**3.2 Chemical structures of prebiotics**

Prebiotics are typically comprised of oligomers of 6-carbon and/or 5-carbon sugars, with different bonding structures, and different chain lengths (see **Figure 1** for examples) [11].

Fructans from inulin are typically linear oligomers primarily made up of fructose monomers connected by β-2,1 bonds. Fructans from agave tend to have a more complex structure with multiple side branches and β-2,6 linkages in addition to β-2,1 bonds [12]. FFn-type fructans such as inulin are comprised entirely of fructose subunits, whereas GFn-type fructans (typically shorter chain oligosaccharides) may have a glucose subunit connected by an α-2,1 bond onto the main fructan chain. These short-chain GFn-type FOS molecules are typically produced by enzymatically adding fructose subunits onto sucrose (glucose-fructose) as the starting substrate. In contrast, short chain FFn FOS would be produced by hydrolysis of inulin (long chain FFn) using endoinulinases [11, 12].

Galactooligosaccharides (GOS) may also be comprised exclusively of galactose subunits, or it may have a glucose terminal subunit arising from use of lactose (glucose-galactose) as the initial substrate to produce GOS using β-galactosidases. The galactose subunits may be connected by β-1,3, β-1,4, or β-1,6 bonds, and may include branched structures, depending upon the type of β-galactosidase [11, 13].

Xylooligosaccharides (XOS) are primarily comprised of xylose subunits connected by β-1,4 bonds, although longer chain XOS may contain branches of arabinose subunits, acetyl groups, or uronic acids (originally present in the xylan source material) that can influence their functionality. XOS that includes arabinose sub-groups are frequently referred to as arabinoxylanoligosaccharides, or AXOS [11, 14].

Mannooligosaccharides are derived from mannan in biomass, and thus are typically made up of mannose subunits connected by β-1,4 bonds [15]. Isomaltooligosaccharides (IMOS) contain glucose subunits, but vary in their bonding structure (affected by manufacturing method). α-1,6 bonds are typical, with either linear or branched structures [11].

Complex starch structures that resist breakdown by pancreatic enzymes into glucose can persist into the colon – thus leading to the concept of "resistant starch" as a prebiotic. Resistant starch (RS) is available in four forms, depending upon method of manufacture and bond structure [16, 17]. Like starch, most of the bonds are of the α-1,4 variety; the presence of other types of bonds may confer "resistance". RS1 is conventional starch that may be trapped within whole grains. RS2 is typically starch with more complex branching or bond structures, rendering it less accessible to amylase. RS3 is produced when starch undergoes retrogradation, i.e., cooked starch is cooled below its gelatinization temperature. RS4 starches have undergone chemical modification, usually by acidification or cross-linking [16].

The microbial ability to utilize specific substrates is dependent upon the enzymes and transporters encoded within the cells. Some microbes can utilize a broad set of substrates, while others are more selective. This also points to the selectivity of the prebiotic; preferably, the prebiotic preferentially feeds beneficial bacteria, with limited growth of undesirable bacteria. **Table 1** summarizes the key hydrolytic enzymes and the corresponding substrates/reactions [19]. These

**55**

**Figure 1.**

and other carbohydrates.

*Chemical and bonding structures of prebiotics.*

*Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived…*

enzymes may be extracellular, or intracellular, which can influence substrate utili-

There are various types of transport systems that move prebiotics into the cell, although microbes are likely to only possess a subset, targeted towards a narrower set of substrates. Some transport systems are specific to (certain) monomers, while others target specific oligosaccharides. Chemical structures must be matched to the structure of the transport system in order to be transported into the cell. **Table 2** summarizes key membrane transport systems involved in utilization of prebiotics

From a selectivity perspective, it is advantageous if the substrate is used intracellularly – thus, the requisite transport system plus intracellular fructofuranosidases

zation; intracellular enzymes require a corresponding transporter system.

*DOI: http://dx.doi.org/10.5772/intechopen.89484*

*Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived… DOI: http://dx.doi.org/10.5772/intechopen.89484*


#### **Figure 1.**

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

enzymes and transport systems responsible for their utilization.

inulin (long chain FFn) using endoinulinases [11, 12].

either linear or branched structures [11].

**3.2 Chemical structures of prebiotics**

for examples) [11].

AXOS [11, 14].

to utilize these prebiotics. Such prebiotics are less likely to directly feed undesirable bacteria if these bacteria do not have the right transport systems and enzymes. Below, we describe the different chemical structures of prebiotics, along with the

Prebiotics are typically comprised of oligomers of 6-carbon and/or 5-carbon sugars, with different bonding structures, and different chain lengths (see **Figure 1**

Fructans from inulin are typically linear oligomers primarily made up of fructose monomers connected by β-2,1 bonds. Fructans from agave tend to have a more complex structure with multiple side branches and β-2,6 linkages in addition to β-2,1 bonds [12]. FFn-type fructans such as inulin are comprised entirely of fructose subunits, whereas GFn-type fructans (typically shorter chain oligosaccharides) may have a glucose subunit connected by an α-2,1 bond onto the main fructan chain. These short-chain GFn-type FOS molecules are typically produced by enzymatically adding fructose subunits onto sucrose (glucose-fructose) as the starting substrate. In contrast, short chain FFn FOS would be produced by hydrolysis of

Galactooligosaccharides (GOS) may also be comprised exclusively of galactose

subunits, or it may have a glucose terminal subunit arising from use of lactose (glucose-galactose) as the initial substrate to produce GOS using β-galactosidases. The galactose subunits may be connected by β-1,3, β-1,4, or β-1,6 bonds, and may include branched structures, depending upon the type of β-galactosidase [11, 13]. Xylooligosaccharides (XOS) are primarily comprised of xylose subunits connected by β-1,4 bonds, although longer chain XOS may contain branches of arabinose subunits, acetyl groups, or uronic acids (originally present in the xylan source material) that can influence their functionality. XOS that includes arabinose sub-groups are frequently referred to as arabinoxylanoligosaccharides, or

Mannooligosaccharides are derived from mannan in biomass, and thus are typically made up of mannose subunits connected by β-1,4 bonds [15].

Isomaltooligosaccharides (IMOS) contain glucose subunits, but vary in their bonding structure (affected by manufacturing method). α-1,6 bonds are typical, with

Complex starch structures that resist breakdown by pancreatic enzymes into glucose can persist into the colon – thus leading to the concept of "resistant starch" as a prebiotic. Resistant starch (RS) is available in four forms, depending upon method of manufacture and bond structure [16, 17]. Like starch, most of the bonds are of the α-1,4 variety; the presence of other types of bonds may confer "resistance". RS1 is conventional starch that may be trapped within whole grains. RS2 is typically starch with more complex branching or bond structures, rendering it less accessible to amylase. RS3 is produced when starch undergoes retrogradation, i.e., cooked starch is cooled below its gelatinization temperature. RS4 starches have undergone chemical modification, usually by acidification or cross-linking [16]. The microbial ability to utilize specific substrates is dependent upon the enzymes and transporters encoded within the cells. Some microbes can utilize a broad set of substrates, while others are more selective. This also points to the selectivity of the prebiotic; preferably, the prebiotic preferentially feeds beneficial bacteria, with limited growth of undesirable bacteria. **Table 1** summarizes the key hydrolytic enzymes and the corresponding substrates/reactions [19]. These

**54**

*Chemical and bonding structures of prebiotics.*

enzymes may be extracellular, or intracellular, which can influence substrate utilization; intracellular enzymes require a corresponding transporter system.

There are various types of transport systems that move prebiotics into the cell, although microbes are likely to only possess a subset, targeted towards a narrower set of substrates. Some transport systems are specific to (certain) monomers, while others target specific oligosaccharides. Chemical structures must be matched to the structure of the transport system in order to be transported into the cell. **Table 2** summarizes key membrane transport systems involved in utilization of prebiotics and other carbohydrates.

From a selectivity perspective, it is advantageous if the substrate is used intracellularly – thus, the requisite transport system plus intracellular fructofuranosidases


#### **Table 1.**

*Key enzymes for carbohydrate utilization.*

would have an advantage (i.e., FOS that can be transported intracellularly would have an advantage over FOS/inulin that can only be processed via extracellular enzymes). The restricted capacity of most transporter systems precludes use of long chain fructans by many *Lactobacillus* species [11].

#### **3.3 Types of microbes with enzyme/transport systems of various types**

Microbes that have fructofuranosidase encoded extracellularly are able to use long-chain FFN-type FOS, and inulin. Some species of *Lactobacillus* (casei, paracasei) and *Streptococcus* are examples [11]. The resulting short chain fructans may be transported intracellularly for utilization. Mao et al. [23] identified 19 strains from human feces that were capable of metabolizing FOS, including multiple strains of *E. coli*, *Enterobacter cloacae*, *Bifidobacterium* spp., and *Lactobacillus* spp. Additional bacteria also proved capable of growth on FOS. This includes additional strains of *E. coli and Bifidobacteria*, along with several strains of *Streptococcus, Clostridia, Roseburia, Klebsiella*, and *Enterococcus* [23].

According to Rossi et al. [24], virtually all *Bifidobacteria* are able to grow on short-chain FOS. However, most *Bifidobacteria* grew poorly on inulin (only 8 out of 55), because most of the fructofuranosidases are intracellular, and inulin cannot be transported intracellularly. Scott et al. [25] made similar observations when inulin with a DP >25 were fed to *Bifidobacteria*. However, in a mixed culture system, inulin may be broken down into shorter chain FFn-FOS, and then used by *Bifidobacteria*. Thus, growth of *Bifidobacteria* in the presence of inulin is primarily due to crossfeeding in the presence of other microbes that act as primary degraders, rather than direct feeding by inulin.

Different species of *Bifidobacteria* contain ABC transporters, sucrose permeases, fructose PTS transporters, and MFS transporters. The type(s) of available transporters dictate substrate utilization, whether GFn-type short chain FOS, FFn-type FOS, or analogous substrates. Although several strains of *Bifidobacteria* can utilize GOS, there are significant differences between strains [26]. Certain strains of *B. breve* and *B. longum* contain an extracellular galactanase that breaks down long-chain GOS and galactan in plant fiber, producing di- and

**57**

on α-glucan [11].

*phosphotransferase.*

*Key Transmembrane transport systems.*

**Table 2.**

*Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived…*

**Transport system Target molecules Examples/implications**

There are many variations of the ABC transporter. Transporters in the CUT1 class work on sucrose, lactose, maltose, FOS, maltodextrins, XOS, and other oligosaccharides. Transporters in the CUT2 class generally transport monomers such as arabinose, xylose, ribose,

Such a system is present in *L. acidophilus*, for utilization of short-chain FOS; *Bifidobacteria* have an ABC transporter specific to XOS; various enterobacteria, including *E. coli*, have CUT1 and CUT2 transporters for maltose and various

Allows *L. plantarum* to utilize shortchain FOS synthesized from sucrose (GFn-type), but FFn FOS, which lacks the sucrose structure, cannot be used by *L. plantarum*

Present in many bacteria, fungi, yeasts, plants, animals, humans; key

*L. rhamnosus GG* contains a fructose PTS transporter, which allows growth on various types of FOS and

*L. gasseri* contains a lactose PTS

LacS is stated to be the sole transporter for GOS, with specificity for β-galactosides [22].

for energy homeostasis

inulin

transporter [22]

6-carbon sugars

glucose [20, 21]

their structure.

[11, 21]

Fructose PTS transporters Transport system targets

Lactose PTS transporters Transport system targets the

LacS and LacY permeases MFS-type transport systems

Sucrose permease Transport of substrates with a

type FOS.

Transport system is highly specific to compounds that incorporate sucrose as part of

A major transporter system with various types, allowing intracellular transport of glucose, lactose, xylose, oligosaccharides, FFn-type FOS

the fructose component of substrates, thus allowing use of fructose, sucrose, inulin, and both FFn and GFn-type FOS.

lactose component of substrates

enabling transport of molecules with lactose module. The LacS transport system allows a microbe to use lactose, GOS (with lactose terminus), and lactitol. LacS and LacY differ based upon source family.

sucrose module, such as GFn-

*CUT = Carbohydrate Uptake Transporter; FFn = FOS comprised of "n" fructose (F) subunits and a fructose terminal unit; GFn = FOS containing "n" fructose (F) subunits and a terminal glucose (G); PTS = phosphoenolpyruvate* 

tri-saccharides that can be transported into the cell and converted into galactose via intracellular β-galactosidase [27]. *B. lactis* BI-04 contains lactose permease and ABC transport systems, along with β-galactosidase [28], that enable utilization of GOS. *L. acidophilus* has the ability to utilize many different prebiotics, with various monomeric subunits and bond structures [28]. Such broad utilization is due to a multiplicity of molecular transport systems and hydrolytic enzymes, including up to nine different enzymes from the GH13 family that act

*DOI: http://dx.doi.org/10.5772/intechopen.89484*

ATP-dependent binding cassette (ABC-type) transporter system

Sucrose phosphoenolpyruvate phosphotransferase (PTS) transport system

Major facilitator superfamily (MFS) transporter system

*Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived… DOI: http://dx.doi.org/10.5772/intechopen.89484*


*CUT = Carbohydrate Uptake Transporter; FFn = FOS comprised of "n" fructose (F) subunits and a fructose terminal unit; GFn = FOS containing "n" fructose (F) subunits and a terminal glucose (G); PTS = phosphoenolpyruvate phosphotransferase.*

#### **Table 2.**

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

**Enzyme Substrates, reactions**

α-amylase, glucoamylase,

β-endoglucanase, cellobiohydrolase,

β-galactanase, β-galactosidase,

*Key enzymes for carbohydrate utilization.*

pullulanase

β-glucosidase

α-galactosidase

**Table 1.**

would have an advantage (i.e., FOS that can be transported intracellularly would have an advantage over FOS/inulin that can only be processed via extracellular enzymes). The restricted capacity of most transporter systems precludes use of long

Converts starch and RS into glucose

Converts cellulose and β-gluco-oligosaccharides into cellobiose, then

Converts galactan into β-GOS, then galactose; β-galactosidase can also remove galactose subunits that are present as side chains in

Inulinase, β-fructofuranosidase Converts inulin into FOS, and FOS into fructose/glucose

xylose

glucose

xylan/XOS β-mannanase, β-mannosidase Converts MOS into mannobiose, then mannose α-arabinofuranosidase Releases arabinose from side chains of xylan and AXOS

β-galactosidases Converts lactose into glucose and galactose; aids individuals with lactose intolerance [18] β-Endoxylanases, β-xylosidases Converts XOS into short chain XOS (sc-XOS) and xylobiose, then

Microbes that have fructofuranosidase encoded extracellularly are able to use long-chain FFN-type FOS, and inulin. Some species of *Lactobacillus* (casei, paracasei) and *Streptococcus* are examples [11]. The resulting short chain fructans may be transported intracellularly for utilization. Mao et al. [23] identified 19 strains from human feces that were capable of metabolizing FOS, including multiple strains of *E. coli*, *Enterobacter cloacae*, *Bifidobacterium* spp., and *Lactobacillus* spp. Additional bacteria also proved capable of growth on FOS. This includes additional strains of *E. coli and Bifidobacteria*, along with several strains of *Streptococcus, Clostridia,* 

According to Rossi et al. [24], virtually all *Bifidobacteria* are able to grow on short-chain FOS. However, most *Bifidobacteria* grew poorly on inulin (only 8 out of 55), because most of the fructofuranosidases are intracellular, and inulin cannot be transported intracellularly. Scott et al. [25] made similar observations when inulin with a DP >25 were fed to *Bifidobacteria*. However, in a mixed culture system, inulin may be broken down into shorter chain FFn-FOS, and then used by *Bifidobacteria*. Thus, growth of *Bifidobacteria* in the presence of inulin is primarily due to crossfeeding in the presence of other microbes that act as primary degraders, rather than

Different species of *Bifidobacteria* contain ABC transporters, sucrose permeases, fructose PTS transporters, and MFS transporters. The type(s) of available transporters dictate substrate utilization, whether GFn-type short chain FOS, FFn-type FOS, or analogous substrates. Although several strains of *Bifidobacteria* can utilize GOS, there are significant differences between strains [26]. Certain strains of *B. breve* and *B. longum* contain an extracellular galactanase that breaks down long-chain GOS and galactan in plant fiber, producing di- and

**3.3 Types of microbes with enzyme/transport systems of various types**

chain fructans by many *Lactobacillus* species [11].

*Roseburia, Klebsiella*, and *Enterococcus* [23].

direct feeding by inulin.

**56**

*Key Transmembrane transport systems.*

tri-saccharides that can be transported into the cell and converted into galactose via intracellular β-galactosidase [27]. *B. lactis* BI-04 contains lactose permease and ABC transport systems, along with β-galactosidase [28], that enable utilization of GOS. *L. acidophilus* has the ability to utilize many different prebiotics, with various monomeric subunits and bond structures [28]. Such broad utilization is due to a multiplicity of molecular transport systems and hydrolytic enzymes, including up to nine different enzymes from the GH13 family that act on α-glucan [11].

Starch, owing to its high DP and complex branched structure, would be degraded in the presence of extracellular enzymes that can act on α-1,4 and α-1,6 linkages between glucose subunits. Certain *Bifidobacteria*, including *B. pseudolongum* and *B. breve* have the necessary enzymes for extracellular starch utilization [29].

Microbes such as the *L. acidophilus* cluster (including *L. johnsonii*, *L. helveticus, L. reuteri* and *L. plantarum*) contain LacS permease and β-galactosidase which allow these microbes to transport GOS into the cell, then break it down into glucose and galactose for metabolism [28].

Several *Bifidobacteria* contain the hydrolytic enzymes needed to break down the β-1,4 linkages present in xylooligosaccharides (XOS) and XOS with arabinose side groups (AXOS). Key enzymes include β-xylosidase and β-xylanase, the latter which breaks down longer chain XOS into shorter chains, ultimately xylobiose, that may be converted into xylose using β-xylosidase. AXOS requires arabinofuranosidase enzymes to process the arabinose side group. Some carbohydrate esterases may also be present to deal with acetyl or feruloyl side groups. The enzymes may be intracellular or extracellular; intracellular enzymes also require transporters such as an ABC transport system to act on the longer chain oligomers. Ejby et al. [30] noted that ABC transporters specific to XOS are exclusive to *Bifidobacteria*. *B. lactis, B. breve*, and *B. bifidum* are among the many species of *Bifidobacteria* that have the requisite enzymes and transport systems for utilization of short and longer chain XOS and AXOS. Crossfeeding of *Bifidobacteria* is aided by *Bacteroides* and *Prevotella*, which act as primary degraders that break down insoluble xylan in plant fiber into soluble oligosaccharides.

The wide variation in structures of prebiotics, along with the different transport and enzyme systems, ultimately dictate the selectivity of the prebiotic in a mixed culture. Monoculture systems provide some useful insights into the utilization of prebiotics by various substrates. Makelainen et al. [31] conducted a thorough study of the growth of >15 microbes, some beneficial, some pathogenic, in the presence of 11 different carbohydrate sources. Aggregate growth over 24 hours was reported as the area under the curve of DP600 measurements. **Figures 2**–**6** show growth of various probiotics and pathogenic microbes on glucose, FOS, GOS, scXOS, and XOS, respectively. As expected, all bacteria grew well on glucose (**Figure 2**), consistent with the widespread ability of microbes to utilize simple 6-carbon sugars.

Low DP FFn-FOS proved to be a good substrate for several strains of *Bifidobacteria*, along with *L. paracasei* and *L. acidophilus*, but was also used by *E. coli EHEC*, *S. epidermis*, and *C. perfringens*, among pathogenic bacteria tested. GOS also grew well on *Bifidobacteria* and *Lactobacilli*, but also proved to be an excellent substrate for several pathogenic bacteria, including *E. coli* and *C. perfringens*.

Consistent with the unique chemical structure and enzyme/transporter requirements, there was less microbial growth on scXOS and XOS. Fewer strains of *Bifidobacteria* utilized XOS, along with select strains of Lactobacillus. Li et al., in a study using 29 *Lactobacillus* strains and 35 strains of *Bifidobacterium*, observed that all *Bifidobacterium* strains tested grew on a high dose of XOS, and 30 of 35 strains grew on low dose XOS [32]. They also noted that *Lactobacillus* strains were able to utilize XOS, albeit with fewer strains and at lower efficiency compared to *Bifidobacteria*.

However, importantly, Makelainen et al. [31] noted minimal growth of pathogenic bacteria in the presence of XOS (**Figures 5** and **6**), consistent with a much higher selectivity of XOS for beneficial bacteria. This is a key advantage in a mixed microbial environment such as the GI tract. *Bifidobacteria* and *Lactobacillus* species have to compete with many more bacteria for FOS and GOS, which thus increases the dose required for efficacy. XOS, conversely, is better targeted to *Bifidobacteria*, and in a mixed culture, could be efficacious at a lower dose.

The aggregate area under the curve data reported by Makelainen et al. [31] do not, however, capture changes in growth rates, which can vary over time. Similarly,

**59**

**Figure 3.**

*Makelainen et al. [31].*

**Figure 2.**

*Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived…*

*Microbial growth on glucose (positive control). Data from Makelainen et al. [31].*

any issues with viability of microbes could be masked by rapid early growth, which may not be sustained. **Figure 7** illustrates growth of a strain of *B. breve* on FOS, XOS, and inulin, showing temporal effects. A noteworthy observation is that viability of *B. breve* decreased significantly after ~12–16 hours if grown on FOS or inulin, whereas growth on XOS sustained *B. breve* for a longer period, even up to 48 hours.

*Microbial growth on FFn FOS (DP 2–7). FFn = FOS comprised exclusively of fructose (F) subunits. Data from* 

*DOI: http://dx.doi.org/10.5772/intechopen.89484*

### *Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived… DOI: http://dx.doi.org/10.5772/intechopen.89484*

**Figure 2.** *Microbial growth on glucose (positive control). Data from Makelainen et al. [31].*

#### **Figure 3.**

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

galactose for metabolism [28].

Starch, owing to its high DP and complex branched structure, would be degraded

Microbes such as the *L. acidophilus* cluster (including *L. johnsonii*, *L. helveticus, L. reuteri* and *L. plantarum*) contain LacS permease and β-galactosidase which allow these microbes to transport GOS into the cell, then break it down into glucose and

Several *Bifidobacteria* contain the hydrolytic enzymes needed to break down the β-1,4 linkages present in xylooligosaccharides (XOS) and XOS with arabinose side groups (AXOS). Key enzymes include β-xylosidase and β-xylanase, the latter which breaks down longer chain XOS into shorter chains, ultimately xylobiose, that may be converted into xylose using β-xylosidase. AXOS requires arabinofuranosidase enzymes to process the arabinose side group. Some carbohydrate esterases may also be present to deal with acetyl or feruloyl side groups. The enzymes may be intracellular or extracellular; intracellular enzymes also require transporters such as an ABC transport system to act on the longer chain oligomers. Ejby et al. [30] noted that ABC transporters specific to XOS are exclusive to *Bifidobacteria*. *B. lactis, B. breve*, and *B. bifidum* are among the many species of *Bifidobacteria* that have the requisite enzymes and transport systems for utilization of short and longer chain XOS and AXOS. Crossfeeding of *Bifidobacteria* is aided by *Bacteroides* and *Prevotella*, which act as primary degraders

in the presence of extracellular enzymes that can act on α-1,4 and α-1,6 linkages between glucose subunits. Certain *Bifidobacteria*, including *B. pseudolongum* and *B.* 

*breve* have the necessary enzymes for extracellular starch utilization [29].

that break down insoluble xylan in plant fiber into soluble oligosaccharides.

and in a mixed culture, could be efficacious at a lower dose.

The aggregate area under the curve data reported by Makelainen et al. [31] do not, however, capture changes in growth rates, which can vary over time. Similarly,

The wide variation in structures of prebiotics, along with the different transport and enzyme systems, ultimately dictate the selectivity of the prebiotic in a mixed culture. Monoculture systems provide some useful insights into the utilization of prebiotics by various substrates. Makelainen et al. [31] conducted a thorough study of the growth of >15 microbes, some beneficial, some pathogenic, in the presence of 11 different carbohydrate sources. Aggregate growth over 24 hours was reported as the area under the curve of DP600 measurements. **Figures 2**–**6** show growth of various probiotics and pathogenic microbes on glucose, FOS, GOS, scXOS, and XOS, respectively. As expected, all bacteria grew well on glucose (**Figure 2**), consistent with the widespread ability of microbes to utilize simple 6-carbon sugars. Low DP FFn-FOS proved to be a good substrate for several strains of *Bifidobacteria*, along with *L. paracasei* and *L. acidophilus*, but was also used by *E. coli EHEC*, *S. epidermis*, and *C. perfringens*, among pathogenic bacteria tested. GOS also grew well on *Bifidobacteria* and *Lactobacilli*, but also proved to be an excellent substrate for several pathogenic bacteria, including *E. coli* and *C. perfringens*. Consistent with the unique chemical structure and enzyme/transporter requirements, there was less microbial growth on scXOS and XOS. Fewer strains of *Bifidobacteria* utilized XOS, along with select strains of Lactobacillus. Li et al., in a study using 29 *Lactobacillus* strains and 35 strains of *Bifidobacterium*, observed that all *Bifidobacterium* strains tested grew on a high dose of XOS, and 30 of 35 strains grew on low dose XOS [32]. They also noted that *Lactobacillus* strains were able to utilize XOS, albeit with fewer strains and at lower efficiency compared to *Bifidobacteria*. However, importantly, Makelainen et al. [31] noted minimal growth of pathogenic bacteria in the presence of XOS (**Figures 5** and **6**), consistent with a much higher selectivity of XOS for beneficial bacteria. This is a key advantage in a mixed microbial environment such as the GI tract. *Bifidobacteria* and *Lactobacillus* species have to compete with many more bacteria for FOS and GOS, which thus increases the dose required for efficacy. XOS, conversely, is better targeted to *Bifidobacteria*,

**58**

*Microbial growth on FFn FOS (DP 2–7). FFn = FOS comprised exclusively of fructose (F) subunits. Data from Makelainen et al. [31].*

any issues with viability of microbes could be masked by rapid early growth, which may not be sustained. **Figure 7** illustrates growth of a strain of *B. breve* on FOS, XOS, and inulin, showing temporal effects. A noteworthy observation is that viability of *B. breve* decreased significantly after ~12–16 hours if grown on FOS or inulin, whereas growth on XOS sustained *B. breve* for a longer period, even up to 48 hours.

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

#### **Figure 4.**

*Microbial growth on GGan GOS (DP 3–5). GGan GOS is comprised of n subunits of galactose (Ga) with a terminal glucose (G) subunit. Data from Makelainen et al. [31].*

#### **Figure 5.**

*Microbial growth on short chain XOS (scXOS; DP 2–5). Data from Makelainen et al. [31].*

This may have important implications in terms of sustaining key microbes in the digestive tract.

In the next section, we describe health impacts of prebiotics, with a particular emphasis on studies with XOS, AXOS, and MOS, due to their distinct chemical structures and selectivity for beneficial bacteria.

**61**

**Figure 7.**

**Figure 6.**

**4. Health impacts of prebiotics**

*Comparative growth of B. breve on FOS, XOS, and inulin.*

*Microbial growth on XOS (DP 2–10). Data from Makelainen et al. [31].*

Stimulating the growth of beneficial bacteria with prebiotics can lead to a cascade of health effects, as illustrated in **Figure 8**. Much of the historical focus had been on *Bifidobacterium spp*. and *Lactobacillus spp.*, although more recently, beneficial health outcomes have been associated with other microbial species as well. Short chain fatty acids, primarily acetate, propionate, and butyrate, along with lactate, are produced by fermentation of non-digestible fibers and prebiotics, potentially reducing the pH within the colon. This can promote absorption of minerals such as

*Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived…*

*DOI: http://dx.doi.org/10.5772/intechopen.89484*

*Functional Attributes and Health Benefits of Novel Prebiotic Oligosaccharides Derived… DOI: http://dx.doi.org/10.5772/intechopen.89484*

**Figure 6.**

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

This may have important implications in terms of sustaining key microbes in the

*Microbial growth on GGan GOS (DP 3–5). GGan GOS is comprised of n subunits of galactose (Ga) with a* 

*terminal glucose (G) subunit. Data from Makelainen et al. [31].*

*Microbial growth on short chain XOS (scXOS; DP 2–5). Data from Makelainen et al. [31].*

structures and selectivity for beneficial bacteria.

In the next section, we describe health impacts of prebiotics, with a particular emphasis on studies with XOS, AXOS, and MOS, due to their distinct chemical

**60**

digestive tract.

**Figure 5.**

**Figure 4.**

*Microbial growth on XOS (DP 2–10). Data from Makelainen et al. [31].*

**Figure 7.** *Comparative growth of B. breve on FOS, XOS, and inulin.*
