1 Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
2 Laboratory Microbiology Gent Culture Collection, Ghent University, B-9000 Gent, Belgium
Correspondence
Luc De Vuyst
ldvuyst{at}vub.ac.be
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ABSTRACT |
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INTRODUCTION |
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Lactobacillus amylovorus DCE 471, an isolate from corn steep liquor, has been shown to produce the bacteriocin amylovorin L471 (Callewaert et al., 1999). This bacteriocin has been identified as being small, thermostable and strongly hydrophobic, with antagonistic activity towards closely related strains (De Vuyst et al., 1996b
). The homofermentative producer strain has also been reported to be a fast and strong acidifier that confers a competitive advantage (De Vuyst et al., 1996a
, b
). The growth and bacteriocin production kinetics of L. amylovorus DCE 471 in MRS medium have been the subject of previous investigations (De Vuyst et al., 1996a
, b
; Lejeune et al., 1998
; Callewaert et al., 1999
). Recently, it was reported that the temperature and pH conditions that prevail during sourdough fermentations correspond to the range of conditions for good growth, acidification and bacteriocin production by L. amylovorus DCE 471 (Messens et al., 2002
). Therefore, this strain may be useful as a starter culture in the competitive cereal environment, provided it withstands particular environmental stresses. L. amylovorus DCE 471, however, was found to exhibit biphasic growth kinetics when cultivated under suboptimal growth or stress conditions. These growth patterns are characterized by two distinct exponential-growth phases and two separate bacteriocin-production phases.
In this study, the growth limits and the stress hardening capacity of L. amylovorus DCE 471 were explored from a kinetic point of view. First, biokinetic parameters characteristic for growth and bacteriocin production were calculated to describe the influence of environmental stress caused by unfavourable temperature, pH and salt conditions that resulted in biphasic fermentation kinetics. Second, the biphasic fermentation kinetics were explained by a stress response and stress resistance that coincided with an altered colony and cell morphology.
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METHODS |
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Inoculum preparation.
This consisted of two steps. First, 10 ml SSM was inoculated with 0·1 ml of a freshly prepared L. amylovorus culture and incubated at 37 °C for 12 h. This pre-culture was used to inoculate 90 ml SSM. After incubation at 37 °C for 12 h, this second pre-culture was used to inoculate the fermenter.
Fermentation experiments.
In vitro fermentation experiments were carried out in a 15 l laboratory fermenter (Biostat C; B. Braun Biotech International) with a working volume of 10 l. Preparation of the fermenter and on-line control of the fermentation process (temperature, pH, agitation) were performed as described previously (Messens et al., 2002).
The influence of sublethal environmental conditions on cell growth and bacteriocin production by L. amylovorus DCE 471 was assessed. Therefore, the strain was cultivated at a constant pH of 5·4 and a temperature of 28 and 31 °C, a constant temperature of 37 °C and a constant pH of 6·4, and in the presence of 3 % (w/v) NaCl at a constant temperature of 37 °C and a constant pH of 5·4. All experiments performed under these conditions resulted in biphasic kinetics for growth, sugar consumption and bacteriocin production.
Assays.
At regular time intervals, samples were aseptically withdrawn from the fermentation vessel and immediately cooled on ice. The optical density of the samples was measured at 600 nm (Uvikon 923; Kontron Instruments). Determination of biomass concentration (X), colony-forming units (c.f.u.), total lactic acid concentration (L), residual maltose (M) and fructose (F) concentrations, and bacteriocin activity levels (B) were performed as described elsewhere (De Vuyst et al., 1996a, b
; Lejeune et al., 1998
; Messens et al., 2002
). In brief, biomass (as cell dry mass; CDM) was determined gravimetrically after membrane filtration. Bacteriocin activity was determined by a twofold critical dilution method. Activity was expressed in arbitrary units (AU) per ml or mega arbitrary units (MAU) per litre. The lactic acid concentration and the residual fructose and maltose concentrations were determined by HPLC using a Waters chromatograph. The standard deviations for the maltose, fructose, lactic acid and CDM measurements were 0·040, 0·035, 0·025 and 0·11 g l-1, respectively.
Modelling.
The mathematical equations represented in Table 1 were used to describe growth and product formation by L. amylovorus DCE 471. These equations were integrated with the Euler integration technique in Microsoft Excel 2000. To avoid unrealistic fitting solutions without physiological relevance, and computational solving problems (e.g. convergence problems), all parameters needed for the modelling were estimated by manual adjustment until a good visual fit of the curves was obtained.
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rep-PCR fingerprinting.
Total DNA from cells obtained through microcentrifugation (13 000 r.p.m., 15 min) of 1·5 ml samples was extracted as described previously (Gevers et al., 2001). PCR amplifications were performed with a DNA thermal cycler GeneAmp PCR System 9600 (Perkin Elmer) as described previously (Versalovic et al., 1994
), using Goldstar DNA polymerase (Eurogentec) and the primer (GTG)5 (5'-GTGGTGGTGGTGGTG-3'). Electrophoresis of the PCR products was performed in a 1·5 % agarose gel (15x20 cm) for 16 h at a constant voltage of 2 V cm-1 in 1xTAE buffer (40 mM Tris/acetate, 1 mM EDTA, pH 8·0) at 4 °C. The rep-PCR profiles were observed after staining of the gel with ethidium bromide and visualization under UV light, followed by digital image capturing using a CCD camera (Fotodyne). The resulting fingerprints were analysed using the BIONUMERICS version 2.0 software package (Applied Maths). The similarity among digitized profiles was calculated using the Pearson correlation. A mean linkage (UPGMA) dendrogram was derived from the profiles.
PFGE of chromosomal DNA.
The preparation of genomic DNA was performed in situ in agarose blocks by the method of Hung & Bandziulis (1990) with slight modifications. At regular intervals, cells were harvested from 10 ml fermentation liquor by microcentrifugation (13 000 r.p.m., 15 min), washed with 50 mM EDTA (pH 8·5) and suspended in 300 µl per OD600 unit of the same buffer. After pre-heating 1·1 % low-melting-point (LMP) agarose prepared in 50 mM EDTA (pH 8·5) and cooling to 45 °C, 125 µl of the cell suspension was mixed with 750 µl LMP agarose. The mixture was solidified in a plug mould for at least 15 min at 4 °C. The plugs were incubated overnight at 37 °C in lysis buffer (50 mM EDTA, pH 8·5; 0·05 % N-laurylsarcosine; 2 mg lysozyme ml-1; 12·5 U mutanolysin ml-1). Proteinase K treatment was performed overnight at 50 °C in NDS buffer (10 mM Tris, pH 8·0; 1 % SDS; 0·5 M EDTA, pH 8·5; 2 mg proteinase K ml-1). Plugs containing lysed cells were washed six times in 50 mM EDTA (pH 8·5) at room temperature. Before restriction enzyme digestion, the plugs were soaked in TE buffer (10 mM Tris-base, 1 mM EDTA, pH 8·0) for 1 h and then slowly shaken for 1 h in an appropriate restriction enzyme buffer. Restriction enzyme digestion with SmaI was performed overnight at 30 °C. Electrophoresis was carried out with a CHEF mapper (Bio-Rad) in 1·1 % PFGE-certified agarose (Bio-Rad) and using 0·5xTBE electrophoresis buffer (0·045 M Tris/borate, 0·001 M EDTA). The switch time was 230 s, the current was 5·3 V cm-1, the temperature was 14 °C and the running time was 24 h. The agarose gel was further treated and analysed as described above, except that the similarity among digitized profiles was calculated using the dice correlation.
Scanning electron microscopy.
To prevent crystallization of the salt in the fermentation liquor, samples (1·5 ml) were microcentrifuged (13 000 r.p.m. for 15 min) first. The recovered pellet was resuspended in 1·5 ml sterile water, and appropriate dilutions were prepared. Then, 100 µl of cell suspension were smeared onto the surface of a microscope slide, which was carefully passed through a heating source without boiling the liquor. The samples were coated with carbon. Cells were examined and photographed at a magnification of 2000 with a JEOL-JSM 6400 electron microscope (JEOL, Tokyo, Japan) operating at a voltage of 20 kV.
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RESULTS |
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DISCUSSION |
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It is remarkable that a higher specific bacteriocin production was observed in the first growth phase compared to the second one. This indicates a stress response of the L. amylovorus culture, stress to which the bacteria are subjected at the start of the fermentation. It has been observed before that bacteriocin production is stimulated by stress conditions, resulting in lower growth rates, lower cell yields and relatively high bacteriocin activity levels (De Vuyst et al., 1996a; Uguen et al., 1999
). Stress caused by a shift of fermentation temperature from 37 to 30 °C has been shown to enhance the specific bacteriocin production by L. amylovorus DCE 471 after adaptation to this suboptimal value (Lejeune et al., 1998
). Yet, De Vuyst et al. (1996a)
observed two types of colony morphologies that existed as heterogeneous populations in MRS medium under stable environmental conditions. Therefore, stress is certainly responsible for the fermentation kinetics observed in the SSM used, given the shift in colony morphology. Whitley & Marshall (1999)
described that L. amylovorus NCFB 2745 exhibits rough (R) and smooth (S) colony forms. The different morphology is not due to genetic variability in the population but to a relatively stable switch in phenotypic expression, depending on different environmental conditions such as incubation temperature, pH, anaerobiosis and media composition. For instance, smooth L. amylovorus NCFB 2745 colonies are found on agar plates after five transfers in MRS medium containing glucose and citrate. Smooth cultures revert to rough ones by culturing under aerobic conditions. The rough type ferments glucose homofermentatively, while the smooth type shows patterns of fermentation that are typical of a heterofermentative Lactobacillus, producing carbon dioxide and ethanol in addition to lactate. However, De Vuyst et al. (1996a)
observed a 100 % homofermentative behaviour when growing L. amylovorus DCE 471 in MRS medium. Also, in SSM no switch from homo- to heterofermentation could be observed.
Stress-induced morphological changes have been described previously for several non-sporulating Gram-negative and Gram-positive bacteria. Furthermore, it is known that bacterial cells can rely on mechanisms for survival and resistance against multiple stresses. For instance, upon growth under acidic conditions or exposure to heat shock, a set of inducible responses resulting in a complex regulated expression of genes, in which sigma factors are involved, leads to tolerance or an increased resistance (Foster, 1995; Haldenwang, 1995
; Segal & Ron, 1998
). This process, along with other stress responses, is known to induce major changes in physiology and morphology (Morita, 1993
). A variety of stresses, such as a lowered pH, are known to lead to shrinkage of Escherichia coli cells due to the induction of the bolA morphogene (Santos et al., 1999
). Dramatic morphological changes and a severe decrease in the viability of Propionibacterium freudenreichii and Enterococcus faecalis are seen upon their exposure to extreme acidic growth conditions (Hartke et al., 1998
; Giard et al., 2000
; Jan et al., 2001
). In the case of L. amylovorus DCE 471, the filamentous growth of the more resistant biotype during the second growth phase might be ascribed to an enhanced stress resistance, resulting in a normal multiplication of the surviving cells but hampered cell division. Stress caused by the presence of salt, often present in fermenting foods, has previously been shown to induce an altered morphology of food-borne pathogens as well. For instance, it has been shown that Listeria monocytogenes became filamentous upon cultivation in the presence of 10·0 % salt because of a hampered cell multiplication, but nevertheless was able to survive for long periods at high salt concentrations (Brzin, 1975
). In the case of L. amylovorus DCE 471, salt sensitivity may be the reason why part of the original cell population starved during the intermediate stationary phase. Similarly, other suboptimal and stress-inducing environmental conditions may lead to biphasic growth coinciding with an altered cell and colony morphology.
From an industrial point of view, biphasic profiles observed for growth and bacteriocin production cannot be considered as advantageous since type II sourdoughs are characterized by short fermentation times and need a strong acidification at the beginning of the process. Furthermore, this feature does not contribute to an enhanced competitiveness of the strain since only low biomass concentrations, producing moderate amylovorin L471 levels, are observed during the first growth phase. On the other hand, the temperature dependence of sugar consumption by L. amylovorus DCE 471 observed during this study may improve the competitiveness of this strain. A temperature of 28 °C promoted the use of maltose by L. amylovorus DCE 471. Fast maltose consumption coincided with high bacteriocin activity levels. Both phenomena may hamper the development of other maltose-dependent micro-organisms in the same ecosystem. This is interesting from a physiologicalecological point of view, because maltose is the most important energy source for L. amylovorus in a cereal environment.
In this study, the biphasic growth patterns of L. amylovorus DCE 471 were modelled and elucidated in an SSM. It was shown that unfavourable growth conditions caused by low temperatures, high pH and high salt concentrations resulted in biphasic growth patterns. A morphological change coincided with an increased resistance of the bacterial population. However, the underlying molecular mechanism responsible for these phenomena still has to be elucidated in the case of L. amylovorus DCE 471.
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ACKNOWLEDGEMENTS |
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Received 16 July 2002;
revised 16 October 2002;
accepted 24 December 2002.
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