• Leroy, F. & De Vuyst, L. (2001). Growth of the bacteriocin-producing Lactobacillus sakei Strain CTC 494 in MRS broth is strongly reduced due to nutrient exhaustion: a nutrient depletion model for the growth of lactic acid bacteria. Applied and Environmental Microbiology, 67, 4407-4413.

  Although commercial MRS broth has been designed to allow excellent growth of lactobacilli, most of these bacteria are still subjected to a self-inhibiting process. The most likely explanation is the accumulation of lactic acid or other toxic end products and the depletion of nutrients. In this study, the self-inhibition of Lactobacillus sakei CTC 494 was analyzed in a kinetic way, and a nutrient depletion model was set up to describe the growth inhibition process. This simple model has considerable advantages compared to commonly used descriptive models such as the logistic growth equation. It offers a better fit and a more realistic description of the growth data by taking into account both growth inhibition due to lactic acid production and changes in growth rates due to nutrient depletion. Depending on the fermentation conditions, in MRS broth there appears to be a strong decrease of the specific growth rate over time. Some undefined compounds present in the complex nitrogen source of MRS broth appear to be of crucial importance because of their limited availability. Moreover, nutrient availability affects bacteriocin production through its effect on cell growth as well on the bacteriocin production per cell. A plateau value for the bacteriocin production by L. sakei CTC 494 was observed.

• Leroy, F., Verluyten, J., Neysens, P., Degeest, B., Messens, W. & De Vuyst, L. (2001). Modelling the kinetics of functional starter cultures to improve food fermentation processes. Acta Horticulturae, 566, 363-368.

  Functional starter cultures of lactic acid bacteria are defined as strains that are used as starter cultures for food fermentation, and that are able to express certain functional properties which give an added value to the end-product (e.g. bacteriocin producers, exopolysaccharide producers, probiotic strains). A key problem is that in some cases strains that display promising behaviour under optimal laboratory conditions do not express their functional properties in an actual food environment. Predictive modelling techniques, which are usually applied to estimate the outgrowth, inactivation or toxin production of food spoilers and pathogens, may be successfully applied to investigate the application potential of functional starter cultures, and to unravel the mutual influences between a food environment and bacterial functionality. As a case study, the biokinetic features of several functional lactic acid bacteria (Lactobacillus sakei CTC 494, Lactobacillus curvatus LTH 1174, Lactobacillus amylovorus DCE 471, Enterococcus faecium RZS C5, and Streptococcus thermophilus LY03) were studied through primary, secondary, and tertiary modelling as a function of environmental characteristics which are typical for certain fermented foods (e.g. yoghurt, fermented sausage and sourdough). Several conclusions could be drawn with respect to starter selection and fermentation technology. For instance, the meat isolates Lb. sakei CTC 494 and Lb. curvatus LTH 1174 turned out to be promising novel bacteriocin-producing starters for sausage fermentation, whereas Lb. amylovorus DCE 471 seemed more suitable as a starter for industrial sourdough fermentation. E. faecium RZS C5 has potential to be used as a bacteriocin-producing coculture for food fermentations. Moreover, modelling indicated how exopolysaccharide production by S. thermophilus LY03 in yoghurt may be optimised by adjusting the process conditions.

• Leroy, F., Verluyten, J., Messens, W. & De Vuyst, L. (2002). Modelling contributes to the understanding of the different behaviour of bacteriocin-producing strains in a meat environment. International Dairy Journal, 12, 247-253.

Bacteriocins from lactic acid bacteria are potent antibacterial agents that enable the producer cells to selectively and efficiently inhibit a part of the competing background flora, including several spoilage bacteria and foodborne pathogens. The use of bacteriocinogenic lactic acid bacterium strains, either as starters or as protective cultures, is very limited in the food industry, possibly because many questions about the in situ functionality of such strains remained unanswered. A modelling approach was developed to study the functionality of lactic acid bacterium bacteriocin producers in a meat environment. The kinetic properties of three potential bacteriocin-producing starter cultures for sausage fermentation, namely Lactobacillus sakei CTC 494, Lactobacillus curvatus LTH 1174, and Lactobacillus amylovorus DCE 471 were compared in MRS, used as a meat simulation medium. Moreover, the application possibilities of the bacteriocin-producing Enterococcus faecium RZS C5 strain were evaluated. It turned out that growth and bacteriocin production by Lb. sakei CTC 494 and Lb. curvatus LTH 1174 are adapted to the meat environment, whereas Lb. amylovorus DCE 471 is kinetically not able to dominate the meat fermentation process. Furthermore, the use of bacteriocin-producing enterococci as functional cocultures seems tot be attractive.

• Leroy, F., Degeest, B. & De Vuyst, L. (2002). A novel area of predictive modelling: describing the functionality of beneficial mircoorganisms in foods. International Journal of Food Microbiology, 73, 251-259.

  Predicitive microbiology generally focuses on the potential outgrowth of spoilage bacteria and foodborne pathogens in foods. Little attention has been paid to the biokinetics of beneficial foodgrade microorganisms, such as lactic acid bacteria. The latter is commonly used in the food fermentation industry, mainly for the in situ production of the antimicrobial lactic acid to extend the shelf life of the food. Furthermore, many strains show additional industrial potential as novel starter cultures since they produce functional metabolites, such as bacteriocins and exopolysaccharides. The production of these functional metabolite has been demonstrated during in vitro experiments, but in many cases these novel starter cultures seem to be less efficient when applied in a food system. A modelling approach may contribute to a better understanding of the tight relation between the food environment and bacterial functionality. Primary modelling can be applied to fit the experimental data concerning cell growth, sugar metabolism, and the production of functional metabolites for a given set of environmental conditions. This led to conclusions concerning the growth-associated production of bacteriocin and exopolyasaccharides, the inactivation of these molecules when cell growth levels off, and a minimum cell concentration to trigger on bacteriocin production. Examples deal with the production of the bacteriocin sakacin K by the natural fermented sausage isolate Lactobacillus sakei CTC 494, and the production of heteropolysaccharides by the yoghurt starter culture Streptococcus thermophilus LY03. Secondary modelling of biokinetic parameters quantifies the production of bacteriocin and exopolysaccharides in function of environmental factors. As an example, the specific bacteriocin production by Lb. sakei CTC 494 decreases with increasing sodium chloride concentrations. Furthermore, since the assessment of functionality is frequently hampered by the nature of the food system, mathematical modelling techniques may help to predict the functional behaviour of novel lactic acid bacteria starter cultures in a food matrix, and hence quantify in situ production. For example, a model may simulate cell growth and exopolysaccharide production of S. thermophilus LY03 in a milk environment, where direct measurements are difficult to perform.

• De Vuyst, L., Schrijvers, V., Paramithiotis, S., Hoste, B., Vancanneyt, M., Swings,J., Kalantzopoulos, G., Tsakalidou, E. & Messens, W. (2002). The biodiversity of Lactic acid bacteria in greek traditional wheat sourdoughs is reflected in both composition and metabolite formation. Applied and Environmental Microbiology, 68, 6059-6069.

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's 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 Greek traditional wheat sourdoughs. 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 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 occured 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.

• Messens, W. & De Vuyst, L. (2002). Inhibitory substances produced by Lactobacilli isolated from sourdoughs—a review. International Journal of Food Microbiology, 72 (1-2), 31-43.

  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.

• Messens, W., Neysens, P., Vansieleghem, W., Vanderhoeven, J. & De Vuyst, L (2002). Modeling growth and bacteriocin production by Lactobacillus amylovorus DCE 471 in response to temperature and pH values used for sourdough fermentations. Applied and Environmental Microbiology, 68, 1431-1435.

The biokinetics of cell growth of Lactobacillus amylovorys DCE 471 and bacteriocin production by this strain were investigated as a function of the temperatures (28 to 44°C) and pH walues (pH 4.2 to 6.4) that are characteristic of a sourdough fermentation process. The influence of temperature and pH on microbial behavior is described by using a successfully validated predictive model.

• Leroy, F. & De Vuyst, L (2003). A combined model to predict the functionality of the bacteriocin-producing Lactobacillus sakei strain CTC 494. Applied and Environmental Microbiology, 69, 1093-1099.

  The use of bacteriocin-producing lactic acid bacteria for improved food fermentation processes seems promising. However, lack of fundamental knowledge about the functionality of bacteriocin-producing strains under food fermentation conditions hampers their industrial use. Predictive microbiology or mathematical estimation of microbial behaviour in food ecosystems may help to overcome this problem. In this study, a combined model was developed that was able to estimate, from a given initial situation of temperature, pH, and nutrient availability, the growth and self-inhibition dynamics of a bacteriocin-producing Lactobacillus sakei CTC 494 culture in (modified) MRS broth. Moreover, the drop in pH induced by lactic acid production and the bacteriocin activity toward Listeria as an indicator organism were modeled. Self-inhibition was due to the depletion of nutrients as well as to the production of lactic acid. Lactic acid production resulted in a pH drop, an accumulation of toxic undissociated lactic acid molecules, and a shift in the dissociation degree of the growth-inhibiting buffer components. The model was validated experimentally.

• Neysens, P., Messens, W., Gevers, D., Swings, J. & De Vuyst, L (2003). Biphasic kinetics of growth and bacteriocin production with Lactobacillus amylovorus DCE 471 occur under stress conditions. Microbiology, 149, 1073-1082.

  Micro-organisms used during the production of fermented foods are subjected to several abiotic stresses. Microbial survival during these processes strongly depends on the ability of the cells to adapt and become more tolerant to the environmental conditions. Cultivation of Lactobacillus amylovorus DCE 471, a potential strain for use during type II sourdough fermentations, at low temperatures, unfavourable pH and high salt concentrations resulted in biphasic growth patterns. In addition, two seperate bacteriocins peaks, as well as a dramatic change in cellular morphology, were observed. In general, an increase of the specific bacteriocin production occured during the second growth phase. Finally, the observed sugar consumption profiles were affected by the applied fermentation temperature. Moreover, the highest bacteriocin activity occured during maltose consumption at a low constant temperature of 28° C and a constant pH of 5.4. Plate counts from both growth phases revealed the existence of two colony types. Irregular colonies were found to outnumber smoother colonies during the first growth phase, while the second growth phase was characterized by a greater number of smooth colonies. Electron microscopy was used to investigate the observed morphological switch at the single-cell level. Single, rod-shaped cells changed into elongated cells that grew in chains. Colony and cell morphology changes coincided with the biphasic growth pattern.

• Neysens, P., Messens, W.& De Vuyst, L (2003). Effect of sodium chloride on growth and bacteriocin production by Lactobacillus amylovorus DCE 471. International Journal of Food Microbiology, in press.

  A kinetic investigation of the effect of sodium chloride on cell growth of Lactobacillus amylovorus DCE 471 and amylovorin L471 production was carried out through in vitro experiments using a temperature and pH prevailing during sourdough fermentations. Sodium chloride interfered both with cell growth and bacteriocin production. Biomass formation and amylovorin L471 production decreased in the presence of increasing salt concentrations. Maximum bacteriocin activities were observed after the addition of 10 g/l of NaCl, while the maximum specific growth rate reached an optimum at 5g/l of NaCl. High salt concentrations (20-40 g/l) resulted in biphasic fermentation profiles. Based on these results, incorporation of 5-10 g/l of sodium chloride in the water phase of type II sourdough preparations might be beneficial to enhance bacterial growth and amylovorin L471 production, and so contribute to the competitiveness of the strain in a sourdough environment.

last updated : 9-04-2003