**3.1. Homo EPS biosynthesis**

Homo EPS are synthesized outside the cell by specific glycosyltransferase (GTF) or fructosyltransferase (FTF) enzymes (commonly named glucansucrases or fructan-sucrases). Homo-EPS producing LAB also use extracellular GTF enzymes to synthesize high-molecular mass α-glucans from sucrose. This process uses sucrose as a specific substrate, and the energy required for the process comes from sucrose hydrolysis. There is no energy requirement for EPS-production other than for enzyme biosynthesis because EPS synthesis by GTF or FTF does not involve active transport processes or the use of activated carbohydrate precursors. Therefore, large amounts of sucrose can easily be converted to EPS. *Lb. sanfranciscensis* produces up to 40 g/l levan and 25 g/l 1-kestose during growth in the presence of 160 g/l sucrose [63].

```
Glucan synthesis reactions catalysed by GTF can be written as follows (Fig. 1): 
sucrose + H2O → glucose + fructose 
sucrose + acceptor carbohydrate → oligosaccharide + fructose 
sucrose + glucan (n) → glucan (n+1) + fructose
```
Although GTF enzymes have a high degrees of similarity, lactobacilli produce a broad spectrum of glucans, including polymers with α-1,6 linkages (dextran), α-1,3 linkages (mutan), and both α-1,6 and α-1,4 linkages (alternan). The relative molecular weight of glucans from lactobacilli range from 1 × 106 Da to 5 × 107 Da [6]. In addition, GTF enzymes are not saturated by their substrate, and transfer reactions exceed the sucrose hydrolysis under sucrose concentrations above 100 mM [64].

**Figure 1.** The dextran synthesis by GTF (dextran sucrase).

520 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

amino acids alone had no effect on EPS production.

EPS production.

**3. EPS biosynthesis by LAB** 

the presence of 160 g/l sucrose [63].

sucrose + H2O → glucose + fructose

sucrose + glucan (n) → glucan (n+1) + fructose

**3.1. Homo EPS biosynthesis** 

and glucose was a more effective carbon source than galactose, sucrose, maltose, fructose, and raffinose [53]. EPS production by *Lb. casei* CG11 was investigated in basal minimum medium containing galactose, glucose, lactose, sucrose, maltose, and melibiose; glucose was the most efficient carbon source, and lactose and galactose were the least efficient ones [61]. EPS production by *Lb. rhamnosus* C83 was investigated in a chemically defined medium containing different carbon sources (glucose, fructose, mannose, and maltose) at different concentrations. Mannose at 40 g/l was by far the most efficient carbon source. Furthermore, increased Mg, Mn and Fe concentrations stimulated EPS production in synthetic media [59]. In addition, Macedo et al. [54] reported about the importance of salts in culture media and the strong positive effect of salts and amino acids on *Lb. rhamnosus* RW-9595M growth and EPS production. The addition of salts and amino acids largely increased EPS production (to 2775 mg/l) in whey permeate supplemented with yeast extract, although the addition of

It has been shown that an optimal ratio between the carbon and nitrogen is absolutely necessary to achieve high EPS yields [62]. The production of EPS by *Str. thermophilus* LY03 is modulated by both the absolute quantities and the ratio of carbon to nitrogen (C/N ratio). The carbon source is converted into lactic acid to produce energy as well as to synthesize the cell wall and EPS, and nitrogen is necessary for the synthesis of essential cell components. Therefore, a higher C/N ratio and sufficient quantities of both carbon and nitrogen increase

Homo EPS are synthesized outside the cell by specific glycosyltransferase (GTF) or fructosyltransferase (FTF) enzymes (commonly named glucansucrases or fructan-sucrases). Homo-EPS producing LAB also use extracellular GTF enzymes to synthesize high-molecular mass α-glucans from sucrose. This process uses sucrose as a specific substrate, and the energy required for the process comes from sucrose hydrolysis. There is no energy requirement for EPS-production other than for enzyme biosynthesis because EPS synthesis by GTF or FTF does not involve active transport processes or the use of activated carbohydrate precursors. Therefore, large amounts of sucrose can easily be converted to EPS. *Lb. sanfranciscensis* produces up to 40 g/l levan and 25 g/l 1-kestose during growth in

Although GTF enzymes have a high degrees of similarity, lactobacilli produce a broad spectrum of glucans, including polymers with α-1,6 linkages (dextran), α-1,3 linkages (mutan), and both α-1,6 and α-1,4 linkages (alternan). The relative molecular weight of

Glucan synthesis reactions catalysed by GTF can be written as follows (Fig. 1):

sucrose + acceptor carbohydrate → oligosaccharide + fructose

The GTF enzymes of streptococci are generally produced constitutively. In contrast, the GTF enzymes of *Leuconostoc* species are specifically induced by sucrose. For example, GTF expression in *Leuc. mesenteroides* is low in the presence of carbon sources other than sucrose and is increased by the addition of sucrose [5]. GTF expression during sucrose fermentation is 10-15-fold higher than that measured during glucose fermentation in *Leuc. mesenteroides* Lcc4. In fed-batch fermentation with both glucose and sucrose, GTF activity was similar to that obtained with sucrose alone. These results show that GTF expression is low in the presence of glucose alone, and that GTF activity is significantly induced by sucrose. A sucrose concentration of 20 g/l is sufficient to ensure the induction of enzyme synthesis, and higher concentrations (up to 60 g/l) do not lead to a further increase in enzyme synthesis [65].

```
The fructan synthesis reaction catalyzed by FTF can be written as follows: 
sucrose + H2O → fructose + glucose 
sucrose + acceptor carbohydrate → oligosaccharide + glucose 
sucrose + fructan (n) → fructan (n+1) + glucose
```
Fructans generally have a relative molecular weight exceeding 5 × 106 Da. Similar to GTFs, FTFs are not saturated by their substrate, namely, sucrose, and transfer reactions exceed the rate of sucrose hydrolysis for sucrose concentrations above 200 mM [5]. FTFs such as Lev, Inu, and LevS from lactobacilli exhibit pH optima of between 5.0 and 5.5. The optimum temperature for enzymes from the thermophilic *Lb. reuteri* is higher (50°C) than that of the *Lb. sanfranciscensis* enzyme (35°C – 40°C) [5].

### **3.2. Hetero EPS biosynthesis**

Hetero EPS are not synthesized by extracellular enzymes, but are instead synthesized by a complex sequence of interactions involving intracellular enzymes. EPS are made by polymerization of repeating units, and these repeating units are built by a series of addition of sugar nucleotides at the cytoplasmic membrane. Sugars are the starting materials for the synthesize sequence. LAB strains can utilize various monosaccharides and disaccharides as energy sources, via some well-studied sugar uptake systems include primary transport systems, direct coupling of sugar translocation to ATP hydrolysis via a transport-specific ATPase; secondary sugar transport systems, coupling of sugar transport to the transport of ions or other solutes, both as symport and antiport transport systems; and group translocation systems, coupling of sugar transport to phosphorylation via the phosphoenolpyruvate (PEP) dependent phosphotransferase system (PTS; Fig. 2) [8]. Polysaccharides must be hydrolyzed before uptake. For example, starch is hydrolyzed by α-amylase, and the raction products are subsequently hydrolyzed by the enzymes described above.

Exopolysaccharides of Lactic Acid Bacteria for Food and Colon Health Applications 523

**Figure 2.** Pathway of lactose fermentation in lactic acid bacteria.

for EPS biosynthesis. The addition of the repeating unit is completed by the action of glycosyl transferase on the sugar residue attached to C55-polyprenyl phosphate. Therefore, the type and number of glycosyl transferases available determine the range of repeating units in hetero-EPS. C55-polyprenyl phosphate is also involved in bacterial cell wall biosynthesis, and therefore, cell wall biosynthesis and EPS synthesis compete for this

*Lc. lactis* strains possess a lactose-specific PEP-PTS sugar transport system that imports extracellular lactose, resulting in increased intracellular lactose-6-phosphate. Lactose-6 phosphate is then hydrolyzed, and the galactose-6-phosphate moiety is metabolized by the tagatose-6P pathway (Fig. 2).

*Lb. delbrueckii* subsp. *bulgaricus* and *Str. thermophilus* are generally galactose-negative and take up lactose via a lactose/galactose antiport transport system. The glucose moiety of imported lactose is fermented by these strains, while the galactose moiety is excreted via the lactose/galactose antiport system.

After the addition of a hetero-EPS repeating unit, the unit is exported through the cell membrane and becomes polymerized into the final hetero-EPS. Hence, several enzymes and proteins are involved in the biosynthesis and secretion of heterotype EPS, and the enzymes and proteins involved in these processes may not be unique to hetero-EPS anabolism.

Sugars taken into the cell are converted into sugar nucleotides. Iintracellular monosaccharides are converted to sugar nucleotide substrates for polymerization reactions, including UDP (uridine diphosphate), dNTP (thymidine diphosphate), and GDP (guanosine diphosphate). Such polymerization reactions are catalyzed by glycosyl pyrophosphorylases.

Glu-1P (Gal-1P) + UTP → UDP-Glu (UDP-Gal) + pyrophosphate

UDP-glucose is then converted to UDP-galactose by epimerases such as UDP-glucose-4 epimerase. This reaction is reversible.

UDP-glucose ↔ UDP-galactose

Glycosidic linkages are formed on membranes in the cytoplasm. A sugar moiety is transferred to C55-polyprenyl phosphate, a carrier lipid and component of the membrane, by priming glycosyl transferases. This transfer triggers the addition of a repeating unit to the hetero-EPS molecule. Disruption of the priming glycosyl transferase gene generates non-EPS-producing mutants [66]. Thus, priming glycosyl transferases are thought to be crucial

**Figure 2.** Pathway of lactose fermentation in lactic acid bacteria.

subsequently hydrolyzed by the enzymes described above.

Hetero EPS are not synthesized by extracellular enzymes, but are instead synthesized by a complex sequence of interactions involving intracellular enzymes. EPS are made by polymerization of repeating units, and these repeating units are built by a series of addition of sugar nucleotides at the cytoplasmic membrane. Sugars are the starting materials for the synthesize sequence. LAB strains can utilize various monosaccharides and disaccharides as energy sources, via some well-studied sugar uptake systems include primary transport systems, direct coupling of sugar translocation to ATP hydrolysis via a transport-specific ATPase; secondary sugar transport systems, coupling of sugar transport to the transport of ions or other solutes, both as symport and antiport transport systems; and group translocation systems, coupling of sugar transport to phosphorylation via the phosphoenolpyruvate (PEP) dependent phosphotransferase system (PTS; Fig. 2) [8]. Polysaccharides must be hydrolyzed before uptake. For example, starch is hydrolyzed by α-amylase, and the raction products are

*Lc. lactis* strains possess a lactose-specific PEP-PTS sugar transport system that imports extracellular lactose, resulting in increased intracellular lactose-6-phosphate. Lactose-6 phosphate is then hydrolyzed, and the galactose-6-phosphate moiety is metabolized by the

*Lb. delbrueckii* subsp. *bulgaricus* and *Str. thermophilus* are generally galactose-negative and take up lactose via a lactose/galactose antiport transport system. The glucose moiety of imported lactose is fermented by these strains, while the galactose moiety is excreted via the

After the addition of a hetero-EPS repeating unit, the unit is exported through the cell membrane and becomes polymerized into the final hetero-EPS. Hence, several enzymes and proteins are involved in the biosynthesis and secretion of heterotype EPS, and the enzymes

Sugars taken into the cell are converted into sugar nucleotides. Iintracellular monosaccharides are converted to sugar nucleotide substrates for polymerization reactions, including UDP (uridine diphosphate), dNTP (thymidine diphosphate), and GDP (guanosine diphosphate). Such polymerization reactions are catalyzed by glycosyl pyrophosphorylases.

UDP-glucose is then converted to UDP-galactose by epimerases such as UDP-glucose-4-

Glycosidic linkages are formed on membranes in the cytoplasm. A sugar moiety is transferred to C55-polyprenyl phosphate, a carrier lipid and component of the membrane, by priming glycosyl transferases. This transfer triggers the addition of a repeating unit to the hetero-EPS molecule. Disruption of the priming glycosyl transferase gene generates non-EPS-producing mutants [66]. Thus, priming glycosyl transferases are thought to be crucial

and proteins involved in these processes may not be unique to hetero-EPS anabolism.

Glu-1P (Gal-1P) + UTP → UDP-Glu (UDP-Gal) + pyrophosphate

**3.2. Hetero EPS biosynthesis** 

tagatose-6P pathway (Fig. 2).

lactose/galactose antiport system.

epimerase. This reaction is reversible. UDP-glucose ↔ UDP-galactose

> for EPS biosynthesis. The addition of the repeating unit is completed by the action of glycosyl transferase on the sugar residue attached to C55-polyprenyl phosphate. Therefore, the type and number of glycosyl transferases available determine the range of repeating units in hetero-EPS. C55-polyprenyl phosphate is also involved in bacterial cell wall biosynthesis, and therefore, cell wall biosynthesis and EPS synthesis compete for this

substrate. The repeating unit is exported through the bacterial membrane, and is polymerized to become a hetero-EPS (Fig. 3).

Exopolysaccharides of Lactic Acid Bacteria for Food and Colon Health Applications 525

mutants. Tsuda et al. generated the EPS-producing mutant strain 301102S from the non-EPS-producing *Lb. plantarum* 301102 following exposure to the mutagens acridine orange and novobiocin [4]. The activities of α-phosphoglucomutase (PGM), UDP-glucose pyrophosphorylase (UGP), and UDP-galactose 4-epimerase (UGE) were measured in parental and mutant strains by using the method of Mozzi [68], and were found to be almost the same for both [Tsuda & Miyamoto, unpublished data]. Next, priming glycosyl transferase genes in parental and mutant strains were amplified with the thermal cycler. Primers were designed to amplify a priming glycosyl transferase gene referring to complete *Lb. plantarum* WCFS1 genome sequenced [69]. PCR products were subjected to restriction digestion, which allowed identification of putative priming glycosyl transferase gene. PCR products were also applied to single strand conformation polymorphism (SSCP) analysis for detecting point mutations. However, both parental and mutant strains had the same priming glycosyl transferase gene sequence, and similar levels of activities of the PGM, UGP, and UGE enzymes. Thus, although priming glycosyl transferases are essential, other factors may also be necessary for EPS production, and a mutation affecting EPS production may occure in another gene. Morona et al. reported that an autophosphorylating protein-tyrosine kinase is essential for encapsulation in *Streptococcus pneumoniae* [70]. A point mutation in the gene encoding the autophosphorylating protein-tyrosine kinase affecting the ATP-binding domain resulted

in loss of EPS production.

ingredients in foods.

**4. Polysaccharides and oligosaccharides for colon health** 

EPS produced by LAB have various functional roles in human or animal health including immunomodulatory properties, antiviral activity, antioxidant activity, and antihypertensive activity [1, 55, 71, 72], and have also been used as food additives for texture improvement. These properties have been extensively reviewed [8, 9, 56, 73, 74]. Besides these properties, prebiotics based on LAB and oligosaccharides have other health benefits. Prebiotics are usually non-digestible oligosaccharides that selectively stimulate the growth and activity of a limited number of bacterial species in the colon, such as bifidobacteria and lactobacilli, and therefore, improve host health. Detrimental bacteria may form substances such as ammonia, hydrogen sulfide, indles, and amines that are noxious to the host. However, beneficial bacteria such as bifidobacteria and lactobacilli inhibit the proliferation of detrimental bacteria, and their cell components stimulate the host immune system [75]. Gastrointestinal microflora consist of approximately 1014 colony forming units (cfu)/g of various types of both detrimental and beneficial bacteria, and the numbers and composition vary greatly along the gastrointestinal tract. The balance of the gastrointestinal micro flora influences different aspects of host health such as bowel movement, tympanites flatulence, and the absorption of nutrients. Many factors may upset this balance, including stress, consumption of antibiotics, infection, food poisoning, and the natural ageing process. To redress this balance, the growth and activities of beneficial bacteria may be enhanced by specific

**Figure 3.** Outline of biosynthesis of hetero EPS. PGM: α-phosphoglucomutase, UGP: UDP-glucose pyrophospholyrase UGE: UDP-galactose 4-epimerase, TGP: dTDP-glucose pyrophospholyrase TRS: dTDP-rhamnose synthetic enzyme system, PMI: phosphomannoisomerase PMM: phosphomannomutase, GMP: GDP-mannose pyrophospholyrase
