Exopolysaccharides
production by Lactobacillus bulgaricus:
A review
ABSTRACT
The
production of exopolysaccharides 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 30oC.
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.
Keyword: Lactobacillus
bulgaricus, exopolysaccharide
INTRODUCTION
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.
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.
PRODUCTION
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.
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).
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 30oC. 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.
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.
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 37oC after 24 h of fermentation.
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.
COMPOSITION
AND STRUCTURE
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.
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).
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).
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).
BIOSYNTHESIS
Zourari
et al. (1992) suggested that L. delbrueckii subsp. bulgaricus, lactose uptake takes place via a lactose
permease. Inside the cell, β-galactosidase cleaves lactose to form glucose
and galactose. The latter is exchanged
with lactose via a lactose-galactose antiport system. Malaka and Abustam (2004) reported that
during the early exponential phase of growth of L. delbrueckii subsp. bulgaricus RR, there was no EPS
biosynthesis. Production of EPS started
at the end of exponential phase, but most EPS was produced at the end of growth
and approximately 90% of glucose was consumed in the stationary phase (Petry et al., 2000).
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).
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).
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).
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).
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).
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.
APPLICATION OF EPS
EPS
have a variety of industrial applications, including their use as biothickeners
in foods. EPS produced by LAB contribute
significantly to the structure and viscosity of fermented milk product (Malaka,
1997; Malaka et al., 1996; Malaka et al., 1997; Malaka and Baco, 2000;
Boels et al., 2003). Polysaccharides
may function in foods as viscosifying agents, stabilizers, emulsifiers, gelling
agents, or water binding agents (van den Berg et al., 1995). EPS of
bacteria origin have unique rheological properties because of their capability
of forming very viscous solutions at low concentrations and their pseudoplastic
nature (Yang et al., 1999). The EPS produced by food-grade LAB have been
considered as a new generation of food thickeners to improve the rheological
properties of foods (Robijn, 1996).
Perry et al. (1997) reported
that using EPS starter cultures consisting of specific strains of L. bulgaricus increases moisture
retention and improve the melt of lower-fat mozzarella cheese. Hess et
al. (1997) demonstrated that yoghurt made with EPS-producing strains of L. delbrueckii subsp. bulgaricus were less susceptible to
syneresis, required less force to penetrate the gel, and were more extensible
than yoghurt made with nonropy strain (non EPS-producing strains). The EPS appear to interact with the surface
of bacterial cells and is associated with the protein matrix, affecting the
viscosity and stability of the milk gel (Schellhaass and Morris, 1985).
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).
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.
CONCLUSIONS
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.
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).
The
EPS produced by foodgrade LAB, such as L.
bulgaricus have been considered as a new generation of food thickeners to
improve the rheological properties of foods.
On the other hand, the EPS have potentials pharmaceutical prospect as
antitumor activity, or antivirus would be develop more extensive in the future
time.
ACKNOWLEDGEMENT
I am indebted to my husband Prof. Sudirman Baco for his suffort and
reading the manuscript.
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Publikasi pada Jurnal INSTEK (Informasi Sains dan Teknologi Kimia) ISSN 1693-5861
No. 2 Tahun 2 : 84-164 (2005)
