L. De Vuyst

Bacteriocins from Lactic Acid Bacteria: Production, Purification, and Food Applications

In fermented foods, lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been purified and characterized. The kinetics of bacteriocin production by LAB in relation to process factors have been studied in detail through mathematical modeling and positive predictive microbiology. Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness), in fermented sausage (anti-listerial effect), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level. The highly promising results of these studies underline the important role that functional, bacteriocinogenic LAB strains may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety. In addition, antimicrobial production by probiotic LAB might play a role during in vivo interactions occurring in the human gastrointestinal tract, hence contributing to gut health.

Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins

The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.

Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota

Sourdough is a microbial ecosystem of lactic acid bacteria (LAB) and yeasts in a matrix of mainly cereal flour and water. Culture-dependent and culture-independent microbiological analysis together with metabolite target analyses of different sourdoughs enabled to understand this complex fermentation process. It is difficult to link the species diversity of the sourdough microbiota with the (geographical) type of sourdough and the flour used, although the type and quality of the latter is the main source of autochthonous LAB in spontaneous sourdough fermentations and plays a key role in establishing stable microbial consortia within a short time. Carbohydrate fermentation targeted towards maltose catabolism, the use of external alternative electron acceptors, amino acid transamination reactions, and/or the arginine deiminase pathway are metabolic activities that favour energy production, cofactor (re)cycling, and/or tolerance towards acid stress, and hence contribute to the competitiveness and dominance of certain species of LAB found in sourdoughs. Also, microbial interactions play an important role. The availability of genome sequences for several LAB species that are of importance in sourdough as well as technological advances in the fields of functional genomics, transcriptomics, and proteomics enable new approaches to study sourdough fermentations beyond the single species level and will allow an integral analysis of the metabolic activities and interactions taking place in sourdough. Finally, the implementation of selected starter cultures in sourdough technology is of pivotal importance for the industrial production of sourdoughs to be used as flavour carrier, texture-improving, or health-promoting dough ingredient.

Microbial ecology of sourdough fermentations: diverse or uniform ?

Sourdough is a specific and stressful ecosystem inhabited by yeasts and lactic acid bacteria (LAB), mainly heterofermentative lactobacilli. On the basis of their inocula, three types of sourdough fermentation processes can be distinguished, namely backslopped ones, those initiated with starter cultures, and those initiated with a starter culture followed by backslopping. Typical sourdough LAB species are Lactobacillus fermentum, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus sanfranciscensis. Typical sourdough yeast species are Candida humilis, Kazachstania exigua, and Saccharomyces cerevisiae. Whereas region specificity is claimed in the case of artisan backslopped sourdoughs, no clear-cut relationship between a typical sourdough and its associated microbiota can be found, as this is dependent on the sampling, isolation, and identification procedures. Both simple and very complex consortia may occur. Moreover, a series of intrinsic and extrinsic factors may influence the composition of the sourdough microbiota. For instance, an influence of the flour (type, quality status, etc.) and the process parameters (temperature, pH, dough yield, backslopping practices, etc.) occurs. In this way, the presence of Lb. sanfranciscensis during sourdough fermentation depends on specific environmental and technological factors. Also, Triticum durum seems to select for obligately heterofermentative LAB species. Finally, there are indications that the sourdough LAB are of intestinal origin.

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801

M. ZAMFIR, R. CALLEWAERT, P.C. CORNEA, L. SAVU, I. VATAFU and L. DE VUYST.1999.Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6·5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram‐negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell‐free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat‐stable (30 min at 121 °C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell‐free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N‐terminal end was blocked.

Applicability of a bacteriocin-producing Enterococcus faecium as a co-culture in Cheddar cheese manufacture

Two strains, Enterococcus faecium RZS C5 and E. faecium DPC 1146, produce listericidal bacteriocins, so-called enterocins. E. faecium RZS C5 was studied during batch fermentation in both a complex medium (MRS) and in milk to understand the influence of environmental factors, characteristic for milk and cheese, on both growth and bacteriocin production. Fermentation conditions were chosen in view of the applicability of in situ enterocin production during Cheddar cheese production. Enterocin production by E. faecium RZS C5 in MRS started in the early logarithmic growth phase, and enterocin activity decreased during the stationary phase. The effect of pH on enterocin production and decrease of activity was as intense as the effect on bacterial growth. Higher enterocin production took place at pH 5.5 compared with pH 6.5. The use of lactose instead of glucose increased the production of enterocin, and at higher lactose concentration, production increased more and loss of activity decreased. The production in skimmed milk compared to MRS was lower and was detected mainly in the stationary phase. When casein hydrolysate was added to the milk, enterocin production was higher and started earlier, indicating the importance of an additional nitrogen source for growth of E. faecium in milk. For co-cultures of E. faecium RZS C5 with the starters used during Cheddar cheese manufacture, no enterocin activity was detected during the milk fermentation. Furthermore, the applicability of E. faecium RZS C5 and E. faecium DPC 1146 strains was tested in Cheddar cheese manufacture on pilot scale. Enterocin production took place from the beginning of the cheese manufacturing and was stable during the whole ripening phase of the cheese. This indicates that both an early and late contamination of the milk or cheese can be combated with a stable, in situ enterocin production. The use of such a co-culture is an additional safety provision beyond good manufacturing practices.

Enterococcus faecium RZS C5, an interesting bacteriocin producer to be used as a co-culture in food fermentation

Enterocins, bacteriocins produced by enterococci, are gaining interest because of their industrial potential. Due to its bacteriocin production, Enterococcus faecium RZS C5, a natural cheese isolate, has a strong activity towards Listeria monocytogenes. For this reason, the strain may be applicable as a bacteriocin-producing co-culture in food fermentation in order to reduce the risk on Listeria outgrowth. The strain displays remarkable bacteriocin production kinetics. Whereas most lactic acid bacteria produce bacteriocin in a growth-associated way until the beginning of the stationary phase, bacteriocin production by E. faecium RZS C5 in MRS broth at controlled pH values below 7.5 is characterised by a boost of bacteriocin activity levels in the very early growth phase. In addition, bacteriocin production kinetics are closely linked to the environmental and cultural conditions. However, no straightforward statement about the effect of environmental stress on bacteriocin production can be made since the effect is dependent on the type of stress applied. Kinetic experiments in milk and on pilot scale, applying Cheddar cheese-making conditions, have indicated that the strain may be effective as a bacteriocin-producing co-culture. Further research is needed to evaluate the use of E. faecium RZS C5 as a co-culture for the production of fermented sausage.

Exopolysaccharide-producing Streptococcus thermophilus strains as functional starter cultures in the production of fermented milks

Relationships between exopolysaccharide (EPS) production (amount, molecular mass and sugar composition of the EPS) by different Streptococcus thermophilus strains as a functional starter culture, and textural characteristics (viscosity) of fermented milk and yoghurt have been studied. Five interesting heteropolysaccharide-producing strains have been tested. Both S. thermophilus LY03 and S. thermophilus CH101 produced the highest amounts of EPS and also displayed the highest apparent viscosities in fermented milk. S. thermophilus ST 111 and S. thermophilus STD differed considerably in EPS yields, but not in apparent viscosities of fermented milk. In addition, S. thermophilus ST 111 displayed a high variability in EPS amounts when cultivated in milk. In milk medium, S. thermophilus LY03 produced two heteropolysaccharides, a high-molecular-mass (HMM) EPS and a low-molecular-mass (LMM) EPS of the same composition (Gal/Glu/GalNAc=3.4:1.4:1.0). S. thermophilus ST 111 produced only a HMM-EPS (Gal/Rha=2.5:1.0), while S. thermophilus CH 101 (Gal/Glu=1.0:1.0), S. thermophilus ST 113 (Gal/Glu/Rha/GalNAc=1.7:3.9:1.5:1.0) and S. thermophilus STD (Gal/Glu/Rha/GalNAc=3.5:6.2:1.2:1.0) produced only LMM-EPS. Both HMM-EPS and LMM-EPS solutions (S. thermophilus LY03) demonstrated a pseudoplastic character; HMM-EPS solutions of 0.2% (m/v) displayed a high consistency as well. Although its production of high EPS amounts, S. thermophilus LY03 resulted in relatively thin yoghurts, so that texture values did not directly correlate with EPS production capacity. Once structure/function relationships are known, one can determine the molecular properties of the isolated and purified EPS (molecular size, structural characteristics) from candidate strains to predict their potential in texture formation. For a final selection of interesting EPS-producing starter strains one should test the EPS production under yoghurt manufacturing conditions.

Bacteriocin Production with Lactobacillus amylovorus DCE 471 Is Improved and Stabilized by Fed-Batch Fermentation

ABSTRACT Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.

Evaluation of the spoilage lactic acid bacteria in modified-atmosphere-packaged artisan-type cooked ham using culture-dependent and culture-independent approaches

Aims:  To investigate microbial diversity and population dynamics of spoilage‐sensitive modified‐atmosphere‐packaged (MAP) artisan‐type cooked ham in relation to storage temperature.