Luc De Vuyst

Recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria

Abstract Microbial exopolysaccharides (EPS) occur as capsules or as secreted slime. They represent a small fraction of todays biopolymer market with factors limiting their use being mainly associated with economical production. Efficient production and reduction in recovery costs requires knowledge of biosynthesis and adoption of appropriate bioprocess technology. EPS from generally recognized as safe food grade microorganisms, particularly lactic acid bacteria (LAB), have potential as food additives or as functional food ingredients with both health and economic benefits. Many different heteropolysaccharides (HePS) are secreted by LAB regarding sugar composition and molecular size but they show few common structural features, which raises questions about the relationship between structure and texture. HePS are made by polymerizing repeating units formed in the cytoplasm. These are assembled at the membrane by specific glycosyltransferases (GTF) through the sequential addition of sugar nucleotides. The latter are delivered as building blocks and attached to the growing repeating unit that is anchored on a lipid carrier. After completion, the repeating unit is externalized and polymerized. The enzymes and proteins involved in biosynthesis and secretion are not necessarily unique to HePS formation. These processes involve a genetic organization that includes specific eps genes and “housekeeping” genes involved in sugar nucleotide biosynthesis. The intentional and controlled use of HePS from LAB or use of strains producing HePS in situ is important in the food industry. However, instability in production and variability of HePS yields are well documented. Therefore, a well understood optimized carbon flux and supply of sugar nucleotides, knowledge of the GTF, and the functional expression of combinations of genes from different origin into stable, industrial strains open interesting ways to polysaccharide engineering.

Antimicrobial Potential of Lactic Acid Bacteria

The preservative effect of lactic acid bacteria during the manufacture and subsequent storage of fermented foods is mainly due to the acidic conditions that they create in the food during their development. This souring effect is primarily due to the fermentative conversion of carbohydrates to organic acids (lactic and acetic acid) with a concomitant lowering of the pH of the food, an important characteristic that leads to an increased shelf-life and safety of the final product. In recent decades, it has become clear that the overall inhibitory action of lactic acid bacteria is due to more complex antagonistic systems produced by the starter cultures. Lactic acid bacteria are capable of producing and excreting inhibitory substances other than lactic and acetic acid. These substances are antagonistic to a wide spectrum of microorganisms, and thus can make significant contributions to their preservative action. They are produced in much smaller amounts than lactic acid and acetic acid, and include formic acid, free fatty acids, ammonia, ethanol, hydrogen peroxide, diacetyl, acetoin, 2,3-butanediol, acetaldehyde, benzoate, bacteriolytic enzymes, bacteriocins and antibiotics, as well as several less well-defined or completely unidentified inhibitory substances (Klaenhammer, 1988; Daeschel, 1989; Lind-gren & Dobrogosz, 1990; Schillinger, 1990; Piard & Desmazeaud, 1991, 1992; Vandenbergh, 1993). Some of these substances display antagonistic activity towards many food spoilage and foodborne pathogenic microorganisms, including psychrotrophic lactobacilli and leuconostocs, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Listeria monocytogenes, Staphyloc-occus aureus, etc. The competitive removal of essential substrates, the accumulation of D-amino acids, a lowering of oxidation-reduction potential and coaggregation may further restrict undesirable microorganisms. Unfortunately, in some instances the antibiosis will be detrimental by inhibition of other desirable lactic strains composing the mixed starter culture.

Inhibitory substances produced by Lactobacilli isolated from sourdoughs--a review.

Several sourdough lactic acid bacteria (LAB) produce inhibitory substances other than organic acids. Bacteriocins (bavaricin A, and plantaricin ST31), a bacteriocin-like inhibitory substance (BLIS C57), and a new antibiotic (reutericyclin) have been discovered. Maximum antimicrobial production was found in the pH range 4.0-6.0. Temperature optima vary strongly. The substances are resistant to heat and acidity, and inactivated by proteolytic enzymes, except for reutericyclin. Bavaricin A and plantaricin ST31 have been purified to homogeneity. Bavaricin A is classified as a class IIa bacteriocin. Reutericyclin is a new tetramic acid. The mode of action of bavaricin A, BLIS C57, and reutericyclin is bactericidal. Some of these substances are active towards some Bacilli, Staphylococci and Listeria strains. Up to now, only the application potential of purified bavaricin A has been examined. More research should be done to study the production, the activity, and the stability of these inhibitory substances in food systems as these often differ from the broths mostly used in this kind of studies. Furthermore, an extensive screening of the sourdough microflora must be performed, in particular towards Bacilli and fungi. This could lead to the discovery of additional inhibitory substances, although it seems that the frequency of isolating bacteriocin-producing sourdough LAB is low. However, potent antimicrobials towards Bacilli as well as antifungal substances will have to be found using rational screening strategies and novel purification and analytical techniques.

Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana

ABSTRACT The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named“ Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like).

Cross-Feeding between Bifidobacterium longum BB536 and Acetate-Converting, Butyrate-Producing Colon Bacteria during Growth on Oligofructose

ABSTRACT In vitro coculture fermentations of Bifidobacterium longum BB536 and two acetate-converting, butyrate-producing colon bacteria, Anaerostipes caccae DSM 14662 and Roseburia intestinalis DSM 14610, with oligofructose as the sole energy source, were performed to study interspecies interactions. Two clearly distinct types of cross-feeding were identified. A. caccae DSM 14662 was not able to degrade oligofructose but could grow on the fructose released by B. longum BB536 during oligofructose breakdown. R. intestinalis DSM 14610 could degrade oligofructose, but only after acetate was added to the medium. Detailed kinetic analyses of oligofructose breakdown by the last strain revealed simultaneous degradation of the different chain length fractions, in contrast with the preferential degradation of shorter fractions by B. longum BB536. In a coculture of both strains, initial oligofructose degradation and acetate production by B. longum BB536 took place, which in turn also allowed oligofructose breakdown by R. intestinalis DSM 14610. These and similar cross-feeding mechanisms could play a role in the colon ecosystem and contribute to the combined bifidogenic/butyrogenic effect observed after addition of inulin-type fructans to the diet.

The Biodiversity of Lactic Acid Bacteria in Greek Traditional Wheat Sourdoughs Is Reflected in Both Composition and Metabolite Formation

ABSTRACT Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of bakers yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.

Nisin, A Lantibiotic Produced by Lactococcus Lactis Subsp. Lactis: Properties, Biosynthesis, Fermentation and Applications

Nisin, which has been known for about five decades, is a lanthionine-containing bacteriocin produced by certain Lactococcus lactis subsp. lactis strains. A number of reviews have been published dealing with various aspects of nisin (Berridge, 1953; Hawley, 1957; Schaller, 1960; Hawley, 1962; Marth, 1966; Jarvis & Morisetti, 1969; Polanowski, 1972; Baranova & Egorov, 1973; Lipinska, 1977; Hurst, 1978, 1981, 1983; Rayman & Hurst, 1984; Bucci et al., 1990; Fowler & Gasson, 1991). This chapter attempts to cover all aspects of nisin, with the main emphasis on its properties, biosynthesis and fermentative production, and on its applications. However, the large number of publications on nisin precludes complete coverage of the literature; omissions or partial coverage of literature data should therefore not be considered as a slight to the authors. The genetics of nisin will be discussed in Chapter 6.

Biodiversity of Exopolysaccharides Produced by Streptococcus thermophilus Strains Is Reflected in Their Production and Their Molecular and Functional Characteristics

ABSTRACT Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as Streptococcus thermophilus and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium. In addition, the acidification rates of the different strains were compared with their milk clotting behaviors. The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening. EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium. Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium. An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa. A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa). Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished. Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method. All of the EPS from the S. thermophilus strains tested were classified into six groups according to their monomer compositions. Apart from galactose and glucose, other monomers, such as (N-acetyl)galactosamine, (N-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents. Three strains were found to produce EPS containing (N-acetyl)glucosamine, which to our knowledge was never found before in an EPS from S. thermophilus. Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures. Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures.

Cross-feeding between bifidobacteria and butyrate-producing colon bacteria explains bifdobacterial competitiveness, butyrate production, and gas production

Inulin-type fructans are not digested and reach the human colon intact, where they are selectively fermented by the colon microbiota, in particular bifidobacteria. As a result, they are converted, directly or indirectly, to short-chain fatty acids and other organic acids, as well as gases, and lead to both bifidogenic and butyrogenic health-promoting effects. Bifidobacteria display phenotypic variation on strain level as to their capacity to degrade inulin-type fructans. Also, different chain lengths of inulin-type fructans may stimulate different subgroups within the bifidobacterial population. The end-metabolites of inulin-type fructan degradation by bifidobacteria reflect their growth rates on these polymers. Other colon bacteria are also able to degrade inulin-type fructans, as is the case for lactobacilli, Bacteroides, certain enterobacteria, and butyrate producers. Bacterial cross-feeding mechanisms in the colon lay at the basis of overall butyrate production, a functional characteristic of several colon bacteria that is always accompanied by gas production. Finally, specificity of polysaccharide use by the colon microbiota may determine diet-induced alterations in the microbiota and consequent metabolic effects.

Characterization of the Antagonistic Activity of Lactobacillus amylovorus DCE 471 and Large Scale Isolation of Its Bacteriocin Amylovorin L471

Summary Lactobacillus amylovorus DCE 471, isolated from corn steep liquor, produces a bacteriocin, called amy-lovorin L471. The strain displays two different colony morphologies. The protein profiles of both colony types show a very high visual similarity to the type strain of the Lactobacillus acidophilus DNA homology group A3. Amylovorin L471 is identified as a small, thermostable and strongly hydrophobic bacteriocin displaying antagonistic activity towards closely related strains. Production of amylovorin is growth- associated indicating primary metabolite kinetics. It is maximally produced at a controlled pH of 5.0–5.4. Two simple and upscaleable isolation procedures seem to be promising for large-scale isolation of bac- teriocins: organic solvent (chloroform/methanol) extraction and precipitation, and expanded bed adsorption.