statistically significant reduction of greater than 50% was observed in the *E. faecalis* group compared to placebo [71].

While these findings suggest that probiotics and the AMPs produced may benefit patients with acne, larger randomized controlled clinical trials are needed. Further studies will elucidate the most efficacious strains, preparations, and treatment regimens for the treatment of acne and potential uses in other conditions.

## **5. Conclusion**

humans, plants, and insects, that act as a first line of defense against natural antimicrobial agents [63]. These peptides are extremely small, are anti-inflammatory, and have been shown to exhibit properties against bacteria, fungi, viruses, and tumors. They have even shown the ability to overcome bacterial resistance because it is difficult to develop complete resistance to AMPs, making them potential candidates for future therapeutic medications [64, 65]. Besides being produced by many eukaryotic organisms, numerous bacteria have been found to produce AMPs. These bacterial AMPs, called bacteriocidins, have been isolated from about 50 various bacterial species, especially lactic acid-producing bacteria [66–68]. Some researchers refer to bacteriocidins as only those produced by Gram-positive bacteria, which are further classified into two subgroups: lantibiotics (class I) and nonlantibiotics (class II). Many of the lactic acid bacteria produce AMPs in the nonlantibiotics group. AMPs produced by Gramnegative bacteria are sometimes referred to as microcins and classified further into two groups: class I and class II [68]. For simplicity, the general term "bacteriocidins" will be used here.

Compared to AMPs produced by eukaryotic organisms, bacteriocidins have a narrower spectrum of activity, only capable of targeting a few species but have the advantage of being more potent. Bacteriocidins are active at pico- to nanomolar concentrations compared to micromolar concentrations required when produced by eukaryotes. Bacteriocidins are bactericidal,

There have been multiple studies performed observing the effects of AMPs on many disorders, including acne vulgaris. In 2006, Bowe et al. discovered that a normal oral flora bacterium, *Streptococcus salivarius,* was capable of inhibiting the growth of *P. acnes* by producing a bacteriocidin called bacteriocin-like inhibitory substances (BLIS). While BLIS is responsible for inhibiting group A streptococcus (GAS), a pathogenic bacterium responsible for causing many upper respiratory infections, its activity against *P. acnes* had not previously been evaluated. In this *in vitro* study, oral swabs were taken from 106 subjects and cultured for the growth of *S. salivarius.* Out of 106, 33 specimens yielded growth of *S. salivarius* and were available for assays of *P. acnes* and GAS. Results found 11 (33.3%) inhibited the growth of *P. acnes* and 13 (39.4%) inhibited the growth of GAS. Although these results focused only on *in vitro* activity, this study demonstrated the potential use of BLIS or BLIS-producing

A similar study in 2009 by Kang et al. demonstrated the effects of the bacterium, *Enterococcus faecalis* SL-5 (a very common inhabitant of the human gastrointestinal tract) and its effect on *P. acnes*. He conducted *in vitro* and *in vivo* studies. In the *in vitro* aspect of the study, *E. faecalis* proved to be bacteriocidal to *P. acnes* due to a bacteriocidin named ESL5. In the clinical trial, 70 subjects with mild-to-moderate acne were enrolled in an 8-week double-blind, randomized, placebo-controlled phase III study. Subjects were randomized into the probiotic or placebo group. Those in the experimental group applied a lotion containing ESL5 to the areas of the face involved with acne twice per day, and the control group applied a placebo lotion twice daily. After 8 weeks of application, a decrease in the number of comedones was seen in the probiotic group compared to the placebo group; however, these results were not statistically significant. In the inflammatory lesion counts, a statistically significant reduction of greater

causing pore formation in cell membranes [69].

100 Probiotics - Current Knowledge and Future Prospects

bacteria in future as acne topical treatment formulations [70].

than 50% was observed in the *E. faecalis* group compared to placebo [71].

In summary, oral and topical probiotics are emerging as an exciting treatment option or adjuvant treatment for acne. Although additional research needs to be performed, the clinical trials conducted so far have continued to provide evidence that probiotics can improve acne, along with multiple other inflammatory disorders, with very limited adverse effects. In the upcoming years, probiotic formulations have the potential to be a fundamental component of acne treatment and may augment the efficacy of current treatments today.

#### **Conflict of interest**

No conflict of interest to be reported by the authors.

## **Author details**

Caitlin F. Porubsky<sup>2</sup> , Alexandria B. Glass2 , Victoria Comeau2 , Christopher Buckley1,2, Marcus B. Goodman2 and Mary-Margaret Kober1 \*

\*Address all correspondence to: mmkober@gmail.com

1 Riverchase Dermatology, Naples, FL, USA

2 Department of Dermatology, Philadelphia College of Osteopathic Medicine, Roswell, GA, USA

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**Chapter 6**

**Provisional chapter**

*Lactobacillus* **Species in Breast Milk**

*Lactobacillus* **Species in Breast Milk**

DOI: 10.5772/intechopen.72639

*Lactobacillus* species, present in the microbiota of breast milk, is a probiotic that deserves significant attention. It has a beneficial effect on the composition of the intestinal microflora and the intestinal immune system. In infants who were having *Lactobacillus fermentum*, a lower incidence of gastrointestinal and respiratory infections was noticed, in contrast to the control group. The significant anti-inflammatory effect of *L. fermentum* can be utilized to prevent and treat mastitis in breastfeeding women. It has also been shown to have a better clinical effect than classic antibiotics. Moreover, the higher share of *L. fermentum* in intestinal microflora of children with normal weight compared to obese

Microbiota is a substantial collection of genetic and bioactive materials responsible for building and regulating our defense systems. Bacteria and their intestinal microbial proportions modulate the immune system, greatly affecting the health and illness of an individual. Gastrointestinal flora is in close and continuous contact with epithelial and immune cells. This constant stimulation is essential for the development and functioning of the immune system [1]. These types of bacteria that colonize the guts of a newborn determine how the system develops, acting as an important antigenic stimulus for developing the immune response.

In the last 20 years, probiotics, bifidobacteria, *Lactobacilli*, microorganisms, and gastrointestinal flora, all of which can modulate the aspects of both natural and acquired immune responses in the host and thus affect human health, have become of prime importance. This importance is, of course, widely emphasized commercially. However, the actual effects and actions of individual probiotic strains vary, and it is very important to know what specific

ones opens other potential possibilities of the use of this probiotic.

**Keywords:** *Lactobacillus*, microbiota, probiotics, mastitis, obesity

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

http://dx.doi.org/10.5772/intechopen.72639

Martin Gregora

**Abstract**

**1. Introduction**

Martin Gregora


#### **Chapter 6**

**Provisional chapter**

## *Lactobacillus* **Species in Breast Milk**

*Lactobacillus* **Species in Breast Milk**

#### Martin Gregora Martin Gregora Additional information is available at the end of the chapter

[61] Pavicic T, Wollenweber U, Farwick M, Korting HC. Anti-microbial and -inflammatory activity and efficacy of phytosphingosine: An in vitro and in vivo study addressing acne

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[67] Harder J, Tsuruta D, Murakami M, Kurokawa I. What is the role of antimicrobial peptides (AMP) in acne vulgaris? Experimental Dermatology. 2013;**22**(6):386-391

[68] Hassan M, Kjos M, Nes IF, Diep DB, Lotfipour F. Natural antimicrobial peptides from bacteria: Characteristics and potential applications to fight against antibiotic resistance.

[69] Nissen-Meyer J, Nes IF.Ribosomally synthesized antimicrobial peptides: Their function, structure, biogenesis, and mechanism of action. Archives of Microbiology. 1997;**167**(2/3):

[70] Bowe WP, Filip JC, DiRienzo JM, Volgina A, Margolis DJ. Inhibition of propionibacterium acnes by bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus

[71] Kang BS, Seo JG, Lee GS, Kim JH, Kim SY, Han YW, et al. Antimicrobial activity of enterocins from *Enterococcus faecalis* SL-5 against *Propionibacterium acnes*, the causative agent in acne vulgaris, and its therapeutic effect. Journal of Microbiology. 2009;**47**(1):101-109

vulgaris. International Journal of Cosmetic Science. 2007;**29**(3):181-190

potential for inflammatory acne vulgaris. PLoS One. 2013;**8**(8):e72923

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106 Probiotics - Current Knowledge and Future Prospects

e1-e8

67-77

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72639

#### **Abstract**

*Lactobacillus* species, present in the microbiota of breast milk, is a probiotic that deserves significant attention. It has a beneficial effect on the composition of the intestinal microflora and the intestinal immune system. In infants who were having *Lactobacillus fermentum*, a lower incidence of gastrointestinal and respiratory infections was noticed, in contrast to the control group. The significant anti-inflammatory effect of *L. fermentum* can be utilized to prevent and treat mastitis in breastfeeding women. It has also been shown to have a better clinical effect than classic antibiotics. Moreover, the higher share of *L. fermentum* in intestinal microflora of children with normal weight compared to obese ones opens other potential possibilities of the use of this probiotic.

DOI: 10.5772/intechopen.72639

**Keywords:** *Lactobacillus*, microbiota, probiotics, mastitis, obesity

#### **1. Introduction**

Microbiota is a substantial collection of genetic and bioactive materials responsible for building and regulating our defense systems. Bacteria and their intestinal microbial proportions modulate the immune system, greatly affecting the health and illness of an individual. Gastrointestinal flora is in close and continuous contact with epithelial and immune cells. This constant stimulation is essential for the development and functioning of the immune system [1]. These types of bacteria that colonize the guts of a newborn determine how the system develops, acting as an important antigenic stimulus for developing the immune response.

In the last 20 years, probiotics, bifidobacteria, *Lactobacilli*, microorganisms, and gastrointestinal flora, all of which can modulate the aspects of both natural and acquired immune responses in the host and thus affect human health, have become of prime importance. This importance is, of course, widely emphasized commercially. However, the actual effects and actions of individual probiotic strains vary, and it is very important to know what specific

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

probiotics are considered in order to determine their effects. Bacteria colonize vast areas of the mucous membranes and they are also present in important body fluids like breast milk. The mother's vaginal flora and breast milk are clearly among the most important sources of bacteria for the newborn. Varying studies have reported differing quantities of live bacteria in breast milk, but most studies report median numbers of 102 –10<sup>3</sup> and a range of 10<sup>1</sup> –10<sup>7</sup> colony forming units per ml of breast milk [2]. The infant who receives 300–700 ml of milk per day receives a large amount of these bacteria at the same time. The microbiota of milk, like that of mucous membranes, is individual and changeable. The probiotic bacteria present in mucous membranes and breast milk includes *Lactobacillus fermentum*. The expected pathway by which *Lactobacilli* is received into the milk is enteromammary transport through the dendritic cells [3]. This type of transport is still a controversial subject; however, various studies suggest that dendritic cells can pick up bacteria located in the intestinal lumen and transfer them to the lamina propria. Once the bacteria get inside the dendritic cells, they can penetrate the mammary glands and other mucosal surfaces.

This strain is also able to colonize the mammary glands when administered to nursing mothers in capsule form. A similar effect on the health of children has been described in other probiotic strains. A multicenter, randomized, double-blind, placebo-controlled trial [4] on 126 healthy children aged 12–48 months with *Lactobacillus paracasei* (66 infants in the experimental group and 60 infants in the placebo group) showed a lower incidence of respiratory and gastrointestinal tract infections in the experimental group than in the control group (**Table 2**). An immunostimulatory effect was observed, consisting of a significant increase in the production of innate and acquired immunity peptides. Innate immunity peptides, produced by epithelial cells, Paneth cells, neutrophils, and macrophages, act as endogenous antimicrobial substances and defend the body against a broad range of pathogens (bacteria, fungi, protozoa, and viruses).

**Table 1.** *Lactobacillus fermentum* administered to 6-month-old infants over a 6-month period versus *Lactobacilli*-free

Total infections 189 142 30 Gastrointestinal infections 33 19 46 Respiratory infections 134 106 26 • Upper respiratory 121 94 27 • Lower respiratory 13 12 13

control group [8].

Acute gastroenteritis, *n* (%) (number of episodes)

Rhinitis, *n* (%) (number of episodes)

Otitis media, *n* (%) (number of episodes)

Pharyngitis, *n* (%) (number of episodes)

Laryngitis, *n* (%) (number of episodes)

Tracheitis, *n* (%) (number of episodes) **Control group Experimental group Incidence rate decrease (%)**

*Lactobacillus* Species in Breast Milk

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http://dx.doi.org/10.5772/intechopen.72639

Another bacterium isolated from breast milk that has a positive effect on diseases in infants and children is *L. reuteri*. The mechanism of action of *L. reuteri* strains has been evaluated in *in vitro* and animal studies. One of the best-documented mechanisms is their antimicrobial activity. *L. reuteri* strains produce reuterin, a broad-spectrum antibacterial substance that can

> 12 (18.2) (19)

> 22 (33.3) (44)

8 (12.1) (11)

13 (19.7) (22)

6 (9.1) (7)

11 (16.7) (16)

0.007

0.438

0.151

0.007

0.029

0.048

**Disease Control group Experimental group** *p*

24 (40.0) (28)

24 (40.0) (50)

13 (21.7) (17)

25 (41.7) (30)

14 (23.3) (14)

19 (31.7) (30)

**Table 2.** Common infectious diseases observed during the study period [4].

#### **2.** *Lactobacillus* **species and infectious diseases of infants**

Respiratory and gastrointestinal tract infections are a significant problem for young children attending daycare centers or preschool, especially in the winter season. Common infectious diseases are facilitated by a general immaturity of the immune system and of the respiratory and gastrointestinal tract function [4]. An increased number of acute diseases translate into a significant financial burden for both the family and society. The increased costs are related to medical care visits and medication as well as to time away from work and/or for payment for someone to look after a sick child [5].

The most widely used probiotic species, which belong to the genera *Lactobacillus* and *Bifidobacterium*, have shown clinically significant benefits in the treatment and prevention of childhood diarrheal and allergic diseases in at-risk populations such as allergic families, hospitalized patients, or children in daycare centers. In a study in which *Lactobacillus reuteri* was administered for 3 months in 336 otherwise healthy children attending daycare centers, it was shown that during the administration and for the next 3 months, the number of episodes of diarrhea has significantly decreased [6]. The effects of probiotics in preventing respiratory tract infections are also receiving increasing attention. In accordance with the same study mentioned earlier, the number of respiratory tract infections in the 336 children has also significantly decreased at 3 and 6 months after the administration of the probiotics [6]. There are many sources of confusion concerning probiotic intervention in children. First, the mode of probiotic administration in the general child population is challenging. Second, the selection of a specific probiotic strain or a probiotic mixture is crucial for the possible beneficial effects. The duration of breastfeeding and the use of infant formula also affect the outcome [7]. Several clinical studies have been carried out to investigate bacteria isolated from human milk. In a 6-month study [8] with 91 infants in the control group and 97 infants in the *L. fermentum* group, a reduction in the total number of infections, especially gastrointestinal tract and respiratory infections, was observed in the probiotics group (**Table 1**). *L. fermentum* was selected for the study for safety and for its anti-infective and immunomodulatory properties.


probiotics are considered in order to determine their effects. Bacteria colonize vast areas of the mucous membranes and they are also present in important body fluids like breast milk. The mother's vaginal flora and breast milk are clearly among the most important sources of bacteria for the newborn. Varying studies have reported differing quantities of live bacteria in

forming units per ml of breast milk [2]. The infant who receives 300–700 ml of milk per day receives a large amount of these bacteria at the same time. The microbiota of milk, like that of mucous membranes, is individual and changeable. The probiotic bacteria present in mucous membranes and breast milk includes *Lactobacillus fermentum*. The expected pathway by which *Lactobacilli* is received into the milk is enteromammary transport through the dendritic cells [3]. This type of transport is still a controversial subject; however, various studies suggest that dendritic cells can pick up bacteria located in the intestinal lumen and transfer them to the lamina propria. Once the bacteria get inside the dendritic cells, they can penetrate the mam-

Respiratory and gastrointestinal tract infections are a significant problem for young children attending daycare centers or preschool, especially in the winter season. Common infectious diseases are facilitated by a general immaturity of the immune system and of the respiratory and gastrointestinal tract function [4]. An increased number of acute diseases translate into a significant financial burden for both the family and society. The increased costs are related to medical care visits and medication as well as to time away from work and/or for payment for

The most widely used probiotic species, which belong to the genera *Lactobacillus* and *Bifidobacterium*, have shown clinically significant benefits in the treatment and prevention of childhood diarrheal and allergic diseases in at-risk populations such as allergic families, hospitalized patients, or children in daycare centers. In a study in which *Lactobacillus reuteri* was administered for 3 months in 336 otherwise healthy children attending daycare centers, it was shown that during the administration and for the next 3 months, the number of episodes of diarrhea has significantly decreased [6]. The effects of probiotics in preventing respiratory tract infections are also receiving increasing attention. In accordance with the same study mentioned earlier, the number of respiratory tract infections in the 336 children has also significantly decreased at 3 and 6 months after the administration of the probiotics [6]. There are many sources of confusion concerning probiotic intervention in children. First, the mode of probiotic administration in the general child population is challenging. Second, the selection of a specific probiotic strain or a probiotic mixture is crucial for the possible beneficial effects. The duration of breastfeeding and the use of infant formula also affect the outcome [7]. Several clinical studies have been carried out to investigate bacteria isolated from human milk. In a 6-month study [8] with 91 infants in the control group and 97 infants in the *L. fermentum* group, a reduction in the total number of infections, especially gastrointestinal tract and respiratory infections, was observed in the probiotics group (**Table 1**). *L. fermentum* was selected for the study for safety and for its anti-infective and immunomodulatory properties.

–10<sup>3</sup>

and a range of 10<sup>1</sup>

–10<sup>7</sup>

colony

breast milk, but most studies report median numbers of 102

**2.** *Lactobacillus* **species and infectious diseases of infants**

mary glands and other mucosal surfaces.

108 Probiotics - Current Knowledge and Future Prospects

someone to look after a sick child [5].

**Table 1.** *Lactobacillus fermentum* administered to 6-month-old infants over a 6-month period versus *Lactobacilli*-free control group [8].

This strain is also able to colonize the mammary glands when administered to nursing mothers in capsule form. A similar effect on the health of children has been described in other probiotic strains. A multicenter, randomized, double-blind, placebo-controlled trial [4] on 126 healthy children aged 12–48 months with *Lactobacillus paracasei* (66 infants in the experimental group and 60 infants in the placebo group) showed a lower incidence of respiratory and gastrointestinal tract infections in the experimental group than in the control group (**Table 2**). An immunostimulatory effect was observed, consisting of a significant increase in the production of innate and acquired immunity peptides. Innate immunity peptides, produced by epithelial cells, Paneth cells, neutrophils, and macrophages, act as endogenous antimicrobial substances and defend the body against a broad range of pathogens (bacteria, fungi, protozoa, and viruses).

Another bacterium isolated from breast milk that has a positive effect on diseases in infants and children is *L. reuteri*. The mechanism of action of *L. reuteri* strains has been evaluated in *in vitro* and animal studies. One of the best-documented mechanisms is their antimicrobial activity. *L. reuteri* strains produce reuterin, a broad-spectrum antibacterial substance that can


**Table 2.** Common infectious diseases observed during the study period [4].

inhibit the growth of a wide spectrum of microorganisms such as Gram-positive or -negative bacteria, yeast, fungi, and parasites. *L. reuteri* strains may also regulate immune response. The results of 14 studies involving controlled trials and one systematic review indicate that the use of *L. reuteri* may be considered in the management of acute gastroenteritis as an adjunct to rehydration. There is also some evidence that *L. reuteri* is effective in reducing the incidence of diarrhea in children attending daycare centers [9]. *Lactobacillus rhamnosus*, a probiotic strain of human origin, also influences immune response both specifically by stimulating antibody production and non-specifically by enhancing the phagocytic activity of the blood leucocytes. It can promote the recovery from rotavirus diarrhea and can reduce the incidence of diarrhea associated with the use of antibiotics. In a randomized, double-blind, placebo-controlled study with 571 healthy children aged 1–6 years, there was a 17% relative reduction in the number of children with respiratory infections with complications and lower respiratory tract infections and a 19% relative reduction in antibiotic treatments for respiratory infection [10].

#### **3.** *L. fermentum* **in the treatment of mastitis**

Mastitis is a common disease during lactation, affecting 3–33% of lactating mothers. Inflammation of the mammary glands usually has an infectious origin involving staphylococci, streptococci, and/or Corynebacterium. Traditionally, *Staphylococcus aureus* has been considered the main etiological agent of acute mastitis, although *Staphylococcus epidermidis* is emerging as the leading cause of chronic mastitis. Multidrug resistance and/or the formation of biofilms are very common among clinical isolates of these two staphylococcal species. This explains why mastitis is difficult to treat with antibiotics and why it constitutes one of the main reasons to cease breastfeeding. In this context, the development of new strategies based on probiotics, as alternatives or complements to antibiotic therapy for the management of mastitis, is particularly appealing. The anti-inflammatory effect of *L. fermentum* can be successfully used to prevent and treat mastitis in a breastfeeding woman. Given as a nutritional supplement to a woman with breast inflammation, it demonstrated a better clinical effect than conventional antibiotics. Moreover, a higher proportion of *L. fermentum* in breast milk is beneficial to the child by favorably modulating the child's intestinal microflora, with beneficial consequences for the immune system and health. A study [11] of 352 women with symptoms of mastitis demonstrated a beneficial effect of treatment with lactobacilli. The women were divided into three different groups: one group using *L. fermentum* and one group using *Lactobacillus salivarius*, both strains isolated from human milk and a third group receiving antibiotics. After 21 days, a reduction in the number of the main etiological agents causing mastitis (*S. epidermidis*, *S. aureus*, and *Streptococcus mitis*) was observed. This reduction was greater in the probiotic groups (**Figure 1**). The groups in which *Lactobacilli* were used also experienced greater pain reduction (**Figure 2**). A similar study of 225 women with severe mastitis caused by staphylococci demonstrated a beneficial effect of *L. fermentum* treatment. There was a faster retreat than in the control group treated with antibiotics. Mastitis relapse was more common among the women treated with antibiotics (31% versus 10%). The principle of the antibacterial action of *L. fermentum* could be explained by its high ability to adhere

to epithelial cells and inhibit the adhesion of pathogenic bacteria by producing antimicrobial

**Figure 2.** Breast pain score at the beginning (day 0) and at the end (day 21) of the trial. Pain is expressed as extremely

Obesity is viewed as one of the more important public health problems of our time, and the velocity of propagation is highest in children. This can lead to a vicious circle: obese children often become obese adults, and maternal obesity overnourishes the fetus, thereby programming adult size and health with a heightened risk of obesity later in life. Recent scientific

**4. Individually different microflora of normal-weight and obese** 

**Figure 1.** Bacterial counts from breast milk at the beginning (day 0) and at the end (day 21) of the trial [11].

) and by its effect on increased mucin production. The action of

*Lactobacillus* Species in Breast Milk

111

http://dx.doi.org/10.5772/intechopen.72639

compounds (lactic acid, H2

painful (0) to no pain (10) [11].

*L. fermentum* is immunostimulatory.

O2

**individuals and the role of probiotics**

**Figure 1.** Bacterial counts from breast milk at the beginning (day 0) and at the end (day 21) of the trial [11].

inhibit the growth of a wide spectrum of microorganisms such as Gram-positive or -negative bacteria, yeast, fungi, and parasites. *L. reuteri* strains may also regulate immune response. The results of 14 studies involving controlled trials and one systematic review indicate that the use of *L. reuteri* may be considered in the management of acute gastroenteritis as an adjunct to rehydration. There is also some evidence that *L. reuteri* is effective in reducing the incidence of diarrhea in children attending daycare centers [9]. *Lactobacillus rhamnosus*, a probiotic strain of human origin, also influences immune response both specifically by stimulating antibody production and non-specifically by enhancing the phagocytic activity of the blood leucocytes. It can promote the recovery from rotavirus diarrhea and can reduce the incidence of diarrhea associated with the use of antibiotics. In a randomized, double-blind, placebo-controlled study with 571 healthy children aged 1–6 years, there was a 17% relative reduction in the number of children with respiratory infections with complications and lower respiratory tract infections and a 19% relative reduction in antibiotic treatments for respiratory infection [10].

Mastitis is a common disease during lactation, affecting 3–33% of lactating mothers. Inflammation of the mammary glands usually has an infectious origin involving staphylococci, streptococci, and/or Corynebacterium. Traditionally, *Staphylococcus aureus* has been considered the main etiological agent of acute mastitis, although *Staphylococcus epidermidis* is emerging as the leading cause of chronic mastitis. Multidrug resistance and/or the formation of biofilms are very common among clinical isolates of these two staphylococcal species. This explains why mastitis is difficult to treat with antibiotics and why it constitutes one of the main reasons to cease breastfeeding. In this context, the development of new strategies based on probiotics, as alternatives or complements to antibiotic therapy for the management of mastitis, is particularly appealing. The anti-inflammatory effect of *L. fermentum* can be successfully used to prevent and treat mastitis in a breastfeeding woman. Given as a nutritional supplement to a woman with breast inflammation, it demonstrated a better clinical effect than conventional antibiotics. Moreover, a higher proportion of *L. fermentum* in breast milk is beneficial to the child by favorably modulating the child's intestinal microflora, with beneficial consequences for the immune system and health. A study [11] of 352 women with symptoms of mastitis demonstrated a beneficial effect of treatment with lactobacilli. The women were divided into three different groups: one group using *L. fermentum* and one group using *Lactobacillus salivarius*, both strains isolated from human milk and a third group receiving antibiotics. After 21 days, a reduction in the number of the main etiological agents causing mastitis (*S. epidermidis*, *S. aureus*, and *Streptococcus mitis*) was observed. This reduction was greater in the probiotic groups (**Figure 1**). The groups in which *Lactobacilli* were used also experienced greater pain reduction (**Figure 2**). A similar study of 225 women with severe mastitis caused by staphylococci demonstrated a beneficial effect of *L. fermentum* treatment. There was a faster retreat than in the control group treated with antibiotics. Mastitis relapse was more common among the women treated with antibiotics (31% versus 10%). The principle of the antibacterial action of *L. fermentum* could be explained by its high ability to adhere

**3.** *L. fermentum* **in the treatment of mastitis**

110 Probiotics - Current Knowledge and Future Prospects

**Figure 2.** Breast pain score at the beginning (day 0) and at the end (day 21) of the trial. Pain is expressed as extremely painful (0) to no pain (10) [11].

to epithelial cells and inhibit the adhesion of pathogenic bacteria by producing antimicrobial compounds (lactic acid, H2 O2 ) and by its effect on increased mucin production. The action of *L. fermentum* is immunostimulatory.

#### **4. Individually different microflora of normal-weight and obese individuals and the role of probiotics**

Obesity is viewed as one of the more important public health problems of our time, and the velocity of propagation is highest in children. This can lead to a vicious circle: obese children often become obese adults, and maternal obesity overnourishes the fetus, thereby programming adult size and health with a heightened risk of obesity later in life. Recent scientific advances point to systemic low-grade inflammation and local gut microbiota as contributing factors for overnutrition. The gut microbiota enables the hydrolysis of indigestible polysaccharides into easily absorbable monosaccharides and the activation of lipoprotein lipase by direct action on the villous epithelium. Consequently, glucose is rapidly absorbed and fatty acids are excessively stored, with both processes boosting weight gain.

**5. The effect of probiotics deserves further clinical trials**

tions; however, confirmation studies are still needed.

Address all correspondence to: martygora@seznam.cz

guests? Frontiers in Endocrinology. 2014;**5**:91

randomized controlled trial. Nutrients. 2017;**9**(7):pii E669

domized controlled trial. Pediatrics. 2014;**133**:e904

mothers. PLoS One. 2016;**11**(8):e0160856

The Department of Paediatrics, Strakonice Hospital, Strakonice, Czech Republic

[1] Pluznick JL. Gut microbes and host physiology: What happens when you host billions of

[2] Sakwinska O, Moine D, Delley M, et al. Microbiota in breast milk of Chinese lactating

[3] Rodríguez JM. The origin of human milk bacteria: Is there a bacterial entero-mammary pathway during late pregnancy and lactation? Advances in Nutrition. 2014;**5**(6):779-784 [4] Corsello G, Carta M, Marinello R, et al. Preventive effect of cow's milk fermented with *Lactobacillus paracasei* CBA L74 on common infectious diseases in children: A multicenter

[5] Hojsak I, Snovak N, Abdovic S, et al. *Lactobacillus* GG in the prevention of gastrointestinal and respiratory tract infections in children who attend day care centers: A randomized, double-blind, placebo-controlled trial. Clinical Nutrition. 2010;**23**(3):312-316 [6] Gutierrez-Castrellon P. Diarrhea in preschool children and *Lactobacillus reuteri*: A ran-

[7] Taipale TJ, Pienihakkinen K, Isolauri E, et al. Bifidobacterium animalis subsp. lactis BB-12 in reducing the risk of infections in early childhood. Pediatric Research. 2016;**79**(1-1):65-69 [8] Maldonaldo J, Cañabate F, Sempere L, et al. Human milk probiotic *Lactobacillus fermentum* CECT5716 reduces the incidence of gastrointestinal and upper respiratory tract infections in infants. Journal of Pediatric Gastroenterology and Nutrition. 2012;**54**(1):55-61

**Author details**

Martin Gregora

**References**

The mucosal microbiota is formed by millions of bacteria. The *Lactobacillus* species are undoubtedly important bacteria for the development of humoral and cellular immunity. However, in the human gut, they are only a part of a huge mosaic where each particle has its place and function. After decades of research, probiotics are still an open chapter of great and unimagined opportunities to influence the immune system and to treat some of civilization's diseases. Most of these diseases are multifactorial. Influencing the mucosal microflora seems to be a promising step. Available data suggest that some probiotics such as *L. fermentum*, *L. reuteri*, *L. paracasei*, and *L. rhamnosus* may have some effect on community-acquired infec-

*Lactobacillus* Species in Breast Milk

113

http://dx.doi.org/10.5772/intechopen.72639

Bacterial milk composition in obese mothers differs from the bacterial milk composition of mothers with standard body weight [11]. Since breast milk is one of the most important means of colonizing infants with bacteria, there is an idea that there is a relationship between obesity and the transmission of microbial flora from mother to infant. It is known that obese infants and obese children generally have very different microbial flora from infants who are lean and healthy (**Figure 3**). The results reported by Kalliomäki et al. suggested that gut microbiota deviations predispose individuals toward energy storage and obesity. The genus *Bifidobacterium*, affecting both the quantity and quality of the microbiota during the first year of life, was shown to be higher in children who remained normal weight than in children developing overweight. The microbiota aberrancy during infancy in children becoming overweight was also associated with a greater number of *S. aureus* than in children remaining normal weight as assessed by real-time qRT-PCR. These findings imply that high numbers of probiotics and low numbers of *S. aureus* in infancy may provide protection against overweight and obesity development.

Perhaps it would be advisable to think about intervention in cases of obese mothers. When is the right time for such an intervention? We know that some bacteria are transmitted from mother to infant. For an obese mother, it would be most helpful to choose an appropriate intervention before or during pregnancy, in any case before giving birth. If the microbial flora has already been transferred to the infant, it could be optimized during breastfeeding through specific probiotics. *L. fermentum*, a strain isolated directly from breast milk in the form of a food supplement, is available as a possible solution. Whether the expected effect of normalization of the intestinal microflora can be produced by such a solution should be confirmed by further studies.

**Figure 3.** Bacterial counts in fecal samples analyzed by fluorescent *in situ* hybridization during infancy (6–12 months) [12].

### **5. The effect of probiotics deserves further clinical trials**

The mucosal microbiota is formed by millions of bacteria. The *Lactobacillus* species are undoubtedly important bacteria for the development of humoral and cellular immunity. However, in the human gut, they are only a part of a huge mosaic where each particle has its place and function. After decades of research, probiotics are still an open chapter of great and unimagined opportunities to influence the immune system and to treat some of civilization's diseases. Most of these diseases are multifactorial. Influencing the mucosal microflora seems to be a promising step. Available data suggest that some probiotics such as *L. fermentum*, *L. reuteri*, *L. paracasei*, and *L. rhamnosus* may have some effect on community-acquired infections; however, confirmation studies are still needed.

#### **Author details**

advances point to systemic low-grade inflammation and local gut microbiota as contributing factors for overnutrition. The gut microbiota enables the hydrolysis of indigestible polysaccharides into easily absorbable monosaccharides and the activation of lipoprotein lipase by direct action on the villous epithelium. Consequently, glucose is rapidly absorbed and fatty

Bacterial milk composition in obese mothers differs from the bacterial milk composition of mothers with standard body weight [11]. Since breast milk is one of the most important means of colonizing infants with bacteria, there is an idea that there is a relationship between obesity and the transmission of microbial flora from mother to infant. It is known that obese infants and obese children generally have very different microbial flora from infants who are lean and healthy (**Figure 3**). The results reported by Kalliomäki et al. suggested that gut microbiota deviations predispose individuals toward energy storage and obesity. The genus *Bifidobacterium*, affecting both the quantity and quality of the microbiota during the first year of life, was shown to be higher in children who remained normal weight than in children developing overweight. The microbiota aberrancy during infancy in children becoming overweight was also associated with a greater number of *S. aureus* than in children remaining normal weight as assessed by real-time qRT-PCR. These findings imply that high numbers of probiotics and low numbers of *S. aureus* in infancy may provide protection against overweight and obesity development.

Perhaps it would be advisable to think about intervention in cases of obese mothers. When is the right time for such an intervention? We know that some bacteria are transmitted from mother to infant. For an obese mother, it would be most helpful to choose an appropriate intervention before or during pregnancy, in any case before giving birth. If the microbial flora has already been transferred to the infant, it could be optimized during breastfeeding through specific probiotics. *L. fermentum*, a strain isolated directly from breast milk in the form of a food supplement, is available as a possible solution. Whether the expected effect of normalization of the intestinal

microflora can be produced by such a solution should be confirmed by further studies.

**Figure 3.** Bacterial counts in fecal samples analyzed by fluorescent *in situ* hybridization during infancy (6–12 months) [12].

acids are excessively stored, with both processes boosting weight gain.

112 Probiotics - Current Knowledge and Future Prospects

#### Martin Gregora

Address all correspondence to: martygora@seznam.cz

The Department of Paediatrics, Strakonice Hospital, Strakonice, Czech Republic

#### **References**


[9] Urbańska M, Szajewska H. The efficacy of *Lactobacillus reuteri* DSM 17938 in infants and children: A review of the current evidence. European Journal of Pediatrics. 2014; **173**:1327-1337

**Chapter 7**

Provisional chapter

**Probiotics Consumption Increment through the Use of**

DOI: 10.5772/intechopen.72362

Probiotics have been taking value over the last years due to its benefits in human health. Researchers have been looking for options in order to increase probiotics consumption, and one of the more nutritional choices is to use whey as a substrate in fermented beverages. Whey is a by-product liquid obtained during cheese processing. It is an economic source of protein, which provides multiple properties in foods. The main objective of this chapter was to carry out a complete review of important researches related to whey-based fermented beverages production. Researches show that probiotic microorganisms have the ability to grow in whey properly, in such a way that they reach high concentrations, needed to achieve the probiotic effect that consumers are looking for. Certain substances, such as fruit pulps and carboxymethyl cellulose, have been used to improve viscosity, flavor among other important characteristics. Sensorial evaluations have been performed in order to assess consumers' impression, and they have been pleasantly accepted. Average shelf-life is 21 days. Through this review, it is known that whey is an excellent alternative to increment probiotic consumption, not only because it is an outstanding substrate for probiotic micro-organism's growth but also due to its excel-

Keywords: whey, fermented beverage, acid lactic bacteria, probiotic, organoleptic

During the last decades, the use of probiotics has been increasing, due to their important benefits in human health. Kollath, in [1], first defined the term "probiotic," when he suggested the term to denote all organic and inorganic food complexes as "probiotics," in contrast to harmful antibiotics, for the purpose of upgrading such food complexes as supplements. In

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Probiotics Consumption Increment through the Use of

**Whey-Based Fermented Beverages**

Whey-Based Fermented Beverages

Mónica S. Molero and Wilfido J. Briñez

Mónica S. Molero and Wilfido J. Briñez

http://dx.doi.org/10.5772/intechopen.72362

lent sensorial characteristics.

characteristics

1. Introduction

Abstract

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **Probiotics Consumption Increment through the Use of Whey-Based Fermented Beverages** Probiotics Consumption Increment through the Use of Whey-Based Fermented Beverages

DOI: 10.5772/intechopen.72362

Mónica S. Molero and Wilfido J. Briñez Mónica S. Molero and Wilfido J. Briñez

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72362

#### Abstract

[9] Urbańska M, Szajewska H. The efficacy of *Lactobacillus reuteri* DSM 17938 in infants and children: A review of the current evidence. European Journal of Pediatrics. 2014;

[10] Hatakka K, Savilahti E, Ponka A, et al. Effect of long term consumption of probiotic milk on infections in children attending day care centers: Double blind, randomised trial.

[11] Arroyo R, Martin V, Maldonado A, et al. Treatment of infectious mastitis during lactation: Antibiotics versus oral administration of lactobacilli isolated from breast milk.

[12] Kalliomäki M, Collado MC, Salminen S, et al. Early differences in fecal microbiota composition in children may predict overweight. The American Journal of Clinical Nutrition.

**173**:1327-1337

2008;**87**:534-538

BMJ. 2001;**322**(7298):1327

114 Probiotics - Current Knowledge and Future Prospects

Clinical Infectious Diseases. 2010;**50**(12):1551-1558

Probiotics have been taking value over the last years due to its benefits in human health. Researchers have been looking for options in order to increase probiotics consumption, and one of the more nutritional choices is to use whey as a substrate in fermented beverages. Whey is a by-product liquid obtained during cheese processing. It is an economic source of protein, which provides multiple properties in foods. The main objective of this chapter was to carry out a complete review of important researches related to whey-based fermented beverages production. Researches show that probiotic microorganisms have the ability to grow in whey properly, in such a way that they reach high concentrations, needed to achieve the probiotic effect that consumers are looking for. Certain substances, such as fruit pulps and carboxymethyl cellulose, have been used to improve viscosity, flavor among other important characteristics. Sensorial evaluations have been performed in order to assess consumers' impression, and they have been pleasantly accepted. Average shelf-life is 21 days. Through this review, it is known that whey is an excellent alternative to increment probiotic consumption, not only because it is an outstanding substrate for probiotic micro-organism's growth but also due to its excellent sensorial characteristics.

Keywords: whey, fermented beverage, acid lactic bacteria, probiotic, organoleptic characteristics

#### 1. Introduction

During the last decades, the use of probiotics has been increasing, due to their important benefits in human health. Kollath, in [1], first defined the term "probiotic," when he suggested the term to denote all organic and inorganic food complexes as "probiotics," in contrast to harmful antibiotics, for the purpose of upgrading such food complexes as supplements. In

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1965, Lilly et al. [2] used the term "probiotic" to describe those substances secreted by an organism that stimulate the growth of another. Since then, this definition has been evolving remarkably, so that today, probiotics are defined as microbial dietary supplements, viable, selected, which when are introduced in sufficient amount, affect human organism beneficially through their effects on the intestinal tract [3]. On the other hand, Vasudha and Mishra [4] define them as alive microbial supplements, which beneficially affect the host by improving its intestinal microbial balance.

2. Relevant aspects related to the use of whey as a substrate for the

The knowledge of whey physicochemical characterization is an important step in the use of this by-product in the dairy industry for different industrial processes. For this reason, most of the studies related to the use of whey propose a physico-chemical characterization in order to evaluate whether it meets the standards required to be used in technological processes.

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In a very recent research, Molero et al. [13], carried out a physico-chemical characterization of whey obtained by cheese making process applying an artisanal method. It consisted of the determination of pH, titrated acidity, total solids, fat, protein according to the Venezuelan Standard COVENIN, and determination of lactose and minerals by analytical difference. The values obtained were statistically analyzed using a statistical package. The results classify whey as sweet, with excellent nutritional characteristics and attractive to be used in food technology for probiotic production, protein-fermented beverages, among other applications. In an interesting research, Tirado et al. [14] carried out a physico-chemical characterization of whey derived from the production of coastal cheese. Fat analyzes were performed by Gerber method, lactose by the Lane and Eynon method (AOAC 923.09, 920.183b); the protein was analyzed by the Kjedahl method (AOAC 920,152); total solids by spectrophotometry; pH was determined by the method established in AOAC 945.10/90 and the acidity expressed as a percentage of lactic acid according to the Colombian Technical Standard. The values obtained were: fat 0%, lactose 3.69%, protein 2.29%, total solids 6.28%, acidity 0.08% lactic acid, and pH 6.5.

Linares et al. [15] showed similar results in the physico-chemical characterization of sweet whey samples, obtaining a pH of 6.84; acidity titrated of 0.11% (% lactic acid); protein between 0.6 and 1%; and ash 0.6%. De Paula et al. [16] performed a physico-chemical characterization of whey obtained from the manufacture of coastal cheese. This characterization was carried out using the following methods: acidity (AOAC 947.05/90), pH (AOAC 981.12/90), soluble solids (AOAC 932.12/90), total solids (AOAC 925,105/90), and lactose (FIL 28a/74). The following results were obtained: acidity (% lactic acid) 0.11; pH 6.58; total solids 6.83%, protein 0.98%; fat

Similarly, Montero et al. [17] carried out a whey fermentation with Lactobacillus (L) for feeding calves in the tropics. Researchers performed a physico-chemical characterization, determining pH, total protein, fat and total solids, tested by the Standard Method for examination of Dairy Products, 2004. The values obtained for these characteristics were in accordance with the

In other studies, Londoño et al. [18] developed a fermented drink of fresh cheese whey inoculated with L. casei. Acidity, pH, viscosity, total solids, protein, fat, ash, lactose, minerals, soluble solids, reducing sugars, and moisture were determined for this purpose. Acidity, pH,

fermented beverages formulation

0.4%; and lactose 4.54%.

Colombian standards established for this type of analysis.

2.1. Whey physico-chemical characterization

Dairy products have become a healthy alternative to increase probiotics consumption, developing fermented beverages based on milk, whey or their mixture. Whey has been less used than milk. However, it has wonderful physico-chemical characteristics that make it an excellent substrate to be used in the development of fermented beverages.

Whey is a green translucent liquid obtained by separating milk clot in cheese making process [5]. Its composition and characteristics depend on the technological process used and the type of milk. It is composed of 5% lactose, 93% water, 0.85% protein, 0.53% minerals, and 0.36% fat [6].

Its characteristics correspond to a fluid of yellowish green color, turbid, fresh taste, weakly sweet, acidic, with a content of nutrients of 5.5–7% that come from milk. It retains about 55% of total milk ingredients like lactose, soluble proteins, lipids, and mineral salts [7]. Whey is a byproduct of high energetic and nutritional quality. For human being, it serves as an important source of vitamins, proteins, and carbohydrates.

Some statistical studies indicate that a significant portion of this waste is discarded to tributaries, resulting in an environmental problem due to its high biochemical oxygen demand. It physically and chemically affects the soil structure, decreasing the yield of agricultural crops, and polluting water because it depletes dissolved oxygen [7].

For the reasons explained above, dairy industry has been looking for alternatives for the use of this by-product, which is a high pollutant; however, it has a great nutritional value. Among the products of successful acceptance are fermented dairy drinks, refreshing beverages [8], protein concentrates [9], infant formulas [10], and others.

The processing of whey for beverages production began in the 1960s, and Rivella was the first fermented drink prepared from whey, made in Switzerland [11]. Whey products improve texture, reduce flavor and color, emulsify, stabilize, improve flow properties, and show many other functional properties that increase the quality of the products [12].

The main objective of this chapter is to carry out a complete review about fermented beverages based on whey inoculated with probiotics micro-organisms that have been produced around the world over the last years, focusing specially in important aspects such as sensorial and microbiological quality, shelf-life, and probiotic effects, showing that probiotics consumption can be increased through the use of whey as a substrate in this type of formulation, promoting it as a useful dairy by-product due to its excellent sensorial characteristics and its contribution in high quality organoleptic foods.

## 2. Relevant aspects related to the use of whey as a substrate for the fermented beverages formulation

#### 2.1. Whey physico-chemical characterization

1965, Lilly et al. [2] used the term "probiotic" to describe those substances secreted by an organism that stimulate the growth of another. Since then, this definition has been evolving remarkably, so that today, probiotics are defined as microbial dietary supplements, viable, selected, which when are introduced in sufficient amount, affect human organism beneficially through their effects on the intestinal tract [3]. On the other hand, Vasudha and Mishra [4] define them as alive microbial supplements, which beneficially affect the host by improving its

Dairy products have become a healthy alternative to increase probiotics consumption, developing fermented beverages based on milk, whey or their mixture. Whey has been less used than milk. However, it has wonderful physico-chemical characteristics that make it an excel-

Whey is a green translucent liquid obtained by separating milk clot in cheese making process [5]. Its composition and characteristics depend on the technological process used and the type of milk. It is composed of 5% lactose, 93% water, 0.85% protein, 0.53% minerals, and 0.36% fat [6]. Its characteristics correspond to a fluid of yellowish green color, turbid, fresh taste, weakly sweet, acidic, with a content of nutrients of 5.5–7% that come from milk. It retains about 55% of total milk ingredients like lactose, soluble proteins, lipids, and mineral salts [7]. Whey is a byproduct of high energetic and nutritional quality. For human being, it serves as an important

Some statistical studies indicate that a significant portion of this waste is discarded to tributaries, resulting in an environmental problem due to its high biochemical oxygen demand. It physically and chemically affects the soil structure, decreasing the yield of agricultural crops,

For the reasons explained above, dairy industry has been looking for alternatives for the use of this by-product, which is a high pollutant; however, it has a great nutritional value. Among the products of successful acceptance are fermented dairy drinks, refreshing beverages [8], protein

The processing of whey for beverages production began in the 1960s, and Rivella was the first fermented drink prepared from whey, made in Switzerland [11]. Whey products improve texture, reduce flavor and color, emulsify, stabilize, improve flow properties, and show many

The main objective of this chapter is to carry out a complete review about fermented beverages based on whey inoculated with probiotics micro-organisms that have been produced around the world over the last years, focusing specially in important aspects such as sensorial and microbiological quality, shelf-life, and probiotic effects, showing that probiotics consumption can be increased through the use of whey as a substrate in this type of formulation, promoting it as a useful dairy by-product due to its excellent sensorial characteristics and its contribution

lent substrate to be used in the development of fermented beverages.

source of vitamins, proteins, and carbohydrates.

concentrates [9], infant formulas [10], and others.

in high quality organoleptic foods.

and polluting water because it depletes dissolved oxygen [7].

other functional properties that increase the quality of the products [12].

intestinal microbial balance.

116 Probiotics - Current Knowledge and Future Prospects

The knowledge of whey physicochemical characterization is an important step in the use of this by-product in the dairy industry for different industrial processes. For this reason, most of the studies related to the use of whey propose a physico-chemical characterization in order to evaluate whether it meets the standards required to be used in technological processes.

In a very recent research, Molero et al. [13], carried out a physico-chemical characterization of whey obtained by cheese making process applying an artisanal method. It consisted of the determination of pH, titrated acidity, total solids, fat, protein according to the Venezuelan Standard COVENIN, and determination of lactose and minerals by analytical difference. The values obtained were statistically analyzed using a statistical package. The results classify whey as sweet, with excellent nutritional characteristics and attractive to be used in food technology for probiotic production, protein-fermented beverages, among other applications.

In an interesting research, Tirado et al. [14] carried out a physico-chemical characterization of whey derived from the production of coastal cheese. Fat analyzes were performed by Gerber method, lactose by the Lane and Eynon method (AOAC 923.09, 920.183b); the protein was analyzed by the Kjedahl method (AOAC 920,152); total solids by spectrophotometry; pH was determined by the method established in AOAC 945.10/90 and the acidity expressed as a percentage of lactic acid according to the Colombian Technical Standard. The values obtained were: fat 0%, lactose 3.69%, protein 2.29%, total solids 6.28%, acidity 0.08% lactic acid, and pH 6.5.

Linares et al. [15] showed similar results in the physico-chemical characterization of sweet whey samples, obtaining a pH of 6.84; acidity titrated of 0.11% (% lactic acid); protein between 0.6 and 1%; and ash 0.6%. De Paula et al. [16] performed a physico-chemical characterization of whey obtained from the manufacture of coastal cheese. This characterization was carried out using the following methods: acidity (AOAC 947.05/90), pH (AOAC 981.12/90), soluble solids (AOAC 932.12/90), total solids (AOAC 925,105/90), and lactose (FIL 28a/74). The following results were obtained: acidity (% lactic acid) 0.11; pH 6.58; total solids 6.83%, protein 0.98%; fat 0.4%; and lactose 4.54%.

Similarly, Montero et al. [17] carried out a whey fermentation with Lactobacillus (L) for feeding calves in the tropics. Researchers performed a physico-chemical characterization, determining pH, total protein, fat and total solids, tested by the Standard Method for examination of Dairy Products, 2004. The values obtained for these characteristics were in accordance with the Colombian standards established for this type of analysis.

In other studies, Londoño et al. [18] developed a fermented drink of fresh cheese whey inoculated with L. casei. Acidity, pH, viscosity, total solids, protein, fat, ash, lactose, minerals, soluble solids, reducing sugars, and moisture were determined for this purpose. Acidity, pH, total solids, protein, fat, ash, soluble solids, were determined using AOAC methods; lactose was determined using the Teles reagent method; reducing sugars by the method of 3-amino-5 nirosalicylic acid; mineral content was determined by spectrophotometric method of atomic absorption and viscosity by the Brookfield method. The values obtained were found within the established standards for these parameters.

The scientific interest in bacteria as protective agents against different diseases comes from the observation of Metchnikoff, who at the beginning of the twentieth century, emphasized the longevity and good health of the Bulgarian peasants, who consumed large quantities of

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The observations of multiple scientists such as Trapp et al. [30] assumed that consumption of large quantities of foods rich in lactic acid bacteria, eliminated toxin-forming bacteria, while raising the proportion of lactic acid bacteria and intestinal flora, improved health and increased life expectancy. Since then, and throughout almost a hundred years of study, various authors have endeavored to know different functions of beneficial micro-organisms that populate the

Lilly et al. [2] used the term "probiotics" to describe those substances secreted by an organism that stimulates the growth of another, as opposed to the term "antibiotic," understood as any chemical compound used to eliminate or inhibit the growth of infectious organisms. Parker [31] was the first to use "probiotic" referring to organisms and substances that contribute to

The definition of probiotics has evolved remarkably, so that today, they are defined as viable, microbial selected dietary supplements that, when they are introduced in sufficient amounts, affect the human organism through their effects on the intestinal tract [3]. Probiotics must meet some basic requirements to be selected in the development of commercial probiotic products. The most important requirements are: the probiotic micro-organism survives in the product, the physical and genetic stability during product storage is guaranteed, and all its essential properties that evidence its health benefits after consumption, are maintained during manufacture and storage of the product [32]. Laws et al. [33] states that the essential criteria for

Many researches have been carried out with this class of micro-organisms, producing drinks of high microbiological and sensorial quality. Following the same idea, Molero et al. [34] formulated a probiotic fermented beverage based on whey, using a mixed culture of L. acidophilus and commercial yoghurt culture: Streptococcus (S) thermophilus and L. bulgaricus. Tirado et al. [14] produced a fermented whey milk drink using S. salivarius ssp. thermophilus and L. casei.

On the other hand, Linares et al. [15] evaluated the effect of different proportions of citrus pulp on the sensorial acceptability of a fermented and protein drink made from residual whey. For this purpose they used a lyophilized lactic culture of S. thermophilus, L. delbrueckii ssp. bulgaricus, and L. casei. Likewise, Martínez et al. [35] formulated a fermented cheese-whey drink adding maracuyá pulp. For this purpose, lactic ferments were used for direct inoculation: S. thermophilus, L. delbrueckii sub bulgaricus, and Lactococcus lactis sub lactis. Similarly, Vela et al. [36] developed a probiotic whey-based fermented beverage with mango pulp and almonds, using isolated colonies of L. casei. In another study, Fiorentini et al. [37] evaluated the influence of different combinations of probiotic bacteria and different fermentation temperatures on the physico-chemical characteristics of fermented lactic beverages based on soybean and whey. For this purpose, a lyophilized probiotic culture was used, composed of L. acidophilus and Bifidobacterium (B) bifidum. A second

primer selection include acidification, aroma, taste, stability, and texture.

culture was prepared with B. lactis and S. thermophilus.

yoghurt [29].

digestive tract.

intestinal balance.

On the other hand, Miranda et al. [19] carried out a physico-chemical characterization of sweet and acid whey produced in the cheese complex of Bayamo (Cuba). The authors determined acidity, pH, density, and fat content, following the guidelines of the Ministry of Agriculture of Cuba; lactose was determined by the phenol-sulfuric method; dry extract, crude protein, calcium and phosphorus were tested according to internationally recommended methods. The acid whey was distinguished by a lower pH and a higher acidity than the sweet. No significant differences were observed between the two varieties of whey for the remaining characteristics tested. All of them were within the specifications of quality established by the Cuban norm. Low acidity of whey benefits its quality, because it allows a better use for human and animal feeding. It is great important to know about dry extract in the evaluation of the quality of cheese whey as raw material, because it would indicate its water content: a greater amount of water makes whey has less nutritional value.

In this order of ideas, Sepúlveda et al. [20] developed a fermented beverage with the use of fresh whey with the addition of Maracuyá pulp. For this purpose, a physico-chemical characterization was performed, determining pH, viscosity, total solids, protein content, fat, and ash by AOAC methods; lactose was determined by the reactive method of Teles; calcium, sodium, and potassium were determined using the spectrophotometric method of atomic absorption. When comparing composite ranges obtained with data reported by Amiot et al. [21], Scott [22], Posati and Orr [23], and Morales et al. [24], similarities were observed with measures reported for total solids, lactose and protein; under these levels of composition a substrate for fermentation was guaranteed, influencing in the performance of beverage processing properly. On the other hand, fatty content in whey is directly linked to the cheese manufacturing conditions. The values obtained in this trial were considerably lower than that reported by Scott [22], who states that fat content for sweet whey ranges from 0.2 to 0.7%. The pH obtained was slightly higher than that reported by Spreer [25] for this type of whey.

From the studies explained above, it is a fact that whey's physico-chemical characteristics vary depending on the composition of the milk, the cheese making process and the type of cheese, which could determine the ultimate destination of this by-product.

#### 2.2. Microbial cultures used to produce fermented beverages based on whey

The cultures most likely used are lactic acid bacteria (LAB), which play an important role in fermentation processes. They are widely used in food industry because of their involvement in texture, taste, smell and aroma, and development of fermented foods [26].

LABs may be contained in a group of micro-organisms named lactic cultures or starters [27]. They are used in dairy industry for fermented milks production, cheeses, butter, and other products that are required to be fermented [28]. LABs were referred to as probiotics in the 1960s. The scientific interest in bacteria as protective agents against different diseases comes from the observation of Metchnikoff, who at the beginning of the twentieth century, emphasized the longevity and good health of the Bulgarian peasants, who consumed large quantities of yoghurt [29].

total solids, protein, fat, ash, soluble solids, were determined using AOAC methods; lactose was determined using the Teles reagent method; reducing sugars by the method of 3-amino-5 nirosalicylic acid; mineral content was determined by spectrophotometric method of atomic absorption and viscosity by the Brookfield method. The values obtained were found within the

On the other hand, Miranda et al. [19] carried out a physico-chemical characterization of sweet and acid whey produced in the cheese complex of Bayamo (Cuba). The authors determined acidity, pH, density, and fat content, following the guidelines of the Ministry of Agriculture of Cuba; lactose was determined by the phenol-sulfuric method; dry extract, crude protein, calcium and phosphorus were tested according to internationally recommended methods. The acid whey was distinguished by a lower pH and a higher acidity than the sweet. No significant differences were observed between the two varieties of whey for the remaining characteristics tested. All of them were within the specifications of quality established by the Cuban norm. Low acidity of whey benefits its quality, because it allows a better use for human and animal feeding. It is great important to know about dry extract in the evaluation of the quality of cheese whey as raw material, because it would indicate its water content: a greater

In this order of ideas, Sepúlveda et al. [20] developed a fermented beverage with the use of fresh whey with the addition of Maracuyá pulp. For this purpose, a physico-chemical characterization was performed, determining pH, viscosity, total solids, protein content, fat, and ash by AOAC methods; lactose was determined by the reactive method of Teles; calcium, sodium, and potassium were determined using the spectrophotometric method of atomic absorption. When comparing composite ranges obtained with data reported by Amiot et al. [21], Scott [22], Posati and Orr [23], and Morales et al. [24], similarities were observed with measures reported for total solids, lactose and protein; under these levels of composition a substrate for fermentation was guaranteed, influencing in the performance of beverage processing properly. On the other hand, fatty content in whey is directly linked to the cheese manufacturing conditions. The values obtained in this trial were considerably lower than that reported by Scott [22], who states that fat content for sweet whey ranges from 0.2 to 0.7%. The pH obtained was slightly

From the studies explained above, it is a fact that whey's physico-chemical characteristics vary depending on the composition of the milk, the cheese making process and the type of cheese,

The cultures most likely used are lactic acid bacteria (LAB), which play an important role in fermentation processes. They are widely used in food industry because of their involvement in

LABs may be contained in a group of micro-organisms named lactic cultures or starters [27]. They are used in dairy industry for fermented milks production, cheeses, butter, and other products that are required to be fermented [28]. LABs were referred to as probiotics in the 1960s.

established standards for these parameters.

118 Probiotics - Current Knowledge and Future Prospects

amount of water makes whey has less nutritional value.

higher than that reported by Spreer [25] for this type of whey.

which could determine the ultimate destination of this by-product.

2.2. Microbial cultures used to produce fermented beverages based on whey

texture, taste, smell and aroma, and development of fermented foods [26].

The observations of multiple scientists such as Trapp et al. [30] assumed that consumption of large quantities of foods rich in lactic acid bacteria, eliminated toxin-forming bacteria, while raising the proportion of lactic acid bacteria and intestinal flora, improved health and increased life expectancy. Since then, and throughout almost a hundred years of study, various authors have endeavored to know different functions of beneficial micro-organisms that populate the digestive tract.

Lilly et al. [2] used the term "probiotics" to describe those substances secreted by an organism that stimulates the growth of another, as opposed to the term "antibiotic," understood as any chemical compound used to eliminate or inhibit the growth of infectious organisms. Parker [31] was the first to use "probiotic" referring to organisms and substances that contribute to intestinal balance.

The definition of probiotics has evolved remarkably, so that today, they are defined as viable, microbial selected dietary supplements that, when they are introduced in sufficient amounts, affect the human organism through their effects on the intestinal tract [3]. Probiotics must meet some basic requirements to be selected in the development of commercial probiotic products. The most important requirements are: the probiotic micro-organism survives in the product, the physical and genetic stability during product storage is guaranteed, and all its essential properties that evidence its health benefits after consumption, are maintained during manufacture and storage of the product [32]. Laws et al. [33] states that the essential criteria for primer selection include acidification, aroma, taste, stability, and texture.

Many researches have been carried out with this class of micro-organisms, producing drinks of high microbiological and sensorial quality. Following the same idea, Molero et al. [34] formulated a probiotic fermented beverage based on whey, using a mixed culture of L. acidophilus and commercial yoghurt culture: Streptococcus (S) thermophilus and L. bulgaricus. Tirado et al. [14] produced a fermented whey milk drink using S. salivarius ssp. thermophilus and L. casei.

On the other hand, Linares et al. [15] evaluated the effect of different proportions of citrus pulp on the sensorial acceptability of a fermented and protein drink made from residual whey. For this purpose they used a lyophilized lactic culture of S. thermophilus, L. delbrueckii ssp. bulgaricus, and L. casei. Likewise, Martínez et al. [35] formulated a fermented cheese-whey drink adding maracuyá pulp. For this purpose, lactic ferments were used for direct inoculation: S. thermophilus, L. delbrueckii sub bulgaricus, and Lactococcus lactis sub lactis. Similarly, Vela et al. [36] developed a probiotic whey-based fermented beverage with mango pulp and almonds, using isolated colonies of L. casei.

In another study, Fiorentini et al. [37] evaluated the influence of different combinations of probiotic bacteria and different fermentation temperatures on the physico-chemical characteristics of fermented lactic beverages based on soybean and whey. For this purpose, a lyophilized probiotic culture was used, composed of L. acidophilus and Bifidobacterium (B) bifidum. A second culture was prepared with B. lactis and S. thermophilus.

In other studies, Pescuma et al. [38] developed fermented functional beverages based on whey, using lactic acid bacteria: strains of L. acidophilus, L. subsp. bulgaricus, and S. thermophilus. A similar approach was given by Legarová and Kousimska [39] who formulated a whey-based drink using the same starter culture.

from 3 to 35 mm in diameter, contains lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc), acetic acid bacteria, and yeast mixture, coupled with casein and sugar through a matrix of

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Yeasts, such as Kluyveromyces marxianus, have also been used as crops. This yeast has been isolated from fruit, cheese, yoghurt [53], milk [54], and has been used in whey processing. It has the ability to hydrolyze lactose and ferment sugars efficiently [55]. Cóndor et al. [56] prepared a beverage from cheese whey using immobilized Kluyveromyces Marxianus cells. Padín and Díaz [57] worked on the alcoholic fermentation of whey, using this yeast and organic solvents as extractants. Dragone et al. [58] performed the characterization of volatile components in an alcoholic beverage produced from whey with Kluyveromyces marxianus

2.3. Technological process followed for whey-based probiotic fermented beverages

Table 1 summarizes the review of whey-based fermented beverages. It tells the technological process used in drinks manufacture. It is interesting to observe how the use of probiotic microorganism plays an important role. The tendency is to use this type of bacteria and the reason is

In the development of these beverages, authors have used additives in order to improve some organoleptic characteristics, for example oligofructose, hydrocolloids, processed fruits, and others. Regarding technological process, fermentation is the essence of the drink production. However, it can be changes related to raw material (e.g., whey powder, liquid whey, combination with soymilk or whole milk), prior bacteria isolation, among others. Dairy industry has thus diversified methods for producing whey-based beverages. In the following sections, the acceptance of these drinks can be evidenced based on the sensorial evaluations and their

In the process of making dairy drinks, it is essential that they have adequate sensory properties to ensure they are accepted by consumers. Sensory quality researches have been increased over

In this order of ideas, Molero et al. [59] carried out a sensory evaluation of probiotic fermented beverages based on whey. Four treatments were developed using combinations of two stabilizers, carboxymethyl cellulose and unflavored gelatin and two starter cultures: L. acidophilus and a mixed culture with L. acidophilus and yoghurt micro-organisms. For these, a sensory evaluation in three phases was designed. In a first phase test, acceptance-rejection was applied to an untrained 30 people in order to select the essence of fruits that best suited to drinks. In second place, preference test was performed with three concentrations of sugar, applied to 30 people panel. In a third phase, smell, taste, overall acceptance, and consistency of the four treatments were evaluated using an acceptance degree test with 5-point hedonic scale to an untrained panel of 100 people. Fruit essence largely accepted by the panelists was the coconut.

polysaccharides called kefirán [52].

its potential benefit to human health.

probiotic character based on the viable count.

2.4. Fermented whey-based beverage sensorial quality

ATCC22.

production

the recent years.

Katechaki et al. [40] performed a research related to thermal drying of L. delbrueckii subsp. bulgaricus and its efficient use as starter culture in whey fermentation and in cheese process. This micro-organism is a thermophilic LAB, able to ferment lactose, glucose, and fructose [12]. It was isolated from Bulgarian yoghurt, from Germany. Montero et al. [17] used L. casei to ferment whey to feed calves, the culture contained 1 <sup>10</sup><sup>7</sup> CFU/ml. De Castro et al. [41] evaluated the effect of the incorporation of oligofructose on the properties of fermented probiotic lactic beverages using a probiotic culture composed of L. acidophilus, B., and S. thermophilus. Oligofructose is an oligosaccharide, obtained from the enzymatic hydrolysis of insulin [42]. It is a prebiotic whose use can bring functional benefits and can affect the sensory properties of the products significantly [43].

Similarly, Londoño et al. [18] worked on a fresh cheese fermented drink formulation, inoculated with L. casei as a probiotic culture. Other cultures used were L. delbrueckii subsp. bulgaricus and S. salivarius subsp. thermophilus.

In another research, Gallardo et al. [44] evaluated taste and sensation in the mouth of beverages made from whey with addition of hydrocolloids. The functionality of hydrocolloids at low concentrations is that it enhances viscosity and prevents particles sedimentation. It also contributes to the microstructural properties of meals, based on its ability to confer structure to the continuous phase of the substrate, which depends on their solubility in water and/or their intermolecular associations [45]. Beverages were prepared with a commercial yoghurt starter culture, which consisted of L. delbrueckii ssp. bulgaricus and S. thermophilus.

Hernández et al. [46] worked on the preparation of a probiotic drink based on whey, using cultures of L. reuteri and B. bifidum. The first was preserved on LBS (Lactobacillus) agar at 4C. Three subcultures were performed consecutively prior to the use of the strain in the experiment with 1% inoculum. The second was preserved in MRS medium. Two subcultures were performed before use the strain in the experiment with 1% of inoculum.

Following the same idea, Dalev et al. [47] evaluated the sensory quality of whey-based probiotic beverages. A probiotic culture was prepared with strains of B. breve ATCC 15700, B. infantis ATCC 15697, B. animalis/lactis J38, L. plantarum W42, L. plantarum IB, L. casei Lc and S. thermophilus.

Sepulveda et al. [20] prepared a fermented beverage with the use of fresh whey with the addition of Maracuyá pulp, using a traditional lactic acid culture, in a 1:1 ratio of S. thermophilus and L. bulgaricus. Oliveira et al. [48] developed a fermented lactic drink using four probiotic cultures using S. thermophilus and L. delbrueckii ssp. bulgaricus; L. acidophilus and L. rhamnosus.

Kéfir has also been used as a starter culture in the production of beverages from whey [49–51]. Kefir is made by inoculating milk with kefir grains. This grain is irregular and its size varies from 3 to 35 mm in diameter, contains lactic acid bacteria (Lactobacillus, Lactococcus, Leuconostoc), acetic acid bacteria, and yeast mixture, coupled with casein and sugar through a matrix of polysaccharides called kefirán [52].

Yeasts, such as Kluyveromyces marxianus, have also been used as crops. This yeast has been isolated from fruit, cheese, yoghurt [53], milk [54], and has been used in whey processing. It has the ability to hydrolyze lactose and ferment sugars efficiently [55]. Cóndor et al. [56] prepared a beverage from cheese whey using immobilized Kluyveromyces Marxianus cells. Padín and Díaz [57] worked on the alcoholic fermentation of whey, using this yeast and organic solvents as extractants. Dragone et al. [58] performed the characterization of volatile components in an alcoholic beverage produced from whey with Kluyveromyces marxianus ATCC22.

#### 2.3. Technological process followed for whey-based probiotic fermented beverages production

Table 1 summarizes the review of whey-based fermented beverages. It tells the technological process used in drinks manufacture. It is interesting to observe how the use of probiotic microorganism plays an important role. The tendency is to use this type of bacteria and the reason is its potential benefit to human health.

In the development of these beverages, authors have used additives in order to improve some organoleptic characteristics, for example oligofructose, hydrocolloids, processed fruits, and others. Regarding technological process, fermentation is the essence of the drink production. However, it can be changes related to raw material (e.g., whey powder, liquid whey, combination with soymilk or whole milk), prior bacteria isolation, among others. Dairy industry has thus diversified methods for producing whey-based beverages. In the following sections, the acceptance of these drinks can be evidenced based on the sensorial evaluations and their probiotic character based on the viable count.

#### 2.4. Fermented whey-based beverage sensorial quality

In other studies, Pescuma et al. [38] developed fermented functional beverages based on whey, using lactic acid bacteria: strains of L. acidophilus, L. subsp. bulgaricus, and S. thermophilus. A similar approach was given by Legarová and Kousimska [39] who formulated a whey-based

Katechaki et al. [40] performed a research related to thermal drying of L. delbrueckii subsp. bulgaricus and its efficient use as starter culture in whey fermentation and in cheese process. This micro-organism is a thermophilic LAB, able to ferment lactose, glucose, and fructose [12]. It was isolated from Bulgarian yoghurt, from Germany. Montero et al. [17] used L. casei to ferment whey to feed calves, the culture contained 1 <sup>10</sup><sup>7</sup> CFU/ml. De Castro et al. [41] evaluated the effect of the incorporation of oligofructose on the properties of fermented probiotic lactic beverages using a probiotic culture composed of L. acidophilus, B., and S. thermophilus. Oligofructose is an oligosaccharide, obtained from the enzymatic hydrolysis of insulin [42]. It is a prebiotic whose use can bring functional benefits and can affect the sensory properties of the

Similarly, Londoño et al. [18] worked on a fresh cheese fermented drink formulation, inoculated with L. casei as a probiotic culture. Other cultures used were L. delbrueckii subsp. bulgaricus and

In another research, Gallardo et al. [44] evaluated taste and sensation in the mouth of beverages made from whey with addition of hydrocolloids. The functionality of hydrocolloids at low concentrations is that it enhances viscosity and prevents particles sedimentation. It also contributes to the microstructural properties of meals, based on its ability to confer structure to the continuous phase of the substrate, which depends on their solubility in water and/or their intermolecular associations [45]. Beverages were prepared with a commercial yoghurt starter

Hernández et al. [46] worked on the preparation of a probiotic drink based on whey, using cultures of L. reuteri and B. bifidum. The first was preserved on LBS (Lactobacillus) agar at 4C. Three subcultures were performed consecutively prior to the use of the strain in the experiment with 1% inoculum. The second was preserved in MRS medium. Two subcultures were

Following the same idea, Dalev et al. [47] evaluated the sensory quality of whey-based probiotic beverages. A probiotic culture was prepared with strains of B. breve ATCC 15700, B. infantis ATCC 15697, B. animalis/lactis J38, L. plantarum W42, L. plantarum IB, L. casei Lc and

Sepulveda et al. [20] prepared a fermented beverage with the use of fresh whey with the addition of Maracuyá pulp, using a traditional lactic acid culture, in a 1:1 ratio of S. thermophilus and L. bulgaricus. Oliveira et al. [48] developed a fermented lactic drink using four probiotic cultures using S. thermophilus and L. delbrueckii ssp. bulgaricus; L. acidophilus and

Kéfir has also been used as a starter culture in the production of beverages from whey [49–51]. Kefir is made by inoculating milk with kefir grains. This grain is irregular and its size varies

culture, which consisted of L. delbrueckii ssp. bulgaricus and S. thermophilus.

performed before use the strain in the experiment with 1% of inoculum.

drink using the same starter culture.

120 Probiotics - Current Knowledge and Future Prospects

products significantly [43].

S. salivarius subsp. thermophilus.

S. thermophilus.

L. rhamnosus.

In the process of making dairy drinks, it is essential that they have adequate sensory properties to ensure they are accepted by consumers. Sensory quality researches have been increased over the recent years.

In this order of ideas, Molero et al. [59] carried out a sensory evaluation of probiotic fermented beverages based on whey. Four treatments were developed using combinations of two stabilizers, carboxymethyl cellulose and unflavored gelatin and two starter cultures: L. acidophilus and a mixed culture with L. acidophilus and yoghurt micro-organisms. For these, a sensory evaluation in three phases was designed. In a first phase test, acceptance-rejection was applied to an untrained 30 people in order to select the essence of fruits that best suited to drinks. In second place, preference test was performed with three concentrations of sugar, applied to 30 people panel. In a third phase, smell, taste, overall acceptance, and consistency of the four treatments were evaluated using an acceptance degree test with 5-point hedonic scale to an untrained panel of 100 people. Fruit essence largely accepted by the panelists was the coconut.


Author Micro-organism Special additives Fermentative process

Kluyveromyces marxianus Organic solvents Reconstituted whey powder at 20% w/

Probiotics Consumption Increment through the Use of Whey-Based Fermented Beverages

L. casei 100 ml of culture was taken to inoculate

Inverted sugar syrup; maracuyá pulp; carboxymethyl cellulose

L. reuteri y Bifidobacterium bifidum. Reconstituted whey (7%) with addition

(CMC)

Incubation a 37C for 24 h

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w. 100 ml of reconstituted whey 10% v/ v were inoculated with a Kluyveromyces marxianus culture, incubated at 30C. After 8 h 100 ml of selected solvents were added (oleic acid and soybean oil) in a ratio of 1:1 by maintaining them under the same experimental conditions for 30 h

900 ml of whey in a beaker and incubated for 24 h at 39C. Fermented whey was poured into 9 l of fresh whey and fermented 24 h at 39C in a convection oven. The 10 l of fermented whey were poured into 90 l of fresh whey and rested 24 h at room

sucrose was heat treated at 95C for 5 min while liquid whey with

oligofructose was heated to 65C for 30 min. The temperature of the mixture was lowered to 40C. The beverage was made with the addition of the lyophilized culture at 8.3 mg/100 ml. Fermentation occurred at 40C. pH of 4.6 was monitored. The beverage was

maintaining a pH of 5.8 and stirring for 3–5 min. Subsequently, the beverage was flavored with the addition of maracuya pulp, packed and stored at

of 7% sucrose and 0.4% pectin. Three treatments were applied by inoculation of probiotic strains in different ratios. Incubation at 37C and storage at 4C

inoculation (2% v/v) with the starter culture at 42C until reaching a pH of

Fermentation at 37C for 24 h until reaching a pH of 4.4–4.6. Drinks were cooled and supplemented with

temperature

then cooled to 4C

Inoculation was performed

Oligofructose Pasteurized milk with commercial

4C

for 30 days

Hydrocolloids Fermentation was carried out by

4.6

Soy milk Equal amounts of whey and soy milk.

processed fruits

Pescuma et al. [38]

Padín and Díaz [57]

Montero et al. [17]

De Castro et al. [41]

Londoño et al. [18]

Hernández et al. [46]

Gallardo et al. [44]

Dalev et al. [47]

L. acidophilus, L. bulgaricus y Streptococcus thermophilus

L. acidophilus, Bifidobacterium y Streptococcus salivarius thermophilus

L. casei. Other cultures: L. delbrueckii bulgaricus and Streptococcus salivarius

Commercial yoghurt starter culture: L. delbrueckii bulgaricus and Streptococcus thermophilus

Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis/ lactis, L. plantarum, L. casei, and Streptococcus thermophilus

thermophilus


Author Micro-organism Special additives Fermentative process

Whey was pasteurized at 65C for 20 min and cooled to 38C. Inoculation of micro-organisms. Fermentation was carried out controlling pH until 4.5

Mixture of whey, skim milk powder and sugar was pasteurized at 63C for 30 min and cooling to 43C. Inoculation of micro-organisms in equal proportions. Fermentation was carried out for 3 h at 40C controlling pH up to 5.

previously pasteurized at 70C for 30 min and fermented at 42C for 5 h. White sugar was added in order to standardize at 14 Brix. The beverage obtained was pasteurized at 80C for 15 s and packed at the same

Five different treatments with different Maracuya pulp percentages (5; 7,5; 10; 12 y 15%) and final value of 14

150 ml of pasteurized whey was taken and inoculated with Lactobacillus casei. They were incubated at 35C for 48 h. pH was measured. Fully mature pulp was added in a 1:1 ratio. 12.5% (w/v) of previously crushed almonds were

water and inoculated in 250 ml of whey, with a temperature of 25C for

Whey powder was dissolved in water in a 1:1 ratio right before use. Mixture of 40% whole milk, 30% whey of mozzarella cheese, 30% water-soluble extract of soybean and 10% of sugar. Heat treatment at 90C for 5 min, cooling to fermentation temperature (37C). Incubation until reaching a pH between 4.5 and 5. Cooling at 20C, homogenized, distributed in plastic bottles and stored at 7C for 21 days

Incubation at 43C and cooling at 4C.

Drying was carried out in convection ovens at 35, 45, and 55C for 10 h. After drying remaining moisture was removed with further drying at 102C. Fermentation was performed at 37C

Brix

Citrus fruit pulps Fruit juice mixed with whey,

Pasteurized maracuya

indica var. Tommy atkin) and almonds (Amygdalus

Kéfir grains Kefir grains washed with distilled

Water-soluble soybean

subsp. Bulgaricus drying

pulp

communis)

extract

Lactobacillus casei Mango pulp (Magnifera

temperature

added

72 h

for 3 days

Molero et al. [13, 34]

Linares et al. [15]

Martínez et al. [35]

Vela et al. [36]

Teixeira et al. [51]

Fiorentini et al. [37]

Legarová et al. [39]

Katechaki et al. [40]

Lactobacillus acidophilus and commercial yoghurt culture

122 Probiotics - Current Knowledge and Future Prospects

Lactic culture: Streptococcus thermophilus, Lactobacillus delbrueckii sub. bulgaricus, and Lactobacillus casei

Streptococcus thermophilus, Lactobacillus delbrueckii sub. bulgaricus y Lactococcus lactis

Probiotic freeze-dried cultured of L. acidophilus y Bifidobacterium bifidum

Commercial yoghurt starter culture:

L. delbrueckii bulgaricus Lactobacillus delbrueckii

L. delbrueckii bulgaricus y Streptococcus thermophilus

thermophilus y Lactobacillus casei ssp.

Tirado [14] Streptococcus salivarius ssp.

casei


with a group of 20 untrained judges. The scale used was structured with scores from 6 to 10; being 6 "I dislike much," 7 "I do not like," 8 "I do not like or dislike me," 9 "I like," and 10 "I

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Legarová et al. [39] evaluated whey-based fermented beverages using the commercial yoghurt culture of S. thermophilus and L. bulgaricus to determine the effect of milk content on sensorial properties. They evaluated cold samples (4–6C) using a panel of 15 people, an unstructured linear scale was designed. The samples prepared with whey and milk obtained the highest scores in all the descriptors, evidencing that a higher percentage of milk in the mixture, higher

Similarly, De Castro et al. [41] analyzed the sensorial acceptance of whey based fermented beverages with different concentrations of oligofructose (2 and 5%). They evaluated the samples after 72 h of preparation, using: (a) an untrained panel of 36 people, who were asked which drink they liked the most or did not like; and (b) an untrained panel of 50 people in one test of acceptability, using a structured hedonistic scale of 9 points (1—I do not like anything, 9 —I like it very much) and a test of intention to buy using a scale of 5 points (1—definitely not going to buy it; 5—I will definitely buy it). Panelists preferred 2 and 5% oligofructose beverages compared to the control drink (without oligofructose), where acidity was an attribute mentioned by 41% of judges. Furthermore, in terms of acceptability, the average score for both drinks was over 7 points, evidencing that the variation in the oligofructose content added did

On the other hand, Londoño et al. [18] carried out a sensorial evaluation of a fermented beverage made from fresh whey, using L. casei and commercial yoghurt culture of L. bulgaricus and S. thermophilus. They performed a drink acceptability test, using a panel of 80 people, considering a 9 points hedonic scale. The drink had a "like" rating. Pescuma et al. [38] evaluated the noble properties of whey-based functional beverages using LABs, which were made using commercial yoghurt culture of L. bulgaricus and S. thermophilus and a probiotic culture of L. acidophilus. They

Gallardo et al. [44] used a highly trained panel in the sensory evaluation of a whey-based fermented beverage with and without addition of hydrocolloids, with commercial yoghurt as an inoculum (0.02%).They performed the sensory evaluation under controlled conditions, following the ISO Standard 8589 (1988), using samples of 30 mL, in triplicate, on separate days. They used a 100 mm unstructured linear scale to measure the sensation of panelists leaving the sample for a maximum of 3 s before swallowing. According to the results, the addition of hydrocolloids affected the perception of the viscosity of fermented beverages

In other studies, Hernández et al. [46] performed the sensory analysis on a reconstituted wheybased probiotic drink (7%) and pasteurized (80C, 3 min) using three combinations (treatments) of L. reuteri and B. bifidum as inoculum T1: L. reuteri 1% and B. bifidum 0.5%, T2: L. reuteri 1% and B. bifidum 1%, T3: L. reuteri 2% and B. bifidum 0.5%. They used the triangle test and a panel of 10 people (8 women and 2 men) between 22 and 27 years old. They performed 12 tests per session, determining differences between treatments using a "d" value defined as

performed an acceptability test of the fermented drink, obtaining a grade of "I like."

substantially, evidencing the lack of a greasy sensation in the mouth.

like it a lot." The drink got a "I like" rating.

indexes of organoleptic quality in the drink.

not affect acceptance of the beverages.

Table 1 Whey-based fermented beverage technological process review

The preferred concentration of sugar by the panelists was 6%. The best evaluated treatments for consistency and general acceptance were those who had carboxymethyl cellulose as a stabilizer. The best drink evaluated in reference to the taste was the one containing carboxymethyl cellulose and L. acidophilus. The four drinks were equally qualified in relation to the smell.

Similarly, Valencia et al. [60] carried out a sensorial evaluation to nutritional drinks based on pumpkin and whey, enriched with oats and passion fruit. They evaluated 12 beverage formulations considering color, aroma, taste, and acceptability. They performed the test with a panel of 26 school-aged children; each child received three 100 mL samples. They used a questionnaire with expression faces of pleasure or displeasure, corresponding with a hedonic scale of 1–5, being 1 the lowest score and 5, the highest score. They found significant differences between the results obtained for the samples analyzed, but all of them were very well accepted by the panelists.

De Paula et al. [16] performed a sensory evaluation of fermented beverage fermented from whey with and without Maracuyá pulp. They performed an order-preference test, using a panel of 59 consumer catheters, using note 1 for the most preferred and 5 for the least preferred. They coded the samples and presented them randomly in 50 mL beakers, finding that the combination of whey with passion fruit flavor was the most preferred beverage. In addition, they indicated that the panelists described the product as very good, novel, and interesting.

In another research, Vela et al. [36] evaluated a probiotic beverage based on whey with addition of Mango and Almond pulp, in a ratio of 1:1, using L. casei as a probiotic micro-organism. They applied a hedonic scale preference test to assess the level of impact on smell, taste, and texture with a group of 20 untrained judges. The scale used was structured with scores from 6 to 10; being 6 "I dislike much," 7 "I do not like," 8 "I do not like or dislike me," 9 "I like," and 10 "I like it a lot." The drink got a "I like" rating.

Legarová et al. [39] evaluated whey-based fermented beverages using the commercial yoghurt culture of S. thermophilus and L. bulgaricus to determine the effect of milk content on sensorial properties. They evaluated cold samples (4–6C) using a panel of 15 people, an unstructured linear scale was designed. The samples prepared with whey and milk obtained the highest scores in all the descriptors, evidencing that a higher percentage of milk in the mixture, higher indexes of organoleptic quality in the drink.

Similarly, De Castro et al. [41] analyzed the sensorial acceptance of whey based fermented beverages with different concentrations of oligofructose (2 and 5%). They evaluated the samples after 72 h of preparation, using: (a) an untrained panel of 36 people, who were asked which drink they liked the most or did not like; and (b) an untrained panel of 50 people in one test of acceptability, using a structured hedonistic scale of 9 points (1—I do not like anything, 9 —I like it very much) and a test of intention to buy using a scale of 5 points (1—definitely not going to buy it; 5—I will definitely buy it). Panelists preferred 2 and 5% oligofructose beverages compared to the control drink (without oligofructose), where acidity was an attribute mentioned by 41% of judges. Furthermore, in terms of acceptability, the average score for both drinks was over 7 points, evidencing that the variation in the oligofructose content added did not affect acceptance of the beverages.

On the other hand, Londoño et al. [18] carried out a sensorial evaluation of a fermented beverage made from fresh whey, using L. casei and commercial yoghurt culture of L. bulgaricus and S. thermophilus. They performed a drink acceptability test, using a panel of 80 people, considering a 9 points hedonic scale. The drink had a "like" rating. Pescuma et al. [38] evaluated the noble properties of whey-based functional beverages using LABs, which were made using commercial yoghurt culture of L. bulgaricus and S. thermophilus and a probiotic culture of L. acidophilus. They performed an acceptability test of the fermented drink, obtaining a grade of "I like."

The preferred concentration of sugar by the panelists was 6%. The best evaluated treatments for consistency and general acceptance were those who had carboxymethyl cellulose as a stabilizer. The best drink evaluated in reference to the taste was the one containing carboxymethyl cellulose and L. acidophilus. The four drinks were equally qualified in relation

Maracuyá pulp (Passiflora edulis) and Carboxymethyl

Kluyveromyces marxianus Fermentation at 30C for 7 days,

cellulose

Author Micro-organism Special additives Fermentative process

Complete and skimmed pasteurized milk mixed with skim milk powder to obtain 130 g/l of total solids and 26 g/l of fat, supplemented with 20 g/l of casein hydrolyzate. Heat treatment at 90C for 10 min cooled to 4C and stored 24 h prior to use. Fermentation with incubation at 42C until reaching

It was incubated 2 or 3 h, maintaining the temperature until reaching a pH of 4.6. Fermentation was stopped with a fast cooling of 4C. Agitation to ensure that maracuya pulp and vitamin dosage were well incorporated into the

evaluating the fermentation kinetics

Brix and pH

with the reading of <sup>o</sup>

a pH of 4.3

mixture

Probiotics cultures: yoghurt culture of Streptococcus thermophilus y L. delbrueckii bulgaricus; L. rhamnosus

124 Probiotics - Current Knowledge and Future Prospects

Streptococcus thermophilus y

Table 1 Whey-based fermented beverage technological process review

L. bulgaricus.

Similarly, Valencia et al. [60] carried out a sensorial evaluation to nutritional drinks based on pumpkin and whey, enriched with oats and passion fruit. They evaluated 12 beverage formulations considering color, aroma, taste, and acceptability. They performed the test with a panel of 26 school-aged children; each child received three 100 mL samples. They used a questionnaire with expression faces of pleasure or displeasure, corresponding with a hedonic scale of 1–5, being 1 the lowest score and 5, the highest score. They found significant differences between the results obtained for the samples analyzed, but all of them were very well accepted

De Paula et al. [16] performed a sensory evaluation of fermented beverage fermented from whey with and without Maracuyá pulp. They performed an order-preference test, using a panel of 59 consumer catheters, using note 1 for the most preferred and 5 for the least preferred. They coded the samples and presented them randomly in 50 mL beakers, finding that the combination of whey with passion fruit flavor was the most preferred beverage. In addition, they indicated that the panelists described the product as very good, novel, and interesting.

In another research, Vela et al. [36] evaluated a probiotic beverage based on whey with addition of Mango and Almond pulp, in a ratio of 1:1, using L. casei as a probiotic micro-organism. They applied a hedonic scale preference test to assess the level of impact on smell, taste, and texture

to the smell.

Oliveira et al. [48]

Sepulveda et al. [20]

Cóndor et al. [56]

by the panelists.

Gallardo et al. [44] used a highly trained panel in the sensory evaluation of a whey-based fermented beverage with and without addition of hydrocolloids, with commercial yoghurt as an inoculum (0.02%).They performed the sensory evaluation under controlled conditions, following the ISO Standard 8589 (1988), using samples of 30 mL, in triplicate, on separate days. They used a 100 mm unstructured linear scale to measure the sensation of panelists leaving the sample for a maximum of 3 s before swallowing. According to the results, the addition of hydrocolloids affected the perception of the viscosity of fermented beverages substantially, evidencing the lack of a greasy sensation in the mouth.

In other studies, Hernández et al. [46] performed the sensory analysis on a reconstituted wheybased probiotic drink (7%) and pasteurized (80C, 3 min) using three combinations (treatments) of L. reuteri and B. bifidum as inoculum T1: L. reuteri 1% and B. bifidum 0.5%, T2: L. reuteri 1% and B. bifidum 1%, T3: L. reuteri 2% and B. bifidum 0.5%. They used the triangle test and a panel of 10 people (8 women and 2 men) between 22 and 27 years old. They performed 12 tests per session, determining differences between treatments using a "d" value defined as the difference between intensities for two products valued in standard deviation. The triangle test results showed sensorial differences between treatments T1 and T2; however, T3 was different from the first two. To confirm this difference, they performed a test in which 109 consumers participated, finding that 56% expressed differences by T3, 34.86% by T2 and 9.17% showed no preference, selecting T3 as final product, which after a descriptive test, was cataloged with "very good" organoleptic characteristics.

MRS and M17, both at acidic pH (2.4) and neutral pH (7.2), finding that shelf-life of the product was 21 days. In addition, they did not observe marked variability in the results of the

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Similarly, Hernández et al. [46] performed a viable micro-organisms count to a beverage made from whey inoculated with L. reuteri 2% and Bifidobacterium bifidum 0.5%. Count was during storage at 4C for 30 days. They counted L. reuteri on a modified medium of LBS agar, B. bifidum on MRS agar and total population of viable micro-organisms in MRS medium (pH = 5.5), finding

In other research, Sepúlveda et al. [20] carried out an evaluation of the physicochemical characteristics during the storage of a beverage prepared with whey with addition of Maracuyá pulp. As the days went by, a decrease in pH and an increase in acidity were observed, suggesting a shelf-life of no more than 21 days. Oliveira et al. [48] performed a microbiological analysis of four whey-based drinks inoculated with probiotic cultures at 1, 7, 14, 21, and 28 days of storage at 4C. They used MRS-bile agar for counting L. acidophilus, MRS agar for L. delbrueckii ssp. bulgaricus and L. rhamnosus and agar for S. thermophilus. Although the probiotic count decreased during storage (28 days), they found that the beverages contained, on average, 5.3 <sup>10</sup><sup>6</sup> CFU/mL of probiotics after 28 days of storage. In addition, they observed that, on average, pH remained at 4.5 after the first day of storage, showing a decrease between 0.14 and 0.32 units during the first week, decreasing slightly (less than 0.12

Cóndor et al. [56] performed a microbiological evaluation of a whey-based beverage using immobilized Kluyveromyces Marxianus cells, during storage at room temperature and under refrigeration. They counted viable aerobic mesophiles, numbering of total coliforms and count of molds and yeasts, according to Peruvian Technical Standard 202.083 (1988). After storage viable aerobic mesophilic micro-organisms count and molds and yeasts (CFU/ml) were less than 10, while total coliforms (NMP/ml) were less than 3.0, indicating that the beverage was microbiologically acceptable. They also observed acidity and pH profile up to the fifth month

Whey has been used for probiotic fermented beverages development significantly. It has excellent physico-chemical characteristics that make it become an excellent substrate, allowing probiotic bacteria growth in such a way that they reach high concentrations, achieving the probiotic effect and all of its health benefits. On the other hand, whey-based probiotic beverages have extraordinary organoleptic characteristic and they are widely accepted by consumers. An average shelf-life of these beverages is 21 days under refrigeration conditions, ensuring probiotics benefits during this period of time. Throughout this review, it is shown that probiotic consumption can be increased through the use of whey not only due to its excellent physico-chemical characteristics, but also due to its ability to develop beverages with

of evaluation, both at room temperature (22C) and under refrigeration (4C).

high sensorial characteristics and due to its excellent acceptance.

that the beverage fulfilled the criterion of probiotic foods in an acceptable manner.

pH units) up to 28 days of storage, considering it stable.

physico-chemical tests.

3. Conclusions

Following the same idea, Dalev et al. [47] conducted a qualitative sensory analysis of fermented whey and soy milk beverages to five probiotic beverages based on whey and soy milk: (1) unfermented soy milk (control drink), (2) fermented drink with equal volume of whey and soy milk. Starter culture: B. breve and L. casei, (3) fermented drink with equal volume of whey and soy milk. Starter culture: B. infantis and S. thermophilus, (4) fermented drink with equal volume of whey and soy milk. Starter culture: B. animalis and L. plantarum, and (5) fermented drink with equal volume of whey and soy milk. Starter culture: B. breve, L. plantarum, and S. thermophilus.

They performed the sensory evaluation using the Quantitative Description Analysis (QDA) method [61]. They selected descriptors or attributes to be evaluated and used a 10 cm unstructured linear scale, shown in monitors, converting the results on a numerical scale (from 0 to 10 units) expressing them in conventional units. They employed a panel of six people (four women and two men) trained according to the International Standards (ISO 1993), with at least 1 year of experience in descriptive tests of different foods, who received varied samples of 20 mL, each in triplicate. The results showed highly significant differences in attributes such as soy milk odor, cereal odor, fermented taste, strawberry odor, sweet taste, and after taste. They concluded that the addition of processed fruits helps to improve the characteristics of the beverages substantially, and therefore, the qualification as organoleptic quality. Soy milk has been used to prepare products such as yoghurt, but its poor organoleptic characteristics have been responsible for a very low acceptance by consumers.

#### 2.5. Whey-based fermented beverages shelf-life

It is a fact that there are physico-chemical factors that can influence micro-organism survival in fermented beverages, being the most important acidity, temperature, oxygen concentration, type of inoculum and storage conditions. Theoretically, it is expected that for a reasonable time, the product will maintain the characteristics that define it as probiotic, so that quality can be guaranteed to the consumer. For this reason, many researches have evaluated the beverages shelf-life.

In this order of ideas, Fiorentini et al. [37] performed a viable lactic bacteria count in fermented beverages prepared from whey and soybean addition, after 7, 14, and 21 days storage under refrigeration at 7C. They performed a selective count of L. acidophilus on modified MRS agar, with aerobic incubation and a count of B. bifidum on MRS agar modified with anaerobic incubation, both at 37C for 72 h, finding that the number of alive micro-organisms in the fermented drink was 10<sup>7</sup> UFC/ml. Londoño et al. [18] evaluated the viability of the beverage based on fresh whey inoculated with L. casei. They performed counts on two types of agar, MRS and M17, both at acidic pH (2.4) and neutral pH (7.2), finding that shelf-life of the product was 21 days. In addition, they did not observe marked variability in the results of the physico-chemical tests.

Similarly, Hernández et al. [46] performed a viable micro-organisms count to a beverage made from whey inoculated with L. reuteri 2% and Bifidobacterium bifidum 0.5%. Count was during storage at 4C for 30 days. They counted L. reuteri on a modified medium of LBS agar, B. bifidum on MRS agar and total population of viable micro-organisms in MRS medium (pH = 5.5), finding that the beverage fulfilled the criterion of probiotic foods in an acceptable manner.

In other research, Sepúlveda et al. [20] carried out an evaluation of the physicochemical characteristics during the storage of a beverage prepared with whey with addition of Maracuyá pulp. As the days went by, a decrease in pH and an increase in acidity were observed, suggesting a shelf-life of no more than 21 days. Oliveira et al. [48] performed a microbiological analysis of four whey-based drinks inoculated with probiotic cultures at 1, 7, 14, 21, and 28 days of storage at 4C. They used MRS-bile agar for counting L. acidophilus, MRS agar for L. delbrueckii ssp. bulgaricus and L. rhamnosus and agar for S. thermophilus. Although the probiotic count decreased during storage (28 days), they found that the beverages contained, on average, 5.3 <sup>10</sup><sup>6</sup> CFU/mL of probiotics after 28 days of storage. In addition, they observed that, on average, pH remained at 4.5 after the first day of storage, showing a decrease between 0.14 and 0.32 units during the first week, decreasing slightly (less than 0.12 pH units) up to 28 days of storage, considering it stable.

Cóndor et al. [56] performed a microbiological evaluation of a whey-based beverage using immobilized Kluyveromyces Marxianus cells, during storage at room temperature and under refrigeration. They counted viable aerobic mesophiles, numbering of total coliforms and count of molds and yeasts, according to Peruvian Technical Standard 202.083 (1988). After storage viable aerobic mesophilic micro-organisms count and molds and yeasts (CFU/ml) were less than 10, while total coliforms (NMP/ml) were less than 3.0, indicating that the beverage was microbiologically acceptable. They also observed acidity and pH profile up to the fifth month of evaluation, both at room temperature (22C) and under refrigeration (4C).

#### 3. Conclusions

the difference between intensities for two products valued in standard deviation. The triangle test results showed sensorial differences between treatments T1 and T2; however, T3 was different from the first two. To confirm this difference, they performed a test in which 109 consumers participated, finding that 56% expressed differences by T3, 34.86% by T2 and 9.17% showed no preference, selecting T3 as final product, which after a descriptive test, was

Following the same idea, Dalev et al. [47] conducted a qualitative sensory analysis of fermented whey and soy milk beverages to five probiotic beverages based on whey and soy milk: (1) unfermented soy milk (control drink), (2) fermented drink with equal volume of whey and soy milk. Starter culture: B. breve and L. casei, (3) fermented drink with equal volume of whey and soy milk. Starter culture: B. infantis and S. thermophilus, (4) fermented drink with equal volume of whey and soy milk. Starter culture: B. animalis and L. plantarum, and (5) fermented drink with equal volume of whey and soy milk. Starter culture: B. breve,

They performed the sensory evaluation using the Quantitative Description Analysis (QDA) method [61]. They selected descriptors or attributes to be evaluated and used a 10 cm unstructured linear scale, shown in monitors, converting the results on a numerical scale (from 0 to 10 units) expressing them in conventional units. They employed a panel of six people (four women and two men) trained according to the International Standards (ISO 1993), with at least 1 year of experience in descriptive tests of different foods, who received varied samples of 20 mL, each in triplicate. The results showed highly significant differences in attributes such as soy milk odor, cereal odor, fermented taste, strawberry odor, sweet taste, and after taste. They concluded that the addition of processed fruits helps to improve the characteristics of the beverages substantially, and therefore, the qualification as organoleptic quality. Soy milk has been used to prepare products such as yoghurt, but its poor organoleptic characteristics have

It is a fact that there are physico-chemical factors that can influence micro-organism survival in fermented beverages, being the most important acidity, temperature, oxygen concentration, type of inoculum and storage conditions. Theoretically, it is expected that for a reasonable time, the product will maintain the characteristics that define it as probiotic, so that quality can be guaranteed to the consumer. For this reason, many researches have evaluated the beverages

In this order of ideas, Fiorentini et al. [37] performed a viable lactic bacteria count in fermented beverages prepared from whey and soybean addition, after 7, 14, and 21 days storage under refrigeration at 7C. They performed a selective count of L. acidophilus on modified MRS agar, with aerobic incubation and a count of B. bifidum on MRS agar modified with anaerobic incubation, both at 37C for 72 h, finding that the number of alive micro-organisms in the fermented drink was 10<sup>7</sup> UFC/ml. Londoño et al. [18] evaluated the viability of the beverage based on fresh whey inoculated with L. casei. They performed counts on two types of agar,

cataloged with "very good" organoleptic characteristics.

been responsible for a very low acceptance by consumers.

2.5. Whey-based fermented beverages shelf-life

shelf-life.

L. plantarum, and S. thermophilus.

126 Probiotics - Current Knowledge and Future Prospects

Whey has been used for probiotic fermented beverages development significantly. It has excellent physico-chemical characteristics that make it become an excellent substrate, allowing probiotic bacteria growth in such a way that they reach high concentrations, achieving the probiotic effect and all of its health benefits. On the other hand, whey-based probiotic beverages have extraordinary organoleptic characteristic and they are widely accepted by consumers. An average shelf-life of these beverages is 21 days under refrigeration conditions, ensuring probiotics benefits during this period of time. Throughout this review, it is shown that probiotic consumption can be increased through the use of whey not only due to its excellent physico-chemical characteristics, but also due to its ability to develop beverages with high sensorial characteristics and due to its excellent acceptance.

#### Author details

Mónica S. Molero\* and Wilfido J. Briñez

\*Address all correspondence to: mmolero@fing.luz.edu.ve

University of Zulia, Maracaibo, Venezuela

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**Chapter 8**

**Provisional chapter**

**Probiotics and Ruminant Health**

**Probiotics and Ruminant Health**

DOI: 10.5772/intechopen.72846

Probiotics are viable microorganisms with beneficial health effects for humans and animals. They are formulated into many functional foods and animal feed. There is a growing research interest in the application and benefits of probiotics in ruminant production. Several recent studies have evaluated the potential of probiotics in animal nutrition and health. In this chapter, we have reviewed current research on the benefits of probiotics on gut microbial communities in ruminants and their impact on ruminant production,

The gastrointestinal tract of domestic ruminant animals mainly cattle, sheep and goat are inhabited by diverse and complex microbial communities including bacteria, protozoa, fungi, archaea and viruses. In the last three decades, there have been numerous research studies to characterize the gut and rumen microbiota population and understand their importance on ruminant nutrition and health. In dairy cows, the rumen, which is the main fermentation chamber contains different microbial communities; about 100 billion bacteria, protozoa, methanogens and other anaerobic fungi [1, 2]. The major microbial groups in the rumen include *Prevotella*, *Selenomonas*, *Streptococcus*, *Lactobacillus* and *Megasphaera.* The rumen is also predominately inhabited by fiber-degrading bacteria such as *Fibrobacter*, *Ruminococcus*, *Butyrivibrio* and *Bacteroides* [2]. These native microbial groups have important function in the digestion and fermentation of dietary polysaccharides by the host [3]. In addition, the rumen microbial population must be balance and healthy for efficient digestion of feed and impact animal health [4]. In ruminants, variation in the rumen microbiota between individual animals has

**Keywords:** probiotic, ruminant health, gut microbiota, immune response

Sarah Adjei-Fremah, Kingsley Ekwemalor, Mulumebet Worku and Salam Ibrahim

Sarah Adjei-Fremah, Kingsley Ekwemalor, Mulumebet Worku and Salam Ibrahim

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

http://dx.doi.org/10.5772/intechopen.72846

health and overall wellbeing.

**Abstract**

**1. Introduction**


**Chapter 8**

**Provisional chapter**

## **Probiotics and Ruminant Health**

**Probiotics and Ruminant Health**

Sarah Adjei-Fremah, Kingsley Ekwemalor, Mulumebet Worku and Salam Ibrahim Mulumebet Worku and Salam Ibrahim Additional information is available at the end of the chapter

Sarah Adjei-Fremah, Kingsley Ekwemalor,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72846

#### **Abstract**

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10.1051/lait:1989420

132 Probiotics - Current Knowledge and Future Prospects

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Probiotics are viable microorganisms with beneficial health effects for humans and animals. They are formulated into many functional foods and animal feed. There is a growing research interest in the application and benefits of probiotics in ruminant production. Several recent studies have evaluated the potential of probiotics in animal nutrition and health. In this chapter, we have reviewed current research on the benefits of probiotics on gut microbial communities in ruminants and their impact on ruminant production, health and overall wellbeing.

DOI: 10.5772/intechopen.72846

**Keywords:** probiotic, ruminant health, gut microbiota, immune response

#### **1. Introduction**

The gastrointestinal tract of domestic ruminant animals mainly cattle, sheep and goat are inhabited by diverse and complex microbial communities including bacteria, protozoa, fungi, archaea and viruses. In the last three decades, there have been numerous research studies to characterize the gut and rumen microbiota population and understand their importance on ruminant nutrition and health. In dairy cows, the rumen, which is the main fermentation chamber contains different microbial communities; about 100 billion bacteria, protozoa, methanogens and other anaerobic fungi [1, 2]. The major microbial groups in the rumen include *Prevotella*, *Selenomonas*, *Streptococcus*, *Lactobacillus* and *Megasphaera.* The rumen is also predominately inhabited by fiber-degrading bacteria such as *Fibrobacter*, *Ruminococcus*, *Butyrivibrio* and *Bacteroides* [2]. These native microbial groups have important function in the digestion and fermentation of dietary polysaccharides by the host [3]. In addition, the rumen microbial population must be balance and healthy for efficient digestion of feed and impact animal health [4]. In ruminants, variation in the rumen microbiota between individual animals has

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been reported. This variation is dependent on animal age, health status and environmental factors [5–8]. There is a growing research interest in the application of beneficial microbes/ probiotics in ruminant production to help balance the gut microbiota, and as possible alternative to antibiotic use through improved gut health.

include, non-pathogenic, resistance to gastric juice and bile, anatgonize pathogenic bacteria, genetically stable, and exhibit stable qualities during processing, storage and delivery, viable at high populations [16]. In the USA, there are regulatory considerations by the Food and Drug Authority for safety evaluation of microorganisms used as probiotic. The specific micro-

There are different microbial species used as probiotics in ruminants which include bacteria, yeast, etc. **Table 1** presents a list of microorganism targets commonly used as probiotics in ruminants feeds and this includes bacteria species belonging to the genera *Bacillus*, *Enterococcus*, *Lactobacillus*, *Pediococcus*, *Streptococcus* and yeast strains such as *Saccharomyces cerevisiae* and *Kluyveromyces* [29]. The most common commercial probiotics products for ruminants consist of live yeast (*Saccharomyces cerevisiae*). Although, majority of these strains are nonpathogenic and safe, others especially *Bacillus cereus* produces enterotoxins which may not be safe [29]. The use of yeast and fungal probiotics are more effective in adult ruminants, whereas probiotic containing bacteria species have high efficacy in pre-ruminant

> *Enterococcus* **species**

*L. lactis B. adolescentis Streptococcus Salivarius* subsp*. thermophilus*

*L. reuteri B. longum E. faecalis Sporolactobacillus inulinus*

*L. brevis B. infantis Saccharomyces boulardii*

*L. johnsonii B. lactis Clostridium botyricum L. crispatus B. animalis Escherichia coli*

*L. fermentum B. bifidum Lactococcus lactis* subsp*. lactis L. amylovorus Lactococcus lactis* subsp*. cremoriss*

*L. rhamnosus Propionibacterium freudenreichii*

*L. gallinarum Aspergillus niger L. plantarum Kluyveromyces fragilis L. casei Kluyveromyces marxianus L. salivarius Saccharomyces pastorianus*

**Table 1.** Common probiotic microorganisms use for ruminant (Adapted from [16, 17, 27, 28]).

*L. paracasei B. breve E. faecium Bacillus cereus*

**Other species**

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*Pediococcus acidilactici*

*dextranicum*

*Aspergillus oryzae*

*Leuconostoc mesenteroides* subsp*.* 

organism should have "Generally Regarded As Safe" (GRAS) status [26].

**1.2. Different types of probiotic microorganisms**

*Lactobacillus* **species** *Bifidobacterium*

*L. delbrueckii* subsp. *bulgaricus*

*L. helveticus L. acidophilus* **species**

Probiotics are defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" [9]. Probiotics are widely recognized as nonpathogenic microbes with health benefits [10]. The beneficial health effects of probiotics are related to their immunomodulatory activity in the gut by stimulating the secretion of immune modulators such as cytokines and IgA in intestinal mucosa [11]. In ruminants, probiotics are administered to target the rumen (main site of feed digestion) where they have an effect on rumen fermentation especially on feed digestibility and degradability and rumen microbiota [12]. Probiotic positively affect celluloysis and synthesis of microbial protein during digestion [13], and stabilizes rumen pH and lactate levels. In addition, probiotics are able to enhance nutrient absorption [14]. Direct-fed probiotic have been shown to reduce ruminal acidosis [15].

Lactic-acid bacteria strains such as *Lactobacillus*, *Bifidobacterium, Bacillus, Saccharomyces* and *Enterococcus* are commonly used as probiotics in functional foods and animal feed [16–20]. *Lactobacillus* and *Bifidobacterium* species have been shown to provide protection against enteric infection. These beneficial microbes consist of different species of microorganisms such as bacteria and yeast and they may be used as single or multi-strain. The multi-strain probiotics have a broad spectrum effect from the different strains against infections [21], and could increase their beneficial effects of probiotics due to their synergistic adhesion effect [22].

Probiotics are typically used to improve gastrointestinal health, reduce diarrhea, bloating and protect against infectious diseases [23]. Several researchers have reported the benefits of oral administration of probiotics to ruminants. Probiotics regulate and balance gut microbes, promote growth and development of animals, and improve the host resistance to diseases [24]. Recent studies suggest that utilization of probiotics as feed supplement for ruminants improves growth performance, production, and enhance health and overall wellbeing of the animals. Applications of probiotics have been shown to reduce the negative environmental impact such as methane emission associated with ruminant production. In this chapter, we have reviewed current research on the benefits of probiotics on gut microbial communities in ruminants and their impact on ruminant production, health and overall wellbeing.

#### **1.1. Selection of probiotic strain**

It is important to select the suitable strain of a microorganism for use as probiotic. The suitable potential probiotic strain is considered as an inhabitant of the host organism and has the ability to adhere and colonize the epithelial cells of the gut. Also, the potential probiotic microbe should be able to grow and survive in the host [25]. Microbial strains used as probiotics are required not to affect the indigenous gut microbiota population of the host. Other important requirement for the potential probiotic strain is to be able to adapt to the environment of the gut and locate a suitable niche in the rumen (such as epithelium, fluid or feed), and exerts positive effects on the host [8]. Other Safety criteria and characteristic of probiotics to consider include, non-pathogenic, resistance to gastric juice and bile, anatgonize pathogenic bacteria, genetically stable, and exhibit stable qualities during processing, storage and delivery, viable at high populations [16]. In the USA, there are regulatory considerations by the Food and Drug Authority for safety evaluation of microorganisms used as probiotic. The specific microorganism should have "Generally Regarded As Safe" (GRAS) status [26].

#### **1.2. Different types of probiotic microorganisms**

been reported. This variation is dependent on animal age, health status and environmental factors [5–8]. There is a growing research interest in the application of beneficial microbes/ probiotics in ruminant production to help balance the gut microbiota, and as possible alterna-

Probiotics are defined as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" [9]. Probiotics are widely recognized as nonpathogenic microbes with health benefits [10]. The beneficial health effects of probiotics are related to their immunomodulatory activity in the gut by stimulating the secretion of immune modulators such as cytokines and IgA in intestinal mucosa [11]. In ruminants, probiotics are administered to target the rumen (main site of feed digestion) where they have an effect on rumen fermentation especially on feed digestibility and degradability and rumen microbiota [12]. Probiotic positively affect celluloysis and synthesis of microbial protein during digestion [13], and stabilizes rumen pH and lactate levels. In addition, probiotics are able to enhance nutrient absorption [14]. Direct-fed probiotic have been shown to reduce ruminal acidosis [15]. Lactic-acid bacteria strains such as *Lactobacillus*, *Bifidobacterium, Bacillus, Saccharomyces* and *Enterococcus* are commonly used as probiotics in functional foods and animal feed [16–20]. *Lactobacillus* and *Bifidobacterium* species have been shown to provide protection against enteric infection. These beneficial microbes consist of different species of microorganisms such as bacteria and yeast and they may be used as single or multi-strain. The multi-strain probiotics have a broad spectrum effect from the different strains against infections [21], and could increase their beneficial effects of probiotics due to their synergistic adhesion effect [22].

Probiotics are typically used to improve gastrointestinal health, reduce diarrhea, bloating and protect against infectious diseases [23]. Several researchers have reported the benefits of oral administration of probiotics to ruminants. Probiotics regulate and balance gut microbes, promote growth and development of animals, and improve the host resistance to diseases [24]. Recent studies suggest that utilization of probiotics as feed supplement for ruminants improves growth performance, production, and enhance health and overall wellbeing of the animals. Applications of probiotics have been shown to reduce the negative environmental impact such as methane emission associated with ruminant production. In this chapter, we have reviewed current research on the benefits of probiotics on gut microbial communities in

ruminants and their impact on ruminant production, health and overall wellbeing.

It is important to select the suitable strain of a microorganism for use as probiotic. The suitable potential probiotic strain is considered as an inhabitant of the host organism and has the ability to adhere and colonize the epithelial cells of the gut. Also, the potential probiotic microbe should be able to grow and survive in the host [25]. Microbial strains used as probiotics are required not to affect the indigenous gut microbiota population of the host. Other important requirement for the potential probiotic strain is to be able to adapt to the environment of the gut and locate a suitable niche in the rumen (such as epithelium, fluid or feed), and exerts positive effects on the host [8]. Other Safety criteria and characteristic of probiotics to consider

**1.1. Selection of probiotic strain**

tive to antibiotic use through improved gut health.

134 Probiotics - Current Knowledge and Future Prospects

There are different microbial species used as probiotics in ruminants which include bacteria, yeast, etc. **Table 1** presents a list of microorganism targets commonly used as probiotics in ruminants feeds and this includes bacteria species belonging to the genera *Bacillus*, *Enterococcus*, *Lactobacillus*, *Pediococcus*, *Streptococcus* and yeast strains such as *Saccharomyces cerevisiae* and *Kluyveromyces* [29]. The most common commercial probiotics products for ruminants consist of live yeast (*Saccharomyces cerevisiae*). Although, majority of these strains are nonpathogenic and safe, others especially *Bacillus cereus* produces enterotoxins which may not be safe [29]. The use of yeast and fungal probiotics are more effective in adult ruminants, whereas probiotic containing bacteria species have high efficacy in pre-ruminant


**Table 1.** Common probiotic microorganisms use for ruminant (Adapted from [16, 17, 27, 28]).

calves. In pre-ruminants since their rumen is not yet developed, probiotic species administered targets the small intestine, help balance the gut microbiota, and reduce pathogen colonization of the host [30].

**Probiotics type Ruminant Performance effect References** *Lactobacillus casei ssp casei* Calves Weight gain [47] Lactate-utilizing/or lactate-producing bacteria Cattle Improve feed efficiency,

Calf-specific probiotic (six *Lactobacillus* species) Veal calves Reduced diarrhea

Live yeast Beef cattle Improve average daily

Yeast Dairy cows Increased milk yield and

*Lactobacillus casei* Zhang and *Lactobacillus plantarum* P-8 Dairy cows Improve quality

*Bacillus licheniformis* and *Bacillus subtilis* Sheep Reduced lamb mortality

Mushroom-based probiotic **(**Coriolus versicolor) Goats No effect on body

Commercial probiotic Meat Goats Improved average daily

**Table 2.** Summary of benefits of probiotics on growth and production performance of ruminants.

FasTrack Microbial pack (*Lactobacillus acidophilus*, *Saccharomyces cerevisiae*, *Enterococcus faecium*, *Aspergillus oryzae, fructooligosaccharide*, active dry yeast culture

*Lactobacillus acidophilus, Saccharomyces cerevisiae, S.* 

Probiotic mixture (*Bifidobacteriumlongum*, *Bifidobacterium breve*, *Lactobacillus acidophilus*, *Lactobacillus reuteri* and

Multi-strain Probiotic (*Lactobacillus acidophilus*, *Saccharomyces cerevisiae*, *Enterococcus faecium*, *Aspergillus oryzae, fructooligosaccharide*, active dry yeast culture)

*boulardii, Propionibacterium freudenreichii*

*Lactobacillus rhamnosus*)

increase in daily gain

Decreased fecal coliform

gain, final weight, feed intake, feed to gain ratio

Increase feed efficiency Reduced ruminal acidosis

and quantity of milk production

Dairy cows Improve body weight [42, 43]

Increased milk production

content of milk

weight, PCV, White blood cells differential

weight, PCV, White blood cells differential

volume and FAMACHA

Increased average daily milk yield per ewe

Improved fat and protein

rate

Goat No effect on body

count

count

gain

Goats Improved Packed cell

scores

Lactating cows

[48]

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[49]

[50]

[51, 52]

[24]

[53]

[54]

[36]

[37]

[35]

[41]

(2.5%)

counts

quality

Prebiotic are defined as "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth/or activity of one or a limited number of bacteria in the colon" [31]. Prebiotics are commonly dietary fiber and have effect on both the upper and lower GI tract. In the upper GI tract, prebiotics are able to withstand digestion, delay gastric removal, decrease glucose absorption and stimulate the release of intestinal hormonal peptides. The main prebiotics used in animal diet are carbohydrates and oligosaccharide. Non-digestible oligosaccharides used include oligofructose, inulin, lactulose, galactooligosaccharide, transgalactooligosaccharide [16, 32].

Synbiotics on the other hand are products that contain a mixture of probiotic and prebiotics. The host benefit from the synergistic effect of probiotic and prebiotic. Results from studies done have demonstrated the promising effect of synbiotics in reducing the numbers of food borne pathogens [33].

#### **1.3. Administration of probiotics**

There are different route of administration of probiotics. These sites include the oral cavity, intestines, vagina and the skin [34]. In ruminants, probiotics are usually administered orally [35–43]. A study by Deng et al. [44, 45] utilized intravaginal infusion as mode of administering probiotics (containing a lactic acid bacteria mixture) to periparturient cows.

#### **2. Probiotics and ruminant growth and production performance**

#### **2.1. Effect of probiotic on growth performance**

Utilization of probiotics (either dry or live) as natural feed additives have been shown to favorably improve animal performance and welfare, via modulation of gut microbial community which is essential in ensuring host homeostasis [46]. Probiotic have positive effect on growth rate and production performance of animals when administered as single or multi-strain feed supplement (**Table 2**). Oral administration of probiotic has been shown to improve feed intake, daily weight gain and overall weight gain in sheep, goats, and cattle [38–43, 47, 49, 51, 52]. The population of beneficial microbes such as *Lactobacillus* and *Bifidobacteria* are low in neonatal calves, but studies have shown that supplementation with probiotics containing these microbes increases their growth [55]. In dairy cows, probiotic composed of live yeast increased food intake, improved feed efficiency, improved average daily gain and overall total weight. Additionally, probiotic increased milk yield and quality [51, 52].

In small ruminants such as goats and sheep, treatment with commercial probiotic improved average daily gain [35]. Gyenai et al. [36], Ekwemalor et al. [37] and Ekwemalor et al. [41], reported contrary results where there was no effect of probiotics on body weight. These different


calves. In pre-ruminants since their rumen is not yet developed, probiotic species administered targets the small intestine, help balance the gut microbiota, and reduce pathogen

Prebiotic are defined as "non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth/or activity of one or a limited number of bacteria in the colon" [31]. Prebiotics are commonly dietary fiber and have effect on both the upper and lower GI tract. In the upper GI tract, prebiotics are able to withstand digestion, delay gastric removal, decrease glucose absorption and stimulate the release of intestinal hormonal peptides. The main prebiotics used in animal diet are carbohydrates and oligosaccharide. Non-digestible oligosaccharides used include oligofructose, inulin, lactulose, galactooligosac-

Synbiotics on the other hand are products that contain a mixture of probiotic and prebiotics. The host benefit from the synergistic effect of probiotic and prebiotic. Results from studies done have demonstrated the promising effect of synbiotics in reducing the numbers of food

There are different route of administration of probiotics. These sites include the oral cavity, intestines, vagina and the skin [34]. In ruminants, probiotics are usually administered orally [35–43]. A study by Deng et al. [44, 45] utilized intravaginal infusion as mode of administering

Utilization of probiotics (either dry or live) as natural feed additives have been shown to favorably improve animal performance and welfare, via modulation of gut microbial community which is essential in ensuring host homeostasis [46]. Probiotic have positive effect on growth rate and production performance of animals when administered as single or multi-strain feed supplement (**Table 2**). Oral administration of probiotic has been shown to improve feed intake, daily weight gain and overall weight gain in sheep, goats, and cattle [38–43, 47, 49, 51, 52]. The population of beneficial microbes such as *Lactobacillus* and *Bifidobacteria* are low in neonatal calves, but studies have shown that supplementation with probiotics containing these microbes increases their growth [55]. In dairy cows, probiotic composed of live yeast increased food intake, improved feed efficiency, improved average daily gain and overall total weight.

In small ruminants such as goats and sheep, treatment with commercial probiotic improved average daily gain [35]. Gyenai et al. [36], Ekwemalor et al. [37] and Ekwemalor et al. [41], reported contrary results where there was no effect of probiotics on body weight. These different

probiotics (containing a lactic acid bacteria mixture) to periparturient cows.

**2. Probiotics and ruminant growth and production performance**

colonization of the host [30].

136 Probiotics - Current Knowledge and Future Prospects

borne pathogens [33].

**1.3. Administration of probiotics**

charide, transgalactooligosaccharide [16, 32].

**2.1. Effect of probiotic on growth performance**

Additionally, probiotic increased milk yield and quality [51, 52].

**Table 2.** Summary of benefits of probiotics on growth and production performance of ruminants.

observations reported may be due to difference in the probiotic composition used, amount use, specific activity of the probiotic strains and variation in the breeds of goats used in their individual studies. This because studies have shown that different probiotic strains may have different effects depending on their capabilities and enzymatic activities different host species [34]. In the study by Gyenai et al. [36] Spanish Boer kid-goats were drenched with a probiotic mixture consisting of *Bifidobacterium longum*, *Bifidobacterium breve*, *Lactobacillus acidophilus*, *Lactobacillus reuteri* and *Lactobacillus rhamnosus*. The commercial probiotic used by Ekwemalor et al. [41] composed of *Lactobacillus acidophilus*, *Saccharomyces cerevisiae*, *Enterococcus faecium*, *Aspergillus oryzae, fructooligosaccharide*, active dry yeast culture. Whitley et al. [35] used a commercial probiotic containing active dry yeast and lactic acid-producing bacteria, including *Lactobacillus acidophilus* and *Enterococcus faecium*. In addition, Whitley et al. [35] tested the probiotic on Boer crossbred meat goats (50–75% Boer of genetic background) however, Spanish Boer goats were used in the studies by Gyenai et al. [36] and Ekwemalor et al. [41]. Furthermore, two indicators of anemic condition in goats, Packed cell volume and FAMACHA have been reported to be affected by probiotic treatments [41].

nutrients, and/or the synthesis of organic acids and bacteriocins that create an environment

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The beneficial health effect of probiotics have been partly attributed to the ability of probiotic bacteria to modulate the immune system, increasing both innate and adaptive immune response [67, 68]. Research evidence obtained from various in vivo and in vitro studies have demonstrated that probiotic promote gut health via stimulation of the innate immune response [69]. Different probiotic bacteria including Lactobacillus casei, *Lactobacillus casei* strain Shirota, *Streptococcus thermophilus*, *Lactobacillus fermentum* and yeast have been tested to elicit an immune response [67, 70]. Oral administration of *Lactobacillus casei* activated immune cells of the innate immune response, and increased the expression of innate immune receptor, TLR2 [70]. In a similar study in mice, administration of *Lactobacillus casei* strain Shirota (LcS) enhanced innate immune response by stimulating or inhibiting the production of TH1/ TH2 cytokines [67, 71]. Ghadimi et al. [71] reported that probiotic enhanced secretion of IFN-γ (a TH1 cytokine) and inhibited the stimulation of TH2 cytokines such as IL4 and IL5. Research findings by Yan and Polk [72] showed that immunomodulatory effect of a probiotic (*Lactobacillus rhamnosus* GG) in preventing cytokine-induced apoptosis in intestinal epithelial cells. Results from their study indicated that the probiotic inhibited activation of the p38/mitogen activated protein kinase which is a pro apoptotic kinase induced by cytokines TNF, IL-1α, or IFN-γ. Furthermore, the *Lactobacillus rhamnosus* GG probiotic activated Akt/protein kinase B (anti-apoptotic) in colon cells from mice and humans. In an *in vitro* study, a multiple probiotic formulation has been demonstrated to activate NF-κB and stimulate production of TNF-α in epithelial [69]. Studies done in vivo and ex vivo have demonstrated the effect of probiotics treatment on the inflammasome [73]. The inflammasome found in various immune cells (macrophages and dendritic cells) and intestinal epithelial cells consists of cytosolic proteins such as NOD-like receptors, apoptosis-associated speck-like protein containing a CARD domain and caspase-1 (the serine protease). Activation of inflammasome receptors further leads to activation of caspase-1 and Interleukin (IL)-1β and IL-18. Activation and secretion and of the inflammatory cytokines Interleukin (IL)-1β and IL-18 stimulates and enhance the antimicrobial effect of immune cells against intracellular pathogens infection and also activates cell death of inflammasome-activated cells [74–76]. Studies have reported a potential role of the inflammasomes in the development of chronic intestinal inflammation [73, 76]. In ruminants such as bovine, the probiotic *Lactobacillus rhamnosus* GR-1 have been shown to amend *E. coli* induced inflammation in primary bovine mammary epithelial cells. Findings from their study showed that probiotic pretreatment impaired the activation ASC-independent NLRP3 inflammasome, and decreased protein expression of NLRP3 (NOD -like receptor family member

pyrin domain-containing protein 3) and caspase 1 induced by *E. coli* [77].

The molecular impact of oral probiotic supplementation on systemic expression of genes associated with innate immune response in blood have been reported for ruminant species; cows [38, 42, 43], and goats [41] as shown in **Table 3**. Probiotics have been reported to activate pathways immunity and homeostasis including Toll-like receptor pathway, Wnt signaling pathway,

unfavorable for pathogen development [49, 63–66].

**4. Probiotics and immunity**

#### **2.2. Effects of probiotics in milk**

Use of probiotics as feed supplements for ruminants have beneficial influence on milk production, milk quality and functional components such as protein and fat content [24, 54]. Studies have shown that probiotic dairy products are safe for large-scale consumption [56]. A study conducted by Yu et al. [57] showed that dairy cows treated with probiotic species *Aspergillus oryzae* ad *Saccharomyces cerevisiae* increased milk production and milk proteins. Also Sun et al. [58] and Qiao et al. [59] found that probiotics containing *Bacillus subtilis* improved the milk yield and rumen fermentation of dairy cows. Stein et al. [51] and Stella et al. [60] reported that probiotics improved the feed utilization rate, the milk yield and component profiles, and increase the dry matter intake in dairy cow. Xu et al. [24], also reported that probiotic application could reduce udder inflammation and increase milk yield while suppressing somatic cell count. Sun et al. [58] and Lehloenya et al. [61] reported that probiotic administration to dairy cows increased the milk production and simultaneously improved the milk fat, protein and lactose yield, accompanied by a decrease in milk somatic cell count. These positive effects of probiotics on milk production and milk quality characteristics are attributed to the subsequent effects of probiotics on the number of cellulolytic and fiber-degrading bacteria as well as changes in the volatile fatty acid in the rumen [54].

#### **3. Molecular mechanism of action of probiotics**

The mode of action of probiotics in the host organism include: regulation of intestinal microbial homeostasis, stabilization of the gastrointestinal barrier function, expression of bacteriocins enzymatic activity inducing absorption and nutrition, immunomodulatory effects, inhibition of procarcinogenic enzymes and interference with the ability of pathogens to colonize and infect the mucosa [62]. In ruminants, the mechanism of probiotics metabolism is dependent on the strain of microorganism used. Probiotic bacteria can serve to decrease the severity of infection via a number of mechanisms including competition for receptors and nutrients, and/or the synthesis of organic acids and bacteriocins that create an environment unfavorable for pathogen development [49, 63–66].

#### **4. Probiotics and immunity**

observations reported may be due to difference in the probiotic composition used, amount use, specific activity of the probiotic strains and variation in the breeds of goats used in their individual studies. This because studies have shown that different probiotic strains may have different effects depending on their capabilities and enzymatic activities different host species [34]. In the study by Gyenai et al. [36] Spanish Boer kid-goats were drenched with a probiotic mixture consisting of *Bifidobacterium longum*, *Bifidobacterium breve*, *Lactobacillus acidophilus*, *Lactobacillus reuteri* and *Lactobacillus rhamnosus*. The commercial probiotic used by Ekwemalor et al. [41] composed of *Lactobacillus acidophilus*, *Saccharomyces cerevisiae*, *Enterococcus faecium*, *Aspergillus oryzae, fructooligosaccharide*, active dry yeast culture. Whitley et al. [35] used a commercial probiotic containing active dry yeast and lactic acid-producing bacteria, including *Lactobacillus acidophilus* and *Enterococcus faecium*. In addition, Whitley et al. [35] tested the probiotic on Boer crossbred meat goats (50–75% Boer of genetic background) however, Spanish Boer goats were used in the studies by Gyenai et al. [36] and Ekwemalor et al. [41]. Furthermore, two indicators of anemic condition in goats, Packed cell volume and FAMACHA have been reported to be affected by

Use of probiotics as feed supplements for ruminants have beneficial influence on milk production, milk quality and functional components such as protein and fat content [24, 54]. Studies have shown that probiotic dairy products are safe for large-scale consumption [56]. A study conducted by Yu et al. [57] showed that dairy cows treated with probiotic species *Aspergillus oryzae* ad *Saccharomyces cerevisiae* increased milk production and milk proteins. Also Sun et al. [58] and Qiao et al. [59] found that probiotics containing *Bacillus subtilis* improved the milk yield and rumen fermentation of dairy cows. Stein et al. [51] and Stella et al. [60] reported that probiotics improved the feed utilization rate, the milk yield and component profiles, and increase the dry matter intake in dairy cow. Xu et al. [24], also reported that probiotic application could reduce udder inflammation and increase milk yield while suppressing somatic cell count. Sun et al. [58] and Lehloenya et al. [61] reported that probiotic administration to dairy cows increased the milk production and simultaneously improved the milk fat, protein and lactose yield, accompanied by a decrease in milk somatic cell count. These positive effects of probiotics on milk production and milk quality characteristics are attributed to the subsequent effects of probiotics on the number of cellulolytic and fiber-degrading bacteria as well

The mode of action of probiotics in the host organism include: regulation of intestinal microbial homeostasis, stabilization of the gastrointestinal barrier function, expression of bacteriocins enzymatic activity inducing absorption and nutrition, immunomodulatory effects, inhibition of procarcinogenic enzymes and interference with the ability of pathogens to colonize and infect the mucosa [62]. In ruminants, the mechanism of probiotics metabolism is dependent on the strain of microorganism used. Probiotic bacteria can serve to decrease the severity of infection via a number of mechanisms including competition for receptors and

probiotic treatments [41].

**2.2. Effects of probiotics in milk**

138 Probiotics - Current Knowledge and Future Prospects

as changes in the volatile fatty acid in the rumen [54].

**3. Molecular mechanism of action of probiotics**

The beneficial health effect of probiotics have been partly attributed to the ability of probiotic bacteria to modulate the immune system, increasing both innate and adaptive immune response [67, 68]. Research evidence obtained from various in vivo and in vitro studies have demonstrated that probiotic promote gut health via stimulation of the innate immune response [69]. Different probiotic bacteria including Lactobacillus casei, *Lactobacillus casei* strain Shirota, *Streptococcus thermophilus*, *Lactobacillus fermentum* and yeast have been tested to elicit an immune response [67, 70]. Oral administration of *Lactobacillus casei* activated immune cells of the innate immune response, and increased the expression of innate immune receptor, TLR2 [70]. In a similar study in mice, administration of *Lactobacillus casei* strain Shirota (LcS) enhanced innate immune response by stimulating or inhibiting the production of TH1/ TH2 cytokines [67, 71]. Ghadimi et al. [71] reported that probiotic enhanced secretion of IFN-γ (a TH1 cytokine) and inhibited the stimulation of TH2 cytokines such as IL4 and IL5. Research findings by Yan and Polk [72] showed that immunomodulatory effect of a probiotic (*Lactobacillus rhamnosus* GG) in preventing cytokine-induced apoptosis in intestinal epithelial cells. Results from their study indicated that the probiotic inhibited activation of the p38/mitogen activated protein kinase which is a pro apoptotic kinase induced by cytokines TNF, IL-1α, or IFN-γ. Furthermore, the *Lactobacillus rhamnosus* GG probiotic activated Akt/protein kinase B (anti-apoptotic) in colon cells from mice and humans. In an *in vitro* study, a multiple probiotic formulation has been demonstrated to activate NF-κB and stimulate production of TNF-α in epithelial [69]. Studies done in vivo and ex vivo have demonstrated the effect of probiotics treatment on the inflammasome [73]. The inflammasome found in various immune cells (macrophages and dendritic cells) and intestinal epithelial cells consists of cytosolic proteins such as NOD-like receptors, apoptosis-associated speck-like protein containing a CARD domain and caspase-1 (the serine protease). Activation of inflammasome receptors further leads to activation of caspase-1 and Interleukin (IL)-1β and IL-18. Activation and secretion and of the inflammatory cytokines Interleukin (IL)-1β and IL-18 stimulates and enhance the antimicrobial effect of immune cells against intracellular pathogens infection and also activates cell death of inflammasome-activated cells [74–76]. Studies have reported a potential role of the inflammasomes in the development of chronic intestinal inflammation [73, 76]. In ruminants such as bovine, the probiotic *Lactobacillus rhamnosus* GR-1 have been shown to amend *E. coli* induced inflammation in primary bovine mammary epithelial cells. Findings from their study showed that probiotic pretreatment impaired the activation ASC-independent NLRP3 inflammasome, and decreased protein expression of NLRP3 (NOD -like receptor family member pyrin domain-containing protein 3) and caspase 1 induced by *E. coli* [77].

The molecular impact of oral probiotic supplementation on systemic expression of genes associated with innate immune response in blood have been reported for ruminant species; cows [38, 42, 43], and goats [41] as shown in **Table 3**. Probiotics have been reported to activate pathways immunity and homeostasis including Toll-like receptor pathway, Wnt signaling pathway,


In dairy cows, oral probiotic supplementation had systemic effect on differential global gene expression. Probiotic treatment targeted 87 bovine pathways including Wnt signaling pathway, inflammatory response pathway, toll-like receptor signaling pathway, prostaglandin synthesis and regulation pathway and B cell receptor signaling pathway. Probiotic treatment modulated the expression of genes associated with innate immunity and homeostasis such as receptors TLR2, TLR6, TLR7, TLR8; cytokines, IL16, IL6, IL10RA; Wnt signaling genes Wnt8A, Wnt5A, Wnt10B, Kremens; and transcription regulators MAP4K3 and MAP3K8 [38, 42, 43].

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Probiotic have been widely used in cattle production for both dairy and beef cows at all developmental stages and growth. Studies have shown the beneficial effect of direct-fed microbials or probiotic bacteria including *Lactobacillus* and *Bifidobacteria* on growth, production performance (milk production, milk functional components and milk composition) and immune response of dairy cows, beef cattle, neonatal calves, and periparturient cows [38, 43, 51, 52, 55]. Furthermore, probiotic supplementation showed potential effect to decrease ruminal acidosis in feedlot cattle and dairy cows, and also improved immune response in stressed calved [48]. In dairy cows, probiotic increased food intake, improved feed efficiency, and improved average daily gain. Additionally, probiotic increased milk yield and quality [51, 52]. There are limited studies on the effect of probiotics on beef cattle compared to the research conducted on dairy cows. However, the use of probiotic yeasts to improve beef production has been vari-

Studies by Krehbiel et al. [48] have shown that probiotics are effective decreasing fecal shedding of Escherichia coli O157:H7 in infected calves. Research findings reported by Sherman et al. [79], demonstrated that treatment of intestinal cells with lactic acid producing bacteria reduced epithelial injury due to *E. coli* O157:H7 and *E. coli* O127:H6 exposure. Therefore, probiotic is used as one of the many strategies to reduce shedding of *E. coli* o157:H7 and non-

Utilizing probiotics as functional food supplement have been encouraged in goat production [81]. Various commercial probiotic products consisting of either single strains or mixture of strains such as *Lactobacillus reuteri* DDL 19, *Lactobacillus alimentarius* DDL 48, *Enterococcus faecium* DDE 39 and *Bifidobacterium bifidum* DDBA have been tested in goats [35, 81]. Probiotic administration significantly increased body weight, and modified microflora by increasing the number of lactic acid bacteria and *Bifidobacteria* of goats. In addition, probiotic treatment reduced fecal mutagenicity by 60%, which is an indication of the protective influence of probiotics in goats [81]. Whitley et al. [35], observed no effect of probiotics on growth performance, diet digestibility, carcass traits, or fecal microbial populations in meat goats, although

able, possibly due to the diet composition, strain of yeast or yeast viability [78].

**5. Application of probiotic in ruminant**

**5.1. Probiotics and cattle**

O157:H7 in ruminants [80].

**5.2. Probiotics and goats**

**Table 3.** Effect of probiotics on innate immune response gene expression in ruminants.

innate and adaptive immune response pathway [38, 41–43]. A study by Ekwemalor et al. [41] showed that oral probiotic administration may exhibit systemic effect in goat blood, by modulating the expression of genes associated with immunity and homeostasis. In goats, probiotic treatments induced the expression of 32 innate immunity genes and 48 genes in the Wnt signaling pathway. Furthermore, treatment of goats with a mushroom based probiotic in an vivo study trial resulted in serum increase in pro-inflammatory cytokines such as interferon production regulator (IFNr), Rantes and Granulocyte-Colony Stimulating Factor (GCSF). But the level of granulocyte macrophage colony stimulating factor (GM-CSF) reduced [37].

In dairy cows, oral probiotic supplementation had systemic effect on differential global gene expression. Probiotic treatment targeted 87 bovine pathways including Wnt signaling pathway, inflammatory response pathway, toll-like receptor signaling pathway, prostaglandin synthesis and regulation pathway and B cell receptor signaling pathway. Probiotic treatment modulated the expression of genes associated with innate immunity and homeostasis such as receptors TLR2, TLR6, TLR7, TLR8; cytokines, IL16, IL6, IL10RA; Wnt signaling genes Wnt8A, Wnt5A, Wnt10B, Kremens; and transcription regulators MAP4K3 and MAP3K8 [38, 42, 43].

## **5. Application of probiotic in ruminant**

#### **5.1. Probiotics and cattle**

**Innate immune response parameters Genes Ruminant type References**

TLR8 TLR6 TLR7

IL6 IL1B IFNB1 CCL2 CCL3 CCL19 IL16 IL10RA

CXCR1 CCL2 CXCL8

CXCR3

WNT5A WNT10B KREMENS DVL1 PRICKLE3

innate and adaptive immune response pathway [38, 41–43]. A study by Ekwemalor et al. [41] showed that oral probiotic administration may exhibit systemic effect in goat blood, by modulating the expression of genes associated with immunity and homeostasis. In goats, probiotic treatments induced the expression of 32 innate immunity genes and 48 genes in the Wnt signaling pathway. Furthermore, treatment of goats with a mushroom based probiotic in an vivo study trial resulted in serum increase in pro-inflammatory cytokines such as interferon production regulator (IFNr), Rantes and Granulocyte-Colony Stimulating Factor (GCSF). But the level

Goats Dairy cow

Goats Dairy cow

Cattle [43]

Goats [37]

[37, 42, 43]

Dairy cow Goats

[37] [38, 43]

[37] [43]

Toll-like receptors TLR2

140 Probiotics - Current Knowledge and Future Prospects

Cytokines IL4

Chemokines CXCR2

Th1 marker STAT4

Wnt signaling WNT8A

**Table 3.** Effect of probiotics on innate immune response gene expression in ruminants.

of granulocyte macrophage colony stimulating factor (GM-CSF) reduced [37].

Probiotic have been widely used in cattle production for both dairy and beef cows at all developmental stages and growth. Studies have shown the beneficial effect of direct-fed microbials or probiotic bacteria including *Lactobacillus* and *Bifidobacteria* on growth, production performance (milk production, milk functional components and milk composition) and immune response of dairy cows, beef cattle, neonatal calves, and periparturient cows [38, 43, 51, 52, 55]. Furthermore, probiotic supplementation showed potential effect to decrease ruminal acidosis in feedlot cattle and dairy cows, and also improved immune response in stressed calved [48]. In dairy cows, probiotic increased food intake, improved feed efficiency, and improved average daily gain. Additionally, probiotic increased milk yield and quality [51, 52]. There are limited studies on the effect of probiotics on beef cattle compared to the research conducted on dairy cows. However, the use of probiotic yeasts to improve beef production has been variable, possibly due to the diet composition, strain of yeast or yeast viability [78].

Studies by Krehbiel et al. [48] have shown that probiotics are effective decreasing fecal shedding of Escherichia coli O157:H7 in infected calves. Research findings reported by Sherman et al. [79], demonstrated that treatment of intestinal cells with lactic acid producing bacteria reduced epithelial injury due to *E. coli* O157:H7 and *E. coli* O127:H6 exposure. Therefore, probiotic is used as one of the many strategies to reduce shedding of *E. coli* o157:H7 and non-O157:H7 in ruminants [80].

#### **5.2. Probiotics and goats**

Utilizing probiotics as functional food supplement have been encouraged in goat production [81]. Various commercial probiotic products consisting of either single strains or mixture of strains such as *Lactobacillus reuteri* DDL 19, *Lactobacillus alimentarius* DDL 48, *Enterococcus faecium* DDE 39 and *Bifidobacterium bifidum* DDBA have been tested in goats [35, 81]. Probiotic administration significantly increased body weight, and modified microflora by increasing the number of lactic acid bacteria and *Bifidobacteria* of goats. In addition, probiotic treatment reduced fecal mutagenicity by 60%, which is an indication of the protective influence of probiotics in goats [81]. Whitley et al. [35], observed no effect of probiotics on growth performance, diet digestibility, carcass traits, or fecal microbial populations in meat goats, although an effect on the average daily gain was observed. Research findings by Gyenai et al. [36] supports the use of probiotics in goats to enhance microbial retention in the rumen.

**Author details**

Sarah Adjei-Fremah1

**References**

University, Greensboro, NC, USA

State University, Greensboro, NC, USA

, Kingsley Ekwemalor1

1 Department of Animal Sciences, North Carolina Agricultural and Technical State

born-Dill: Institute for Microbiology and Biochemistry; 1995. pp. 101-125

2 Department of Family and Consumer Sciences, North Carolina Agricultural and Technical

[1] Wallace RJ, Newbold CJ. Microbial feed additives for ruminants. In: Fuller R, Heidt P, Rusch V, van der Waaij D. Probiotics: Prospects of Use in Opportunistic Infections. Her-

[2] Mackie R, Aminov B, White C, McSweeney C. Molecular ecology and diversity in gut microbial ecosystems. In: Cronjé PB, editor. Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction. London: CAB International; 2000. pp. 61-77 [3] Zoetendal EG, Collier CT, Koike S, Mackie RI, Gaskins HR. Molecular ecological analysis of the gastrointestinal microbiota: A review. The Journal of Nutrition. 2004;**134**:465-472

[4] Stover MG, Watson RR, Collier RJ. Pre-and probiotic supplementation in ruminant livestock production. In: Probiotics, Prebiotics, and Synbiotics: Bioactive Foods in Health

[5] Mueller S, Saunier K, Hanisch C, Norin E, Alm L, Midtvedt T, et al. Differences in fecal microbiota in different European study populations in relation to age, gender, and country: A cross-sectional study. Applied and Environmental Microbiology.

[6] Abt MC, Artis D. The intestinal microbiota in health and disease: The influence of microbial products on immune cell homeostasis. Current Opinion in Gastroenterology.

[7] Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI.Worlds within worlds: Evolution of the vertebrate gut microbiota. Nature Reviews Microbiology. 2008;**6**(10):776-788 [8] Uyeno Y, Shigemori S, Shimosato T. Effect of probiotics/prebiotics on cattle health and

[9] FAO/WHO. (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Cordoba: Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation Report

productivity. Microbes and Environments. 2015;**30**(2):126-132

\*Address all correspondence to: worku@ncat.edu

Promotion. Amsterdam: Elsevier Inc.; 2015

2006;**72**:1027-1033

2009;**25**(6):496

, Mulumebet Worku1

\* and Salam Ibrahim<sup>2</sup>

http://dx.doi.org/10.5772/intechopen.72846

Probiotics and Ruminant Health

143

There is an increasing market demand for nonfat goat milk and milk products such as yoghurt containing probiotics. A nonfat yoghurt has been developed from goat milk and is enriched with probiotic strains *Lactobacillus acidophilus* and *Bifidobacterium spp.* [82]. Other probiotic microbes have been used to develop different types of fermented drinking milk form goat milk. *Lactobacillus acidophilus* LA-5, *Bifidobacterium animalis* subsp. *lactis* BB-12 novel putative probiotic *Propionibacterium jensenii* 702 co-culturing in goat milk affected their viability and physico-chemical properties of the milk [83]. In a similar study, goat milk fermented with *Lactobacillus fermentum* ME-3 and tested in healthy human subject reduced peroxidized lipoproteins levels, decreased 8-isoprostanes, improved total antioxidant activity and demonstrated an anti-atherogenic effects. The population and activity of lactic acid bacteria in milk was affected after fermentation with *Lactobacillus fermentum* ME-3 [84].

#### **5.3. Probiotics for sheep**

In sheep production, probiotics have been applied to improve feed digestion and gut health. Two probiotics, *Saccharomyces cerevisiae* and *Aspergillus oryzae tested in* sheep had no effect on Nitrogen digestibility *and* net microbial protein flow in the duodenum [85]. In another study, a probiotic mixture containing *Bacillus licheniformis* and *Bacillus subtilis* administered in ewes at late pregnancy and lactation reduced mortality in young lambs. In addition, probiotic treatment increased daily milk yield per ewe and fat and protein content of milk were also increased [54]. In a study by Rigobelo et al. [86], administration of a probiotic mixture containing *Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus* and *Enterococcus faecium* to sheep infected with a non-O157 Shiga toxin-producing *Escherichia coli* (a foodborne pathogen of humans) reduced the fecal shedding of the pathogen. Probiotic treatment in sheep has beneficial effect on rumen methanogenesis, energy retention and Nitrogen utilization. In particular adding yeast culture and β1–4 galacto-oligosaccharides decreased methane emission in sheep [87] Propionibacteriaand lactobacilli-based probiotics were tested in sheep modified the bacterial population have been suggested to be useful to reduce the incidence of butyric and propionic subacute ruminal acidosis in sheep [88].

A study conducted in sheep showed that probiotic microorganisms are been used to improve food safety for consumers. Delcenserie et al. [89], found in their study that the presence of *Bifidobacteria choerinum* may be used as an indicator of fecal contamination of mutton. The study findings suggest that detection and identification of Bifidobacteria correlated with *E. coli* numbers can be used to improve hygienic quality during mutton processing.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

an effect on the average daily gain was observed. Research findings by Gyenai et al. [36] sup-

There is an increasing market demand for nonfat goat milk and milk products such as yoghurt containing probiotics. A nonfat yoghurt has been developed from goat milk and is enriched with probiotic strains *Lactobacillus acidophilus* and *Bifidobacterium spp.* [82]. Other probiotic microbes have been used to develop different types of fermented drinking milk form goat milk. *Lactobacillus acidophilus* LA-5, *Bifidobacterium animalis* subsp. *lactis* BB-12 novel putative probiotic *Propionibacterium jensenii* 702 co-culturing in goat milk affected their viability and physico-chemical properties of the milk [83]. In a similar study, goat milk fermented with *Lactobacillus fermentum* ME-3 and tested in healthy human subject reduced peroxidized lipoproteins levels, decreased 8-isoprostanes, improved total antioxidant activity and demonstrated an anti-atherogenic effects. The population and activity of lactic acid bacteria in milk

In sheep production, probiotics have been applied to improve feed digestion and gut health. Two probiotics, *Saccharomyces cerevisiae* and *Aspergillus oryzae tested in* sheep had no effect on Nitrogen digestibility *and* net microbial protein flow in the duodenum [85]. In another study, a probiotic mixture containing *Bacillus licheniformis* and *Bacillus subtilis* administered in ewes at late pregnancy and lactation reduced mortality in young lambs. In addition, probiotic treatment increased daily milk yield per ewe and fat and protein content of milk were also increased [54]. In a study by Rigobelo et al. [86], administration of a probiotic mixture containing *Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus* and *Enterococcus faecium* to sheep infected with a non-O157 Shiga toxin-producing *Escherichia coli* (a foodborne pathogen of humans) reduced the fecal shedding of the pathogen. Probiotic treatment in sheep has beneficial effect on rumen methanogenesis, energy retention and Nitrogen utilization. In particular adding yeast culture and β1–4 galacto-oligosaccharides decreased methane emission in sheep [87] Propionibacteriaand lactobacilli-based probiotics were tested in sheep modified the bacterial population have been suggested to be useful to reduce the incidence of butyric and propionic subacute rumi-

A study conducted in sheep showed that probiotic microorganisms are been used to improve food safety for consumers. Delcenserie et al. [89], found in their study that the presence of *Bifidobacteria choerinum* may be used as an indicator of fecal contamination of mutton. The study findings suggest that detection and identification of Bifidobacteria correlated with *E. coli*

numbers can be used to improve hygienic quality during mutton processing.

ports the use of probiotics in goats to enhance microbial retention in the rumen.

was affected after fermentation with *Lactobacillus fermentum* ME-3 [84].

**5.3. Probiotics for sheep**

142 Probiotics - Current Knowledge and Future Prospects

nal acidosis in sheep [88].

**Conflict of interest**

The authors declare no conflict of interest.

Sarah Adjei-Fremah1 , Kingsley Ekwemalor1 , Mulumebet Worku1 \* and Salam Ibrahim<sup>2</sup>

\*Address all correspondence to: worku@ncat.edu

1 Department of Animal Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC, USA

2 Department of Family and Consumer Sciences, North Carolina Agricultural and Technical State University, Greensboro, NC, USA

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**Chapter 9**

**Provisional chapter**

**Probiotics, an Alternative Measure to Chemotherapy in**

The use of chemotherapy in treating and enhancing the growth of fish has been widely criticized due to its negative environmental consequence. Hence, the use of probiotics which are bio-friendly seems to be a promising alternative. Therefore, the importance of probiotics in fish production was critically reviewed in line with their growth rate, disease treatment, and immune boosting. It was, however, realized that probiotics such as *Lactobacillus fermentum* and *Saccharomyces cerevisiae* cultured from maize slurry and palm wine, respectively, could serve as good probiotics, which could enhance faster growth rate and wound-healing rate. Probiotics are, therefore, recommended to the fish farmers

**Probiotics, an Alternative Measure to Chemotherapy in** 

DOI: 10.5772/intechopen.72923

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Aquaculture is an ancient occupation of man in which its fast growth due to rapid development has given birth to modern equipment and technology leading to its intensification and commercialization. This development has placed disease problems on the threatening side, making it to be a major constraint to the culture of many aquatic species and consequential impediment on economic and social development in many countries [1]. Owing to the artificial conditions posed by intensive rearing, farmed fish is more susceptible to disease agents than fish in natural aquatic environments [2]. Fish diseases constitute the major limiting factor in aquaculture production since the disease causative agents thrive well in water. Various types of bacterial diseases in fish have been encountered in fresh water fishes across the globe [3]. Jakhar et al. [4] reported that bacterial pathogens cause heavy mortality in both cultured

**Fish Production**

**Abstract**

**1. Introduction**

**Fish Production**

Olumuyiwa Ayodeji Akanmu

Olumuyiwa Ayodeji Akanmu

http://dx.doi.org/10.5772/intechopen.72923

and wild fish/shell species over the world.

Additional information is available at the end of the chapter

so as to increase the profitability of the aquaculture business.

**Keywords:** *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, probiotics

Additional information is available at the end of the chapter

[89] Delcenserie V, Loncaric D, Bonaparte C, Upmann M, China B, Daube G, Gavini F. Bifidobacteria as indicators of faecal contamination along a sheep meat production chain. Journal of Applied Microbiology. 2008;**104**(1):276-284

**Provisional chapter**

#### **Probiotics, an Alternative Measure to Chemotherapy in Fish Production Fish Production**

**Probiotics, an Alternative Measure to Chemotherapy in** 

DOI: 10.5772/intechopen.72923

Olumuyiwa Ayodeji Akanmu

[88] Lettat A, Nozière P, Silberberg M, Morgavi DP, Berger C, Martin C. Rumen microbial and fermentation characteristics are affected differently by bacterial probiotic supplementation during induced lactic and subacute acidosis in sheep. BMC Microbiology. 2012;**12**(1):142

[89] Delcenserie V, Loncaric D, Bonaparte C, Upmann M, China B, Daube G, Gavini F. Bifidobacteria as indicators of faecal contamination along a sheep meat production

chain. Journal of Applied Microbiology. 2008;**104**(1):276-284

150 Probiotics - Current Knowledge and Future Prospects

Olumuyiwa Ayodeji Akanmu Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72923

#### **Abstract**

The use of chemotherapy in treating and enhancing the growth of fish has been widely criticized due to its negative environmental consequence. Hence, the use of probiotics which are bio-friendly seems to be a promising alternative. Therefore, the importance of probiotics in fish production was critically reviewed in line with their growth rate, disease treatment, and immune boosting. It was, however, realized that probiotics such as *Lactobacillus fermentum* and *Saccharomyces cerevisiae* cultured from maize slurry and palm wine, respectively, could serve as good probiotics, which could enhance faster growth rate and wound-healing rate. Probiotics are, therefore, recommended to the fish farmers so as to increase the profitability of the aquaculture business.

**Keywords:** *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, probiotics

#### **1. Introduction**

Aquaculture is an ancient occupation of man in which its fast growth due to rapid development has given birth to modern equipment and technology leading to its intensification and commercialization. This development has placed disease problems on the threatening side, making it to be a major constraint to the culture of many aquatic species and consequential impediment on economic and social development in many countries [1]. Owing to the artificial conditions posed by intensive rearing, farmed fish is more susceptible to disease agents than fish in natural aquatic environments [2]. Fish diseases constitute the major limiting factor in aquaculture production since the disease causative agents thrive well in water. Various types of bacterial diseases in fish have been encountered in fresh water fishes across the globe [3]. Jakhar et al. [4] reported that bacterial pathogens cause heavy mortality in both cultured and wild fish/shell species over the world.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

However, prevention and control of diseases have led to a substantial increase in the use of broadspectrum chemotherapeutics, which has been reported to cause development of resistant bacteria, reduction in yield, and introduction of potential hazard to public health, the environment, and killing of the microbial flora in the digestive tracts, which is beneficial to the fish [5]. The development of resistant bacterial genes as a result of exposure to antimicrobial agents has not only made the drugs applied useless, but has also made the animals treated with it not safe for human consumption; therefore, it turned the treatment exercise to a wasteful process, which eventually makes this to be a major disadvantage of using synthetic antibiotics in aquaculture [6]. The success of modern aquaculture among others hinges on the use of biological control agents for diseases, and this depends on the fact of microbial antagonism [3] and triggering immune response to disease challenge.

include: nonviable, which are dried probiotics; freeze-dried, which are probiotics that thrive well at freezing point; fermentation probiotics, which are produced through fermentation; and viable probiotics, which are living probiotics with guaranteed shelf life [12]. Probiotics have been demonstrated to have potentials for enhancing fish immunity [13], growth [14], wound healing [15], and are eco-friendly [16]. A successful probiotic is expected to be antagonistic to pathogens, by producing antimicrobial substances, which are harmful to the pathogens. In addition, the probiotics should have the capacity to colonize the fish by adhesion and produce important substances like vitamins, which has beneficial effect on the host, in the

Probiotics, an Alternative Measure to Chemotherapy in Fish Production

http://dx.doi.org/10.5772/intechopen.72923

153

Probiotics such as *Lactobacillus, Saccharomyces species* and their combinations have been found useful in aquaculture production [15, 18]. The administration of diets fortified with these probiotics have improved growth in *Oreochromis niloticus* [14], increased immunity in *Cyprinus carpio* [19], improved wound healing in *Clarias gariepinus* [20], and in *Heterobranchus bidorsalis* [15]. It would be of interest to understand the applicability of this bio-technique in advancing the fate of aquaculture and food security. Maintenance of hygiene and especially, chemotherapeutics are widely used as interventions on control of diseases of aquatic animals. However, intensive use of chemicals had contributed to the development of resistant strains of pathogens. Hence, there is the need for natural preventives for improving resistance in fisheries and aquaculture. Meanwhile, a large percentage of culture systems still depend mostly on the use of chemotherapeutic agents in treating and controlling the widespread of these diseases. The abuse of chemotherapeutics in fish farming has led to development of drug-resistant bacteria and multiple antibiotic-resistant in the aquaculture industry [21]. This approach has sometimes resulted in the spread of epizootic diseases and severe economic losses. Moreover, chemotherapy may kill or inhibit the normal micro flora in the digestive tract, which is beneficial to fish [22]. Therefore, there is an urgent need to develop alternative approach to the

The principal objectives of the food security through aquaculture can only be achieved in the face of increase in growth and survival, feed efficiency, and disease resistance of culturable fish species, which reflects positively on production costs. The use of probiotics, which control pathogens through a variety of mechanisms that targets these attributes are viewed as an alternative to the use of antimicrobial agents [23] but the potentials of this technique have to be tested in many indigenous culturable fish species. With increasing demand for eco-friendly aquaculture, the use of probiotics in aquaculture is now widely accepted [24]. Positive effects of applying certain beneficial bacteria in aquaculture have also been well documented [25].

**2. Standards considered in selecting microorganisms as probiotics**

For a microorganism to be considered as a good probiotics candidate, it should be able to exhibit these properties: antagonistic properties through the production of antimicrobial materials such as hydrogen peroxide [26] or siderophores [27]. They should be able to colonize other microorganisms in the fish organ through adhesion [17]. The microorganisms are expected to be viable for long period of time under storage [28]. Adhesion is one of the most

form of growth promotion or protecting the fish against bacterial pathogens [17].

indiscriminate use of antibiotics in fish production.

Generally, the immune system of aquatic organisms is affected by periodic and unexpected changes in their environment. Adverse environmental situations may acutely or chronically, stress the fish, altering some of their biochemical parameters and suppressing their innate and adaptive immune responses [7]. This triggers nonspecific defense mechanisms, which plays important role at all stages of infection. Fish, particularly, depends more heavily on these nonspecific defense mechanisms than mammals. Therefore, there has been an increasing interest in boosting the nonspecific immune system of fish for the treatment and prophylactic measure against disease in the last decade using biological and eco-friendly approach [8].

Probiotics are microbial dietary adjuvant that beneficially affect the host physiology by modulating mucosal and systemic immunity, as well as improve nutritional and microbial balance in the intestinal tract [9]. These biological agents have been utilized for disease control, supplements to improve growth, and in some cases as a means of replacing antimicrobial compounds in aquaculture. Probiotics have proven to inhibit the growth of pathogens through production of antagonistic compounds, competition for attachment sites, nutrients, and alterations of enzymatic activity of pathogens, immune-stimulatory functions, and nutritional benefits such as improvement in digestibility and utilization in feed [10]. Hence, the concept of utilizing probiotics in animal feed, particularly, poultry and fish, is fast gaining acceptance [11]. The objective of prevention and control of disease can be achieved by the use of probiotics. Probiotics are characterized by their ability to adhere and colonize the gastro intestinal tract (GIT) of the hosts and able to replicate to high numbers. These organisms must be able to produce antimicrobial substances and withstand the acidic environment of the GIT of the host animals. Probiotics are known to play an important role in developing innate immunity among the fishes; therefore, help them to fight against any pathogenic bacteria as well as against environmental stressors [11].

Probiotics can be introduced into culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. The use of probiotics will prove a new eco-friendly alternative measure for sustainable aquaculture. A wide range of gram positive bacteria have been evaluated as probiotics. This includes *Aspergillus oryzae, Lactobacillus, Bacillus, Micrococcus, Carnobacterium, Enterococcus, Streptococcus, and Saccharomyces species*. The products of probiotics could be administered through water or incorporated in feed, either singly or in combination [11]. Administration of the probiotics proved harmless to the host as well as human being; it also results in improved resistance to infectious diseases in the hosts. However, the dimensions of the effects of probiotics have to be assessed for different fish species. Probiotics could be prepared in different types which include: nonviable, which are dried probiotics; freeze-dried, which are probiotics that thrive well at freezing point; fermentation probiotics, which are produced through fermentation; and viable probiotics, which are living probiotics with guaranteed shelf life [12]. Probiotics have been demonstrated to have potentials for enhancing fish immunity [13], growth [14], wound healing [15], and are eco-friendly [16]. A successful probiotic is expected to be antagonistic to pathogens, by producing antimicrobial substances, which are harmful to the pathogens. In addition, the probiotics should have the capacity to colonize the fish by adhesion and produce important substances like vitamins, which has beneficial effect on the host, in the form of growth promotion or protecting the fish against bacterial pathogens [17].

However, prevention and control of diseases have led to a substantial increase in the use of broadspectrum chemotherapeutics, which has been reported to cause development of resistant bacteria, reduction in yield, and introduction of potential hazard to public health, the environment, and killing of the microbial flora in the digestive tracts, which is beneficial to the fish [5]. The development of resistant bacterial genes as a result of exposure to antimicrobial agents has not only made the drugs applied useless, but has also made the animals treated with it not safe for human consumption; therefore, it turned the treatment exercise to a wasteful process, which eventually makes this to be a major disadvantage of using synthetic antibiotics in aquaculture [6]. The success of modern aquaculture among others hinges on the use of biological control agents for diseases, and this depends on the fact of microbial antagonism [3] and triggering immune response to disease challenge.

152 Probiotics - Current Knowledge and Future Prospects

Generally, the immune system of aquatic organisms is affected by periodic and unexpected changes in their environment. Adverse environmental situations may acutely or chronically, stress the fish, altering some of their biochemical parameters and suppressing their innate and adaptive immune responses [7]. This triggers nonspecific defense mechanisms, which plays important role at all stages of infection. Fish, particularly, depends more heavily on these nonspecific defense mechanisms than mammals. Therefore, there has been an increasing interest in boosting the nonspecific immune system of fish for the treatment and prophylactic mea-

sure against disease in the last decade using biological and eco-friendly approach [8].

fight against any pathogenic bacteria as well as against environmental stressors [11].

Probiotics can be introduced into culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. The use of probiotics will prove a new eco-friendly alternative measure for sustainable aquaculture. A wide range of gram positive bacteria have been evaluated as probiotics. This includes *Aspergillus oryzae, Lactobacillus, Bacillus, Micrococcus, Carnobacterium, Enterococcus, Streptococcus, and Saccharomyces species*. The products of probiotics could be administered through water or incorporated in feed, either singly or in combination [11]. Administration of the probiotics proved harmless to the host as well as human being; it also results in improved resistance to infectious diseases in the hosts. However, the dimensions of the effects of probiotics have to be assessed for different fish species. Probiotics could be prepared in different types which

Probiotics are microbial dietary adjuvant that beneficially affect the host physiology by modulating mucosal and systemic immunity, as well as improve nutritional and microbial balance in the intestinal tract [9]. These biological agents have been utilized for disease control, supplements to improve growth, and in some cases as a means of replacing antimicrobial compounds in aquaculture. Probiotics have proven to inhibit the growth of pathogens through production of antagonistic compounds, competition for attachment sites, nutrients, and alterations of enzymatic activity of pathogens, immune-stimulatory functions, and nutritional benefits such as improvement in digestibility and utilization in feed [10]. Hence, the concept of utilizing probiotics in animal feed, particularly, poultry and fish, is fast gaining acceptance [11]. The objective of prevention and control of disease can be achieved by the use of probiotics. Probiotics are characterized by their ability to adhere and colonize the gastro intestinal tract (GIT) of the hosts and able to replicate to high numbers. These organisms must be able to produce antimicrobial substances and withstand the acidic environment of the GIT of the host animals. Probiotics are known to play an important role in developing innate immunity among the fishes; therefore, help them to Probiotics such as *Lactobacillus, Saccharomyces species* and their combinations have been found useful in aquaculture production [15, 18]. The administration of diets fortified with these probiotics have improved growth in *Oreochromis niloticus* [14], increased immunity in *Cyprinus carpio* [19], improved wound healing in *Clarias gariepinus* [20], and in *Heterobranchus bidorsalis* [15]. It would be of interest to understand the applicability of this bio-technique in advancing the fate of aquaculture and food security. Maintenance of hygiene and especially, chemotherapeutics are widely used as interventions on control of diseases of aquatic animals. However, intensive use of chemicals had contributed to the development of resistant strains of pathogens. Hence, there is the need for natural preventives for improving resistance in fisheries and aquaculture. Meanwhile, a large percentage of culture systems still depend mostly on the use of chemotherapeutic agents in treating and controlling the widespread of these diseases. The abuse of chemotherapeutics in fish farming has led to development of drug-resistant bacteria and multiple antibiotic-resistant in the aquaculture industry [21]. This approach has sometimes resulted in the spread of epizootic diseases and severe economic losses. Moreover, chemotherapy may kill or inhibit the normal micro flora in the digestive tract, which is beneficial to fish [22]. Therefore, there is an urgent need to develop alternative approach to the indiscriminate use of antibiotics in fish production.

The principal objectives of the food security through aquaculture can only be achieved in the face of increase in growth and survival, feed efficiency, and disease resistance of culturable fish species, which reflects positively on production costs. The use of probiotics, which control pathogens through a variety of mechanisms that targets these attributes are viewed as an alternative to the use of antimicrobial agents [23] but the potentials of this technique have to be tested in many indigenous culturable fish species. With increasing demand for eco-friendly aquaculture, the use of probiotics in aquaculture is now widely accepted [24]. Positive effects of applying certain beneficial bacteria in aquaculture have also been well documented [25].

#### **2. Standards considered in selecting microorganisms as probiotics**

For a microorganism to be considered as a good probiotics candidate, it should be able to exhibit these properties: antagonistic properties through the production of antimicrobial materials such as hydrogen peroxide [26] or siderophores [27]. They should be able to colonize other microorganisms in the fish organ through adhesion [17]. The microorganisms are expected to be viable for long period of time under storage [28]. Adhesion is one of the most important criteria for probiotic bacteria because it is considered a pre-requisite for colonization [29]. Probiotic microorganisms will of course have to be nonpathogenic and nontoxic in order to avoid undesirable side effects when administered to fish. Tests of antagonisms, which include studies of adhesion and in-vitro challenged tests, challenged experiments in which fish treated with friendly bacteria are subjected to pathogens in order to evaluate the efficacy of the probiotics by using survival rate as an indicator are important considerable factors in selecting probiotics [30]. The interest of the probiotic use is centered on terrestrial organisms and the term probiotic inevitably is referred to gram positive bacteria associated with the genus *Lactobacillus species.* Panigrahi et al. [28] submission, however, requires some considerations to humans and terrestrial animals. It could be assumed in aquaculture that the intestinal microbiota does not exist as an entity by itself but there is a constant interaction with the environment and the host functions [31]. The bacteria in the aquatic medium could either be ingested with the feed or when the host drinks water. Terrestrial animals (mammals) inherit an important part of the initially colonizing bacteria through contact with the mother, while aquatic species usually spawn eggs in water, without further contact with their parents. This allows the ambient bacteria to colonize intestinal tract, gills, or skin of newly born animals/larvae, which have not fully developed.

the ability to degrade organic materials, reduce ammonia, and inhibit the growth of pathogens by outcompeting them [33]. Lactic acid bacteria are a heterogeneous group of bacteria that are generally considered safe for use in food and food products. Lactic acid bacteria have been used for lactic acid fermentation of sorghum-or maize-based cereals used as infant weaning foods, for example, Pap (*ogi*) prepared from maize slurry [34]. Lactic acid bacteria are spherical, cocci, coccobacilli, or rods and divide in one plane only with the exception of *Pediococcus species* [35]. Lactic acid bacteria have no strict taxonomic significance although they have been shown by serological techniques and 16S ribosomal RNA cataloging to be phylogenetically related. They share a number of common features as earlier stated. Most of these organisms are aero-tolerant anaerobes, which lack cytochromes and porphyrins. The lack of these two components in their systems explains why they are negative to catalase and oxidase tests [36]. The antibacterial effect of lactic acid bacteria (LAB) is therefore ascribed to its tendency to produce

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155

antibiotics-like substances (bacteriocins) such as *Acidophilin, Lactolin,* and *Lactocidin*.

*Saccharomyces cerevisiae* is budding yeast species commonly used in baking and brewing, owing to its fermenting property or ability. The cells are ovoid in shape, 5–10 μm in diameter. It reproduces by a division process known as budding. All strains of *S. cerevisiae* can grow aerobically on glucose, maltose, and trehalose but cannot grow on lactose and cellobiose. It is a single-celled organism, which can easily be cultured with short generation time of about 1.5–2 hours doubling time at a temperature of 30°C. It contains various immune-stimulating compounds such as β-glucans, nucleic acid, and mannan oligosaccharides, which have been reported to enhance immune response and growth of various fish species [37]. Mesalhy et al. [38] recorded higher growth rate in the study carried out using probiotic-supplemented diets on *Oreochromis niloticus* than those kept on basal diet. It was concluded that addition of *Bacillus subtilis* and *S. cerevisiae* enhanced the growth performance, feed utilization, and mitigated the effects of population density, which is the main growth-inhibiting factor in intensive aquaculture systems. The best food conversion rate (FCR) values were recorded in probiotic-supplemented diets, and it was concluded that the probiotic used improved feed utilization, which practically showed that the probiotic used can reduce the amount of feed necessary for animal growth and thus, reduce the cost of production [37].

*Saccharomyces, Clostridium, Bacillus, Enterococcus, Lactobacillus, Shewanella, Leuconostoc, Lactococcus, Carnobacterium, and Aeromonas species* are the commonly used probiotics in fish culture practices [11]. These probiotics have been reported to produce beneficial results to the host organisms. *Bacillus* species increased survival and production of channel catfish (*Ictalurus punctatus*), improved growth and immunity of Nile tilapia (*Oreochromis niloticus*) was achieved through feeding of diet containing *Bacillus subtilis* and *Rhodopseudomonas*, and rainbow trout (*Oncorhynchus mykiss*) was protected against *Vibrio anguillarum* by *Pseudomonas fluorescens* [9]. Generally, probiotics have demonstrated the ability to increase fish growth by enhancing the feed conversion efficiency, as well as confer protection against harmful bacteria by competitive exclusion, production of organic acids (formic acid, acetic acid, and lactic acid), hydrogen peroxide, and several

other compounds [37]. They can also effectively trigger the fish immune system [37].

Abdul El-Halim et al. [39] discovered that the addition of living yeast in diet improved the performance of *Oreochromis niloticus*. Scholz et al. [40] also reported improved growth and survival of sea bass fry with *S. cerevisiae* and attributed this to adherence ability of *S. cerevisiae* cells to the gut

#### **3. Test for pathogenicity of the selected strains**

Microorganisms considered as probiotic candidates should be scrutinized for pathogenicity on the host animals by challenging the target animals with the probiotic microorganisms. The challenged organisms could be administered to the target species through injection, immersion, or addition into the feed. The test of pathogenicity could either be carried out in-vitro or in-vivo.


#### **4. The probiotics characteristics of Lactic acid bacteria and Yeast**

Lactic acid bacteria are potential probiotic candidates in aquaculture and are also known to be a normal inhabitant in the intestine of healthy fish [14]. Most lactic acid bacteria are harmless, while some strains have been reported to have beneficial effects on fish health and are antagonistic to pathogens [32]. Strains of lactic acid bacteria are the most common microbes employed as probiotics. Most probiotic strains belong to the genus *Lactobacillus*. *Lactobacillus species* have the ability to degrade organic materials, reduce ammonia, and inhibit the growth of pathogens by outcompeting them [33]. Lactic acid bacteria are a heterogeneous group of bacteria that are generally considered safe for use in food and food products. Lactic acid bacteria have been used for lactic acid fermentation of sorghum-or maize-based cereals used as infant weaning foods, for example, Pap (*ogi*) prepared from maize slurry [34]. Lactic acid bacteria are spherical, cocci, coccobacilli, or rods and divide in one plane only with the exception of *Pediococcus species* [35]. Lactic acid bacteria have no strict taxonomic significance although they have been shown by serological techniques and 16S ribosomal RNA cataloging to be phylogenetically related. They share a number of common features as earlier stated. Most of these organisms are aero-tolerant anaerobes, which lack cytochromes and porphyrins. The lack of these two components in their systems explains why they are negative to catalase and oxidase tests [36]. The antibacterial effect of lactic acid bacteria (LAB) is therefore ascribed to its tendency to produce antibiotics-like substances (bacteriocins) such as *Acidophilin, Lactolin,* and *Lactocidin*.

important criteria for probiotic bacteria because it is considered a pre-requisite for colonization [29]. Probiotic microorganisms will of course have to be nonpathogenic and nontoxic in order to avoid undesirable side effects when administered to fish. Tests of antagonisms, which include studies of adhesion and in-vitro challenged tests, challenged experiments in which fish treated with friendly bacteria are subjected to pathogens in order to evaluate the efficacy of the probiotics by using survival rate as an indicator are important considerable factors in selecting probiotics [30]. The interest of the probiotic use is centered on terrestrial organisms and the term probiotic inevitably is referred to gram positive bacteria associated with the genus *Lactobacillus species.* Panigrahi et al. [28] submission, however, requires some considerations to humans and terrestrial animals. It could be assumed in aquaculture that the intestinal microbiota does not exist as an entity by itself but there is a constant interaction with the environment and the host functions [31]. The bacteria in the aquatic medium could either be ingested with the feed or when the host drinks water. Terrestrial animals (mammals) inherit an important part of the initially colonizing bacteria through contact with the mother, while aquatic species usually spawn eggs in water, without further contact with their parents. This allows the ambient bacteria to colonize intestinal tract, gills, or skin of newly born ani-

Microorganisms considered as probiotic candidates should be scrutinized for pathogenicity on the host animals by challenging the target animals with the probiotic microorganisms. The challenged organisms could be administered to the target species through injection, immersion, or addition into the feed. The test of pathogenicity could either be carried out in-vitro or in-vivo. **a. In-vitro antagonism tests:** Common way to screen the candidate probiotics is to perform in-vitro antagonism tests in which the pathogens are exposed to the candidate probiotics or their extracellular products in liquids [32] or solid medium. Depending on the extract arrangement of the tests, candidate probiotics can be selected based on the competition for nutrients [32]. The pre-selection of probiotics candidate based on these in-vitro antago-

**b. In-vivo antagonism tests:** Pathogenicity effects of microorganisms considered as probiotics could also be tested in-vivo to determine the safety level of the tested probiotic candidate.

Lactic acid bacteria are potential probiotic candidates in aquaculture and are also known to be a normal inhabitant in the intestine of healthy fish [14]. Most lactic acid bacteria are harmless, while some strains have been reported to have beneficial effects on fish health and are antagonistic to pathogens [32]. Strains of lactic acid bacteria are the most common microbes employed as probiotics. Most probiotic strains belong to the genus *Lactobacillus*. *Lactobacillus species* have

mals/larvae, which have not fully developed.

154 Probiotics - Current Knowledge and Future Prospects

**3. Test for pathogenicity of the selected strains**

nism tests has often led to the finding of effective probiotics [26].

**4. The probiotics characteristics of Lactic acid bacteria and Yeast**

*Saccharomyces cerevisiae* is budding yeast species commonly used in baking and brewing, owing to its fermenting property or ability. The cells are ovoid in shape, 5–10 μm in diameter. It reproduces by a division process known as budding. All strains of *S. cerevisiae* can grow aerobically on glucose, maltose, and trehalose but cannot grow on lactose and cellobiose. It is a single-celled organism, which can easily be cultured with short generation time of about 1.5–2 hours doubling time at a temperature of 30°C. It contains various immune-stimulating compounds such as β-glucans, nucleic acid, and mannan oligosaccharides, which have been reported to enhance immune response and growth of various fish species [37]. Mesalhy et al. [38] recorded higher growth rate in the study carried out using probiotic-supplemented diets on *Oreochromis niloticus* than those kept on basal diet. It was concluded that addition of *Bacillus subtilis* and *S. cerevisiae* enhanced the growth performance, feed utilization, and mitigated the effects of population density, which is the main growth-inhibiting factor in intensive aquaculture systems. The best food conversion rate (FCR) values were recorded in probiotic-supplemented diets, and it was concluded that the probiotic used improved feed utilization, which practically showed that the probiotic used can reduce the amount of feed necessary for animal growth and thus, reduce the cost of production [37].

*Saccharomyces, Clostridium, Bacillus, Enterococcus, Lactobacillus, Shewanella, Leuconostoc, Lactococcus, Carnobacterium, and Aeromonas species* are the commonly used probiotics in fish culture practices [11]. These probiotics have been reported to produce beneficial results to the host organisms. *Bacillus* species increased survival and production of channel catfish (*Ictalurus punctatus*), improved growth and immunity of Nile tilapia (*Oreochromis niloticus*) was achieved through feeding of diet containing *Bacillus subtilis* and *Rhodopseudomonas*, and rainbow trout (*Oncorhynchus mykiss*) was protected against *Vibrio anguillarum* by *Pseudomonas fluorescens* [9]. Generally, probiotics have demonstrated the ability to increase fish growth by enhancing the feed conversion efficiency, as well as confer protection against harmful bacteria by competitive exclusion, production of organic acids (formic acid, acetic acid, and lactic acid), hydrogen peroxide, and several other compounds [37]. They can also effectively trigger the fish immune system [37].

Abdul El-Halim et al. [39] discovered that the addition of living yeast in diet improved the performance of *Oreochromis niloticus*. Scholz et al. [40] also reported improved growth and survival of sea bass fry with *S. cerevisiae* and attributed this to adherence ability of *S. cerevisiae* cells to the gut and secretion of amylase enzymes, which increased digestibility of the diet. The probiotics used by Marzouk et al. [41] enhanced the growth performance of *Oreochromis niloticus* and suppressed the activity of the pathogenic bacteria in the intestine of the tested fish. The disease outbreak was reportedly prevented in fish with the use of *S. cerevisiae* and *Bacillus subtilis* as probiotics. This could have been possible with the ability of these microorganisms to attach and colonize the intestinal walls of the host animals, which eventually prevent other bacterial from getting access to the intestinal walls [41]. Li et al. [42] described positively influenced growth performance of brewer's yeast (*S. cerevisiae*) and feed efficiency of hybrid striped bass (*Morone chrysops xm saxatilis)* and resistance to *Streptococcus iniae* infection. In addition, results of immune response assays illustrated that brewer's yeast can be administered for relatively long periods without causing immune-suppression.

• Improving feed conversion rate and survival rate of aquatic species.

**5.1. Application of probiotics as biological control agents in aquaculture**

The benefits listed above substantiated [46] who anticipated that bacteria would be found useful both as food and as biological control agents of fish diseases and activators of the rate of nutrient regeneration in aquaculture. Zong-fu et al. [12] stated that potential probiotic microorganisms must be able to colonize the fish intestinal mucosa and produce materials, which are eco-friendly to the host but antagonistic to pathogens. Furthermore, optimal diet utilization by the host animal has been ensured with the use of probiotics, which stimulate the multiplication of gut micro flora in the host fish. It should be noted that an application of probiotics into the water and ponds may also have a positive effect on fish health by improving the water quality, since they modify the bacteria composition of the water and sediments.

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157

Probiotics have been applied in various aspects of aquaculture with promising results, especially in shrimp production [47]*. Luminous vibrio* has also been reported to be completely eliminated from the water column and from the sediment of ponds when probiotic strains selected for their inhibitory effect were used [48]. Hence, disease problems could be overcome by applying probiotic biotechnology, which is an application of microbial ecology [47]. Probiotics are expected to have a direct involvement in nutrients or vitamins [26]. They also enhance the growth of fish [49]. Lack of data on the efficacy of probiotics in commercial aquaculture is still affecting the sustained use of probiotics [50]. Most studies on the effects of probiotics on cultured aquaculture animals have emphasized a reduction in mortality or improved resistance against putative pathogens [51]. Probiotic can be added to the host or its

Probiotics could be provided to animals in different ways depending on the aim and objective of the study. However, the best method of administration is continuous feeding. This would ensure that the probiotics is present in the gut in a large number and able to metabolize and

The need to control the micro biota in hatching incubators through the alternative means in reducing the use of antibiotics needs to be adequately emphasized. Fish larvae may ingest substantial amount of bacteria by grazing on suspended particles and egg debris [52]. Ringo

• Reducing the use of chemotherapy.

ambient environment in several ways such as:

**5.2. Probiotic use in fish eggs, larvae, juvenile, and adult fish**

• Addition to artificial diet • Addition to culture water

• Addition via live food

produce its probiotics effects.

• Bathing

*Lactobacillus fermentum and S. cerevisiae* have also been reported to improve the growth performance and health status of fish species, *Oreochromis niloticus* [37] and *Mystus montanus* [3]. Various studies have been carried out using some bacteria strains as probiotics on fish species such as *Clarias gariepinus* and *Tilapia species,* but there is little or no information on the use of bacteria strains and yeast species as probiotics on indigenous species such as *H. bidorsalis.* Furthermore, information on the immune response of these probiotics on this fish species is not available [43]. *Lactobacillus fermentum* is a common bacteria strain, which has been used on different fish species. The wide occurrence and high antagonistic effects to the pathogens of the *L. fermentum and the S. cerevisiae* made them a good potential in testing for their probiotic ability and immune response. A beneficial effect by application of certain beneficial bacteria in human, pig, cattle, and poultry nutrition has been well documented by Jong [44]. However, the use of such probiotics in aquaculture is a relatively new concept [45]. Zhou et al. [17] reported the use of beneficial bacteria (probiotics) to displace pathogens by competitive processes being used in animal industry as a better remedy than administering antibiotics. This phenomenon is now gaining acceptance for the control of pathogens in aquaculture.

#### **5. Importance of probiotics in aquaculture**

Probiotics have been found beneficial in various ways such as:


and secretion of amylase enzymes, which increased digestibility of the diet. The probiotics used by Marzouk et al. [41] enhanced the growth performance of *Oreochromis niloticus* and suppressed the activity of the pathogenic bacteria in the intestine of the tested fish. The disease outbreak was reportedly prevented in fish with the use of *S. cerevisiae* and *Bacillus subtilis* as probiotics. This could have been possible with the ability of these microorganisms to attach and colonize the intestinal walls of the host animals, which eventually prevent other bacterial from getting access to the intestinal walls [41]. Li et al. [42] described positively influenced growth performance of brewer's yeast (*S. cerevisiae*) and feed efficiency of hybrid striped bass (*Morone chrysops xm saxatilis)* and resistance to *Streptococcus iniae* infection. In addition, results of immune response assays illustrated that brewer's yeast can be administered for relatively long periods without causing immune-suppression. *Lactobacillus fermentum and S. cerevisiae* have also been reported to improve the growth performance and health status of fish species, *Oreochromis niloticus* [37] and *Mystus montanus* [3]. Various studies have been carried out using some bacteria strains as probiotics on fish species such as *Clarias gariepinus* and *Tilapia species,* but there is little or no information on the use of bacteria strains and yeast species as probiotics on indigenous species such as *H. bidorsalis.* Furthermore, information on the immune response of these probiotics on this fish species is not available [43]. *Lactobacillus fermentum* is a common bacteria strain, which has been used on different fish species. The wide occurrence and high antagonistic effects to the pathogens of the *L. fermentum and the S. cerevisiae* made them a good potential in testing for their probiotic ability and immune response. A beneficial effect by application of certain beneficial bacteria in human, pig, cattle, and poultry nutrition has been well documented by Jong [44]. However, the use of such probiotics in aquaculture is a relatively new concept [45]. Zhou et al. [17] reported the use of beneficial bacteria (probiotics) to displace pathogens by competitive processes being used in animal industry as a better remedy than administering antibiotics. This

phenomenon is now gaining acceptance for the control of pathogens in aquaculture.

**5. Importance of probiotics in aquaculture** 

• Eliminating the stressors like NH<sup>3</sup>

156 Probiotics - Current Knowledge and Future Prospects

lation of beneficial gut flora.

Probiotics have been found beneficial in various ways such as:

• Providing additional nutrients thereby reducing feed costs.

• Stabilizing and controlling the microbial populations.

• Maintaining stable water quality parameters.

• Improving feed and make it to be more attractive.

• Preventing bacterial and viral infections.

• Maintaining desired conditions within the culture environment.

, NO<sup>2</sup>

, and NO3.

• Supporting growth through production of vitamins, minerals, nucleic acids, and by stimu-

The benefits listed above substantiated [46] who anticipated that bacteria would be found useful both as food and as biological control agents of fish diseases and activators of the rate of nutrient regeneration in aquaculture. Zong-fu et al. [12] stated that potential probiotic microorganisms must be able to colonize the fish intestinal mucosa and produce materials, which are eco-friendly to the host but antagonistic to pathogens. Furthermore, optimal diet utilization by the host animal has been ensured with the use of probiotics, which stimulate the multiplication of gut micro flora in the host fish. It should be noted that an application of probiotics into the water and ponds may also have a positive effect on fish health by improving the water quality, since they modify the bacteria composition of the water and sediments.

#### **5.1. Application of probiotics as biological control agents in aquaculture**

Probiotics have been applied in various aspects of aquaculture with promising results, especially in shrimp production [47]*. Luminous vibrio* has also been reported to be completely eliminated from the water column and from the sediment of ponds when probiotic strains selected for their inhibitory effect were used [48]. Hence, disease problems could be overcome by applying probiotic biotechnology, which is an application of microbial ecology [47]. Probiotics are expected to have a direct involvement in nutrients or vitamins [26]. They also enhance the growth of fish [49]. Lack of data on the efficacy of probiotics in commercial aquaculture is still affecting the sustained use of probiotics [50]. Most studies on the effects of probiotics on cultured aquaculture animals have emphasized a reduction in mortality or improved resistance against putative pathogens [51]. Probiotic can be added to the host or its ambient environment in several ways such as:


Probiotics could be provided to animals in different ways depending on the aim and objective of the study. However, the best method of administration is continuous feeding. This would ensure that the probiotics is present in the gut in a large number and able to metabolize and produce its probiotics effects.

#### **5.2. Probiotic use in fish eggs, larvae, juvenile, and adult fish**

The need to control the micro biota in hatching incubators through the alternative means in reducing the use of antibiotics needs to be adequately emphasized. Fish larvae may ingest substantial amount of bacteria by grazing on suspended particles and egg debris [52]. Ringo and Gatesoupe [26] added lactic acid bacteria (LAB) to larvae of some fish species and a significant reduction of larval mortality was recorded when the larvae were challenged with pathogenic microorganisms (*Vibrio*). Ref. [32] fed lactic acid bacteria to *Atlantic Cod* fry to look at the effect of lactic acid bacteria on the growth and survival rate of Atlantic Cod fry. The experimental fish were given short term bathing in a bacteria suspension of probiotic [27]. Long term exposure in the rearing water led to the reduction in mortality of fish [9]. Ref. [23] selected several strains with a positive effect on the survival and growth of artemia juvenile.

**5.5. Improvement in fish growth**

**5.6. Improve the hematology of fish**

reduction in level of oxygen (O<sup>2</sup>

(CO<sup>2</sup>

when the level of lactic acid bacteria in the feed was too high.

Inclusion of probiotics in the diets of fish species such as hybrid striped bass (*Morone chrysops xm saxatilis*), *Oreochromis niloticus*, catfish, and carp could improve the growth performance, body length, weight gain, and feed conversion ratio (FCR) of fish species [57]. Probiotics could also improve the body composition of fish fed with it. The addition of probiotics in the fish diets was reported to reduce the mortality rate. Gatesoupe [30] showed that turbot (*Scophthalmus maximus*) larvae fed with rotifers enriched with lactic acid bacteria had improved resistance against pathogenic vibrio infection, while noninfected fish showed slight increase in mortality

The hematological parameters of fish have been reported to be improved with the addition of probiotic bacterial into the diets of the experimental fish. For instance, the red blood cell counts (RBC) and white blood cell (WBC) of experimental fish were reported improved after being fed with probiotic bacterial [58]. Probiotics actively stimulate the proliferation of lymphocytes (both B and T cells) and further immunoglobulin production in fish [59]. Application of hematological techniques is, therefore, valuable in fish biology for the assessment of fish health and stress response. In the hemoglobin, oxygen is bound and released easily by iron (Fe) action contained in the hemoglobin molecule as blood transverse the pulmonary capillaries. Red blood cells (RBC), mean corpuscular hemoglobin (MCH), and hematocrit (HCT) have been reported by Adeyemo et al. [60] to indicate secondary responses of an organism to irritants. O'Neal and Weirich [61] describe decrease in erythrocytes to be the major and reliable indicators of various sources of stress in fish. Decrease in white blood cells (WBC) indicates vulnerability to stress and infection [58]. Decrease in red blood cells (RBC) indicates

) that is returned to the lungs. It also indicates malnutrition in animal. Decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (McHc) indicates anemia [58]. Differential counts of neutrophils and monocytes give the level of protection against bacterial invasion, while lymphocytes determine the level of immunity. High platelet values show that the fish is likely to withstand and get healed quickly from bruises or wounds, which could be acquired from fight or overstocking. Heterophil/Lymphocyte ratio is a reliable indicator of stress associated with injury [62]. Increase in heterophil/lymphocyte (H/L) ratio indicates stress. Probiotics also actively stimulate the proliferation of lymphocytes

Serum biochemistry deals with the level of various enzymes, minerals, and proteins in the blood. Biochemical values are sometimes variably or invariably affected by blood, sex, age, environment, nutritional status, and experimental factors. Serum is the preferred sample for chemistry analysis, although plasma is often used because of the difficulties of obtaining two samples from one animal. The yield of plasma from a sample is usually greater than that of serum. If plasma is used for the analysis, then the auto coagulant should be considered in result interpretation. Choudhury et al. [63] discovered that dietary supplements of ribonucleic acid significantly influenced the total serum, protein, albumin, and globulin of the experimental fish

(both B and T cells) and further immunoglobulin production in fish [59].

), which is being carried to the tissue and carbon dioxide

Probiotics, an Alternative Measure to Chemotherapy in Fish Production

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159

#### **5.3. Improving the immune response of the fish larvae**

The level of immune response exhibits by the host animals greatly depend on the immune stimulants such animal is able to produce. Immune stimulants are produced to resist or combat any foreign body or objects intended to infect such animal. The immune systems of fish larvae are less developed; therefore, depend on nonspecific immune response to fight against infection [21]. Observations obtained in experiments with warm-blooded animals indicate that probiotic (lactic acid bacteria) administered orally increased resistance to enteric infections [49]. There are many reports that bacterial compounds act as immune stimulant in fish; however, it is not clear whether bacteria administered as probiotic could have a beneficial effect on the immune response of cultured aquatic species [8]. The role of lactic acid bacteria (LAB) within the digestive tract of endothermic animals and humans has been extensively studied [30]. Few authors have tested in-vivo, the protection conferred by probiotics in fish experimentally infected with pathogens. Bernet et al. [53] found that *Lactobacillus* strains isolated from rotifers increased the resistance of Turbot larvae against a pathogenic *Vibrio* species. Gildberg at al. [32] demonstrated that *Carnobacterium* divergens decreased the mortality rate of Atlantic Cod fry challenged with *Vibrio anguillarum.* Douillet and Langdon [54] also reported that *Carnobacterium* administered to fry and fingerlings of Atlantic salmon reduced the mortality caused by *Aeromonas salmonicida, Vibrio ordalli, and Yersinia ruckeri.* The role of lactic acid bacteria as immune-modulators improves nonspecific defenses and is well-known for mammals [31]. Villamil [55] stated that this role has to be determined for fish. Most studies with probiotics conducted to date with fish have been undertaken with strains isolated and selected from aquatic environment and cultured animals.

#### **5.4. Improvement of water quality**

Water quality has been recorded to be improved at the addition of probiotics especially, *Bacillus species*. The rationale behind this is that gram positive *bacillus species* are generally efficient in converting organic matters back to CO<sup>2</sup> than gram negative bacteria [8]. Probiotics has also found its usage in water purification, especially with the culture of nitrifying bacteria in bio filters. Nitrifiers are responsible for the oxidation of ammonia to nitrite and subsequently to nitrate. The nitrifying cultures could be added to the ponds or tanks when an incidental increase of ammonia or nitrite levels is observed. Besides ammonia, nitrite toxicity is a common problem in fish culture especially in stagnant pond and re-circulatory system [56].

#### **5.5. Improvement in fish growth**

and Gatesoupe [26] added lactic acid bacteria (LAB) to larvae of some fish species and a significant reduction of larval mortality was recorded when the larvae were challenged with pathogenic microorganisms (*Vibrio*). Ref. [32] fed lactic acid bacteria to *Atlantic Cod* fry to look at the effect of lactic acid bacteria on the growth and survival rate of Atlantic Cod fry. The experimental fish were given short term bathing in a bacteria suspension of probiotic [27]. Long term exposure in the rearing water led to the reduction in mortality of fish [9]. Ref. [23] selected several strains with a positive effect on the survival and growth of artemia

The level of immune response exhibits by the host animals greatly depend on the immune stimulants such animal is able to produce. Immune stimulants are produced to resist or combat any foreign body or objects intended to infect such animal. The immune systems of fish larvae are less developed; therefore, depend on nonspecific immune response to fight against infection [21]. Observations obtained in experiments with warm-blooded animals indicate that probiotic (lactic acid bacteria) administered orally increased resistance to enteric infections [49]. There are many reports that bacterial compounds act as immune stimulant in fish; however, it is not clear whether bacteria administered as probiotic could have a beneficial effect on the immune response of cultured aquatic species [8]. The role of lactic acid bacteria (LAB) within the digestive tract of endothermic animals and humans has been extensively studied [30]. Few authors have tested in-vivo, the protection conferred by probiotics in fish experimentally infected with pathogens. Bernet et al. [53] found that *Lactobacillus* strains isolated from rotifers increased the resistance of Turbot larvae against a pathogenic *Vibrio* species. Gildberg at al. [32] demonstrated that *Carnobacterium* divergens decreased the mortality rate of Atlantic Cod fry challenged with *Vibrio anguillarum.* Douillet and Langdon [54] also reported that *Carnobacterium* administered to fry and fingerlings of Atlantic salmon reduced the mortality caused by *Aeromonas salmonicida, Vibrio ordalli, and Yersinia ruckeri.* The role of lactic acid bacteria as immune-modulators improves nonspecific defenses and is well-known for mammals [31]. Villamil [55] stated that this role has to be determined for fish. Most studies with probiotics conducted to date with fish have been undertaken with strains isolated and selected from aquatic environment and cultured

Water quality has been recorded to be improved at the addition of probiotics especially, *Bacillus species*. The rationale behind this is that gram positive *bacillus species* are generally effi-

also found its usage in water purification, especially with the culture of nitrifying bacteria in bio filters. Nitrifiers are responsible for the oxidation of ammonia to nitrite and subsequently to nitrate. The nitrifying cultures could be added to the ponds or tanks when an incidental increase of ammonia or nitrite levels is observed. Besides ammonia, nitrite toxicity is a common problem in fish culture especially in stagnant pond and re-circulatory system [56].

than gram negative bacteria [8]. Probiotics has

**5.3. Improving the immune response of the fish larvae**

158 Probiotics - Current Knowledge and Future Prospects

juvenile.

animals.

**5.4. Improvement of water quality**

cient in converting organic matters back to CO<sup>2</sup>

Inclusion of probiotics in the diets of fish species such as hybrid striped bass (*Morone chrysops xm saxatilis*), *Oreochromis niloticus*, catfish, and carp could improve the growth performance, body length, weight gain, and feed conversion ratio (FCR) of fish species [57]. Probiotics could also improve the body composition of fish fed with it. The addition of probiotics in the fish diets was reported to reduce the mortality rate. Gatesoupe [30] showed that turbot (*Scophthalmus maximus*) larvae fed with rotifers enriched with lactic acid bacteria had improved resistance against pathogenic vibrio infection, while noninfected fish showed slight increase in mortality when the level of lactic acid bacteria in the feed was too high.

#### **5.6. Improve the hematology of fish**

The hematological parameters of fish have been reported to be improved with the addition of probiotic bacterial into the diets of the experimental fish. For instance, the red blood cell counts (RBC) and white blood cell (WBC) of experimental fish were reported improved after being fed with probiotic bacterial [58]. Probiotics actively stimulate the proliferation of lymphocytes (both B and T cells) and further immunoglobulin production in fish [59]. Application of hematological techniques is, therefore, valuable in fish biology for the assessment of fish health and stress response. In the hemoglobin, oxygen is bound and released easily by iron (Fe) action contained in the hemoglobin molecule as blood transverse the pulmonary capillaries. Red blood cells (RBC), mean corpuscular hemoglobin (MCH), and hematocrit (HCT) have been reported by Adeyemo et al. [60] to indicate secondary responses of an organism to irritants. O'Neal and Weirich [61] describe decrease in erythrocytes to be the major and reliable indicators of various sources of stress in fish. Decrease in white blood cells (WBC) indicates vulnerability to stress and infection [58]. Decrease in red blood cells (RBC) indicates reduction in level of oxygen (O<sup>2</sup> ), which is being carried to the tissue and carbon dioxide (CO<sup>2</sup> ) that is returned to the lungs. It also indicates malnutrition in animal. Decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (McHc) indicates anemia [58]. Differential counts of neutrophils and monocytes give the level of protection against bacterial invasion, while lymphocytes determine the level of immunity. High platelet values show that the fish is likely to withstand and get healed quickly from bruises or wounds, which could be acquired from fight or overstocking. Heterophil/Lymphocyte ratio is a reliable indicator of stress associated with injury [62]. Increase in heterophil/lymphocyte (H/L) ratio indicates stress. Probiotics also actively stimulate the proliferation of lymphocytes (both B and T cells) and further immunoglobulin production in fish [59].

Serum biochemistry deals with the level of various enzymes, minerals, and proteins in the blood. Biochemical values are sometimes variably or invariably affected by blood, sex, age, environment, nutritional status, and experimental factors. Serum is the preferred sample for chemistry analysis, although plasma is often used because of the difficulties of obtaining two samples from one animal. The yield of plasma from a sample is usually greater than that of serum. If plasma is used for the analysis, then the auto coagulant should be considered in result interpretation. Choudhury et al. [63] discovered that dietary supplements of ribonucleic acid significantly influenced the total serum, protein, albumin, and globulin of the experimental fish (*Labeo rohita*). The highest plasma protein concentration was recorded in fish fed 25% yeastbased diet [64]. Kobeisy et al. [65] studied the roles of 0, 5, 10, and 20% dietary live yeast on the serum glucose of *Oreochromis niloticus* for 13 weeks. They recorded a significant increase in the serum glucose concentration, compared to the control group.

Production economics revealed that high stocking density of 40 fingerlings/m<sup>3</sup>

neutrophil numbers, RBC, and WBC (white blood cell) count in the stressed fish.

**5.8. Accelerates wound healing in fish**

**6. Conclusion**

**Author details**

Olumuyiwa Ayodeji Akanmu

University, Osogbo, Osun State, Nigeria

*pinus* is more profitable [82]. Therefore, to increase fish production, appropriate environmental conditions must be provided [83]. Sohrab et al. [84] reported that growth and nutritional indices were appropriate indicators in assessing the impact of the induced stresses in the Caspian roach larvae owing to stocking density. Many studies have mentioned the importance of the hematological indices to evaluate the stress status of fish as a result of stocking density [84]. Hematological parameters such as hematocrit (HCT), hemoglobin (HB), number of red blood cells (RBC), eosinophil (EOS), and heterophil (HET) in Beluga (*Huso huso*) were not affected by increasing stocking density [85]. Binukumari and Anbarasi [86] recorded decreased trend in

Wound occurs when the integrity of any tissue is compromised for instance: skin breaks, muscle tears, burns, or bone fractures. This is very common with scale less fish such as catfish. Fish skin is divided into three layers namely: epidermis, dermis, and hypodermis. The skin is the outer covering of an animal, which forms a barrier against harmful microorganisms and chemicals entering the body. It has the ability to constantly renew itself after injury. It is highly vulnerable to injury owing to its position outside the body of the animal. The process of wound healing depends on how deep the wound is. Healing of wounds is characterized by synthesis of collagen. Wound-healing studies have been carried out on *Heterobranchus bidorsalis* juveniles [15], rainbow trout [87], channel catfish [88], and Nile tilapia [89]. Erazo-Pagador and Din [20] reported *Clarias gariepinus* fed with diets with dietary ascorbic acid had more rapid and complete wound healing. Histological examination by Erazo-Pagador and Din [20] revealed that at 14 days after wounding, fish fed with diets without ascorbic acid had normal epidermis and dermis but muscle tissues were still regenerating, whereas fish fed with diets containing ascorbic acid had normal epidermis, dermis, and muscle tissues. Rapid wound healing is especially important in the intensive culture of African catfish. This is because these species behave aggressively, have no scales, and have strong pectoral spines that can inflict wounds, especially at high stocking densities [20].

Having mentioned all the benefits that could be derived from using probiotics in fish production, it will be very imperative to embrace the use of this eco-friendly method in fish culture.

Department of Fisheries and Wildlife Management, Faculty of Agriculture, Osun State

Address all correspondence to: olumuyiwa.akanmu@uniosun.edu.ng

profit index and best cost ratio. At high stocking density (40 fingerlings/m<sup>3</sup>

gave the highest

161

), raising of *C. garie-*

http://dx.doi.org/10.5772/intechopen.72923

Probiotics, an Alternative Measure to Chemotherapy in Fish Production

#### **5.7. Stress reduction in fish**

Stress is referred to as "the non-specific response of the body to any demand made upon it." Stresses are additives and increase the susceptibility of animals to disease while decreasing their growth rate and feed conversion efficiency [66]. The degree to which stress affects any particular fish is determined largely by the severity of the stress, its duration, and the health of the fish. Reduced or negative growth is commonly observed during stressful periods, while growth rates or derived parameters are often considered reliable indicators of stress and welfare [67]. Fish under intensive culture conditions are exposed to a variety of stressors owing to the economic realities of large scale production [68]. To enhance production, farmers often increase rearing densities beyond system capacities. Rearing at high density can cause stress through deterioration in water quality, overcrowding, or adverse social interactions [69]. High rearing density adversely increases fish susceptibility to disease, possibly as a result of chronically elevated cortisol levels, which have immune-suppressive and catabolic actions in fish [70]. The common symptoms of stress include: gasping at the surface for oxygen, lack of appetite for food, abnormal swimming position, and fish disease. Stocking density is one of the key factors determining profitability and economic sustainability of a fish farm. Meanwhile, farmers often increase rearing densities to intensify production [71] and these suboptimal conditions may result in chronic stress in fish culture [72]. Three types of stress indicators can be detected in fishes: release of corticosteroid hormones (for example cortisol) into blood circulation [73], changes in hematological parameters, and the whole animal performance like growth and survival rate [68].

Hormonal and blood parameters have frequently been used as indicators of stress in sturgeons [74]. Stocking density has been studied in many bony fishes [72]. Stocking density is one of the most important factors in aquaculture because it directly influences survival, growth, behavior, health, feeding, and production of fish under farmed conditions [75]. The effect of stocking density as a major factor affecting fish growth has been the subject of many studies [76]. Hematological parameters are important indices related to response of fish to different environmental conditions. They are considered as important stress indicators in estimating reactions of fish to various environmental conditions and assessment of its general physiological status [77]. The level of hematological and growth indices in fishes is an important parameter to evaluate the stress responses to various environmental conditions [78]. Many studies have confirmed the significance of the hematological parameters to assess the response of organism to the environment condition and their importance for estimating its general health condition and possible effect of exposure to stressors [79]. Stocking density is considered an environmental stressor in aquaculture [80]. This constitutes an important item in any fish culture operation. The result of the improvement in output with respect to stocking density is essential in an intensive production system [81] with the objective of profit maximization.

Production economics revealed that high stocking density of 40 fingerlings/m<sup>3</sup> gave the highest profit index and best cost ratio. At high stocking density (40 fingerlings/m<sup>3</sup> ), raising of *C. gariepinus* is more profitable [82]. Therefore, to increase fish production, appropriate environmental conditions must be provided [83]. Sohrab et al. [84] reported that growth and nutritional indices were appropriate indicators in assessing the impact of the induced stresses in the Caspian roach larvae owing to stocking density. Many studies have mentioned the importance of the hematological indices to evaluate the stress status of fish as a result of stocking density [84]. Hematological parameters such as hematocrit (HCT), hemoglobin (HB), number of red blood cells (RBC), eosinophil (EOS), and heterophil (HET) in Beluga (*Huso huso*) were not affected by increasing stocking density [85]. Binukumari and Anbarasi [86] recorded decreased trend in neutrophil numbers, RBC, and WBC (white blood cell) count in the stressed fish.

#### **5.8. Accelerates wound healing in fish**

(*Labeo rohita*). The highest plasma protein concentration was recorded in fish fed 25% yeastbased diet [64]. Kobeisy et al. [65] studied the roles of 0, 5, 10, and 20% dietary live yeast on the serum glucose of *Oreochromis niloticus* for 13 weeks. They recorded a significant increase in the

Stress is referred to as "the non-specific response of the body to any demand made upon it." Stresses are additives and increase the susceptibility of animals to disease while decreasing their growth rate and feed conversion efficiency [66]. The degree to which stress affects any particular fish is determined largely by the severity of the stress, its duration, and the health of the fish. Reduced or negative growth is commonly observed during stressful periods, while growth rates or derived parameters are often considered reliable indicators of stress and welfare [67]. Fish under intensive culture conditions are exposed to a variety of stressors owing to the economic realities of large scale production [68]. To enhance production, farmers often increase rearing densities beyond system capacities. Rearing at high density can cause stress through deterioration in water quality, overcrowding, or adverse social interactions [69]. High rearing density adversely increases fish susceptibility to disease, possibly as a result of chronically elevated cortisol levels, which have immune-suppressive and catabolic actions in fish [70]. The common symptoms of stress include: gasping at the surface for oxygen, lack of appetite for food, abnormal swimming position, and fish disease. Stocking density is one of the key factors determining profitability and economic sustainability of a fish farm. Meanwhile, farmers often increase rearing densities to intensify production [71] and these suboptimal conditions may result in chronic stress in fish culture [72]. Three types of stress indicators can be detected in fishes: release of corticosteroid hormones (for example cortisol) into blood circulation [73], changes in hematological parameters, and the whole animal performance

Hormonal and blood parameters have frequently been used as indicators of stress in sturgeons [74]. Stocking density has been studied in many bony fishes [72]. Stocking density is one of the most important factors in aquaculture because it directly influences survival, growth, behavior, health, feeding, and production of fish under farmed conditions [75]. The effect of stocking density as a major factor affecting fish growth has been the subject of many studies [76]. Hematological parameters are important indices related to response of fish to different environmental conditions. They are considered as important stress indicators in estimating reactions of fish to various environmental conditions and assessment of its general physiological status [77]. The level of hematological and growth indices in fishes is an important parameter to evaluate the stress responses to various environmental conditions [78]. Many studies have confirmed the significance of the hematological parameters to assess the response of organism to the environment condition and their importance for estimating its general health condition and possible effect of exposure to stressors [79]. Stocking density is considered an environmental stressor in aquaculture [80]. This constitutes an important item in any fish culture operation. The result of the improvement in output with respect to stocking density is essential in an intensive production system [81] with the objective of profit

serum glucose concentration, compared to the control group.

**5.7. Stress reduction in fish**

160 Probiotics - Current Knowledge and Future Prospects

like growth and survival rate [68].

maximization.

Wound occurs when the integrity of any tissue is compromised for instance: skin breaks, muscle tears, burns, or bone fractures. This is very common with scale less fish such as catfish. Fish skin is divided into three layers namely: epidermis, dermis, and hypodermis. The skin is the outer covering of an animal, which forms a barrier against harmful microorganisms and chemicals entering the body. It has the ability to constantly renew itself after injury. It is highly vulnerable to injury owing to its position outside the body of the animal. The process of wound healing depends on how deep the wound is. Healing of wounds is characterized by synthesis of collagen. Wound-healing studies have been carried out on *Heterobranchus bidorsalis* juveniles [15], rainbow trout [87], channel catfish [88], and Nile tilapia [89]. Erazo-Pagador and Din [20] reported *Clarias gariepinus* fed with diets with dietary ascorbic acid had more rapid and complete wound healing. Histological examination by Erazo-Pagador and Din [20] revealed that at 14 days after wounding, fish fed with diets without ascorbic acid had normal epidermis and dermis but muscle tissues were still regenerating, whereas fish fed with diets containing ascorbic acid had normal epidermis, dermis, and muscle tissues. Rapid wound healing is especially important in the intensive culture of African catfish. This is because these species behave aggressively, have no scales, and have strong pectoral spines that can inflict wounds, especially at high stocking densities [20].

#### **6. Conclusion**

Having mentioned all the benefits that could be derived from using probiotics in fish production, it will be very imperative to embrace the use of this eco-friendly method in fish culture.

#### **Author details**

Olumuyiwa Ayodeji Akanmu

Address all correspondence to: olumuyiwa.akanmu@uniosun.edu.ng

Department of Fisheries and Wildlife Management, Faculty of Agriculture, Osun State University, Osogbo, Osun State, Nigeria

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## *Edited by Shymaa Enany*

Probiotic has been used for centuries especially in fermented dairy products since Metchnikoff associated the intake of fermented milk with prolonged life. Probiotics confer many health benefits to humans, animals, and plants when administered in proper amounts. These benefits include the prevention of gastrointestinal infections and antibiotic-associated diarrhea, the reduction of serum cholesterol and allergenic and atopic complaints, and the protection of the immune system. Furthermore, the proper usage of probiotics could suppress *Helicobacter pylori* infection and Crohn's disease, improve inflammatory bowel disease, and prevent cancer.

In this book, we present specialists with experience in the field of probiotics exploring their current knowledge and their future prospects.

Published in London, UK © 2018 IntechOpen © YelenaYemchuk / iStock

Probiotics - Current Knowledge and Future Prospects

Probiotics

Current Knowledge and Future Prospects

*Edited by Shymaa Enany*