Exopolysaccharide production by Lactbacilus bulgaricus

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            The article explain exopolysaccharides production by Lactobacillus bulgaricus by literature studied. The production of exopolysaccharides (EPS) by Lactobacillus bulgaricus, chemical composition, structure, synthesis and application are outlined.  Production of EPS by bacteria is dependent on the temperature and pH of the medium in terms of carbon, nitrogen, mineral and vitamin content.  Optimal EPS production by L. bulgaricus were at 30^oC.  Most of researches concluded that L. bulgaricus produced EPS between 130 – 330 mg/l in suitable environment and medium.  Composition of EPS mainly composed glucose, galactose and rhamnose in variative ratio, but galactose often dominant comparison in another monosaccharide.   Application of EPS in food can increase the quality of product. On the other hand, the possibility that this EPS may be involved in antitumor activity and another pharmaceutical agent need more confirmation.

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            Exopolysaccharides are produced by great variety of bacteria including lactic acid bacteria (LAB) (Broatbent et al., 2003).  The name exopolysaccharides (EPS) as proposed by Sutherland (1972) provides a general term for all these forms of bacterial polysaccharides found outside the cell wall and will be used in this article. 

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            Microorganism produce EPS in two distinct forms: ropy EPS or loose slime that is excreted into surroundings, and capsular EPS that remain adhered to cell surface creating a discrete covering (Zisu and Shah, 2003).  The role of EPS to cell bacteria is not clearly defined.  In the last decade, an increasing interest was observed in the EPS produced by the good-grade LAB, such as L. bulgaricus as starter culture of fermented milk. These polysaccharides play an important role in the rheology, texture and mouth feel of fermented milk. The objective of this article is to review the EPS production by L. bulgaricus.

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The productions of EPS are found in many species of both Gram-positive and Gram-negative bacteria and have been the subject of numerous investigations in varied fields of interest (Schellhass, 1983).  Soil scientists have an interest in EPS doe to the importance of these polysaccharides to soil properties and the possible role of EPS in the symbiotic relationship between different species of Rhizobium and legumes.  The industrial microbiologist has become interested in exploiting microbial EPS production due to increasing interest and need for novel polysaccharides (Malaka, 1997).  Utilizing microorganisms to produce EPS may offer both economic and availability advantage over traditional plant and marine sources. 

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            Most mucoid bacteria produce EPS under all cultural conditions, but the growth conditions must be optimized for maximal production. Lactobacillus bulgaricus capable of forming 250 mg/l of EPS on milk ultra filtrated, while as much as 350 mg/l had been found in milk  (Cerning, 1990; Grobben et al., 1995). 

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            Production of EPS is dependent on the temperature and pH of the medium as well as composition of the medium in terms of carbon and nitrogen source, and mineral and vitamin contents (Gamar-Nourani et al., 1998; Grobben et al., 2000).  The effect of temperature and pH on EPS production is highly variable and is dependent on the strain used and the experimental conditions.  Some workers have found EPS production to be optimal at low temperature (Marshall et al., 1995; van den Berg et al., 1995), while others have shown EPS production to be favored at much higher temperatures (Grobben et al., 1995; De Vuyst et al., 1998).  The optimum pH for EPS production generally ranges between 5 and 7 (De Vuyst et al., 1998).  Schellhaass (1983) reported that growth at pH 6.2 and 6.0 resulted in more polysaccharide production with L. bulgaricus RR  in skim milk dialysate than growth at pH 5.8 and 5.5. Maintenance of higher pH might result in increased exopolysaccharide production by increasing the time the culture was in log phase.  Higher pH results in a longer stationary phase, which would decrease peptidoglycan and teichoic acid synthesis and could result in increased exopolysaccharide production. Malaka and Abustam (2004) have shown that optimal EPS production for L. bulgaricus were at 30^oC.  Shellhaass and Morris (1985), Malaka and Abustam (2004) indicated that lower incubation temperature resulted in slower growth rate, which promoted longer logarithmic and stationary phases.  Gassem et al. (1997) reported that when lactose was hydrolyzed during fermentation, glucose was utilized by the bacteria and galactose remained in the medium. The composition of the medium (Marshall et al., 1995) and carbon source (Gamar et al., 1997) are shown to increase EPS production.  Grobben et al. (1998) have been shown that EPS production by L. bulgaricus enhanced by multiple-vitamin omission. 

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            Many studies showed a decrease in the total EPS amount when incubation times were increased (Cerning et al., 1992; De Vuyst et al., 1998; Ganzel and Novel, 1994; Mozzi et al., 1996).  The decreased EPS level upon prolonged fermentation may be due to an enzymatic degradation (Cerning, 1990; De Vuyst et al., 1998) or a change in the physical parameters of culture (De Vuyst et al., 1998).  Ganzel and Novel (1994) suggested some reversible DNA rearrangements leading to different cell types which differ in exopolymer production capabilities.  However, the possible relationship between EPS production and the factors contributing to EPS degradation have not been investigated yet.

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            Bouzar et al. (1995) have shown that pink colonies of L. bulgaricus produced the highest amount (130 mg/l), the white colonies produced the lowest (70 mg/l), and the parental strain produced an intermediate amount (110 mg/l) of EPS.  Approximately half of the EPS was produced during the exponential growth phase; while Malaka and Abustam (2004) have shown that EPS was produced during the last exponential growth phase go in the stationary growth phase.  Lactobacillus delbrueckii ssp. bulgaricus RR can be grown in protein-hydrolyzed whey and produce EPS (between 313-330 mg/l), although the bacteria is typically weakly proteolitic and grows poorly in un supplemented media (Briczinski and Roberts, 2002).  The level of EPS production obtained by Toba et al. (1992) through fermentation of skim milk by L. delbrueckii sups. bulgaricus NCFB 2483 were 150 mg/l, by Cerning et al. (1986) through fermentation of skim milk with 1% casein by CNRZ 737 were 424 mg/l, and by Kimmel et al. (1998) through optimizing in semi-defined medium by strain RR were 354 mg/l.  Mozzi et al. (1995) have been shown that L. bulgaricus was the best EPS producer among the bacteria assayed (Streptococcus thermophilus, Lactobacillus acidophilus), an amount of 121 mg/l being obtained at 37^oC after 24 h of fermentation.

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            Petry et al. (2000) developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and EPS production of two strains of L. delbrueckii subsp. bulgaricus.  The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as urotic acid and the carbon source.  EPS production was greatest during the stationary phase.  For strain CNRZ 416 without pH-controlled conditions, 10 g of glucose per liter seemed to be optimal carbon source concentration for the highest EPS yield.  The EPS yield of strain CNRZ 1187 appeared not to be affected by the sugar concentration of the medium.  The EPS yield of the CNRZ strain was 175 mg/l.

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            The findings of numerous researches agree in as much as EPS indicate galactose and glucose to be released after hydrolysis of the polymer (Schellhaass, 1983; Cerning et al., 1986), but the molar ratios reported are very different.  Already by Cerning et al. (1986) investigated the composition of slime secreted by a strain of L. bulgaricus and reported that galactose was the most frequently encountered monomer within the polysaccharide, glucose and rhamnose are present in smaller amounts.  Gas liquid chromatography confirmed the HPTLC identification and revealed that the polymer is composed of galactose, glucose and rhamnose in a molar ratio of approximately 4: 1:1.  The polymers produced by the ropy and non-ropy strains have the same sugar components.  The elution volume of the EPS from the CL Sepharose $B column was almost the same as that of dextran of molecular weight of 488.000 Da and a single sharp peak was observed indicating that the EPS consisted of a single polymer.

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There exist three important groups of EPS produced by LAB: (i)α-glucans, mainly composed of α-1,6- and α-1,3-linked glucose residues, namely, dextran produced by Leuconostoc mesenteroides subsp. Mesenteroides and L. mesenteroides subsp. Dextranicum and mutants produced by Streptococcus mutants and S. sobrinus; (ii) fructans, mainly composes of β-2,6-linked fructose molecules, such as levan produced by S. salivarius; (iii) heteropolysaccharides produced by mesophilic (Lactococcus lactis subsp. Lactis and L. lactis subsp cremoris) and thermophilic (L. delbrueckii subsp. bulgaricus, L. helveticus, and S. thermophilus) LAB (Pham et al., 2000).

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            The sugar components of EPS L. bulgaricus are most frequently galactose, glucose and very often rhamnose (Cerning, 1990); galactose and small quantities glucose and rhamnose (Grobben et al., 1997).  The EPS from strain L. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain CNRZ 416 contained galactose, glucose and rhamnose (Petry et al., 2000).

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            These heteropolysaccharides are composed of linear and branched repeating units varying in size from tetra – to heptasaccharides.  The final EPS of high molecular mass   (1 x 10⁶ – 2 x 10⁶ Da) is formed by polymerization of several hundred to a few thousands of these repeating units (Petry et al., 2000).  L. bulgaricus yielded EPS of molecular weight of approximately 500.000, that contained galactose, glucose, and rhamnose in a molar ratio of approximately 4:4:1 (Cerning et al., 1991); 5 : 1 : 1 with predominantly 1, 4 and 1, 3 linkage patterns (Gruter et al., 1993).

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            Exocellular polysaccharides are synthesized in different growth phases and under a variety of conditions, depending on the organism studied (Cerning, 1990).  The process of synthesis involved can be divided into two principal categories based essentially on the site of synthesis and the nature of the precursors, i.e. synthesis outside the cell or at the cell membrane (Sutherland, 1972). 

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In many cases, research interest in biosynthesis of bacterial heteropolysaccharides has been driven by the knowledge that these polymers played an important role in virulence.  Thus, EPS biosynthesis is relatively well defined in Gram-negative pathogenic bacteria such as Escherichia coli, Salmonella, and Haemophilus influenza.  In these gram-negative systems, assembly of the basic repeating unit occurs at the cytoplasmic membrane and involves sequential transfer of sugar nucleotide diphospho precursors to an isoprenoid lipid carrier, undecaprenyl phosphate (Sutherland, 1972).  Once the basic repeating unit is assembled, the lipid-linked intermediates are usually translocated across the membrane and polymerized outside of the cell.  Then, the EPS may be covalently linked to the cell surface to form a capsule, or released into the medium as slime (Broadbent et al., 2003).

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Functional genetic studies and homology comparisons of the EPS-related gene products from some species lactic acid bacteria (maybe included L. bulgaricus) strongly suggest that cellular mechanisms for EPS synthesis in these species are similar to Gram-negative systems that require formation of lipid-linked intermediates (Stingele et al., 1996; Broadbent et al., 2001).  Work by Stingele et al. (1999) has shown that EPS biosynthesis in S. thermophilus involved a lipid carrier, upon which the sugar monomers that comprise the basic repeating unit are assembled.  The biosynthesis of EPS by L. bulgaricus has not been reported yet, but maybe similar with S. thermophilus.  The first step is catalyzed by Eps, a galactosyl-1 phosphate transferase that attaches galactosyl-phosphate to a lipid-phosphate carrier.  This reaction is followed by the action of three other glycosyltransferases, termed EpsG, EpsI, and EpsF, which sequentially attach N-acetylgalactosamine, glucose, and a branching galactose moiety to the basic repeating unit.  Unfortunately, very little is known about EPS polymerization, translocation, or slime formation by L. bulgaricus or any other Gram-positive bacteria.  Amino acid homologies between enzymes involved in these processes in Gram-negative and Gram-positive bacteria suggest that they occur via similar mechanism, but this hypothesis awaits experimental confirmation (Broatbent et al., 2003).

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            Cellular EPS biosynthesis is, therefore, an energy-intensive process that involves enzymes for production of sugar nucleotide precursors, a glycosyl1-1-phosphate transferase that transfers the first sugar group onto a phosphorylated carrier lipid, one or more glycosyltransferases that sequentially add new sugar groups to the growing repeat unit, and additional enzymes that participate in EPS gene regulation, membrane translocation, and polymerization/ chain length determination function (Cerning, 1990).  These reactions commonly involve additional proteins with function that are not unique to this process (Sutherland, 1972).

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            As noted previously, the lipid carrier for EPS assembly in Gram-negative bacteria is undercaprenyl phosphate, and participation of this molecule in EPS synthesis by LAB may be significant because it is also required for assembly of peptidoglycan, teichoid acids, and lipoteichoid acids (Cerning, 1990).  Specifically, if the cellular concentration of UDP is limited, then competition for lipid carrier may be a key limiting factor in EPS biosynthesis.  This hypothesis is supported by the frequent observation that incubation under conditions that stimulate bacterial growth and division reduce EPS production, while growth conditions which reduce for new cell wall synthesis (such as incubation at a suboptimal temperature) tend to enhance EPS production (Sutherland, 1972; Cerning, 1995; Malaka and Abustam, 2004).

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            Competition for sugar nucleotide precursors is another potential limitation to high EPS yields from EPS-producing LAB (Boels et al., 2001).  Many of the sugar nucleotide precursors used for EPS occur as intermediates in sugar catabolic pathways or, like UDP, are needed for the assembly of peptidoglycan and other glycan-containing cellular polymers (Broadbent et al., 2003).  Because low levels of sugar precursors are limiting factors on EPS yields (unpublished data), greater understanding of L. bulgaricus metabolic flux in sugar metabolism may provide new strategies to enhance EPS production.  Several researchers have demonstrated a correlation between EPS production and intracellular activities on enzymes from Leloir (Galactose-metabolism) and glycolytic pathways, but strain to strain variations are also apparent (Escalante et al., 1998; Levander and Radstrom, 2001).  Although the basis for these differences is not yet clear, recently in our research (unpublished data) showed such knowledge can be used to enhance EPS production in L. bulgaricus by metabolic engineering of central carbon metabolism.

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 Furthermore, several reports indicate that they can confer health benefits on consumers arising from their immunogenic and cholesterol-lowering properties (Hess et al., 1997).  As well as live bacteria (probiotics), EPS can improve intestinal balance to promote health; dietary carbohydrates may function as prebiotics, beneficially affecting the colonic microflora (Yang et al., 1999).

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Oda et al. (1983) reported an antitumor EPS produced by L. helveticus.  The slime produced by Bifidobacterium adolescentis had immunomodifying effects on mouse splennocytes (Gomez et al., 1988).  Kitazawa et al. (1992) showed that the slime-forming Lc. Lactis subsp. cremoris KVS 20 had antitumor activity, and the slime contained strong B-cell dependent mitogenic substances.

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            Production of EPS by L. bulgaricus is growth-associated, and it is influenced by medium compositioc, culture temperature and pH.  Variations in carbon sources in growth medium resulted in different yields of EPS produced by L. bulgaricus, product range from 110 mg/l until 330 mg/l.

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            The EPS L. bulgaricus was shown to consist of linear and branched repeating units varying in size from tetra – to heptasaccharides.  The sugar component of the EPS is most frequently galactose, glucose and very often rhamnose in a molar ratio of approximately 4:4:1 (Cerning et al., 1991); 5:1:1 with predominantly 1,4 and 1,3 linkage patterns (Gruter et al., 1993).