Exopolysaccharides from lactic acid bacteria: perspectives and challenges

Some lactic acid bacteria (LAB) secrete a polysaccharide polymer. This extracellular polysaccharide, or "exopolysaccharide" (EPS), is economically important because it can impart functional effects to foods and confer beneficial health effects. LAB have a "Generally Recognized As Safe" (GRAS) classification and are likely candidates for the production of functional EPSs. Current challenges are to improve the productivity of EPSs from LAB and to produce EPSs of a structure and size that impart the desired functionality. The engineering of improvements in these properties will depend on a deep understanding of the EPS biosynthetic metabolism and of how the structure of EPSs relates to a functional effect when incorporated into a food matrix.     

Biosynthesis, characterisation, and design of bacterial exopolysaccharides from lactic acid bacteria

Lactic acid bacteria (LAB) are characterised by their conversion of a large proportion of their carbon feed, fermentable sugars, to lactic acid. However, in addition to lactic acid production, the LAB are able to divert a small proportion of fermentable sugars towards the biosynthesis of exopolysaccharides (EPSs) that are independent of the cell surface and cell wall material. These microbial EPSs when suspended or dissolved in aqueous solution provide thickening and gelling properties, and, as such, there is great interest in using EPSs from food grade microorganisms (such as the LAB that are traditionally used for food fermentations) for use as thickening agents. The current review includes a brief summary of the recent literature describing features of the biosynthetic pathways leading to EPS production. Many aspects of EPS biosynthesis in LAB are still not fully understood and a number of inferences are made regarding the similarity of the pathway to those involved in the synthesis of other cell polysaccharides, e.g., cell wall components. The main body of the review will cover practical aspects concerned with the isolation and characterisation of EPS structures. In the last couple of years, a substantial number of structures have been published and a summary of the common elements of these structures is included as is a suggestion for a system for representing structures. A brief highlight of the attempts that are being made to design ‘tailor’-made polysaccharides using genetic modification and control of metabolic flux is presented.     

Complete Genome Sequence of Streptococcus thermophilus Strain MN-ZLW-002

Streptococcus thermophilus MN-ZLW-002 was originally isolated from traditionally fermented Chinese dairy products. One of the strain-dependent characteristics of this bacterium is its ability to produce exopolysaccharides (EPSs). This study determined and analyzed the genome sequence of MN-ZLW-002. Its complete genome comprised 2,046 genes and 1,848,520 nucleotides with an average GC content of 39%. The EPS cluster of MN-ZLW-002 includes 25 open reading frames (ORFs), and some results indicate a horizontal gene transfer between MN-ZLW-002 and other lactic acid bacteria (LAB).     

Sugar composition and FT-IR analysis of exopolysaccharides produced by microbial isolates from paper mill slime deposits

Thirty exopolysaccharides (EPS) produced by bacteria isolated from biofilms or slimelayers from different paper and board mills in Finland, France and Spain were subjected to size exclusion chromatography and sugar compositional analysis. High performance size exclusion chromatography (HPSEC) analysis revealed that some samples were composed of several molecular weight populations. These samples were fractionated by size exclusion chromatography and pooled accordingly. Principal components analysis (PCA) of the sugar compositions of the different pools indicated the presence of glucans and mannans caused by insufficient removal of the carbon or nitrogen source (yeast extract) from the bacteria growth medium leading to an overestimation of the glucose and mannose level in the sample, respectively. From the point of view of slime problems the EPS populations are the most important for multivariate analysis. Four groups of EPSs have been recognized by PCA analysis: a group of EPSs produced by Enterobacter and related genera similar to the regularly reported colanic acid; a group of Methylobacterium EPSs having high galactose and pyruvate levels and two groups that showed less dense clusters produced by Bacillus and related genera, showing high mannose and/or glucose levels and Klebsiella EPSs that showed galactose with rhamnose as major characteristic sugar moieties. Fourier transform infrared spectroscopy (FT-IR) of the same samples followed by discriminant partial least squares regression (DPLS) and linear discriminant analysis (LDA) showed that, when used with a well-defined training set, FT-IR could be used clustering instead of time-consuming sugar composition analysis. The Enterobacter and Methylobacetrium EPS groups could be recognized clearly. However the fact that this could hardly be done for the other two groups in the dataset indicates the importance of a larger and well-defined training or calibration set. The potential to use FT-IR, as a tool for pattern recognition and clustering with respect to EPS structures produced by micro organisms isolated from a paper mill environment is discussed.     

Advances in bacterial exopolysaccharides: from production to biotechnological applications

A vast number of bacterial extracellular polysaccharides (EPSs) have been reported over recent decades, and their composition, structure, biosynthesis and functional properties have been extensively studied. Despite the great diversity of molecular structures already described for bacterial EPSs, only a few have been industrially developed. The main constraints to full commercialization are their production costs, mostly related to substrate cost and downstream processing. In this article, we review EPS biosynthetic and fermentative processes, along with current downstream strategies. Limitations and constraints of bacterial EPS development are stressed and correlation of bacterial EPS properties with polymer applications is emphasized.     

Detection of extracellular bound proteinase in EPS-producing lactic acid bacteria cultures on skim milk agar

AIMS: Skim milk agar was developed to investigate extracellular cell-bound proteinase in yogurt cultures, Streptococcus thermophilus and Lactobacillus bulgaricus. METHODS AND RESULTS: The Lact. bulgaricus cultures produced more extracellular cell-bound proteinase than did Strep. thermophilus cultures. Strong positive correlations between the size of the exopolysaccharide (EPS) layer and extracellular cell-bound proteinase were found for both Streptococcus and Lactobacillus cultures. CONCLUSION: Strong positive linear relationships existed between the EPS size and colony size and the diameter of clear zone and colony size for Streptococcus cultures, whereas weak positive linear relationships were observed for Lactobacillus cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: These data are useful to validate the relationship between extracellular proteinase and the EPS size of LAB. Also, a convenient medium to detect the presence of extracellular cell-bound proteinase of LAB is valuable for dairy industries.     

Optimization of exopolysaccharide overproduction by Lactobacillus confusus in solid state fermentation under high salinity stress

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.     

Growth and exopolysaccharide (EPS) production by Oenococcus oeni I4 and structural characterization of their EPSs

AIMS: To study the influence of medium constituents on growth, and exopolysaccharide (EPS) production by a strain of Oenococcus oeni. The structure of one of the EPSs has also been characterized. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulfuric acid method. After purification and fractionation of crude EPSs, the sugar composition was determined by GLC-MS of the TMS methyl glycosides. The major polysaccharide is 2-substituted-(1-3)-beta-D-glucan. This structure was determined by methylation analysis and conventional (1)H- and (13)C-nuclear magnetic resonance spectroscopy. In addition, O. oeni synthesized two heteropolysaccharides, although a lesser proportion, constituted by galactose and glucose, and one of them also showed rhamnose. The sugar source has a clear influence on growth and EPS synthesis, and EPS production was not enhanced by adding ethanol or increasing the nitrogen source. EPS biosynthesis starts in the exponential growth phase, and continued during the stationary growth phase. CONCLUSIONS: Higher EPS yields were obtained on cultures grown on glucose + fructose. O. oeni produces a beta-glucan, as the predominant EPS, and it is also able to produce two heteropolysaccharides. Significance and Impact of the Study: This work provides a better understanding of EPS synthesis by O. oeni and shows the first EPS structure described for this species.     

Optimization of Exopolysaccharide Overproduction by Lactobacillus confusus in Solid State Fermentation under High Salinity Stress

It is believed that high concentrations of sodium chloride (NaCl) suppress the biosynthesis of exopolysaccharide (EPS) in lactic acid bacteria (LAB). Nevertheless, overproduction of EPSs due to high salinity stress in solid state fermentation performed on an agar surface was demonstrated in this study using a response surface methodology via a central composite design (CCD). Under optimized conditions with NaCl 4.97% and sucrose 136.5 g/L at 40.79 h of incubation, the EPS yield was 259% (86.36 g/L of EPS), higher than the maximum yield produced with the modified MRS medium containing only 120 g/L of sucrose without NaCl (33.4 g/L of EPS). Biosynthesis of EPS by Lactobacillus confusus TISTR 1498 was independent of biomass production. Our results indicated that high salinity stress can enhance EPS production in solid state fermentation.     

End plate spikes in the human electromyogram. Revision of the fusimotor theory.

'End plate spike' (EPS) is a spontaneous action potential of a normal striated muscle. EPSs are found in local 'active spots' of the muscle. The prevailing hypothesis about the origin of EPSs states that when a needle electrode affects a motor nerve branch near the neuromuscular junction at the end plate zone, an increased leakage of acetylcholine to the synaptic cleft ensues. This elicits postsynaptic action potentials of the muscle fibre which can be recorded as EPSs with the same needle electrode. Thus EPSs are thought to be caused by needle injury or irritation of the motor axon. We suggest that EPSs are action potentials of intrafusal muscle fibres and that 'active spots' are in fact muscle spindles. Waveform analysis reveals three types of EPSs: small EPSs, not propagated outside the active spot either: i) with negative onset; or ii) with short positive initial deflection; and iii) large EPSs resembling propagated motor unit potentials (MUPs) but with a typical EPS firing pattern, distinctly different from that of the MUPs. Study of EPS activation in different manoeuvres associates small EPSs with intrafusal gamma motor units and large MUP-like EPSs with beta motor units.     

Neutral sugar composition of extracellular polysaccharides produced by strains of Butyrivibrio fibrisolvens

The extracellular polysaccharides (EPSs) produced by 37 isolates presently classified as Butyrivibrio species (or more specifically as Butyrivibrio fibrisolvens) were purified from glucose-grown cultures. The neutral sugar compositions of these EPSs were determined by both thin-layer and gas-liquid chromatographic techniques. Results showed that while the neutral sugar composition of the EPS was constant for a given strain, it varied considerably between strains. In addition, several acidic components in the EPS, of both known and unknown structure, were detected artifactually as acetylated lactones, the acetylated alditols derived from these lactone(s), or both. Two novel components, L-altrose and the acidic sugar 4-O-[1-carboxyethyl]-D-galactose, were common constituents of the EPS from some strains of B. fibrisolvens. These and other EPS compositional features were used to sort isolates of B. fibrisolvens into groups which may have taxonomic significance. A scheme for sorting isolates into these groups, and the relative relationships between groups, is proposed.     

Genetics and engineering of microbial exopolysaccharides for food: approaches for the production of existing and novel polysaccharides.

Microbial exopolysaccharides (EPSs) are used in the food industry for their unique properties as viscosifiers, stabilisers, emulsifiers or gelling agents. In recent years, significant progress in the understanding of the genetics and biochemistry of microbial EPS synthesis by both Gram-negative and Gram-positive bacteria has been made. Biosynthesis pathways have been elucidated, and several of the genes involved have been characterised. This knowledge can now be applied to start EPS engineering or to improve EPS production.     

Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains.     

Enhancing the Production and Monosaccharide Composition of Exopolysaccharides of Lactobacillus plantarum VAL6 by Applying Thermal Stress and Increased Carbon Dioxide Concentration

Microbiology - Lactobacilli are able to produce exopolysaccharides (EPSs) with a wide diversity in structure and composition. However, changes in EPS production under environmental challenges are...     

A positive correlation between bacterial autoaggregation and biofilm formation in native Sinorhizobium meliloti isolates from Argentina

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodule formation on roots of alfalfa plants. S. meliloti produces two exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote symbiosis. EPS I and EPS II are secreted in two major fractions that reflect differing degrees of subunit polymerization, designated high- and low-molecular-weight fractions. We reported previously that EPSs are crucial for autoaggregation and biofilm formation in S. meliloti reference strains and isogenic mutants. However, the previous observations were obtained by use of "domesticated" laboratory strains, with mutations resulting from successive passages under unnatural conditions, as has been documented for reference strain Rm1021. In the present study, we analyzed the autoaggregation and biofilm formation abilities of native S. meliloti strains isolated from root nodules of alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all the native isolates revealed a high degree of identity with reference S. meliloti strains. PCR analysis of the expR gene of all the isolates showed that, as in the case of reference strain Rm8530, this gene is not interrupted by an insertion sequence (IS) element. A positive correlation was found between autoaggregation and biofilm formation abilities in these rhizobia, indicating that both processes depend on the same physical adhesive forces. Extracellular complementation experiments using mutants of the native strains showed that autoaggregation was dependent on EPS II production. Our results indicate that a functional EPS II synthetic pathway and its proper regulation are essential for cell-cell interactions and surface attachment of S. meliloti.     

Exopolysaccharides production in Lactobacillus bulgaricus and Lactobacillus casei exploiting microfiltration

The physiology of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus casei, extensively used in the dairy industry, was studied in order to evaluate key parameters in the synthesis of exopolysaccharides and to improve their production through novel fermentation processes. Selected strains were studied in shake flasks and in fermentor experiments using glucose and lactose as main carbon sources and bacto casitone as the only complex component, in a temperature range between 35 and 42°C. The production of exopolysaccharides was monitored and correlated to the growth conditions using both a colorimetric assay and chromatographic methods. Fermentor experiments in batch mode yielded 100 mg l⁻¹ of EPS from L. bulgaricus and 350 mg l⁻¹ from L. casei. Moreover, the use of a microfiltration (MF) bioreactor resulted in exopolysaccharides (EPS) concentrations threefold and sixfold those of batch experiments, respectively. The monosaccharidic composition of the two analyzed polymers differed from those previously reported. The optimization of the production of EPSs using the MF fermentation strategy could permit the use of these molecules produced by generally recognised as safe (GRAS) microorganisms in the place of other polysaccharides in the food industry.     

Analysis of Microbial Composition in Acid Whey for Dairy Fan Making in Yunnan by Conventional Method and 16S rRNA Sequencing

Dairy fan is a traditional milk product made by adding ‘acid juice’ and heating up. It has been consumed by people of minority ethnic region in Yunnan Province of China for over 1,000 years. In order to improve the quality of dairy fan for further industrial-scale production, we investigated the microbial composition, especially Lactic Acid Bacteria (LAB), by conventional method and 16S rRNA sequences analysis in acid whey, which was the starter culture for traditional dairy fan making. Twenty samples of acid whey were collected from Yunnan. Ninety-one phylotypes of LAB were isolated from these samples and identified by conventional method and 16S rRNA sequencing analysis. The results showed that all isolates belonged to three genera and nine different species. Moreover, Lactobacillus helveticus was the predominant population among these samples, mesophilic and thermophilic LAB could be considered as the major microbial composition of acid whey in the Yunnan Province of China. This paper systematically studied the LAB composition of acid whey, which may be valuable for designing starter culture for dairy fan production.     

Changes in biosynthesis of exopolysaccharide in Lactococcus lactis subspecies cremoris treated by moderate pulsed electric field treatment

Metabolome analysis and physicochemical analyses were executed with cell extracts of a Lactococcus lactis subspecies cremoris strain treated by moderate pulsed electric field (PEF) to elucidate the mechanism of enhanced production of exopolysaccharide (EPS) by the treatment. Metabolome analysis by capillary electrophoresis time of flight mass spectrometry annotated 224 metabolites from the cytoplasmic extract of the strain, which, however, showed no significant changes in metabolites related to the EPS production. Electron microscopic observation and chemical analysis of undecaprenoids as carrier of EPS biosynthetic intermediates suggested that PEF treatment dissociated immature EPSs from the intermediates due to the focal electro-condensation of hydrogen ions at the cell surface. Thus, liberated undecaprenyl phosphates were recycled efficiently, which resulted in mass increase of EPS with smaller molecular weight. The study suggested the feasibility of moderate PEF treatment as a food processing technique and revealed the mechanism of enhanced production of EPS by the treatment.     

Lactic acid bacteria from raw milk as potentially beneficial strains to prevent bovine mastitis

Bovine mastitis produces a wide variety of problems in the dairy farm. The treatment of this disease is based on the use of antibiotics which are not always effective. These drugs are also responsible for the presence of residues in the milk and the increase of antibiotic-resistant strains. Probiotic products were proposed as a valid alternative to antibiotic therapies and are also useful for the prevention of infectious syndromes. With the aim of designing a probiotic product to prevent bovine mastitis, lactic acid bacteria (LAB) were isolated from foremilk samples from different dairy farms in Córdoba-Argentina. One hundred and seventeen LAB were isolated and their beneficial characteristics such as the production of inhibitory substances, surface properties and production of exopolysaccharides (EPS) were assessed. Most of them displayed low degree of hydrophobicity, autoaggregation, EPS negative phenotype and were identified as Enterococcus hirae and Pediococcus pentosaceus. Nine LAB strains inhibited three indicator bacteria. Some isolates were pre-selected and genetically identified according to the results obtained. Antibiotic resistance and virulence factors were studied for the assessment of the safety of the strains. The results obtained were compared to those reported previously from samples obtained in the North-western area of the country and some differences were found.     

Chemical Composition of Two Exopolysaccharides from Bacillus thermoantarcticus

The thermophilic bacterium Bacillus thermoantarcticus produces two exocellular polysaccharides (EPS 1 and EPS 2), which can be obtained from the supernatant of liquid cultures by cold-ethanol precipitation, in yields as high as 400 mg liter(sup-1). The EPS fraction was produced with all substrates tested, although a higher yield was obtained with mannose as the carbon and energy source. The EPS content was proportional to the total biomass. On a weight basis, EPS 1 and EPS 2 represented about 27 and 71%, respectively, of the total carbohydrate fraction. EPS 1 is a sulfate heteropolysaccharide containing mannose and glucose in a relative molar proportion of 1.0 and 0.7, respectively. EPS 2 is a sulfate homopolysaccharide containing mannose as the major component. The absolute configurations of hexoses were shown to be d for both EPSs. Nuclear magnetic resonance spectra confirmed the presence of (alpha)-d-mannose and (beta)-d-glucose in EPS 1 and only (alpha)-d-mannose in EPS 2. In addition, (sup1)H nuclear magnetic resonance analysis and chemical analysis indicated the presence of pyruvic acid in EPS 2.