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Keywords:

  • bifidobacteria;
  • gut colonization;
  • stress tolerance;
  • nondigestible carbohydrate metabolism

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Probiotics are live microorganisms that when administered in adequate amounts confer a health benefit on the host. They are mainly bacteria from the genera Lactobacillus and Bifidobacterium. Traditionally, functional properties of lactobacilli have been studied in more detail than those of bifidobacteria. However, many recent studies have clearly revealed that the bifidobacterial population in the human gut is far more abundant than the population of lactobacilli. Although the ‘beneficial gut microbiota’ still remains to be elucidated, it is generally believed that the presence of bifidobacteria is associated with a healthy status of the host, and scientific evidence supports the benefits attributed to specific Bifidobacterium strains. To carry out their functional activities, bifidobacteria must be able to survive the gastrointestinal tract transit and persist, at least transiently, in the host. This is achieved using stress response mechanisms and adhesion and colonization factors, as well as by taking advantage of specific energy recruitment pathways. This review summarizes the current knowledge of the mechanisms involved in facilitating the establishment, colonization, and survival of bifidobacteria in the human gut.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Bifidobacteria are Gram-positive bacteria that, together with Collinsella, constitute the two main members of the Actinobacteria class inhabiting the human gut (Qin et al., 2010). Several studies have shown that bifidobacteria can occur at concentrations higher than 1010 cells per gram of feces, being more abundant in newborns and infants than in adults and elderly people (Arboleya et al., 2011). Also, current metagenomic studies support the notion that bifidobacteria constitute one of the main bacterial populations in healthy individuals (Qin et al., 2010). Their administration has been associated with beneficial effects, such as diarrhea prevention and normalization of gut dysfunction. Therefore, some Bifidobacterium strains have been categorized as probiotics.

To produce their effects, bifidobacteria must be able to survive the gastrointestinal transit and persist during a certain period of time in the gut. Thus, the study of the survival and colonization processes, as well as the metabolic advantages allowing these microorganisms to remain alive and metabolically active in the human gastrointestinal tract (GIT), are of pivotal importance to understand their functional activities. In this regard, the characterization of the mechanisms underlying the interactions of bifidobacteria with the host has been hampered for many years by the lack of molecular tools adapted for this genus. However, the recent development of expression vectors and mutant-generation systems, as well as the tremendous advance of functional genomics and other omics technologies, has allowed the scientific community to tackle the functionality of bifidobacteria using well established in vitro and in vivo models. In this way, the stress responses to physico-chemical gastrointestinal conditions have been elucidated, and the role of specific molecules involved in adhesion and colonization of the intestinal epithelium has been established. Furthermore, the main metabolic routes for assimilation of dietary carbohydrates that are not digested in the upper GIT have been unraveled. These studies have revealed a wide range of strategies that bifidobacteria use to be able to adapt to the specific environmental conditions of the human gut.

Coping with gastrointestinal stress factors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

During their passage through the GIT, orally administered probiotics find an assortment of harsh environmental conditions (Fig. 1). These conditions jeopardize the survival of these beneficial microorganisms, compromising their viability and functionality. These include digestive enzymes, acid pH in the stomach, defensins, and high concentrations of bile salts in the intestine. Understanding Bifidobacterium response to surmount GI stress factors is essential for a rational selection of probiotic strains and the development of a molecular toolbox to improve their performance.

image

Figure 1. Schematic representation of the human GIT and response of Bifidobacterium to various environmental factors (underlined) or ecological niches. Bifidobacterial key proteins or metabolic processes playing a role in these responses are indicated.

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Impact of low pH on the physiology of bifidobacteria

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Strong acidic conditions imposed by gastric juice composition are one of the first barriers that bifidobacteria cope with in the stomach. With the exception of Bifidobacterium animalis, bifidobacterial tolerance to acid is low. Thus, probiotic use requires the isolation of strains with good acid tolerance, which usually display cross-resistances to other technological and gastrointestinal stress factors (Sánchez et al., 2008).

As a result of exposure to sublethal low pH values, bacteria might develop tolerance to subsequent acid stresses, through an assemblage of acid-inducible mechanisms. The molecular mechanisms underlying this acid tolerance response (ATR) in Bifidobacterium have been delineated, and noticeable differences in acid tolerance have become apparent in different strains. To date, only one study has compared the ATR in wild-type and acid-tolerant-derivative Bifidobacterium strains (Sánchez et al., 2008).

Exposure to low pH affects the proton motive force, which results in H+ accumulation inside the cell. Accordingly, acid response in B. animalis subsp. lactis and acid adaptation in Bifidobacterium longum have been associated with an overproduction of subunits of the F0-F1-ATPase, which counteract this H+ accumulation by an increased H+ extrusion activity (Sánchez et al., 2008). Besides, an increase in branched-chain amino acids and ammonia production, which capture protons acting as a cytoplasmic buffer, was also connected to ATR in B. longum. Likewise, acid adaptation has been related to other changes, such as alterations in sulfur amino acid metabolism, although its particular connection to ATR still remains unclear (Sánchez et al., 2008). Changes in surface properties of particular strains affecting mucin adhesion, pathogen displacement, and fermentative capabilities have also been associated with acquisition of acid tolerance (Collado & Sanz, 2006; Sánchez et al., 2008).

A strong effect of environmental conditions, such as availability of fermentable carbon sources or growth phase, on acid tolerance has been found in Bifidobacterium (Sánchez et al., 2008). This highlights the limitations of in vitro models to mimic the conditions that bifidobacteria face in the gut, even though results from in vitro Bifidobacterium acid resistances have been correlated with survival in a bioreactor model of the stomach–intestine passage (Ritter et al., 2009).

Bile adaptation and response mechanisms

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Presence of bile is one of the physiological barriers bifidobacteria have to face in the intestine. Bile acids are the main components of this biological fluid, and they are responsible for its detergent-like antimicrobial properties. Cytoplasmic accumulation of these acids affects the homeostasis of the cell, leading to leakage of ions. Bifidobacterium resistance to this intestinal condition depends on the species, although they can acquire stable resistance phenotypes as a result of bile exposure. This process frequently involves the appearance of cross-resistances to other stress factors, alterations in antibiotic resistance patterns, carbohydrate metabolism, cellular surface architecture and composition, and in their interaction with the intestinal ecosystem, among others (Sánchez et al., 2008).

The cell membrane is one of the main targets of bile, and, accordingly, in vitro studies have shown that surface structures play a key role in Bifidobacterium tolerance and response. Lipid and protein composition of membranes, alongside cell wall functionalities, suffer profound changes in the presence of bile (Ruiz et al., 2009). Alterations in the concentration of enzymes involved in fatty acid synthesis, which correlate with changes in fatty acid composition of Banimalis subsp. lactis, have also been described (Sánchez et al., 2008). Besides this, production of exopolysaccharides (EPS) has been associated with bile tolerance in a number of strains, EPS production being regulated by bile in some cases (Ruas-Madiedo et al., 2009; Fanning et al., 2012).

Efflux pumps are a common detoxification mechanism used by bacteria. To date, two efflux pumps directly related to bile detoxification, BetA and Ctr, have been described in Bifidobacterium (Gueimonde et al., 2009). Interestingly, betA has been shown to be controlled by a bile-inducible promoter, which might contribute to guarantee its expression within the GIT (Gueimonde et al., 2009; Ruiz et al., 2012). A number of Bifidobacterium transporters overexpressed during bile exposure in Bifidobacterium breve UCC2003 have also been found to participate in its tolerance (Ruiz et al., 2012).

Particular aspects of Bifidobacterium response seem to be species dependent. For instance, while most of the glycolytic enzymes were overproduced after bile exposure in B. longum, xylulose-5-phospahte/fructose-6-phosphate phosphoketolase and glyceraldehyde-3-phosphate dehydrogenase are the sole glycolytic enzymes overproduced in B. animalis subsp. lactis. Thus, these observations were correlated with increased glucose consumption in B. longum, but not in B. animalis subsp. lactis (Sánchez et al., 2008). On the other hand, some common aspects of Bifidobacterium responses to bile reflect that bifidobacteria deal with membrane and oxidative stresses. Hence, a complete set of chaperones and proteases were found to be overexpressed to counteract misfolding and aggregation of proteins. Induction of proteins involved in SOS response is also noticeable (Sánchez et al., 2008).

Whereas bile salt hydrolase (BSH), the enzyme responsible for bile salt deconjugation, has been found to be overproduced in bile-resistant strains of B. animalis subsp. lactis, no solid relationship has been found between the presence of the enzyme and the tolerance phenotype in bifidobacteria. However, in vivo studies reported an increase in BSH production in the gut of rabbits, and hence, a role of BSH for bifidobacterial tolerance to bile in the gut might not be ruled out (Sánchez et al., 2008; Yuan et al., 2008).

Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Other intestinal factors that might affect Bifidobacterium survival and functionality in the GIT include digestive enzymes such as pepsin and pancreatin. Both human gastric and pancreatic juices, or simulated ones containing both pepsin and pancreatin, have demonstrated similar effects on bifidobacterial survival in vitro. However, in spite of the viability loss of bifidobacteria in gastric juices, most of the strains appear to possess a natural ability to survive pancreatin and pepsin-containing solutions (Masco et al., 2007). Nonetheless, the effects of digestive enzymes on Bifidobacterium properties are yet to be described. For instance, Wang et al. (2010) have recently shown that in vitro pepsin treatment affects adhesion to intestinal cell lines, which might correlate with the proteinaceous nature of some Bifidobacterium adhesion factors (Guglielmetti et al., 2008; González-Rodríguez et al., 2012).

In the GIT, bifidobacteria also face enteric antimicrobial peptides namely defensins and cathelicidins. They are effectors of the innate immune system in mammals and contribute to the host defense against enteric microbial infections, regulating the microbial communities within the GIT. These compounds have a broad range antimicrobial activity and are likely to affect ingested probiotics. It has been shown that certain mouse defensins are particularly active against pathogens and not against commensal microorganisms (Masuda et al., 2011). However, Bifidobacterium tolerance and response to mammalian defensins have not received much scientific attention, so very little is known about the precise mechanisms involved in these processes. Moreover, it has been reported that certain probiotics stimulate the production of enteric defensins. In a clinical trial, Kabeerdoss et al. (2011) recently showed that administration of B. animalis subsp. lactis Bb12 results in an increased production of beta-2-defensin in humans, which might explain the ability of bifidobacteria to prevent some infections.

Adhesion and intestinal colonization mechanisms: key players

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Adhesion of the microorganisms to the intestinal mucosa is an important feature involved in colonization and related to the ability of the strains to interact with the host. Thus, adhesion to the mucosa has frequently been used as a criterion for the selection of probiotic strains. Although the role of adhesion in probiotic functionality is still controversial, there are studies supporting a relationship between in vitro adhesion and in vivo colonization, and a link between mucosal adhesion and beneficial effects during intestinal inflammation has also been reported. The adhesion ability of the strains also seems to be important for immune modulation and for competitive exclusion of pathogens (Castagliuolo et al., 2005).

The outer region of the intestinal mucosa consists of a mucus layer covering the epithelial cells. This mucus is rich in glycoproteins and glycolipids, providing abundant targets, including carbohydrate moieties, for bacterial adhesion. Some probiotic microorganisms share carbohydrate-binding specificities with enteropathogens providing a clear rationale for the use of probiotics for over-competing pathogens to prevent infection. Thus, adhesion to human intestinal mucus and/or human intestinal epithelial cell lines has been the most commonly used models to evaluate bacterial adhesion. In this regard, the ability of certain bifidobacterial strains to competitively exclude enteropathogens from intestinal cells and human intestinal mucus has been demonstrated.

The mechanisms of bacterial adhesion to the gastrointestinal mucosa are complex and involve both nonspecific phenomena, such as electrostatic forces and hydrophobic interactions, as well as specific phenomena dependent on the presence of bacterial adhesins and mucosal receptors. Therefore, factors such as cell wall properties and composition and the presence of adhesins constitute the most important determinants of the ability of the strain to adhere to the mucosa. Bifidobacteria use both types of mechanisms, specific and nonspecific, to adhere to the intestinal mucosa, involving both nonproteinaceous and proteinaceous molecules. Thus, the adhesion of bifidobacteria to the human intestinal mucosa is a multifaceted process in which molecules of different nature, including proteins, lipids and carbohydrates, seem to play a role (Gueimonde et al., 2007). In addition, intestinal conditions (pH, bile, digestive enzymes) have also been documented to have an effect on adhesion to epithelial cells (de los Reyes-Gavilán et al., 2011).

Several reports indicate that proteinaceous components are involved in bifidobacteria adhesion to the intestinal mucosa. Among them, extracellular proteins play a pivotal role (Table 1). Extracellular proteins comprise proteins that are secreted and either surface-attached or released into the environment. Some of these proteins have been proposed as being responsible for beneficial effects exerted by probiotic bacteria over human health, due to the possibility of a direct interaction with epithelial and immune cells. These effects include pathogen displacement and competition, enhancement of the mucosal barrier and immunomodulation, and the precise molecular mechanism of action and receptors are starting to be understood (Sánchez et al., 2010). Among them, adhesins are extracellular proteins associated with the bacterial cell surface, many of them finally being released to the external milieu. They can be divided into two groups: adhesins with a signal peptide within their amino acid sequence, which are synthesized in the cytoplasm and directed to the protein export machinery, and those that have not and are exported, in most cases, by unknown mechanisms. Among the bifidobacterial adhesins containing an export signal, BopA must be highlighted. BopA was the first adhesin discovered in bifidobacteria. It is a lipoprotein, homologous to some peptidases, involved in the adhesion of Bifidobacterium bifidum to Caco-2 cells (Guglielmetti et al., 2008). Also, B. bifidum and B. longum strains overexpressing bopA showed enhanced adhesion to intestinal epithelial cells, clearly demonstrating a role of BopA in intestinal adhesion (Gleinser et al., 2012). Another group of proteins associated with specific machinery for export outside of the cell is pili-like proteins, recently discovered in B. breve (O'Connell Motherway et al., 2011). Transcriptome analysis of B. breve UCC2003 in a murine model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster essential for efficient gut colonization. Although the specific binding to enterocytes or intestinal mucus of B. breve Tad proteins has not yet been characterized, a role for these proteins in favouring the adhesion of B. breve to the intestinal epithelium has been suggested (O'Connell Motherway et al., 2011). Conservation of the tad genes among other Bifidobacterium genomes seems to indicate a pivotal role of these proteins in several Bifidobacterium species, and further research on these pili will surely give clues as to how bifidobacteria colonize and transiently persist in the human GIT.

Table 1. Proteins of bifidobacteria involved in binding to mucin or other host extracellular matrices, adhesion to human intestinal cells, or colonization of the intestine in animal models
Protein (species)FunctionReferences
BopA (B. bifidum)Lipoprotein involved in the adhesion of bifidobacteria to Caco-2 cells. It shows immunomodulatory activityGuglielmetti et al. (2008)
Transaldolase (B. bifidum)Mucin binding capability and aggregation factorGonzález-Rodríguez et al. (2012)
Tad proteins (B. breve)Essential for efficient in vivo murine gut colonizationO'Connell Motherway et al. (2011)
DnaK (B. animalis subsp. lactis)Human plasminogen receptor whose expression is triggered by bileCandela et al. (2010)
Enolase (B. animalis subsp. lactis)Human plasminogen receptorCandela et al. (2009)
Glutamine synthetase, BSH and phosphoglycetarate mutase (B. animalis subsp. lactis)Binding of human plasminogenCandela et al. (2007)

A second group of adhesins can be found on the bacterial surface, apparently lacking secretion signals, surface-attachment, or binding domains. This group of proteins includes glycolytic, housekeeping, and ribosomal proteins, which once surface-exposed develop other functions. For this reason, they are denominated moonlighting proteins. The list of moonlighting proteins involved in adhesion or in the attachment of mucosal components is large and in continuous growth, and their presence on the surface of bifidobacteria is very common (Candela et al., 2007; Ruiz et al., 2009). Moonlighting proteins that have been shown to have adhesin function, including mucus-binding capability and affinity for different host-related extracellular matrices (i.e. plasminogen and fibronectin), include DnaK, glutamine synthetase, enolase, bile salt hydrolase and phosphoglycerate mutase in B. animalis subsp. lactis, and transaldolase in B. bifidum (Candela et al., 2007, 2009, 2010; González-Rodríguez et al., 2012).

Finally, nonproteinaceous molecules, such as EPS, have also been reported to be involved in the adhesion of probiotics to the intestinal mucosa. EPS are carbohydrate polymers present on the surface of many microorganisms, including bifidobacteria. The role of these EPS in the adhesion to the intestinal mucosa or in the colonization of the gut has been demonstrated using in vitro and in vivo models (Fanning et al., 2012).

Exploitation of metabolic resources typical from the human gut

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Bifidobacteria can utilize host-indigestible complex carbohydrates, which can stimulate their growth and metabolic activity. An important part of their genomes is devoted to the utilization of these carbon sources, indicating their adaptation to the gut environment. Several studies demonstrate that different strains of bifidobacteria prefer oligosaccharides to monosaccharides as carbon sources. The metabolism of oligo- and polysaccharides provides a competitive advantage with respect to other intestinal bacterial groups.

A decade ago, the sequencing of B. longum NCC2705 genome revealed the high presence of glycosyl hydrolases degrading a wide range of di-, tri-, and oligosaccharides. Schell et al. (2002) demonstrated that glycosyl hydrolases and oligosaccharides transporters are organized in clusters, which are structured into preserved modules including sugar-responsive repressors, ABC-type oligosaccharide transporters, and 1–6 genes encoding various types of glycosyl hydrolases. Currently, with more than 20 bifidobacterial genomes available in public databases, we know that these oligosaccharide degradation clusters are a common trait in many Bifidobacterium species (Bottacini et al., 2010). In the following paragraphs, we will focus on some important oligosaccharide utilization pathways of bifidobacteria that partially explain their gut adaptation (Table 2), although we have to bear in mind that they display a plethora of sugar utilization capabilities directed to make use of other carbon sources (Pokusaeva et al., 2011).

Table 2. Enzymes involved in the degradation of nondigestible oligosaccharides and host glycans in bifidobacteria
SpecieEnzymePotential substrateReferences
B. longum subsp. infantisEndo-β-N-acetylglucosaminidasesN-glycans of host glycoproteins and breast milk oligosaccharidesGarrido et al. (2012)
α-sialidaseMilk sialyloligosaccharidesSela et al. (2011)
α-fucosidases, N-acetyl-β-d-hexosaminidases, β-galactosidasesHuman milk oligosaccharidesAsakuma et al. (2011)
B. bifidum α-fucosidases, β-galactosidases, β-N-acetylhexosaminidase, lacto-N-biosidaseHuman milk oligosaccharidesAsakuma et al. (2011)
α-sialidaseMilk sialyloligosaccharidesKiyohara et al. (2011)
β-galactosidasesGalactooligosaccharidesGoulas et al. (2009)
α-sialidases, β-galactosidases, lacto-N-biosidase, α-l-arabinofuranosidase, β-N-acetylhexosaminidase, endo-α-N-acetylgalactosaminidase, α-fucosidaseN-glycans, O-glycans and terminal sugars of host glycans and mucinTurroni et al. (2010, 2011a)
B. longum subsp. longumEndo-α-N-acetylgalactosaminidaseO-glycan of intestinal mucin glycoproteinsKatayama et al. (2005)
α-l-arabinofuranosidaseArabinan, arabinoxylan and arabinooligosaccharidesMargolles & de los Reyes-Gavilán (2003)
β-galactosidases, galactoside symporter, glycosyltransferaseHuman milk oligosaccharides and galactooligosaccharidesGonzález et al. (2008)
B. adolescentis β-xylosidases, exo-oligoxylanase, α-l-arabinofuranosidase, arabinoxylan arabinofuranohydrolaseXylobiose, arabinoxylan, xylooligosaccharides and arabinoxylanooligosaccharidesLagaert et al. (2011)
AmylaseStarch and related polysaccharidesRhim et al. (2006)
B. breve AmylopullulanaseStarch and related polysaccharidesPokusaeva et al. (2011)
β-fructofuranosidaseFructooligosaccharidesPokusaeva et al. (2011)
Cellodextrin-binding protein, cellodextrin permease, β-glucosidaseCellodextrinsPokusaeva et al. (2011)
Permease protein of ABC transporter system for sugars-galactan metabolism, galactoside symporters, β-glucosidase, glycosyltransferases, galactokinase, galactosidases, glutamine synthetase, N-acylglucosamine 2-epimeraseHuman milk oligosaccharidesTurroni et al. (2011b)
B. animalis subsp. lactisSugar-binding protein, sugar permease of ABC transporter system, xylose isomerase, endo-1,4- β-xylanases, β-xylosidases, xylulose kinaseXylooligosaccharidesGilad et al. (2010)
β-fructofuranosidaseFructooligosaccharidesJaner et al. (2004)

In the colon, nondigestible oligosaccharides may be degraded by bifidobacteria to monosaccharides. The monosaccharides will then be processed by the central carbohydrate catabolic pathway of bifidobacteria, the so-called fructose-6-phosphate shunt or bifid shunt. Some of the most abundant dietary nondigestible oligosaccharides present in the lower GIT are the xylooligosaccharides. Recent studies have shown their bifidogenic effect (Gilad et al., 2010). Xylooligosaccharides are transported into the cell by ABC transport systems and then degraded intracellularly to d-xylose through the action of xylosidases. Recently, xylooligosaccharide degrading enzymes, such as exo-oligoxylanases from Bifidobacterium adolescentis, have been characterized (Lagaert et al., 2011). Different studies showed that B. animalis subsp. lactis BB12 is capable of utilizing xylooligosaccharides as a carbon source but cannot ferment xylan, arabinoxylan, or xylose (Gilad et al., 2010). In contrast, other results show that B. longum NCC2705 is capable of fermenting xylose as the only carbon source (Liu et al., 2011). These results suggest specific adaptation capabilities depending on the species and strains and indicate that some species, such as B. longum, can have adaptive advantages over other species less familiar with the human gut environment, such as B. animalis subsp. lactis.

On the other hand, several in vitro and in vivo studies showed that galactooligosaccharides added into the diet can modify the intestinal microbiota profile by increasing the number of bifidobacteria. These galactooligosaccharides are hydrolyzed by β-galactosidases. Noteworthy is the ability of some β-galactosidases from bifidobacteria to produce other classes of prebiotics by transferase activity of galactosyl groups toward lactose and to synthesize transgalacto-oligosaccharides. Recently, several studies open the possibility of using Bifidobacterium strains to produce new prebiotics with species-specific bifidogenic effects (Davis et al., 2011).

Inulin and fructooligosaccharides are nondigestible oligosaccharides present in several fruits and vegetables, and they have been reported to stimulate the bifidobacteria growth. The β-fructofuranosidase is the enzyme responsible for fructan and sucrose hydrolysis. The gene encoding this enzyme was characterized in B. animalis subsp. lactis DSM 10140 (Janer et al., 2004). Short-chain fructooligosaccharides are usually preferred as a substrate for growth, and many species failed to grow on inulin. In a previous study, 18 Bifidobacterium strains were grouped according to their ability to completely, or partially, ferment oligofructose, inulin, or both (Falony et al., 2009).

Starch escapes digestion and enters the colon, representing an excellent carbohydrate source for those gut bacteria able to produce amylolytic enzymes. A few studies have shown the presence of α-amylase and pullulanase activity in various bifidobacteria strains able to utilize starch, amylopectin, and pullulan. One extracellular amylase (AmyB) from B. adolescentis was purified and characterized (Rhim et al., 2006). Also, the apuB gene encoding an extracellular type II amylopullulanase was found in B. breve UCC2003. This gene is essential in starch or long-chain maltooligosaccharides metabolism (Pokusaeva et al., 2011).

Recently, a galacto-N-biose – lacto-N-biose I pathway has been characterized in bifidobacteria. Through this pathway, disaccharides from milk are broken down by active extracellular glycosidases in human milk oligosaccharides (fucosidases, sialidases, lacto-N-biosidases, β-galactosidases) and galacto-N-biose. Furthermore, galacto-N-biose is the core structure of mucin type sugar, originated by active extracellular glycosidases, such as endo-α-N-acetylgalactosaminidase (EngBF) and α-l-fucosidase (AfcA) acting on mucin glycoproteins (Turroni et al., 2011a). Gene expression analysis of B. longum subsp. longum grown in the presence of breast milk has shown an upregulation of genes involved in the galactose/lacto-N-biose pathway (González et al., 2008). On the other hand, lacto-N-biose residues of human milk oligosaccharides might act as a bifidogenic factor in breastfed infants. They are used as a carbohydrate source by strains that usually colonize the infant GIT, such as B. longum subsp. infantis, B. longum subsp. longum, B. bifidum, and B. breve. These oligosaccharides caused no prebiotic effect on strains that are found in adult GIT, such as B. adolescentis, Bifidobacterium catenulatum, Bifidobacterium angulatum, and Bifidobacterium dentium, as well as on other lactic acid and enteric bacteria (Xiao et al., 2010). This prebiotic effect is supported by the information contained in the genomes of bifidobacteria from infant GIT. Typical species from infants (i.e. B. longum subsp. infantis and B. breve) have more genes predicted to be involved in human milk oligosaccharide utilization than species commonly inhabiting adult GIT, which have more genes involved in the metabolism of plant-derived complex carbohydrates (Table 2). Some studies have shown that B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on human milk oligosaccharides as the only carbon source (Ward et al., 2007; Turroni et al., 2011b). In this context, human milk oligosaccharides can play a role in the health-promoting attributes of human breast milk and also an important role in infant diet during and after weaning.

Finally, intestinal mucin is one of the most abundant metabolic resources for bacteria inhabiting the human gut. In this regard, several studies have demonstrated the ability of different bifidobacteria to metabolize human intestinal mucus, such as B. longum, B. breve, and B. bifidum (Ruas-Madiedo et al., 2008). Furthermore, the analysis of the B. bifidum PRL2010 genome sequence has demonstrated its ability to metabolize mucin and suggests that this capacity may contribute to the infant gut colonization (Turroni et al., 2010).

The set of enzymes mentioned above provides bifidobacteria with the ability to degrade carbon sources specifically found in the human gut, and this ability offers a selective advantage over other bacteria less adapted to the use of these metabolic resources. Therefore, these metabolic capabilities confer an advantage for bifidobacteria in terms of survival and colonization in the human GIT.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

Nowadays, Bifidobacterium strains are one of the most frequently commercialized probiotics for human consumption, owing to the accumulated scientific evidence of their beneficial effects. Bifidobacteria are able to exert their effects through direct interaction with host cells, mainly enterocytes and immune cells, and it is generally believed that in order to be able to trigger downstream responses, they must be able to reach the intestine in a functionally active state. In the last decade, research into the physiological responses of bifidobacteria in the presence of different stress factors from the human GIT, as well as the knowledge of their versatility to compete with the gut microbiota for metabolic resources and adhesion sites in their natural niche, has contributed enormously to understanding the success of these bacteria as desirables cohabitants of our body. Also, this knowledge nowadays allows the scientific community to select the strains based on scientific rationale for defined target applications.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References

The authors thank the Spanish Ministry of Science and Innovation (MICINN) and the ‘Plan Nacional I+D+i’ for the financial support of the research work through the projects AGL2010-14952 and RM2010-00012-00-00. B.S. was the recipient of a Juan de la Cierva postdoctoral contract, and I.G.-R. was the recipient of an FPI grant, from MICINN. L.R. had a JAE-Predoctoral contract, financed by CSIC.

References

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  2. Abstract
  3. Introduction
  4. Coping with gastrointestinal stress factors
  5. Impact of low pH on the physiology of bifidobacteria
  6. Bile adaptation and response mechanisms
  7. Other intestinal factors affecting bifidobacterial viability: enzymes and antimicrobial peptides
  8. Adhesion and intestinal colonization mechanisms: key players
  9. Exploitation of metabolic resources typical from the human gut
  10. Conclusions
  11. Acknowledgements
  12. References
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