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Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enterica serovar Typhimurium reveals a role for lactic acid and other inhibitory compounds
Research in Microbiology 157 (2006) 241–247 www.elsevier.com/locate/resmic
Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enterica serovar Typhimurium reveals a role for lactic acid and other inhibitory compounds Lefteris Makras a , Vagelis Triantafyllou a,b , Domitille Fayol-Messaoudi c , Tom Adriany a , Georgia Zoumpopoulou b , Effie Tsakalidou b , Alain Servin c , Luc De Vuyst a,∗ a Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Department of Applied Biological Sciences and Engineering,
Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium b Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural University of Athens (AUA), 11855 Athens, Greece c Pathogènes et Fonctions des Cellules Epithéliales Polarisées, Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510,
Faculté de Pharmacie, Université Paris XI, 92296 Châtenay-Malabry, France Received 8 June 2005; accepted 1 September 2005 Available online 22 September 2005
Abstract Six Lactobacillus strains including commercial probiotic ones (L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota, L. johnsonii La1, L. plantarum ACA-DC 287 and L. rhamnosus GG) were investigated, through batch fermentations under controlled conditions, for their capacity to inhibit Salmonella enterica serovar Typhimurium SL1344. All lactobacilli displayed strong antibacterial activity toward this Gramnegative pathogen and significantly inhibited invasion of the pathogen into cultured human enterocyte-like Caco-2/TC7 cells. By studying the production kinetics of antibacterial activity and applying the appropriate acid and pH control samples during a killing assay, we were able to distinguish between the effect of lactic acid and other inhibitory compounds produced. The antibacterial activity of L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota and L. rhamnosus GG was solely due to the production of lactic acid. The antibacterial activity of L. johnsonii La1 and L. plantarum ACA-DC 287 was due to the production of lactic acid and (an) unknown inhibitory substance(s). The latter was (were) only active in the presence of lactic acid. In addition, the lactic acid produced was responsible for significant inhibitory activity upon invasion of Salmonella into Caco-2/TC7 cells. 2005 Elsevier SAS. All rights reserved.
1. Introduction In the past two decades, the application of probiotics to the prevention and management of gastrointestinal disorders has received much interest [26]. Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer a health benefit upon the host [13]. Lactobacilli are commonly used as probiotics [7]. Well-designed clinical trials support the effectiveness of probiotics in keeping the human colon healthy [16]. However, there exists a lack of information about the underlying mechanisms responsible for the beneficial effects of probiotics [13]. As an example, probiotics can * Corresponding author.
E-mail address: [email protected] (L. De Vuyst). 0923-2508/$ – see front matter 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2005.09.002
function as microbial barriers against gastrointestinal pathogens through competitive exclusion of pathogen binding, modulation of the host’s immune system and production of inhibitory compounds [7,27]. Lactobacilli produce a wide range of antibacterial compounds including sugar catabolites such as organic acids (e.g., lactic acid and acetic acid); oxygen catabolites such as hydrogen peroxide; proteinaceous compounds such as bacteriocins, other low-molecular-mass peptides, and antifungal peptides/proteins; fat and amino acid metabolites such as fatty acids, phenyllactic acid, and OH-phenyllactic acid; and other compounds such as reuterin and reutericyclin [6,7,17,22,25, 27,29]. Bacteriocins produced by lactobacilli are antibacterial peptides or proteins that are inhibitory towards Gram-positive bacteria only [6]. Low-molecular-mass compounds such as lac-
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tic acid have been reported to be inhibitory towards Gramnegative pathogenic bacteria [1,24]. Alternatively, a heat-stable, low-molecular-mass antibacterial substance different from lactic acid and present in the cell-free culture supernatant of L. acidophilus LB kills a wide range of Gram-negative bacteria and inhibits adhesion to and invasion of Caco-2 cells by Salmonella enterica ser. Typhimurium SL1344 [4,5,20]. Although it is clear that the inhibitory activity of lactobacilli towards bacteria such as Salmonella is due simply to production of lactic acid, it has not always been clear whether other antibacterial compounds might also be produced, since the findings concerning such substance(s) have been based on small-scale killing studies without kinetic analyses [2,18]. The aim of the present study was to examine the kinetics of production of compounds with anti-Salmonella activity by lactobacilli during batch fermentations under controlled conditions. To exclude possible interference, low-molecular-mass acid metabolites from sugar and protein catabolism were measured as well. 2. Materials and methods 2.1. Bacterial strains and media Six Lactobacillus strains, including commercial probiotic ones, were used in this study (Table 1). S. enterica serovar Typhimurium SL1344 [14] was provided by Stocker B.A.D (Stanford, CA). All strains were stored as glycerol (25%, v/v) stocks at −80 ◦ C in the appropriate media. To obtain a fresh culture, lactobacilli were propagated twice in de Man–Rogosa–Sharpe (MRS) broth (Difco, Detroit, MI) at 37 ◦ C in a standard incubator for 12 h. The transfer inoculum was always 1% (v/v). The Salmonella strain was transferred once to trypticase soy agar (TSA; Difco) before a colony was picked up for further experimental use. The strain was cultured in Luria–Bertani (LB) broth (Difco) at 37 ◦ C for 18 h. Solid media were prepared by adding 1.5% (w/v) agar to the appropriate broths. 2.2. Preliminary investigation of the anti-Salmonella activity of CFCS of lactobacilli grown on a small scale To investigate the inhibitory activity of the six Lactobacillus strains against S. Typhimurium SL1344, small-scale fermentations in MRS broth (30 ml) were carried out at 37 ◦ C for 18 h in triplicate. After centrifugation (8000 g, 20 min, 4 ◦ C), the cell-free culture supernatant (CFCS) was adjusted to pH 4.5 using 1 N NaOH and filter-sterilized. The CFCS was tested for its anti-Salmonella activity using an in vitro killing assay and an in vitro assay for inhibition of invasion of Salmonella into Caco-2/TC7 cells, and analyzed through high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) (see below). 2.3. Kinetic analysis of the growth and inhibitory activity of lactobacilli during batch fermentation The kinetic analysis of growth and production of antibacterial compounds active towards S. Typhimurium SL1344 by
the six Lactobacillus strains was carried out during laboratory fermentations in 10 l MRS broth in duplicate or triplicate. Fermentations were carried out in a 15-l laboratory fermentor (Biostat C; B. Braun Biotech International, Melsungen, Germany) as described previously [30]. The initial pH of the medium was adjusted to pH 6.5. At predetermined intervals, samples were withdrawn aseptically and used for the analysis of growth and low-molecular-mass metabolites (see below). In addition, CFCS of these samples was adjusted to pH 4.5, filter-sterilized, and tested for anti-Salmonella activity (see below). 2.4. Analysis of microbial growth and low-molecular-mass metabolites Growth was measured by optical density, cell counts and biomass determinations as described previously [30]. The maximum specific growth rate (µmax ) was calculated by linear regression of ln(OD/OD0 ) as a function of time, where OD0 refers to the optical density at the start of the exponential growth phase. The residual glucose levels and lactic acid titers were determined by HPLC analysis as described previously [30], except that an ICSep ICE-ORH-801 column (Interchim, Montluçon, France) was used with 10 mM H2 SO4 as mobile phase at a flow rate of 0.4 ml min−1 . Succinic acid, phenyllactic acid, and OH-phenyllactic acid were determined by LC/MS analysis. A Waters chromatograph (Waters Corp., Milford, MA) coupled with a mass spectrometer (Quattro MicroTM ; Waters Corp.) was used. A capillary column ( 150 × 4.6 mm, 3 µm; Atlantis, Waters Corp.) kept at 35 ◦ C was used with a mobile phase consisting of ultrapure water, acetonitrile (100%, v/v) and ammonium acetate (10 mM) at a flow rate of 0.2 ml min−1 . 2.5. In vitro killing assay for anti-Salmonella activity The inhibitory activity of CFCS towards S. Typhimurium SL1344 was determined by an in vitro killing assay. To obtain mid-logarithmic phase cells, 10 ml of LB broth were inoculated with 200 µl of a fresh Salmonella culture and incubated at 37 ◦ C for 3 h. The culture was centrifuged (5500 g, 20 min, 4 ◦ C), the pellet was washed once with phosphate-buffered saline (PBS; 0.8% NaCl, 0.02% KH2 PO4 , 0.115% Na2 HPO4 , w/v, pH 7.4) and resuspended in Dulbecco’s modified Eagle’s minimum essential medium (DMEM; Invitrogen, Cergy, France). Then, 500 µl of the culture (2 × 108 CFU ml−1 in DMEM) were mixed with 500 µl CFCS or pH control sample and incubated at 37 ◦ C. The pH control sample was MRS broth adjusted to pH 4.5 with 37% (w/v) HCl. The final pH of the incubation medium was always 5.0 ± 0.2. After 4 h of incubation, the surviving S. Typhimurium SL 1344 cells were determined by plating on TSA. The antibacterial activity was expressed as a % of reduction of viable cells (in log CFU ml−1 ) with respect to untreated cells.
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Table 1 CFCS of lactobacilli: antibacterial activity, inhibition of invasion, and products of sugar and amino acid catabolism Lactobacillus straina (source or origin)b
Antibacterial activity (%)c
Inhibition of invasion (%)d
LAe (mM)
L. johnsonii La1 (LC1, Nestlé) L. casei Shirota (Yakult, Yakult Honsha) L. rhamnosus GG (Gefilus, Valio) L. amylovorus DCE 471 (Corn steep liquor [8]) L. acidophilus IBB 801 (Dairy product [33]) L. plantarum ACA-DC 287 (Greek Xynotyri cheese [8])
48 ± 5
46 ± 2
186 ± 4
44 ± 12
44 ± 0
40 ± 10
SAe (mM)
PLAe (µM)
OH-PLAe (µM)
0.87 ± 0.12
250 ± 21
143 ± 32
158 ± 11
0.14 ± 0.08
<50
37 ± 7
155 ± 13
0.40 ± 0.11
52 ± 10
39 ± 7
184 ± 8
0.17 ± 0.09
<50
45 ± 10
29 ± 3
151 ± 18
4.01 ± 0.24
151 ± 23
64 ± 11
51 ± 2
190 ± 14
0.15 ± 0.09
<50
96 ± 14
58 ± 11 76 ± 21 <50 94 ± 30 <50
a Lactobacilli were grown in MRS broth (30 ml) at 37 ◦ C for 18 h. Experiments were performed in triplicate. Values are expressed as means ± standard deviation. b Probiotic strains L. casei Shirota, L. johnsonii La1, and L. rhamnosus GG were isolated from their commercial products. c Antibacterial activity of CFCS (adjusted to pH 4.5) of lactobacilli against S. Typhimurium SL1344 after 4 h of incubation at 37 ◦ C. Antibacterial activity is expressed as % of reduction in viable cells (in log CFU ml−1 ) with respect to untreated cells. d Effect of pretreatment of S. Typhimurium SL1344 with CFCS of lactobacilli upon invasion by this pathogen of cultured human enterocyte-like Caco-2/TC7 cells. Inhibition of invasion is expressed as % reduction in invasion (in log CFU ml−1 ) with respect to the value obtained with cells treated with DMEM. e LA, lactic acid; SA, succinic acid; PLA, phenyllactic acid; OH-PLA, hydroxy-phenyllactic acid.
2.6. In vitro assay for inhibition of invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells The assay for inhibition of invasion of S. Typhimurium SL1344 into cultured human enterocyte-like Caco-2/TC7 cells [3] was conducted as previously reported [4,5,20] with slight modifications. Prior to infection, S. Typhimurium SL1344 cells in the exponential growth phase (2 × 108 CFU ml−1 in DMEM) were mixed with CFCS of the lactobacilli studied, pH control sample, or DMEM, and incubated at 37 ◦ C for 1 h. The pathogenic bacteria were then enumerated, washed twice with PBS, and resuspended in DMEM. The Caco2/TC7 cells, prepared in 24-well tissue culture plates (ATGC, Marne la Valée, France), were washed twice with PBS and infected with the pretreated pathogen. The plates were incubated at 37 ◦ C in an atmosphere of 10% CO2 –90% air for 1 h, and then washed three times with PBS. An antibiotic assay was used to determine the numbers of internalized S. Typhimurium SL1344 cells. The infected Caco-2/ TC7 cells were incubated in DMEM containing gentamicin 100 µg, ml−1 (Sigma-Aldrich, St. Louis, MO), for 1 h. Bacteria that adhered to the Caco-2/TC7 brush border were rapidly killed, whereas those located within the cells survived. The cells were washed twice with PBS and lysed with sterile ultrapure water and S. Typhimurium SL1344 cells were enumerated by plating on TSA. Each assay was conducted in triplicate with three successive passages of the Caco-2/TC7 cells. The inhibition of invasion was expressed as a % of reduction of invasion (in log CFU ml−1 ) with respect to the value obtained with cells treated with DMEM.
trations of an acid alone, or a mixture of acids. The acids were tested in amounts corresponding to their concentrations in the CFCS of the lactobacilli tested. Therefore, lactic acid (concentrations between 75 and 250 mM; Sigma-Aldrich), succinic acid (2.5–15 mM; VWR International, Darmstadt, Germany), phenyllactic acid (500–2500 µM; Sigma-Aldrich) and OHphenyllactic acid (250–1000 µM; Sigma-Aldrich) were added to MRS broth; the liquid was adjusted to pH 4.5 using 1 M NaOH or 37% (w/v) HCl, and filter-sterilized. Also, mixtures of lactic acid (150 mM) plus succinic acid (5, 10, or 15 mM), of lactic acid (150 or 175 mM), plus succinic acid (5 mM), phenyllactic acid (2000 µM), and OH-phenyllactic acid (500 µM), and of succinic acid (5 mM), plus phenyllactic acid (2000 µM), and OH-phenyllactic acid (500 µM) were prepared. The antiSalmonella activity was measured using an in vitro killing assay (see above) performed in triplicate. 2.8. Characteristics of the inhibitory compound(s) produced by L. johnsonii La1 and L. plantarum ACA-DC 287 CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287, obtained after 12, 14 and 16 h of fermentation, was tested for the sensitivity of its antibacterial activity to pepsin, proteinase K, and bacterial protease (Bacillus licheniformis, Streptomyces griseus; Sigma-Aldrich). All proteases were added to the CFCS at a final concentration of 1 mg ml−1 . The heat stability of the antibacterial activity was examined by incubating 1 ml of CFCS at 60, 80 and 100 ◦ C for 15, 30, and 60 min. The antibacterial activity of all treated samples was measured using a killing assay for anti-Salmonella activity (see above).
2.7. Preparation of acid control samples 2.9. Statistics To test the contribution of the different acids produced by lactobacilli to their anti-Salmonella activity, acid control samples were prepared. These samples contained various concen-
Results are expressed as means ± standard deviation. The level of significance was calculated using two-sided Student’s
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t tests for paired or independent samples. In the case of independent samples, the variances of the two compared groups were assumed to be equal. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Preliminary investigation of the anti-Salmonella activity of CFCS of lactobacilli grown on a small scale The incubation of S. Typhimurium SL1344 cells with CFCS (pH 4.5) of all tested lactobacilli resulted in a significant decrease (P < 0.001) in Salmonella viability after 4 h of contact (Table 1). The reduction in viability ranged from 40 ± 10% (L. rhamnosus GG) to 64 ± 11% (L. plantarum ACA-DC 287). The pH control sample gave only a 2 ± 1% reduction in the viability of the pathogen, indicating that the anti-Salmonella activity of the lactobacilli tested was not simply due to a decrease in pH. Concerning the in vitro assay for inhibition of invasion of S. Typhimurium SL1344 into cultured human enterocytelike Caco-2/TC7 cells, the viability of the pathogen was not significantly affected after 1 h of contact (preincubation time) with CFCS (pH 4.5) from the lactobacilli tested. For all strains, inhibition was significant (P < 0.01) compared to inhibition of invasion caused when the pathogen was treated with the pH control sample (Table 1). The main metabolic end product present in the CFCS of all lactobacilli tested was lactic acid in amounts ranging from 150 to 190 mM (Table 1). No acetic acid, formic acid or ethanol were produced. Moreover, phenyllactic acid, OH-phenyllactic acid and succinic acid were found in the CFCS of some of the lactobacilli tested. Relatively high amounts of phenyllactic acid were produced by L. johnsonii La1 and L. acidophilus IBB 801, namely 250 ± 21 and 151 ± 23 µM, respectively. L. acidophilus IBB 801 produced a high amount of succinic acid (4.01 ± 0.24 mM) as well. 3.2. Kinetic analysis of the growth of lactobacilli during batch fermentations Laboratory fermentations with the six Lactobacillus strains followed similar growth patterns. The fermentation and biokinetic parameters are summarized in Table 2. All strains were
fast acidifiers, resulting in a final pH of about 4.5 within 12 h of fermentation. In all cases lactic acid was the main metabolic end product of glucose catabolism. As an example, L. johnsonii La1 cells entered the stationary phase after 10 h of fermentation; at the end of fermentation, the pH was 3.93 ± 0.07 and almost all glucose was consumed and converted into lactic acid (Fig. 1). All lactobacilli tested followed similar lactic acid production patterns (data not shown). In addition, L. johnsonii La1 produced succinic acid (1.2 ± 0.2 mM), phenyllactic acid (923 ± 68 µM), and OH-phenyllactic acid (237 ± 111 µM) too. L. acidophilus IBB 801 produced high amounts of succinic acid (11.6 ± 0.5 mM) in addition to phenyllactic acid (812 ± 35 µM) and OH-phenyllactic acid (168 ± 43 µM). 3.3. Kinetic analysis of the inhibitory activity of lactobacilli during batch fermentations The antibacterial activity of the CFCS, obtained throughout the fermentations, towards S. Typhimurium SL1344 was high for all lactobacilli at the end of fermentation (Fig. 2). The antiSalmonella activity of CFCS of L. johnsonii La1 was already significant (P < 0.001) after 10 h of growth (129 mM lactic acid produced), and the highest reduction (60 ± 3%) in the viability of S. Typhimurium SL1344 was reached after 14 h of fermentation (162 mM lactic acid produced). In all cases, the antibacterial activity of CFCS was evident when the pH of the fermentation medium was lower than 4.5 and hence when a high concentration of lactic acid (above 120 mM) was reached (Figs. 1 and 2). The antibacterial activity of the acid control samples was low when the lactic acid concentration was between 75 and 125 mM, but increased linearly with lactic acid concentrations from 125 to 225 mM (Fig. 2). The antibacterial activity of the CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287 was significantly higher than the antibacterial activity caused by MRS broth containing the same amount of lactic acid as the CFCS tested, indicating the presence of an antibacterial factor close to but different from lactic acid (Fig. 2). For instance, the CFCS of L. johnsonii La1, obtained after 12 h of fermentation and containing 147 ± 6 mM of lactic acid resulted in a reduction of 54 ± 2% of the viability of S. Typhimurium SL1344, whereas MRS broth containing the same amount of lactic acid resulted in a reduction of 21 ± 2%
Table 2 Batch fermentation and biokinetic parameters for growth of lactobacilli Strain L. johnsonii La1 L. plantarum ACA-DC 287 L. casei Shirota L. rhamnosus GG L. amylovorus DCE 471 L. acidophilus IBB 801
Parametera Final pH
ODmax
CDMmax
Cell countsmax
µmax b
3.93 ± 0.07 3.87 ± 0.04 3.90 ± 0.02 3.86 ± 0.08 3.89 ± 0.03 4.08 ± 0.05
10.7 ± 0.6 11.4 ± 0.1 7.9 ± 0.3 10.3 ± 0.7 10.1 ± 0.8 8.4 ± 0.4
2.16 ± 0.19 2.26 ± 0.05 1.79 ± 0.12 3.32 ± 0.13 2.45 ± 0.09 1.91 ± 0.11
9.11 ± 0.09 9.14 ± 0.01 9.34 ± 0.14 8.66 ± 0.33 8.85 ± 0.21 9.04 ± 0.41
0.96(0.99) 0.82(0.99) 0.67(0.98) 0.82(0.99) 0.72(0.99) 0.93(0.99)
a OD −1 −1 max , maximum observed OD600 ; CDMmax , maximum observed biomass (in g CDM l ); cell countsmax , maximum observed cell counts (in log CFU ml ); µmax , maximum specific growth rate (in h−1 ). Values are means of the results of three experiments. Values are expressed as mean ± standard deviation. b Numbers in parentheses are correlation coefficients (r 2 ).
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Fig. 1. Batch fermentation of L. johnsonii La1 in MRS broth with free pH at 37 ◦ C. Left axis: OD600 (2); cell counts (in log CFU ml−1 ) (Q); pH ("). Right axis: glucose consumption (in mM) (1); lactic acid formation (in mM) (!). Results are means of three independent experiments.
Fig. 2. Kinetics of the antibacterial activity of CFCS of lactobacilli against S. Typhimurium SL1344 as a function of lactic acid produced during fermentations with free pH. Killing activity was measured after 4 h of incubation at 37 ◦ C with CFCS (adjusted to pH 4.5) of L. johnsonii La1 (2); L. casei Shirota (Q); L. rhamnosus GG ("); L. acidophilus IBB 801 (1); L. amylovorus DCE 471 (P); and L. plantarum ACA-DC 287 (E). Acid control samples (MRS broth containing increasing amounts of lactic acid) (!). Antibacterial activity is expressed as a percent of reduction of viable cells (in log CFU ml−1 ) with respect to untreated cells. ∗ P < 0.05, ∗∗ P < 0.01, level of significance of the antibacterial activity of CFCS of lactobacilli compared with that of acid control samples. Results are means of three independent experiments.
only. This difference between the antibacterial activity of the CFCS and the acid control samples was significant (P < 0.05) only when the lactic acid concentration in the CFCS was higher than 125 mM for both L. johnsonii La1 and L. plantarum ACADC 287. For the other lactobacilli tested, the antibacterial activity of the CFCS was not significantly higher than that of the control samples, suggesting that their inhibitory effect was only due to the production of lactic acid (Fig. 2). With MRS broth containing various concentrations of succinic acid, phenyllactic acid and OH-phenyllactic acid, alone or in combination, no antibacterial activity was observed. When MRS broth containing lactic acid was tested in combination with the other acids, only the combination with succinic acid
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Fig. 3. Invasion of Caco-2/TC7 cells by S. Typhimurium SL1344 for 1 h at 37 ◦ C. SL1344 cells were pretreated with CFCS (adjusted to pH 4.5) of L. johnsonii La1 obtained through fermentations with free pH (2). Acid control samples were MRS broth containing increasing amounts of lactic acid ("). Inhibition of invasion is expressed as a percent of the reduction in invasion (in log CFU ml−1 ) with respect to the value obtained with cells treated with DMEM. The effect of CFCS and acid control samples upon inhibition of invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells was significant (P < 0.01) in all cases compared with that of pH control samples (MRS medium adjusted to pH 4.5 with HCl). Results are the means of three independent experiments.
(10–15 mM) increased antibacterial activity (40–80%) as compared with that of lactic acid (150 mM) alone, indicating a possible increase in the anti-Salmonella activity in the presence of both lactic acid and succinic acid. Treatment of S. Typhimurium SL1344 with CFCS of L. johnsonii La1, obtained through fermentations with free pH, resulted in strong inhibition of invasion of the pathogen into Caco-2/TC7 cells that increased with fermentation time (Fig. 3). For instance, the CFCS of L. johnsonii La1 obtained after 14 h of fermentation did not significantly decrease the viability of the pathogen after 1 h of contact (6 ± 1% reduction of viability), but considerably inhibited invasion of Caco-2/TC7 cells (51 ± 4%) when compared with inhibition caused by MRS broth adjusted to pH 4.5 (18 ± 3%). The effect of CFCS was no higher than that of acid control samples (Fig. 3), indicating that inhibition of invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells by L. johnsonii La1 was due to production of lactic acid. 3.4. Characteristics of inhibitory compound(s) produced by L. johnsonii La1 and L. plantarum ACA-DC 287 The anti-Salmonella activity of the CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287 was insensitive to all proteases tested. This indicates that bacteriocins were not involved in the anti-Salmonella activity of those strains. The anti-Salmonella activity was not reduced when the samples were heated. For instance, the non-treated CFCS of L. johnsonii La1 obtained after 12 h of fermentation resulted in a reduction of 48 ± 7% of S. Typhimurium SL1344 viability, and a similar reduction (48 ± 8%) was observed when the CFCS was heated at 100 ◦ C for 60 min.
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4. Discussion In this study, a clear distinction could be made between the effects of lactic acid alone and other thus far uncharacterized compounds produced by certain Lactobacillus strains. The CFCS of all lactobacilli tested, including probiotic strains such as L. casei Shirota, L. johnsonii La1, and L. rhamnosus GG, showed strong antibacterial activity against S. Typhimurium SL1344 and significantly inhibited invasion of this pathogen into cultured human enterocyte-like Caco-2/TC7 cells. All strains produced high amounts of lactic acid (above 150 mM). It was further demonstrated that the antibacterial activity of the CFCS of L. amylovorus DCE 471, L. acidophilus IBB 801, L. casei Shirota, and L. rhamnosus GG was solely due to the production of lactic acid. It is largely accepted that weak organic acids such as lactic acid show strong antibacterial activity [1,25]. Similarly, in control experiments that were performed in this study, lactic acid at a concentration of above 125 mM displayed strong antibacterial activity against S. Typhimurium SL1344. Lactic acid is detected in low concentrations in the human colon (<5 mM), where it is produced as an intermediary product of carbohydrate fermentation [11,21,28]. There, it is further converted into short-chain fatty acids, which can reach a total concentration of above 100 mM and thus might exert antibacterial activity against Gram-negative pathogens. Only in a few reports was the production of organic acids suggested to be the major factor in the activity of lactobacilli against Gram-negative bacteria [15,24]. In contrast, in numerous studies, the production of additional antibacterial compounds has been suggested to explain the activity of lactobacilli against Salmonella and Escherichia coli [7,27]. Yet all those studies failed to purify the responsible inhibitory substances; moreover, in most cases, those experiments were performed on a small scale. In the present paper, the production of such antibacterial compounds by L. johnsonii La1 and L. plantarum ACA-DC 287, inhibitory to S. Typhimurium SL1344, was demonstrated through kinetic analysis of the CFCS obtained through controlled laboratory batch fermentations. The anti-Salmonella activity of the CFCS of all lactobacilli tested followed lactic acid production. Nevertheless, the antibacterial activity of the CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287 could not be attributed to lactic acid alone, as the CFCS of both strains displayed killing activities beyond the activity of the lactic acid concentrations present. The anti-Salmonella effect of the non-proteinaceous, heat-stable substances produced by L. johnsonii La1 and L. plantarum ACA-DC 287 was significant only when the pH of the fermentation medium was lower than 4.5 and when the concentration of lactic acid was above 125 mM. It was not found during fermentations with constant pH (unpublished results, L.Makras and L. De Vuyst). It has previously been shown that inhibitory substances such as aromatic and heterocyclic molecules, e.g., mevalonolactone produced by L. plantarum E 76, are active against Gram-negative bacteria only at low pH values and in the presence of lactic acid [23]. Importantly, lactic acid acts as a permeabilizer of the outer membrane of Gramnegative bacteria and thus may trigger the antibacterial activity
of other inhibitory compounds [1]. Furthermore, phenyllactic acid and OH-phenyllactic acid are widely produced by lactobacilli [29] and usually display a broad antifungal spectrum in the concentration range of 100–400 µM [19,29]. Phenyllactic acid, in concentrations ranging from 20 to 80 mM, is also inhibitory towards the Gram-positive bacteria Listeria monocytogenes, Staphylococcus aureus and Enterococcus faecalis, as well as against Gram-negative bacteria such as Providencia stuartii and Klebsiella oxytoca [9,10]. However, in the concentration range in which phenyllactic acid and OH-phenyllactic acid were produced by the Lactobacillus strains used in this study, these compounds showed no antibacterial activity against S. Typhimurium SL1344, nor did they increase the antibacterial activity of lactic acid. Similarly, common lactic acid bacteria metabolites such as diacetyl, acetaldehyde, acetoin and 2,3-butanediol are active towards Gram-negative bacteria only at high concentrations [6]. Inhibition of the invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells by the CFCS of L. johnsonii La1 could be duplicated by MRS broth containing the same amounts of lactic acid as the CFCS. This indicates that high concentrations of lactic acid not only promoted cell death, but also interfered with the invasion mechanisms of the pathogen. These results support the observation of the accumulation of organic acids as a mechanism for inhibition of invasion of Salmonella into intestinal epithelial cells [12,31]. The exact mode of inhibition is still not clear; however, it has recently been demonstrated that acids decrease the expression of genes required for the invasion of Salmonella into human colon carcinoma T84 cells [32]. The results presented here provide evidence that certain lactobacilli, including probiotic strains, strongly inhibit the gastrointestinal pathogen S. Typhimurium SL1344. The activity of most of these Lactobacillus strains was due to the production of lactic acid alone. Also, lactic acid had a significant inhibitory effect on the invasion of human epithelial cells by the pathogen. Nevertheless, the antibacterial activity of L. johnsonii La1 and L. plantarum ACA-DC 287 was due to the combination of lactic acid and one or more hitherto unknown, nonproteinaceous, heat-stable anti-Salmonella compounds which are active at low pH. Acknowledgements This study has been carried out through financial support from the Commission of the European Communities specific RTD program “Quality of Life and Management of Living Resources”, QLK1-2001-01179 “PROPATH”— Molecular analysis and mechanistic elucidation of the functionality of probiotics and prebiotics in the inhibition of pathogenic microorganisms to combat gastrointestinal disorders and to improve human health. It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area. This work was also supported by the Research Council of the Vrije Universiteit Brussel, the Fund for Scientific Research-Flanders, the Flemish Institute for the Encouragement of Scientific and Technological Research in the Industry, and a grant for scien-
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tific exchange between the Flemish Community and INSERM (France). References [1] H.L. Alakomi, E. Skytta, M. Saarela, T. Mattila-Sandholm, K. Latva-Kala, I.M. Helander, Lactic acid permeabilizes Gram-negative bacteria by disrupting the outer membrane, Appl. Environ. Microbiol. 66 (2000) 2001– 2005. [2] M.F. Bernet-Camard, V. Lievin, D. Brassart, J.R. Neeser, A.L. Servin, S. Hudault, The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo, Appl. Environ. Microbiol. 63 (1997) 2747–2753. [3] I. Chantret, A. Rodolosse, A. Barbat, E. Dussaulx, E. Brotlaroche, A. Zweibaum, M. Rousset, Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell-line Caco-2— evidence for glucose-dependent negative regulation, J. Cell Sci. 107 (1994) 213–225. [4] M.H. Coconnier, V. Lievin, M.F. Bernet-Camard, S. Hudault, A.L. Servin, Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB, Antimicrob. Agents Chemother. 41 (1997) 1046–1052. [5] M.H. Coconnier, V. Lievin, M. Lorrot, A.L. Servin, Antagonistic activity of Lactobacillus acidophilus LB against intracellular Salmonella enterica serovar Typhimurium infecting human enterocyte-like Caco-2/TC-7 cells, Appl. Environ. Microbiol. 66 (2000) 1152–1157. [6] L. De Vuyst, E.J. Vandamme (Eds.), Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and Applications, Blackie, London, 1994. [7] L. De Vuyst, L. Avonts, L. Makras, in: C. Remacle, B. Reusens (Eds.), Probiotics, Prebiotics and Gut Health, Woodhead Publishing Ltd., Cambridge, 2004, pp. 416–482. [8] L. De Vuyst, L. Makras, L. Avonts, H. Holo, Q. Yi, A.L. Servin, D. FayolMessaoudi, C. Berger, G. Zoumpopoulou, E. Tsakalidou, D. Sgouras, B. Martinez-Gonzales, E. Panayotopoulou, A. Mentis, D. Smarandache, L. Savu, P. Thonart, I.F. Nes, Antimicrobial potential of probiotic or potentially probiotic lactic acid bacteria, the first results of the international European research project PROPATH of the PROEUHEALTH cluster, Microb. Ecol. Health Dis. 16 (2004) 125–130. [9] V. Dieuleveux, M. Gueguen, Antimicrobial effects of D-3-phenyllactic acid on Listeria monocytogenes in TSB–YE medium, milk, and cheese, J. Food Protect. 61 (1998) 1281–1285. [10] V. Dieuleveux, S. Lemarinier, M. Gueguen, Antimicrobial spectrum and target site of D-3-phenyllactic acid, Int. J. Food Microbiol. 40 (1998) 177– 183. [11] S. Duncan, P. Louis, H.J. Flint, Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product, Appl. Environ. Microbiol. 70 (2004) 5810–5817. [12] J.A. Durant, V.K. Lowry, D.J. Nisbet, L.H. Stanker, D.E. Corrier, S.C. Ricke, Short-chain fatty acids affect cell-association and invasion of HEp-2 cells by Salmonella Typhimurium, J. Environ. Sci. Health B 34 (1999) 1083–1099. [13] FAO/WHO, Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria, Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation Report, Cordoba, 2001. [14] B.B. Finlay, S. Falkow, Salmonella interactions with polarized human intestinal Caco-2 epithelial-cells, J. Infect. Dis. 162 (1990) 1096–1106. [15] L.J. Fooks, G.R. Gibson, In vitro investigations of the effect of probiotics and prebiotics on selected human intestinal pathogens, FEMS Microbiol. Ecol. 39 (2002) 67–75.
247
[16] H.S. Gill, F. Guarner, Probiotics and human health: A clinical perspective, Postgrad. Med. J. 80 (2004) 516–526. [17] I.M. Helander, A. von Wright, T.M. Mattila-Sandholm, Potential of lactic acid bacteria and novel antimicrobials against Gram-negative bacteria, Trends Food Sci. Tech. 8 (1997) 146–150. [18] S. Hudault, V. Lievin, M.F. Bernet-Camard, A.L. Servin, Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection, Appl. Environ. Microbiol. 63 (1997) 513–518. [19] P. Lavermicocca, F. Valerio, A. Visconti, Antifungal activity of phenyllactic acid against molds isolated from bakery products, Appl. Environ. Microbiol. A 69 (2003) 634–640. [20] V. Liévin-Le Moal, R. Amsellem, A.L. Servin, M.H. Coconnier, Lactobacillus acidophilus (strain LB) from the resident adult human gastrointestinal microflora exerts activity against brush border damage promoted by a diarrhoeagenic Escherichia coli in human enterocyte-like cells, Gut 50 (2002) 803–811. [21] S. Macfarlane, G.T. Macfarlane, Regulation of short-chain fatty acid production, Proc. Nutr. Soc. 62 (2003) 67–72. [22] J. Magnusson, K. Strom, S. Roos, J. Sjogren, J. Schnurer, Broad and complex antifungal activity among environmental isolates of lactic acid bacteria, FEMS Microbiol. Lett. 219 (2003) 129–135. [23] M.L. Niku-Paavola, A. Laitila, T. Mattila-Sandholm, A. Haikara, New types of antimicrobial compounds produced by Lactobacillus plantarum, J. Appl. Microbiol. 86 (1999) 29–35. [24] M. Ogawa, K. Shimizu, K. Nomoto, R. Tanaka, T. Hamabata, S. Yamasaki, T. Takeda, Y. Takeda, Inhibition of in vitro growth of Shiga toxinproducing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid, Int. J. Food Microbiol. 68 (2001) 135– 140. [25] A.C. Ouwehand, in: S. Salminen, A. von Wright (Eds.), Antimicrobial Components from Lactic Acid Bacteria, Dekker, New York, 1998, pp. 139–159. [26] A.C. Ouwehand, S. Salminen, E. Isolauri, Probiotics: An overview of beneficial effects, Anton. Leeuw. Int. J. 82 (2002) 279–289. [27] A.L. Servin, Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens, FEMS Microbiol. Rev. 28 (2004) 405–440. [28] D.L. Topping, P.M. Clifton, Short-chain fatty acids and human colonic function: Roles of resistant starch and nonstarch polysaccharides, Physiol. Rev. 81 (2001) 1031–1064. [29] F. Valerio, P. Lavermicocca, M. Pascale, A. Visconti, Production of phenyllactic acid by lactic acid bacteria: An approach to the selection of strains contributing to food quality and preservation, FEMS Microbiol. Lett. 233 (2004) 289–295. [30] R. Van der Meulen, L. Avonts, L. De Vuyst, Short fractions of oligofructose are preferentially metabolized by Bifidobacterium animalis DN-173 010, Appl. Environ. Microbiol. 70 (2004) 1923–1930. [31] F. Van Immerseel, J. De Buck, F. Pasmans, P. Velge, E. Bottreau, V. Fievez, F. Haesebrouck, R. Ducatelle, Invasion of Salmonella enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids, Int. J. Food Microbiol. 85 (2003) 237–248. [32] F. Van Immerseel, J. De Buck, F. Boyen, L. Bohez, F. Pasmans, J. Volf, M. Sevcik, I. Rychlik, F. Haesebrouck, R. Ducatelle, Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with Salmonella enterica serovar Enteritidis, Appl. Environ. Microbiol. 70 (2004) 3582–3587. [33] M. Zamfir, R. Callewaert, P.C. Cornea, L. Savu, I. Vatafu, L. De Vuyst, Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801, J. Appl. Microbiol. 87 (1999) 923–931.
Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enterica serovar Typhimurium reveals a role for lactic acid and other inhibitory compounds Lefteris Makras a , Vagelis Triantafyllou a,b , Domitille Fayol-Messaoudi c , Tom Adriany a , Georgia Zoumpopoulou b , Effie Tsakalidou b , Alain Servin c , Luc De Vuyst a,∗ a Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Department of Applied Biological Sciences and Engineering,
Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium b Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural University of Athens (AUA), 11855 Athens, Greece c Pathogènes et Fonctions des Cellules Epithéliales Polarisées, Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 510,
Faculté de Pharmacie, Université Paris XI, 92296 Châtenay-Malabry, France Received 8 June 2005; accepted 1 September 2005 Available online 22 September 2005
Abstract Six Lactobacillus strains including commercial probiotic ones (L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota, L. johnsonii La1, L. plantarum ACA-DC 287 and L. rhamnosus GG) were investigated, through batch fermentations under controlled conditions, for their capacity to inhibit Salmonella enterica serovar Typhimurium SL1344. All lactobacilli displayed strong antibacterial activity toward this Gramnegative pathogen and significantly inhibited invasion of the pathogen into cultured human enterocyte-like Caco-2/TC7 cells. By studying the production kinetics of antibacterial activity and applying the appropriate acid and pH control samples during a killing assay, we were able to distinguish between the effect of lactic acid and other inhibitory compounds produced. The antibacterial activity of L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota and L. rhamnosus GG was solely due to the production of lactic acid. The antibacterial activity of L. johnsonii La1 and L. plantarum ACA-DC 287 was due to the production of lactic acid and (an) unknown inhibitory substance(s). The latter was (were) only active in the presence of lactic acid. In addition, the lactic acid produced was responsible for significant inhibitory activity upon invasion of Salmonella into Caco-2/TC7 cells. 2005 Elsevier SAS. All rights reserved.
1. Introduction In the past two decades, the application of probiotics to the prevention and management of gastrointestinal disorders has received much interest [26]. Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer a health benefit upon the host [13]. Lactobacilli are commonly used as probiotics [7]. Well-designed clinical trials support the effectiveness of probiotics in keeping the human colon healthy [16]. However, there exists a lack of information about the underlying mechanisms responsible for the beneficial effects of probiotics [13]. As an example, probiotics can * Corresponding author.
E-mail address: [email protected] (L. De Vuyst). 0923-2508/$ – see front matter 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2005.09.002
function as microbial barriers against gastrointestinal pathogens through competitive exclusion of pathogen binding, modulation of the host’s immune system and production of inhibitory compounds [7,27]. Lactobacilli produce a wide range of antibacterial compounds including sugar catabolites such as organic acids (e.g., lactic acid and acetic acid); oxygen catabolites such as hydrogen peroxide; proteinaceous compounds such as bacteriocins, other low-molecular-mass peptides, and antifungal peptides/proteins; fat and amino acid metabolites such as fatty acids, phenyllactic acid, and OH-phenyllactic acid; and other compounds such as reuterin and reutericyclin [6,7,17,22,25, 27,29]. Bacteriocins produced by lactobacilli are antibacterial peptides or proteins that are inhibitory towards Gram-positive bacteria only [6]. Low-molecular-mass compounds such as lac-
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tic acid have been reported to be inhibitory towards Gramnegative pathogenic bacteria [1,24]. Alternatively, a heat-stable, low-molecular-mass antibacterial substance different from lactic acid and present in the cell-free culture supernatant of L. acidophilus LB kills a wide range of Gram-negative bacteria and inhibits adhesion to and invasion of Caco-2 cells by Salmonella enterica ser. Typhimurium SL1344 [4,5,20]. Although it is clear that the inhibitory activity of lactobacilli towards bacteria such as Salmonella is due simply to production of lactic acid, it has not always been clear whether other antibacterial compounds might also be produced, since the findings concerning such substance(s) have been based on small-scale killing studies without kinetic analyses [2,18]. The aim of the present study was to examine the kinetics of production of compounds with anti-Salmonella activity by lactobacilli during batch fermentations under controlled conditions. To exclude possible interference, low-molecular-mass acid metabolites from sugar and protein catabolism were measured as well. 2. Materials and methods 2.1. Bacterial strains and media Six Lactobacillus strains, including commercial probiotic ones, were used in this study (Table 1). S. enterica serovar Typhimurium SL1344 [14] was provided by Stocker B.A.D (Stanford, CA). All strains were stored as glycerol (25%, v/v) stocks at −80 ◦ C in the appropriate media. To obtain a fresh culture, lactobacilli were propagated twice in de Man–Rogosa–Sharpe (MRS) broth (Difco, Detroit, MI) at 37 ◦ C in a standard incubator for 12 h. The transfer inoculum was always 1% (v/v). The Salmonella strain was transferred once to trypticase soy agar (TSA; Difco) before a colony was picked up for further experimental use. The strain was cultured in Luria–Bertani (LB) broth (Difco) at 37 ◦ C for 18 h. Solid media were prepared by adding 1.5% (w/v) agar to the appropriate broths. 2.2. Preliminary investigation of the anti-Salmonella activity of CFCS of lactobacilli grown on a small scale To investigate the inhibitory activity of the six Lactobacillus strains against S. Typhimurium SL1344, small-scale fermentations in MRS broth (30 ml) were carried out at 37 ◦ C for 18 h in triplicate. After centrifugation (8000 g, 20 min, 4 ◦ C), the cell-free culture supernatant (CFCS) was adjusted to pH 4.5 using 1 N NaOH and filter-sterilized. The CFCS was tested for its anti-Salmonella activity using an in vitro killing assay and an in vitro assay for inhibition of invasion of Salmonella into Caco-2/TC7 cells, and analyzed through high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry (LC/MS) (see below). 2.3. Kinetic analysis of the growth and inhibitory activity of lactobacilli during batch fermentation The kinetic analysis of growth and production of antibacterial compounds active towards S. Typhimurium SL1344 by
the six Lactobacillus strains was carried out during laboratory fermentations in 10 l MRS broth in duplicate or triplicate. Fermentations were carried out in a 15-l laboratory fermentor (Biostat C; B. Braun Biotech International, Melsungen, Germany) as described previously [30]. The initial pH of the medium was adjusted to pH 6.5. At predetermined intervals, samples were withdrawn aseptically and used for the analysis of growth and low-molecular-mass metabolites (see below). In addition, CFCS of these samples was adjusted to pH 4.5, filter-sterilized, and tested for anti-Salmonella activity (see below). 2.4. Analysis of microbial growth and low-molecular-mass metabolites Growth was measured by optical density, cell counts and biomass determinations as described previously [30]. The maximum specific growth rate (µmax ) was calculated by linear regression of ln(OD/OD0 ) as a function of time, where OD0 refers to the optical density at the start of the exponential growth phase. The residual glucose levels and lactic acid titers were determined by HPLC analysis as described previously [30], except that an ICSep ICE-ORH-801 column (Interchim, Montluçon, France) was used with 10 mM H2 SO4 as mobile phase at a flow rate of 0.4 ml min−1 . Succinic acid, phenyllactic acid, and OH-phenyllactic acid were determined by LC/MS analysis. A Waters chromatograph (Waters Corp., Milford, MA) coupled with a mass spectrometer (Quattro MicroTM ; Waters Corp.) was used. A capillary column ( 150 × 4.6 mm, 3 µm; Atlantis, Waters Corp.) kept at 35 ◦ C was used with a mobile phase consisting of ultrapure water, acetonitrile (100%, v/v) and ammonium acetate (10 mM) at a flow rate of 0.2 ml min−1 . 2.5. In vitro killing assay for anti-Salmonella activity The inhibitory activity of CFCS towards S. Typhimurium SL1344 was determined by an in vitro killing assay. To obtain mid-logarithmic phase cells, 10 ml of LB broth were inoculated with 200 µl of a fresh Salmonella culture and incubated at 37 ◦ C for 3 h. The culture was centrifuged (5500 g, 20 min, 4 ◦ C), the pellet was washed once with phosphate-buffered saline (PBS; 0.8% NaCl, 0.02% KH2 PO4 , 0.115% Na2 HPO4 , w/v, pH 7.4) and resuspended in Dulbecco’s modified Eagle’s minimum essential medium (DMEM; Invitrogen, Cergy, France). Then, 500 µl of the culture (2 × 108 CFU ml−1 in DMEM) were mixed with 500 µl CFCS or pH control sample and incubated at 37 ◦ C. The pH control sample was MRS broth adjusted to pH 4.5 with 37% (w/v) HCl. The final pH of the incubation medium was always 5.0 ± 0.2. After 4 h of incubation, the surviving S. Typhimurium SL 1344 cells were determined by plating on TSA. The antibacterial activity was expressed as a % of reduction of viable cells (in log CFU ml−1 ) with respect to untreated cells.
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Table 1 CFCS of lactobacilli: antibacterial activity, inhibition of invasion, and products of sugar and amino acid catabolism Lactobacillus straina (source or origin)b
Antibacterial activity (%)c
Inhibition of invasion (%)d
LAe (mM)
L. johnsonii La1 (LC1, Nestlé) L. casei Shirota (Yakult, Yakult Honsha) L. rhamnosus GG (Gefilus, Valio) L. amylovorus DCE 471 (Corn steep liquor [8]) L. acidophilus IBB 801 (Dairy product [33]) L. plantarum ACA-DC 287 (Greek Xynotyri cheese [8])
48 ± 5
46 ± 2
186 ± 4
44 ± 12
44 ± 0
40 ± 10
SAe (mM)
PLAe (µM)
OH-PLAe (µM)
0.87 ± 0.12
250 ± 21
143 ± 32
158 ± 11
0.14 ± 0.08
<50
37 ± 7
155 ± 13
0.40 ± 0.11
52 ± 10
39 ± 7
184 ± 8
0.17 ± 0.09
<50
45 ± 10
29 ± 3
151 ± 18
4.01 ± 0.24
151 ± 23
64 ± 11
51 ± 2
190 ± 14
0.15 ± 0.09
<50
96 ± 14
58 ± 11 76 ± 21 <50 94 ± 30 <50
a Lactobacilli were grown in MRS broth (30 ml) at 37 ◦ C for 18 h. Experiments were performed in triplicate. Values are expressed as means ± standard deviation. b Probiotic strains L. casei Shirota, L. johnsonii La1, and L. rhamnosus GG were isolated from their commercial products. c Antibacterial activity of CFCS (adjusted to pH 4.5) of lactobacilli against S. Typhimurium SL1344 after 4 h of incubation at 37 ◦ C. Antibacterial activity is expressed as % of reduction in viable cells (in log CFU ml−1 ) with respect to untreated cells. d Effect of pretreatment of S. Typhimurium SL1344 with CFCS of lactobacilli upon invasion by this pathogen of cultured human enterocyte-like Caco-2/TC7 cells. Inhibition of invasion is expressed as % reduction in invasion (in log CFU ml−1 ) with respect to the value obtained with cells treated with DMEM. e LA, lactic acid; SA, succinic acid; PLA, phenyllactic acid; OH-PLA, hydroxy-phenyllactic acid.
2.6. In vitro assay for inhibition of invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells The assay for inhibition of invasion of S. Typhimurium SL1344 into cultured human enterocyte-like Caco-2/TC7 cells [3] was conducted as previously reported [4,5,20] with slight modifications. Prior to infection, S. Typhimurium SL1344 cells in the exponential growth phase (2 × 108 CFU ml−1 in DMEM) were mixed with CFCS of the lactobacilli studied, pH control sample, or DMEM, and incubated at 37 ◦ C for 1 h. The pathogenic bacteria were then enumerated, washed twice with PBS, and resuspended in DMEM. The Caco2/TC7 cells, prepared in 24-well tissue culture plates (ATGC, Marne la Valée, France), were washed twice with PBS and infected with the pretreated pathogen. The plates were incubated at 37 ◦ C in an atmosphere of 10% CO2 –90% air for 1 h, and then washed three times with PBS. An antibiotic assay was used to determine the numbers of internalized S. Typhimurium SL1344 cells. The infected Caco-2/ TC7 cells were incubated in DMEM containing gentamicin 100 µg, ml−1 (Sigma-Aldrich, St. Louis, MO), for 1 h. Bacteria that adhered to the Caco-2/TC7 brush border were rapidly killed, whereas those located within the cells survived. The cells were washed twice with PBS and lysed with sterile ultrapure water and S. Typhimurium SL1344 cells were enumerated by plating on TSA. Each assay was conducted in triplicate with three successive passages of the Caco-2/TC7 cells. The inhibition of invasion was expressed as a % of reduction of invasion (in log CFU ml−1 ) with respect to the value obtained with cells treated with DMEM.
trations of an acid alone, or a mixture of acids. The acids were tested in amounts corresponding to their concentrations in the CFCS of the lactobacilli tested. Therefore, lactic acid (concentrations between 75 and 250 mM; Sigma-Aldrich), succinic acid (2.5–15 mM; VWR International, Darmstadt, Germany), phenyllactic acid (500–2500 µM; Sigma-Aldrich) and OHphenyllactic acid (250–1000 µM; Sigma-Aldrich) were added to MRS broth; the liquid was adjusted to pH 4.5 using 1 M NaOH or 37% (w/v) HCl, and filter-sterilized. Also, mixtures of lactic acid (150 mM) plus succinic acid (5, 10, or 15 mM), of lactic acid (150 or 175 mM), plus succinic acid (5 mM), phenyllactic acid (2000 µM), and OH-phenyllactic acid (500 µM), and of succinic acid (5 mM), plus phenyllactic acid (2000 µM), and OH-phenyllactic acid (500 µM) were prepared. The antiSalmonella activity was measured using an in vitro killing assay (see above) performed in triplicate. 2.8. Characteristics of the inhibitory compound(s) produced by L. johnsonii La1 and L. plantarum ACA-DC 287 CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287, obtained after 12, 14 and 16 h of fermentation, was tested for the sensitivity of its antibacterial activity to pepsin, proteinase K, and bacterial protease (Bacillus licheniformis, Streptomyces griseus; Sigma-Aldrich). All proteases were added to the CFCS at a final concentration of 1 mg ml−1 . The heat stability of the antibacterial activity was examined by incubating 1 ml of CFCS at 60, 80 and 100 ◦ C for 15, 30, and 60 min. The antibacterial activity of all treated samples was measured using a killing assay for anti-Salmonella activity (see above).
2.7. Preparation of acid control samples 2.9. Statistics To test the contribution of the different acids produced by lactobacilli to their anti-Salmonella activity, acid control samples were prepared. These samples contained various concen-
Results are expressed as means ± standard deviation. The level of significance was calculated using two-sided Student’s
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t tests for paired or independent samples. In the case of independent samples, the variances of the two compared groups were assumed to be equal. P values less than 0.05 were considered statistically significant. 3. Results 3.1. Preliminary investigation of the anti-Salmonella activity of CFCS of lactobacilli grown on a small scale The incubation of S. Typhimurium SL1344 cells with CFCS (pH 4.5) of all tested lactobacilli resulted in a significant decrease (P < 0.001) in Salmonella viability after 4 h of contact (Table 1). The reduction in viability ranged from 40 ± 10% (L. rhamnosus GG) to 64 ± 11% (L. plantarum ACA-DC 287). The pH control sample gave only a 2 ± 1% reduction in the viability of the pathogen, indicating that the anti-Salmonella activity of the lactobacilli tested was not simply due to a decrease in pH. Concerning the in vitro assay for inhibition of invasion of S. Typhimurium SL1344 into cultured human enterocytelike Caco-2/TC7 cells, the viability of the pathogen was not significantly affected after 1 h of contact (preincubation time) with CFCS (pH 4.5) from the lactobacilli tested. For all strains, inhibition was significant (P < 0.01) compared to inhibition of invasion caused when the pathogen was treated with the pH control sample (Table 1). The main metabolic end product present in the CFCS of all lactobacilli tested was lactic acid in amounts ranging from 150 to 190 mM (Table 1). No acetic acid, formic acid or ethanol were produced. Moreover, phenyllactic acid, OH-phenyllactic acid and succinic acid were found in the CFCS of some of the lactobacilli tested. Relatively high amounts of phenyllactic acid were produced by L. johnsonii La1 and L. acidophilus IBB 801, namely 250 ± 21 and 151 ± 23 µM, respectively. L. acidophilus IBB 801 produced a high amount of succinic acid (4.01 ± 0.24 mM) as well. 3.2. Kinetic analysis of the growth of lactobacilli during batch fermentations Laboratory fermentations with the six Lactobacillus strains followed similar growth patterns. The fermentation and biokinetic parameters are summarized in Table 2. All strains were
fast acidifiers, resulting in a final pH of about 4.5 within 12 h of fermentation. In all cases lactic acid was the main metabolic end product of glucose catabolism. As an example, L. johnsonii La1 cells entered the stationary phase after 10 h of fermentation; at the end of fermentation, the pH was 3.93 ± 0.07 and almost all glucose was consumed and converted into lactic acid (Fig. 1). All lactobacilli tested followed similar lactic acid production patterns (data not shown). In addition, L. johnsonii La1 produced succinic acid (1.2 ± 0.2 mM), phenyllactic acid (923 ± 68 µM), and OH-phenyllactic acid (237 ± 111 µM) too. L. acidophilus IBB 801 produced high amounts of succinic acid (11.6 ± 0.5 mM) in addition to phenyllactic acid (812 ± 35 µM) and OH-phenyllactic acid (168 ± 43 µM). 3.3. Kinetic analysis of the inhibitory activity of lactobacilli during batch fermentations The antibacterial activity of the CFCS, obtained throughout the fermentations, towards S. Typhimurium SL1344 was high for all lactobacilli at the end of fermentation (Fig. 2). The antiSalmonella activity of CFCS of L. johnsonii La1 was already significant (P < 0.001) after 10 h of growth (129 mM lactic acid produced), and the highest reduction (60 ± 3%) in the viability of S. Typhimurium SL1344 was reached after 14 h of fermentation (162 mM lactic acid produced). In all cases, the antibacterial activity of CFCS was evident when the pH of the fermentation medium was lower than 4.5 and hence when a high concentration of lactic acid (above 120 mM) was reached (Figs. 1 and 2). The antibacterial activity of the acid control samples was low when the lactic acid concentration was between 75 and 125 mM, but increased linearly with lactic acid concentrations from 125 to 225 mM (Fig. 2). The antibacterial activity of the CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287 was significantly higher than the antibacterial activity caused by MRS broth containing the same amount of lactic acid as the CFCS tested, indicating the presence of an antibacterial factor close to but different from lactic acid (Fig. 2). For instance, the CFCS of L. johnsonii La1, obtained after 12 h of fermentation and containing 147 ± 6 mM of lactic acid resulted in a reduction of 54 ± 2% of the viability of S. Typhimurium SL1344, whereas MRS broth containing the same amount of lactic acid resulted in a reduction of 21 ± 2%
Table 2 Batch fermentation and biokinetic parameters for growth of lactobacilli Strain L. johnsonii La1 L. plantarum ACA-DC 287 L. casei Shirota L. rhamnosus GG L. amylovorus DCE 471 L. acidophilus IBB 801
Parametera Final pH
ODmax
CDMmax
Cell countsmax
µmax b
3.93 ± 0.07 3.87 ± 0.04 3.90 ± 0.02 3.86 ± 0.08 3.89 ± 0.03 4.08 ± 0.05
10.7 ± 0.6 11.4 ± 0.1 7.9 ± 0.3 10.3 ± 0.7 10.1 ± 0.8 8.4 ± 0.4
2.16 ± 0.19 2.26 ± 0.05 1.79 ± 0.12 3.32 ± 0.13 2.45 ± 0.09 1.91 ± 0.11
9.11 ± 0.09 9.14 ± 0.01 9.34 ± 0.14 8.66 ± 0.33 8.85 ± 0.21 9.04 ± 0.41
0.96(0.99) 0.82(0.99) 0.67(0.98) 0.82(0.99) 0.72(0.99) 0.93(0.99)
a OD −1 −1 max , maximum observed OD600 ; CDMmax , maximum observed biomass (in g CDM l ); cell countsmax , maximum observed cell counts (in log CFU ml ); µmax , maximum specific growth rate (in h−1 ). Values are means of the results of three experiments. Values are expressed as mean ± standard deviation. b Numbers in parentheses are correlation coefficients (r 2 ).
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Fig. 1. Batch fermentation of L. johnsonii La1 in MRS broth with free pH at 37 ◦ C. Left axis: OD600 (2); cell counts (in log CFU ml−1 ) (Q); pH ("). Right axis: glucose consumption (in mM) (1); lactic acid formation (in mM) (!). Results are means of three independent experiments.
Fig. 2. Kinetics of the antibacterial activity of CFCS of lactobacilli against S. Typhimurium SL1344 as a function of lactic acid produced during fermentations with free pH. Killing activity was measured after 4 h of incubation at 37 ◦ C with CFCS (adjusted to pH 4.5) of L. johnsonii La1 (2); L. casei Shirota (Q); L. rhamnosus GG ("); L. acidophilus IBB 801 (1); L. amylovorus DCE 471 (P); and L. plantarum ACA-DC 287 (E). Acid control samples (MRS broth containing increasing amounts of lactic acid) (!). Antibacterial activity is expressed as a percent of reduction of viable cells (in log CFU ml−1 ) with respect to untreated cells. ∗ P < 0.05, ∗∗ P < 0.01, level of significance of the antibacterial activity of CFCS of lactobacilli compared with that of acid control samples. Results are means of three independent experiments.
only. This difference between the antibacterial activity of the CFCS and the acid control samples was significant (P < 0.05) only when the lactic acid concentration in the CFCS was higher than 125 mM for both L. johnsonii La1 and L. plantarum ACADC 287. For the other lactobacilli tested, the antibacterial activity of the CFCS was not significantly higher than that of the control samples, suggesting that their inhibitory effect was only due to the production of lactic acid (Fig. 2). With MRS broth containing various concentrations of succinic acid, phenyllactic acid and OH-phenyllactic acid, alone or in combination, no antibacterial activity was observed. When MRS broth containing lactic acid was tested in combination with the other acids, only the combination with succinic acid
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Fig. 3. Invasion of Caco-2/TC7 cells by S. Typhimurium SL1344 for 1 h at 37 ◦ C. SL1344 cells were pretreated with CFCS (adjusted to pH 4.5) of L. johnsonii La1 obtained through fermentations with free pH (2). Acid control samples were MRS broth containing increasing amounts of lactic acid ("). Inhibition of invasion is expressed as a percent of the reduction in invasion (in log CFU ml−1 ) with respect to the value obtained with cells treated with DMEM. The effect of CFCS and acid control samples upon inhibition of invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells was significant (P < 0.01) in all cases compared with that of pH control samples (MRS medium adjusted to pH 4.5 with HCl). Results are the means of three independent experiments.
(10–15 mM) increased antibacterial activity (40–80%) as compared with that of lactic acid (150 mM) alone, indicating a possible increase in the anti-Salmonella activity in the presence of both lactic acid and succinic acid. Treatment of S. Typhimurium SL1344 with CFCS of L. johnsonii La1, obtained through fermentations with free pH, resulted in strong inhibition of invasion of the pathogen into Caco-2/TC7 cells that increased with fermentation time (Fig. 3). For instance, the CFCS of L. johnsonii La1 obtained after 14 h of fermentation did not significantly decrease the viability of the pathogen after 1 h of contact (6 ± 1% reduction of viability), but considerably inhibited invasion of Caco-2/TC7 cells (51 ± 4%) when compared with inhibition caused by MRS broth adjusted to pH 4.5 (18 ± 3%). The effect of CFCS was no higher than that of acid control samples (Fig. 3), indicating that inhibition of invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells by L. johnsonii La1 was due to production of lactic acid. 3.4. Characteristics of inhibitory compound(s) produced by L. johnsonii La1 and L. plantarum ACA-DC 287 The anti-Salmonella activity of the CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287 was insensitive to all proteases tested. This indicates that bacteriocins were not involved in the anti-Salmonella activity of those strains. The anti-Salmonella activity was not reduced when the samples were heated. For instance, the non-treated CFCS of L. johnsonii La1 obtained after 12 h of fermentation resulted in a reduction of 48 ± 7% of S. Typhimurium SL1344 viability, and a similar reduction (48 ± 8%) was observed when the CFCS was heated at 100 ◦ C for 60 min.
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4. Discussion In this study, a clear distinction could be made between the effects of lactic acid alone and other thus far uncharacterized compounds produced by certain Lactobacillus strains. The CFCS of all lactobacilli tested, including probiotic strains such as L. casei Shirota, L. johnsonii La1, and L. rhamnosus GG, showed strong antibacterial activity against S. Typhimurium SL1344 and significantly inhibited invasion of this pathogen into cultured human enterocyte-like Caco-2/TC7 cells. All strains produced high amounts of lactic acid (above 150 mM). It was further demonstrated that the antibacterial activity of the CFCS of L. amylovorus DCE 471, L. acidophilus IBB 801, L. casei Shirota, and L. rhamnosus GG was solely due to the production of lactic acid. It is largely accepted that weak organic acids such as lactic acid show strong antibacterial activity [1,25]. Similarly, in control experiments that were performed in this study, lactic acid at a concentration of above 125 mM displayed strong antibacterial activity against S. Typhimurium SL1344. Lactic acid is detected in low concentrations in the human colon (<5 mM), where it is produced as an intermediary product of carbohydrate fermentation [11,21,28]. There, it is further converted into short-chain fatty acids, which can reach a total concentration of above 100 mM and thus might exert antibacterial activity against Gram-negative pathogens. Only in a few reports was the production of organic acids suggested to be the major factor in the activity of lactobacilli against Gram-negative bacteria [15,24]. In contrast, in numerous studies, the production of additional antibacterial compounds has been suggested to explain the activity of lactobacilli against Salmonella and Escherichia coli [7,27]. Yet all those studies failed to purify the responsible inhibitory substances; moreover, in most cases, those experiments were performed on a small scale. In the present paper, the production of such antibacterial compounds by L. johnsonii La1 and L. plantarum ACA-DC 287, inhibitory to S. Typhimurium SL1344, was demonstrated through kinetic analysis of the CFCS obtained through controlled laboratory batch fermentations. The anti-Salmonella activity of the CFCS of all lactobacilli tested followed lactic acid production. Nevertheless, the antibacterial activity of the CFCS of L. johnsonii La1 and L. plantarum ACA-DC 287 could not be attributed to lactic acid alone, as the CFCS of both strains displayed killing activities beyond the activity of the lactic acid concentrations present. The anti-Salmonella effect of the non-proteinaceous, heat-stable substances produced by L. johnsonii La1 and L. plantarum ACA-DC 287 was significant only when the pH of the fermentation medium was lower than 4.5 and when the concentration of lactic acid was above 125 mM. It was not found during fermentations with constant pH (unpublished results, L.Makras and L. De Vuyst). It has previously been shown that inhibitory substances such as aromatic and heterocyclic molecules, e.g., mevalonolactone produced by L. plantarum E 76, are active against Gram-negative bacteria only at low pH values and in the presence of lactic acid [23]. Importantly, lactic acid acts as a permeabilizer of the outer membrane of Gramnegative bacteria and thus may trigger the antibacterial activity
of other inhibitory compounds [1]. Furthermore, phenyllactic acid and OH-phenyllactic acid are widely produced by lactobacilli [29] and usually display a broad antifungal spectrum in the concentration range of 100–400 µM [19,29]. Phenyllactic acid, in concentrations ranging from 20 to 80 mM, is also inhibitory towards the Gram-positive bacteria Listeria monocytogenes, Staphylococcus aureus and Enterococcus faecalis, as well as against Gram-negative bacteria such as Providencia stuartii and Klebsiella oxytoca [9,10]. However, in the concentration range in which phenyllactic acid and OH-phenyllactic acid were produced by the Lactobacillus strains used in this study, these compounds showed no antibacterial activity against S. Typhimurium SL1344, nor did they increase the antibacterial activity of lactic acid. Similarly, common lactic acid bacteria metabolites such as diacetyl, acetaldehyde, acetoin and 2,3-butanediol are active towards Gram-negative bacteria only at high concentrations [6]. Inhibition of the invasion of S. Typhimurium SL1344 into Caco-2/TC7 cells by the CFCS of L. johnsonii La1 could be duplicated by MRS broth containing the same amounts of lactic acid as the CFCS. This indicates that high concentrations of lactic acid not only promoted cell death, but also interfered with the invasion mechanisms of the pathogen. These results support the observation of the accumulation of organic acids as a mechanism for inhibition of invasion of Salmonella into intestinal epithelial cells [12,31]. The exact mode of inhibition is still not clear; however, it has recently been demonstrated that acids decrease the expression of genes required for the invasion of Salmonella into human colon carcinoma T84 cells [32]. The results presented here provide evidence that certain lactobacilli, including probiotic strains, strongly inhibit the gastrointestinal pathogen S. Typhimurium SL1344. The activity of most of these Lactobacillus strains was due to the production of lactic acid alone. Also, lactic acid had a significant inhibitory effect on the invasion of human epithelial cells by the pathogen. Nevertheless, the antibacterial activity of L. johnsonii La1 and L. plantarum ACA-DC 287 was due to the combination of lactic acid and one or more hitherto unknown, nonproteinaceous, heat-stable anti-Salmonella compounds which are active at low pH. Acknowledgements This study has been carried out through financial support from the Commission of the European Communities specific RTD program “Quality of Life and Management of Living Resources”, QLK1-2001-01179 “PROPATH”— Molecular analysis and mechanistic elucidation of the functionality of probiotics and prebiotics in the inhibition of pathogenic microorganisms to combat gastrointestinal disorders and to improve human health. It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area. This work was also supported by the Research Council of the Vrije Universiteit Brussel, the Fund for Scientific Research-Flanders, the Flemish Institute for the Encouragement of Scientific and Technological Research in the Industry, and a grant for scien-
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tific exchange between the Flemish Community and INSERM (France). References [1] H.L. Alakomi, E. Skytta, M. Saarela, T. Mattila-Sandholm, K. Latva-Kala, I.M. Helander, Lactic acid permeabilizes Gram-negative bacteria by disrupting the outer membrane, Appl. Environ. Microbiol. 66 (2000) 2001– 2005. [2] M.F. Bernet-Camard, V. Lievin, D. Brassart, J.R. Neeser, A.L. Servin, S. Hudault, The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo, Appl. Environ. Microbiol. 63 (1997) 2747–2753. [3] I. Chantret, A. Rodolosse, A. Barbat, E. Dussaulx, E. Brotlaroche, A. Zweibaum, M. Rousset, Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell-line Caco-2— evidence for glucose-dependent negative regulation, J. Cell Sci. 107 (1994) 213–225. [4] M.H. Coconnier, V. Lievin, M.F. Bernet-Camard, S. Hudault, A.L. Servin, Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB, Antimicrob. Agents Chemother. 41 (1997) 1046–1052. [5] M.H. Coconnier, V. Lievin, M. Lorrot, A.L. Servin, Antagonistic activity of Lactobacillus acidophilus LB against intracellular Salmonella enterica serovar Typhimurium infecting human enterocyte-like Caco-2/TC-7 cells, Appl. Environ. Microbiol. 66 (2000) 1152–1157. [6] L. De Vuyst, E.J. Vandamme (Eds.), Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and Applications, Blackie, London, 1994. [7] L. De Vuyst, L. Avonts, L. Makras, in: C. Remacle, B. Reusens (Eds.), Probiotics, Prebiotics and Gut Health, Woodhead Publishing Ltd., Cambridge, 2004, pp. 416–482. [8] L. De Vuyst, L. Makras, L. Avonts, H. Holo, Q. Yi, A.L. Servin, D. FayolMessaoudi, C. Berger, G. Zoumpopoulou, E. Tsakalidou, D. Sgouras, B. Martinez-Gonzales, E. Panayotopoulou, A. Mentis, D. Smarandache, L. Savu, P. Thonart, I.F. Nes, Antimicrobial potential of probiotic or potentially probiotic lactic acid bacteria, the first results of the international European research project PROPATH of the PROEUHEALTH cluster, Microb. Ecol. Health Dis. 16 (2004) 125–130. [9] V. Dieuleveux, M. Gueguen, Antimicrobial effects of D-3-phenyllactic acid on Listeria monocytogenes in TSB–YE medium, milk, and cheese, J. Food Protect. 61 (1998) 1281–1285. [10] V. Dieuleveux, S. Lemarinier, M. Gueguen, Antimicrobial spectrum and target site of D-3-phenyllactic acid, Int. J. Food Microbiol. 40 (1998) 177– 183. [11] S. Duncan, P. Louis, H.J. Flint, Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product, Appl. Environ. Microbiol. 70 (2004) 5810–5817. [12] J.A. Durant, V.K. Lowry, D.J. Nisbet, L.H. Stanker, D.E. Corrier, S.C. Ricke, Short-chain fatty acids affect cell-association and invasion of HEp-2 cells by Salmonella Typhimurium, J. Environ. Sci. Health B 34 (1999) 1083–1099. [13] FAO/WHO, Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria, Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation Report, Cordoba, 2001. [14] B.B. Finlay, S. Falkow, Salmonella interactions with polarized human intestinal Caco-2 epithelial-cells, J. Infect. Dis. 162 (1990) 1096–1106. [15] L.J. Fooks, G.R. Gibson, In vitro investigations of the effect of probiotics and prebiotics on selected human intestinal pathogens, FEMS Microbiol. Ecol. 39 (2002) 67–75.
247
[16] H.S. Gill, F. Guarner, Probiotics and human health: A clinical perspective, Postgrad. Med. J. 80 (2004) 516–526. [17] I.M. Helander, A. von Wright, T.M. Mattila-Sandholm, Potential of lactic acid bacteria and novel antimicrobials against Gram-negative bacteria, Trends Food Sci. Tech. 8 (1997) 146–150. [18] S. Hudault, V. Lievin, M.F. Bernet-Camard, A.L. Servin, Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection, Appl. Environ. Microbiol. 63 (1997) 513–518. [19] P. Lavermicocca, F. Valerio, A. Visconti, Antifungal activity of phenyllactic acid against molds isolated from bakery products, Appl. Environ. Microbiol. A 69 (2003) 634–640. [20] V. Liévin-Le Moal, R. Amsellem, A.L. Servin, M.H. Coconnier, Lactobacillus acidophilus (strain LB) from the resident adult human gastrointestinal microflora exerts activity against brush border damage promoted by a diarrhoeagenic Escherichia coli in human enterocyte-like cells, Gut 50 (2002) 803–811. [21] S. Macfarlane, G.T. Macfarlane, Regulation of short-chain fatty acid production, Proc. Nutr. Soc. 62 (2003) 67–72. [22] J. Magnusson, K. Strom, S. Roos, J. Sjogren, J. Schnurer, Broad and complex antifungal activity among environmental isolates of lactic acid bacteria, FEMS Microbiol. Lett. 219 (2003) 129–135. [23] M.L. Niku-Paavola, A. Laitila, T. Mattila-Sandholm, A. Haikara, New types of antimicrobial compounds produced by Lactobacillus plantarum, J. Appl. Microbiol. 86 (1999) 29–35. [24] M. Ogawa, K. Shimizu, K. Nomoto, R. Tanaka, T. Hamabata, S. Yamasaki, T. Takeda, Y. Takeda, Inhibition of in vitro growth of Shiga toxinproducing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid, Int. J. Food Microbiol. 68 (2001) 135– 140. [25] A.C. Ouwehand, in: S. Salminen, A. von Wright (Eds.), Antimicrobial Components from Lactic Acid Bacteria, Dekker, New York, 1998, pp. 139–159. [26] A.C. Ouwehand, S. Salminen, E. Isolauri, Probiotics: An overview of beneficial effects, Anton. Leeuw. Int. J. 82 (2002) 279–289. [27] A.L. Servin, Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens, FEMS Microbiol. Rev. 28 (2004) 405–440. [28] D.L. Topping, P.M. Clifton, Short-chain fatty acids and human colonic function: Roles of resistant starch and nonstarch polysaccharides, Physiol. Rev. 81 (2001) 1031–1064. [29] F. Valerio, P. Lavermicocca, M. Pascale, A. Visconti, Production of phenyllactic acid by lactic acid bacteria: An approach to the selection of strains contributing to food quality and preservation, FEMS Microbiol. Lett. 233 (2004) 289–295. [30] R. Van der Meulen, L. Avonts, L. De Vuyst, Short fractions of oligofructose are preferentially metabolized by Bifidobacterium animalis DN-173 010, Appl. Environ. Microbiol. 70 (2004) 1923–1930. [31] F. Van Immerseel, J. De Buck, F. Pasmans, P. Velge, E. Bottreau, V. Fievez, F. Haesebrouck, R. Ducatelle, Invasion of Salmonella enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids, Int. J. Food Microbiol. 85 (2003) 237–248. [32] F. Van Immerseel, J. De Buck, F. Boyen, L. Bohez, F. Pasmans, J. Volf, M. Sevcik, I. Rychlik, F. Haesebrouck, R. Ducatelle, Medium-chain fatty acids decrease colonization and invasion through hilA suppression shortly after infection of chickens with Salmonella enterica serovar Enteritidis, Appl. Environ. Microbiol. 70 (2004) 3582–3587. [33] M. Zamfir, R. Callewaert, P.C. Cornea, L. Savu, I. Vatafu, L. De Vuyst, Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801, J. Appl. Microbiol. 87 (1999) 923–931.