**2.2. Hetero-EPS**

The chemical composition of hetero-EPS shows wide variablity. Hetero-EPS are polymerized repeating units mainly composed of D-glucose, D-galactose, and L-rhamnose. The composition of the monosaccharide subunits and the structure of the repeating units are considered not to be species-specific, except in case of *Lactobacillus kefiranofaciens* subsp. *kefiranofaciens*. This species, isolated from kefir grain, a fermented dairy food from the North Caucasus region, produces large amounts of polysaccharides [24]. Hetero-EPS-producing strains of *Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii,* and *Lactobacillus helveticus,* among others have been identified (Table 2) [25-49]. Heterofermentative LAB such as *Leuc. dextranicum* are well known homo-EPS producers, while homofermentative LAB are well-studied hetero-EPS producers. Heterofermentative in addition to homofermentative LAB can produce EPS. *Lactobacillus fermentum* is an EPSproducing heterofermentative LAB for which the EPS structure has been determined [50]. Figueroa et al. reported that *Lactobacillus brevis* and *Lactobacillus buchneri* showed ropiness on glucose- or sucrose-containing media, although they did not investigate whether such ropiness derived from hetero-EPS or from other slimy substances [51].

The quantities of hetero-EPS produced by LAB vary greatly. EPS production is 50-350 mg/l for *Str. thermophilus*, 80-600 mg/l for *Lc. lactis subsp. cremoris*, 60-150 mg/l for *Lb. delbrueckii*  subsp. *bulgaricus*, 50-60 mg/l for *Lactobacillus casei* [52], and approximately 140 mg/l for *Lactobacillus plantarum* [45, 53]. The highest recorded yields of hetero-EPS are 2775 mg/l for *Lactobacillus rhamnosus* RW-9595M [54] and 2500 mg/l for *Lb. kefiranofaciens* WT-2B [55]. However, the quantities of EPS produced by LAB are much lower than the yields from other industrially important microorganisms such as *Xanthomonas campestris*, which produces 30- 50 g/l xanthan gum [56]. Even so, amounts of EPS produced by LAB are sufficient to exploit for in situ applications. LAB are 'generally recognized as safe' (GRAS) microorganisms, and LAB strain culture would be a useful method to produce EPS for food applications if the LAB could be grown in edible and safe culture media such as whey, and if fermentation conditions were optimized to obtain a high yield.

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

microorganisms adhere to the surface of teeth.

alternan-producing strain [21]

be fructan [11].

**2.2. Hetero-EPS** 

from the proportions of different types of linkages; water-soluble glucans are rich in α-1,6 linkages, while water-insoluble glucans are rich in α-1,3 linkages [17]. Ingestion of mutan has been linked with dental caries, as insoluble mutans can adhere to teeth, thus helping

Alternan has alternate α-1,6 and α-1,3 linkages, and this structure is thought to be responsible for its distinctive physical properties including high solubility and low viscosity. These characteristics provide this glucan with a potential commercial application as a low viscosity texturizer in foods. *Leuc. mesenteroides* NRRL B-1355 was first reported to be an

Levan is an EPS produced from sucrose. It is fructan composed of β-2,6-linked fructose molecules with some β-2,1-linked branches. Incidentally, inulin is a fructan composed of β-2,1-linked fructose molecules with some β-2,6-linked branches. *Str. salivarius, Leuc. mesenteroides,* and *Lactobacillus reuteri* are known to be levan-producing LAB [22-23]. In addition, the EPS produced by *Lactobacillus sanfranciscensis* TMW 1.392 has been reported to

The chemical composition of hetero-EPS shows wide variablity. Hetero-EPS are polymerized repeating units mainly composed of D-glucose, D-galactose, and L-rhamnose. The composition of the monosaccharide subunits and the structure of the repeating units are considered not to be species-specific, except in case of *Lactobacillus kefiranofaciens* subsp. *kefiranofaciens*. This species, isolated from kefir grain, a fermented dairy food from the North Caucasus region, produces large amounts of polysaccharides [24]. Hetero-EPS-producing strains of *Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii,* and *Lactobacillus helveticus,* among others have been identified (Table 2) [25-49]. Heterofermentative LAB such as *Leuc. dextranicum* are well known homo-EPS producers, while homofermentative LAB are well-studied hetero-EPS producers. Heterofermentative in addition to homofermentative LAB can produce EPS. *Lactobacillus fermentum* is an EPSproducing heterofermentative LAB for which the EPS structure has been determined [50]. Figueroa et al. reported that *Lactobacillus brevis* and *Lactobacillus buchneri* showed ropiness on glucose- or sucrose-containing media, although they did not investigate whether such

The quantities of hetero-EPS produced by LAB vary greatly. EPS production is 50-350 mg/l for *Str. thermophilus*, 80-600 mg/l for *Lc. lactis subsp. cremoris*, 60-150 mg/l for *Lb. delbrueckii*  subsp. *bulgaricus*, 50-60 mg/l for *Lactobacillus casei* [52], and approximately 140 mg/l for *Lactobacillus plantarum* [45, 53]. The highest recorded yields of hetero-EPS are 2775 mg/l for *Lactobacillus rhamnosus* RW-9595M [54] and 2500 mg/l for *Lb. kefiranofaciens* WT-2B [55]. However, the quantities of EPS produced by LAB are much lower than the yields from other industrially important microorganisms such as *Xanthomonas campestris*, which produces 30- 50 g/l xanthan gum [56]. Even so, amounts of EPS produced by LAB are sufficient to exploit for in situ applications. LAB are 'generally recognized as safe' (GRAS) microorganisms, and

ropiness derived from hetero-EPS or from other slimy substances [51].

Fermentation conditions using undefined media have been improved to maximize yields. However, a chemically defined medium containing a carbohydrate source, mineral salts, amino acids, vitamins, and nucleic acid bases is more suitable for investigating the influence of different nutrients on LAB growth and EPS biosynthesis. The total yield of EPS produced by LAB depends on the composition of the medium (carbon and nitrogen sources) and the growth conditions, i.e., temperature, pH, and incubation time.

Under conditions of higher temperatures and slower growth, the production of the polymer per cell in *Lb. delbrueckii* subsp. *bulgaricus* NCFB 2772 was greater in milk [57]. Another study investigated the optimum culture conditions for EPS production by *Lb. delbrueckii* subsp. *bulgaricus* RR in semidefined medium [58], and determined the optimum temperature and pH conditions for EPS production to be 36°C - 39°C and pH 4.5 - 5.5. The optimal temperature for EPS production was approximately 40°C for thermophilic LAB strains, and around 25°C for mesophilic LAB. Gamar et al. [59] reported increased slime production at lower incubation temperatures, and an increase in the final EPS concentration in *Lb. rhamnosus* following incubation at 25°C instead of 30°C. The effects of temperature on EPS production in whey were investigated in *Lb. plantarum* [53], and the yield was found to be higher at 25°C than at either 30°C or 37°C. Moreover, an inverse relationship was observed between EPS production per cell and the growth temperature for *Lactobacillus sake* [49], i.e. the lower the temperature, the higher the EPS production per cell. However, the growth rate in the exponential phase decreased at low temperatures. Therefore, the temperature for the maximal production of EPS is based on a balance of cell density and EPS production per cell. Maximal EPS production by *Lb. sake* was obtained under anaerobic conditions at 20°C, although EPS production per cell was higher at 10°C. Therefore, it is possible that severe environmental conditions trigger EPS production as a protective mechanism.

The effects of alterations to the nitrogen and carbon sources used in EPS production have also been investigated. According to early reports, neither LAB growth nor EPS production was specifically linked to the presence of casein or whey proteins in the growth medium. Garcia et al. [57] reported that EPS production by *Lb. delbrueckii* subsp. *bulgaricus* NCFB 2772 increased during the early growth pase in the presence of hydrolyzed casein in milk, while the addition of hydrolyzed casein to MRS medium did not increase EPS production. This strain produced 25 mg/l EPS when grown on fructose in a defined medium, and 80 mg/l EPS when grown on glucose [60]. The optimum Bacto-casitone concentration for EPS production by *Lb. delbrueckii* subsp. *bulgaricus* RR was investigated in semidefined medium [58]. In this study, there was a significant relationship between the Bacto-casitone concentration and EPS production; the higher the casitone concentration, the higher the EPS yield that was obtained. For *Lb. plantarum* grown in whey, yeast extract was a more effective nitrogen source for EPS production than soy peptide, tryptone, peptone, and Lab-Lemco powder,

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 amino acids alone had no effect on EPS production.

Exopolysaccharides of Lactic Acid Bacteria for Food and Colon Health Applications 521

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

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].

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

The fructan synthesis reaction catalyzed by FTF can be written as follows:

sucrose + acceptor carbohydrate → oligosaccharide + glucose

under sucrose concentrations above 100 mM [64].

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

sucrose + H2O → fructose + glucose

sucrose + fructan (n) → fructan (n+1) + glucose

*Lb. sanfranciscensis* enzyme (35°C – 40°C) [5].

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 EPS production.
