Probioticos 2 by micromecuis

Vol. 77, No. 19

Modification of the Technical Properties of Lactobacillus johnsonii NCC 533 by Supplementing the Growth Medium with Unsaturated Fatty Acids䌤 J. A. Muller,1,2 R. P. Ross,1,3,4 W. F. H. Sybesma,2 G. F. Fitzgerald,3 and C. Stanton1,4* Teagasc, Moorepark Food Research Centre, Moorepark, Fermoy, County Cork, Ireland1; Nestle´ Product Technology Centre Konolfingen, Konolfingen, Switzerland2; Department of Microbiology, University College Cork, Cork, County Cork, Ireland3; and Alimentary Pharmabiotic Centre, Cork, Ireland4 Received 20 April 2011/Accepted 27 July 2011

the membrane is essential for the mobility and functionality of embedded proteins and lipids, diffusion of proteins and other molecules across the membrane, and appropriate separation of membranes during cell division. Membrane fluidity can be altered by changing the ratio of saturated to unsaturated fatty acids in the membrane, with the double bond in unsaturated fatty acids preventing lipids from being closely packed and thus increasing fluidity. Bacteria can alter the composition of the membrane fatty acids by a number of processes, including cyclopropanation, cis-trans isomerization, (de)saturation, or the incorporation of free exogenous fatty acids into the membrane (7, 9, 18, 21). It has been reported that bacterial membrane composition changes during acid shock (5, 23), osmolarity fluctuations (46), freeze-drying (31), and exposure to suboptimal growth temperatures (16). Fatty acids are essential nutrients for many lactic acid bacteria (LAB), and supplementation of growth medium with fatty acids can influence the membrane composition and growth rate (20, 49). However, free fatty acids can also be bacteriostatic or bactericidal, depending on the bacterial strain, fatty acid concentration, and degree of saturation (18). Oleic acid, often used in the form of Tween 80, is a known stimulant for LAB growth and has been reported to have protective effects against stress (7, 49). For example, Tween 80 increased survival of Lactobacillus rhamnosus GG after exposure to gastric juice,

Probiotics are defined as “live microorganisms, which when administered in adequate amounts confer a health benefit on the host” (11). Retaining probiotic viability through to the intestine presents major technological and biological hurdles, given the extreme conditions that probiotics encounter during production and storage and in the harsh environment of the stomach. During production and processing, probiotics undergo several stresses such as substrate depletion (end of fermentation) and osmolarity and temperature shock (freeze or spray drying) (25, 35, 43), and yet they must remain viable during storage until consumption (8, 19, 34). When exposed to such stresses, bacteria generally activate a general stress response that involves upregulation of certain genes. This survival mechanism includes synthesis of chaperone proteins (such as groESL and dnaK) (7, 32, 36, 51) and proteases (48) and mechanisms which alter energy maintenance (45). Bacteria can also change the composition of the cell membrane when exposed to stress in order to maintain proper membrane fluidity (homeoviscous adaptation) (38), which is an important factor in coping with stress conditions (9, 47). Proper fluidity of

* Corresponding author. Mailing address: Teagasc, Moorepark Food Research Centre, Moorepark, Fermoy, County Cork, Ireland. Phone: 353 25 42606. Fax: 353 25 42340. E-mail: catherine.stanton@teagasc.ie. 䌤 Published ahead of print on 5 August 2011. 6889

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The aim of this study was to investigate the influence of supplementing growth medium with unsaturated fatty acids on the technical properties of the probiotic strain Lactobacillus johnsonii NCC 533, such as heat and acid tolerance, and inhibition of Salmonella enterica serovar Typhimurium infection. Our results showed that the membrane composition and morphology of L. johnsonii NCC 533 were significantly changed by supplementing a minimal Lactobacillus medium with oleic, linoleic, and linolenic acids. The ratio of saturated to unsaturated plus cyclic fatty acids in the bacterial membrane decreased by almost 2-fold when minimal medium was supplemented with unsaturated fatty acids (10 ␮g/ml). The subsequent acid and heat tolerance of L. johnsonii decreased by 6- and 20-fold when the strain was grown in the presence of linoleic and linolenic acids, respectively, compared with growth in oleic acid (all at 10 ␮g/ml). Following acid exposure, significantly higher (P < 0.05) oleic acid content was detected in the membrane when growth medium was supplemented with linoleic or linolenic acid, indicating that saturation of the membrane fatty acids occurred during acid stress. Cell integrity was determined in real time during stressed conditions using a fluorescent viability kit in combination with flow cytometric analysis. Following heat shock (at 62.5°C for 5 min), L. johnsonii was unable to form colonies; however, 60% of the bacteria showed no cell integrity loss, which could indicate that the elevated heat inactivated vital processes within the cell, rendering it incapable of replication. Furthermore, L. johnsonii grown in fatty acid-enriched minimal medium had different adhesion properties and caused a 2-fold decrease in S. enterica serovar Typhimurium UK1-lux invasion of HT-29 epithelial cells compared with bacteria grown in minimal medium alone. This could be related to changes in the hydrophobicity and fluidity of the membrane. Our study shows that technical properties underlying probiotic survivability can be affected by nutrient composition of the growth medium.

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MATERIALS AND METHODS Bacterial strains and growth conditions. L. johnsonii NCC 533 was provided by Nestle´ Culture Collection for this research study. L. johnsonii was routinely cultured anaerobically in either minimal Lactobacillus medium (MLM⫹) or in de Man-Rogosa-Sharpe (MRS) medium (Difco, Franklin Lakes, NJ) to optical densities at 590 nm (OD590) of 1.5 and 4.0 for log phase and stationary phase, respectively. MLM⫹ was comprised of glucose (20 g/liter; Sigma, St. Louis, MO), tryptone (10 g/liter; Sigma), yeast extract (5 g/liter; Merck, Jersey City, NJ), sodium acetate (5 g/liter; Sigma), dipotassium hydrogen phosphate (26 g/liter; Sigma), diammonium hydrogen citrate (20 g/liter; Sigma), Tween 80 (0.5 g/liter; Sigma), magnesium sulfate (0.2 g/liter; Sigma), and manganese(II)-sulfate (0.05 g/liter; Sigma). Where mentioned, oleic (C18:1 c9), linoleic (C18:2 c9c12), or ␣-linolenic (C18:3 c9c12c15; Sigma) acid was added to MLM⫹ at a rate of 10 ␮g/ml. The number of CFU was determined by serial dilutions in maximum recovery diluent (MRD; Sigma), and subsequently 10 ␮l was spot plated and incubated anaerobically (Anaerocult A; Merck, Jersey City, NJ) for 72 h at 37°C on MRS agar plates. S. enterica serovar Typhimurium UK1-lux was kindly donated by University College Cork ([UCC] Microbiology Department) and used for exclusion experiments. S. Typhimurium UK1-lux has the lux vector p16Slux integrated in the 16S DNA locus of the chromosome, allowing quantification by measurement of luminescence (33). Erythromycin was added at a concentration of 50 ␮g/ml to the growth medium to maintain the lux-expressing Salmonella strain UK1-lux. Before the experiments, UK1-lux was grown in antibiotic-free medium, and luminescence remained stable. UK1-lux was stocked in tryptic soy broth (TSB; Sigma) and glycerol (ratio, 60:40) and stored at ⫺80°C until use. For each experiment, 10 ␮l of UK1-lux stock was streaked on brain heart infusion (BHI; Sigma) agar containing 50 ␮g/ml erythromycin and incubated aerobically for 10 h at 37°C. Subsequently, a luminescent colony was used to inoculate 20 ml of TSB and grown aerobically overnight; this was used to inoculate adherence and competitive exclusion assays. The UK1-lux strain was enumerated by spot plating serial dilutions on BHI agar and incubated aerobically at 37°C for 16 h. When

UK1-lux was cocultured with NCC 533, xylose lysine deoxycholate agar ([XLD] Fluka, St. Louis, MO) was used for selective enumeration. HT-29 cells (ECACC 91072201) were purchased from the European Collection of Cell Cultures (Salisbury, United Kingdom) and routinely cultured using complete medium (CM) comprised of McCoy 5A medium (Sigma) supplemented with fetal bovine serum ([FBS] 100 ml/liter; Sigma), penicillin-streptomycin (10 g/liter; Sigma), and L-glutamate (12 mM; Sigma). HT-29 cells were incubated at 37°C in a 5% CO2 atmosphere. HT-29 cells were stocked in FBS (200 ml/liter), dimethyl sulfoxide ([DMSO] 100 g/liter; Sigma), and McCoy 5A medium (700 ml/liter) supplemented with L-glutamine (0.2 mM; Sigma) and frozen at ⫺196°C until use. Cell integrity. Cell integrity during and after external stress was analyzed using fluorescent nucleic acid staining dyes in combination with flow cytometric analysis. A BD Cell Viability Kit (BD Biosciences, Oxford, United Kingdom), containing the fluorescent nucleic acid dyes thiazole orange ([TO] penetrates and stains all cells; excitation/emission, 480/550 nm) and propidium iodine ([PI] penetrates and stains only cells with compromised membrane; excitation/emission, 515/620 nm), was used. Samples were diluted with MRD to approximately 5 ⫻ 106 CFU/ml and incubated at room temperature for 15 min in the dark with 42 nM TO and 4.3 ␮M PI. Samples were analyzed with a FACSCanto II flow cytometer (BD Biosciences) equipped with three lasers (405, 488, and 633 nm). An air-cooled argon ion laser emitting blue light at 488 nm was used with a standard filter setup. The side scatter signal was used as a trigger signal. The red fluorescence from PI-stained cells was detected through a 556-nm long-pass and a 585/42-nm band-pass filter (D channel). The green fluorescence from the TO-stained cells was detected through a 502-nm long-pass and a 530/30-nm band-pass (E channel) filter. The standard fluorescence-activated cell sorter (FACS) solutions were used according to the manufacturer’s recommendations (BD Biosciences). All analyses were performed using a low flow setting (⬃12 ␮l/min), and the sample concentration was adjusted to maintain the CFU count at approximately 5 ⫻ 106 CFU/ml (⬍1,000 events/s). The data points (10,000 events per sample) were collected and analyzed using BD FacsDiva, version 5.0 (BD Biosciences). Loss of cell integrity was calculated as the percentage of cells that had compromised membranes. Fatty acid analysis. Bacterial cells (100 to 500 mg) were harvested by centrifugation (3,000 ⫻ g for 7 min at 4°C) and washed twice with Dulbecco’s phosphate-buffered saline (dPBS; Sigma). Fatty acids were extracted and methylated using the Anaer1 method of the MIDI microbial identification system (Microbial ID, Newark, NJ) with minor modifications (28). Briefly, saponification was performed using 0.5 ml of methanolic NaOH (3.7 M; Sigma) and 0.5 ml of deionized water. Subsequently, 0.4 mg of internal standard (C13:0; Sigma) was added to each sample (100 to 500 mg). Tubes were then sealed with screw caps, briefly vortexed, and placed in boiling water for 5 min. Following this, the tubes were vortexed and returned to boiling water for 25 min. The methylation step involved the addition of 2 ml of methanolic HCl (3.0 M; Sigma) per sample, followed by brief vortexing and heating in a water bath for 10 min at 80°C. The resulting fatty acid methyl ethers (FAMEs) were extracted using 1.25 ml of extraction solution (1:1 hexane–methyl tert-butyl ether [MTBE]; Sigma). The upper layer was collected and evaporated at 50°C with a constant flow of nitrogen. The FAMEs were then dissolved in 1 ml of hexane and stored at ⫺20°C before analysis by gasliquid chromatography (GLC). The FAMEs were analyzed using a Varian GC3800 gas chromatograph (Varian, Palo Alto, CA) fitted with a CP 7420 column (CP Select CB FAME [Varian]; 100 m; internal diameter, 0.25 mm; film thickness, 0.25 ␮m). Samples (0.5 ␮l) were injected on the column using an Autosampler 8400 and 1079 Injector (Varian). Thirty seconds after injection, a 20% split was initiated for 15 s. The injector was kept at 160°C for 6 s and then increased to 225°C. The column oven was set to 80°C for 8 min and then increased to 200°C (rate, 8.5°C/min) for 77 min. Compounds were detected using a flame ionization detector (FID; at 250°C) while the helium flow through the column was maintained constant at 1.0 ml/min. The retention times were compared with FAME standards (Sigma) to identify the fatty acids present in the sample. The concentrations were then calculated using standard calibration curves and reported as percent FAME. The FAMEs were grouped based on their conformational freedom as follows: saturated fatty acids (SFA) and unsaturated fatty acids plus cyclopropane fatty acids (UFA⫹CFA). Acid shock. L. johnsonii NCC 533 grown in MLM⫹ and in MLM⫹ supplemented with oleic, linolenic, and linolenic acids (all at 10 ␮g/ml) was harvested in log and stationary phases by centrifugation (3,000 ⫻ g for 7 min at 4°C). The harvested cells were then washed twice with dPBS. Following the second wash, the cells were resuspended in phosphoric acid buffer (100 mM) at pH 2.00 for 90 min. The cell integrity was monitored during acid shock. Before and after acid shock, the CFU count and the fatty acid profile were determined according to the methods described above.

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while stearic, elaidic, linoleic, and conjugated linoleic acids did not increase survival (7). Furthermore, a significant membrane desaturation was observed after gastric juice exposure when medium was supplemented with Tween 80. The protective effect of fatty acid supplementation is not limited to LAB. In Escherichia coli, for instance, growth medium enriched with oleic acid increased tolerance to heat and ethanol, while these effects were not observed with linoleic and linolenic acid enrichment (40). The cell membrane can also play a critical role in adhesion to intestinal epithelial cells. Adherence to epithelial cells is one criterion considered to be of importance in order for probiotics to prevent gut colonization or invasion of pathogenic bacteria, such as Bacteroides vulgatus, Clostridium difficile, Salmonella enterica serovar Choleraesuis, Staphylococcus aureus, and certain E. coli strains (1, 3, 6, 26, 37). Thus, by adhering to epithelial cells the probiotics sterically hinder the pathogen from reaching the binding sites needed to initiate invasion (3). The competitive exclusion mode of action could be a result of interference with the bacterial type 1 fimbriae (e.g., in Salmonella) required for initial adherence to the epithelial cells (12). In this respect, it has been shown that free unsaturated fatty acids in the growth medium changed adhesion sites in Caco2 cells and prevented Lactobacillus strains from adhering in vitro (22). However, at low concentrations ␥-linolenic and arachidonic acids promoted growth and adhesion, respectively, of Lactobacillus casei Shirota. This indicates that fatty acids can interfere or promote adhesion of intestinal bacteria. The present study used a nutritional approach to modulate the saturation level in the membrane of Lactobacillus johnsonii and investigated the survival following heat and acid stress.

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VOL. 77, 2011

EFFECT OF UNSATURATED FATTY ACIDS ON LACTOBACILLUS

FIG. 1. (a) Dose-response curve of Tween 80 concentration in MLM (filled blue circles, 0%; filled red triangle, 0.01%; open blue circles, 0.05%; open black squares, 0.1%; green stars, 0.25%). (b) Growth curve of L. johnsonii in MLM⫹ (open blue circles, 0.05% [wt/vol] Tween 80) supplemented with oleic (closed red squares), linoleic (open black triangles), or linolenic (green stars) acid. Values are average of at least three experiments and error bars represent ⫾ SD.

([ANOVA] GraphPad Prism, version 3.03 for Windows; GraphPad Software, San Diego, CA). Differences were considered significant at a P value of ⱕ0.05.

RESULTS Our initial experiments confirmed that L. johnsonii NCC 533 had a growth requirement for oleic acid, and consequently Tween 80 was added to the minimal medium (4). In order to find the lowest concentration needed for uninhibited growth, a dose-response curve with oleic acid was performed (Fig. 1a). Since in these experiments maximum yield (e.g., highest OD) was not the objective, we chose to use the minimal concentration of Tween 80 where the maximum growth rate was achieved. The results demonstrated that supplementation above 5 ␮g/ml of Tween 80 did not increase the growth rate

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Heat shock. L. johnsonii NCC 533 grown in MLM⫹ and in MLM⫹ supplemented with oleic, linoleic, and linolenic acids (all at 10 ␮g/ml) was harvested in log and stationary phases. Subsequently, a heat shock was applied at 58°C or 62.5°C for 5 min to 50-␮l samples using a gradient heat cycler (G-Storm; Genetech, Somerville, MA). The cell integrity and CFU counts were then analyzed before and after heat shock. Furthermore, the fatty acid profile was determined before and after heat shock for log- and stationary-phase cells. For this analysis, 50-ml samples were heat shocked by preheating cells to 58°C in an 80°C water bath and transferring them to a 58°C water bath for 5 min. Cell integrity and CFU counts were comparable in the 50-␮l samples heat shocked using a gradient heat cycler (data not shown). Storage test. Stationary-phase L. johnsonii NCC 533 bacteria grown in MLM⫹ supplemented with oleic, linoleic, and linolenic acids (all at 10 ␮g/ml) were harvested by centrifugation (3,000 ⫻ g for 7 min at 4°C) and resuspended in acidified reconstituted skim milk ([RSM] 200 g/liter). The RSM was acidified with lactic acid (20%, vol/vol; Sigma) to pH 4.5 at 4°C. The acidified RSM samples were stored for 4 weeks at 4°C. Viability of L. johnsonii NCC 533 was measured every week using CFU counts; 0.5 g of sample was added to 0.5 g of MRD and spot plated following serial dilutions. Adherence. A 25-cm2 flask was inoculated with 1 ml of HT-29 frozen stock (passage number 5) and grown to 80% confluency in CM. Cells were washed with dPBS and treated with trypsin (2.5 mg/ml)–0.53 mM EDTA solution (Sigma) at a rate of 0.5 ml per 25 cm2 and incubated for 10 min at 37°C in a 5% CO2 atmosphere. Cells were then transferred to a 12-well plate (at 105 cells per well) in CM, and medium was changed every 2 days until 15 days postconfluence, giving approximately 106 cells per well. Twenty-four hours before the adherence assay, medium was replaced with antibiotic-free CM. Probiotics were grown overnight to stationary phase and harvested by centrifugation (3,500 ⫻ g for 10 min at 4°C) and washed twice with dPBS buffer. Salmonella UK1-lux was grown for 5 h in TSB and resuspended in McCoy 5A medium (supplemented with 12 mM L-glutamate). HT-29 cells were washed with dPBS, and probiotics suspended in McCoy 5A medium (supplemented with 12 mM L-glutamate) were added to each well at a rate of 109 CFU and incubated for 2 h. Following incubation, medium was removed, and wells were washed five times with dPBS and subsequently treated with 0.5 ml of trypsin (2.5 mg/ml)–0.53 mM EDTA solution for release of cells. Bacteria were enumerated using CFU counts in the released HT-29 cells. Salmonella UK1-lux was added at a rate of 106 CFU per well and incubated for 1 h before enumeration. The adherence experiments were assayed in parallel triplicates and repeated three times. Competitive exclusion. Five hours before the competitive exclusion assay, fresh antibiotic-free TSB medium was inoculated with 1% overnight Salmonella UK1-lux (as described above). In this case, the HT-29 cells were prepared as described for the adhesion assay; however, after 1 h of incubation with NCC 533, 106 CFU of Salmonella UK1-lux suspended in McCoy 5A medium (supplemented with 12 mM L-glutamate) was added per well, at which point the luminescence was measured. After 1 h, cells were washed five times with dPBS to remove any nonadhered cells. Next, the cell suspension was incubated for 30 min (at 37°C) in 1 ml of McCoy 5A medium, and luminescence was recorded. To analyze invaded Salmonella, gentamicin (1 mg/liter) was added to 2/3 of the wells, and after 30 min of incubation (at 37°C) luminescence was recorded. Colonyforming units of probiotic and Salmonella bacteria were analyzed by trypsinizing the cells (as described above) and spot plating on MRS agar or XLD agar, respectively. The exclusion experiments were assayed in parallel triplicates and repeated three times. HT-29 cells challenged with just Salmonella or just NCC 533 were the negative and positive controls for these experiments, respectively. The data were normalized to the adhered and invaded Salmonella numbers without preexposure to bacteria, and results are presented as average values ⫾ standard deviation (SD). Adhesion was expressed as the adhesion index, i.e., the amount of adherent bacteria per 100 HT-29 cells, calculated as follows: Aindex ⫽ 100 ⫻ (number of CFU of adhered bacteria/number of HT-29 cells). Luminescence. Luminescence was measured in 12-well plates (Sarstedt, Leicester, United Kingdom) using an IVIS 100 imager (Caliper Life Sciences, Hopkinton, MA). The camera settings were kept constant at 30 s of exposure, medium binning, and F-stop 1. The stage was set to position B, and regions of interest (ROIs) were created around each well. Luminescence was measured in average radiance per well (photons/s/cm2/steradian [RLU]). Microscopy. For differential interference contrast (DIC) microscopy, an Olympus BX51 (Olympus, Hamburg, Germany) fitted with DIC optics and a 60⫻ lens with a numerical aperture of 1.4 was used. Statistical analysis. Heat shock, acid shock, and storage tests were repeated at least three times, whereas the adhesion and competitive exclusion assays were conducted in parallel triplicates and repeated three times. Significant differences were determined by using either paired or unpaired one-way analysis of variance

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TABLE 1. Percentage of FAMEs in L. johnsonii cells harvested at log and stationary phases Percentage of FAMEs by medium and growth phasea Fatty acid designation and/or name

SFA/UFA a b

MLM⫹ and linoleic acid

MLM⫹ and oleic acid

MLM⫹ and linolenic acid

Log

Stationary

Log

Stationary

Log

Stationary

Log

Stationary

3.12 7.22 4.14 0.12 2.41 3.48 3.67 60.20 2.58 0.40 1.08 8.96 0.00 0.15 0.34 2.14 0.00

2.84 5.06* 4.43 0.11 2.12 2.80 4.21* 46.82* 2.44 0.31 1.15 24.90* 0.00 0.45* 0.39 1.98 0.00

1.53 3.51 2.11 0.05 1.52 1.98 1.84 69.98 1.94 0.08 0.68 13.37 0.00 0.06 0.23 1.12 0.00

1.40 2.95 2.50 0.68 1.13 1.94 2.36* 48.28* 1.79 0.05 0.68 34.52* 0.00 0.29 0.23 1.18 0.00

1.89 4.23 2.48 0.00 0.85 2.16 1.96 31.66 1.50 0.47 43.28 7.65 0.00 0.11 0.33 1.42 0.00

1.37 3.01* 2.49 0.27 1.56 2.03 2.05 22.24* 1.23 0.39 43.32 18.63* 0.00 0.13 0.16 1.11* 0.00

1.68 4.86 2.83 0.00 1.02 2.37 2.06 33.06 1.04 0.15 1.00 8.77 38.45 0.00 0.39 1.40 0.83

1.40 3.57* 2.71 0.00 1.64 2.21 2.29 25.25 1.16 0.05 0.90 19.39* 37.62 0.00 0.19 1.20 0.37

0.16

0.12

0.08

0.09

0.07

0.10

0.08

Data are the average of three separate experiments. ⴱ, significant difference in FAMEs between log- and stationary-phase cells (P ⬍ 0.05). CLA, conjugated linoleic acid.

significantly (P ⬎ 0.25). Therefore, 5 ␮g/ml of Tween 80 was added to the base medium (MLM⫹). This base medium was then further supplemented with oleic, linoleic, or linolenic acid at a concentration of 10 ␮g/ml, which was below the determined MIC (MICs of 22, 19, and 18 ␮g/ml of oleic, linoleic, and linolenic acids, respectively). The supplementation of oleic, linoleic, or linolenic acid increased the maximum OD590 of L. johnsonii by 29, 35, or 15%, respectively, compared with no supplementation (Fig. 1b). Interestingly, the membrane composition of L. johnsonii grown on MLM⫹ supplemented with fatty acids was significantly changed compared to that of cells grown on MLM⫹ alone. Membranes of supplemented L. johnsonii in log and stationary phase contained over 30% of the supplemented fatty acid (Table 1). The ratio between saturated fatty acids and unsaturated plus cyclopropane fatty acids (SFA/[UFA⫹CFA]) decreased by 25% from log to stationary phase when the strain was grown in MLM⫹ alone (P ⬍ 0.05). There was no change in the SFA/ (UFA⫹CFA) ratio from log to stationary phase on the addition of the free fatty acids to the growth medium. Nevertheless, the SFA/(UFA⫹CFA) ratio decreased significantly (P ⬍ 0.05) compared with the unsupplemented strain by 50, 44, and 38% in log phase and 33, 42, and 33%, in stationary phase, respectively, when supplemented with oleic, linoleic, and linolenic acids (Fig. 2). Interestingly, there was a significant increase (P ⬍ 0.05) of cyclopropane fatty acid (CFA) from log to stationary phase and a corresponding decrease of oleic acid in all treatments (Table 1). As expected, CFA reached the highest concentration when MLM⫹ was supplemented with its precursor, oleic acid. Conjugated linoleic acid was significantly increased in stationary-phase cells when bacteria were grown in MLM⫹ containing oleic acid. DIC microscopy revealed that the size of L. johnsonii NCC 533 grown in MLM⫹ increased up to 10-fold compared with NCC 533 grown in MRS medium

(Fig. 3), whereas the final cell count of NCC 533 following overnight growth in MLM⫹ was approximately 10-fold lower than with MRS medium. Heat shock. Production of probiotic powders usually involves exposure to a series of stresses such as temperature, osmolarity, and mechanical damage, which can result in viability loss. In order to assess the heat stress tolerance of fatty acid-enriched L. johnsonii NCC 533, the survival of cells was determined following exposure to 58 and 62.5°C for 5 min. Survival was monitored by cell integrity loss and by CFU counts. In addition, the fatty acid profile was analyzed before and after heat exposure at 58°C. The results demonstrated that there was no significant difference in mortality between the

FIG. 2. Ratio of SFA/(UFA⫹CFA) from log-phase (black bars)and stationary-phase (checkered bars)-harvested L. johnsonii bacteria grown in fatty acid-supplemented MLM⫹. OA, oleic acid; LA, linoleic acid; LIA, linolenic acid. Values are the average of at least three experiments, and error bars represent ⫾ SD.

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C14:0 (myristic) C16:0 (palmitic) C16:1 (palmitoleic) C17:0 C17:1 c10 C18:0 (stearic) C18:1 t9 (elaidic) C18:1 c9 (oleic) C18:1 c11 (vaccenic) C17:2 C18:2 c9c12 (linoleic) CFA (cyclopropane) C18:3 c9c12c15 (␣-linolenic) CLA c9t11b C20:1 c11 C18:4 (stearidonic) Unknown

MLM⫹

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TABLE 2. Percentage of live bacteria measured using CFU count and flow cytometry after a 5-min heat shock at 58 and 62.5°C Cell survival (%) by growth phase and detection method after heat shocka Log phase

Supplement CFU

Stationary phase FC

CFU

58°C 62.5°C 58°C 62.5°C 58°C 62.5°C 58°C 62.5°C

MLM⫹ 12.0 Oleic acid 2.1 Linoleic acid 4.1 Linolenic acid 2.3 a

0.0 0.0 0.0 0.0

89.1 85.3 78.0 65.1

19.3 5.7 1.8 0.2

44.4 51.5 18.6 16.5

0.2 0.8 0.0 0.0

56.5 38.6 14.2 15.4

56.2 34.1 5.1 6.9

FC, flow cytometry.

FIG. 3. DIC image of NCC 533 grown in MRS medium (a) and MLM⫹ (b).

brane composition revealed that there was no significant difference in the fatty acid profiles before and after heat shock. Acid shock. To survive passage through the gastrointestinal tract, probiotics need to overcome the low pH encountered in the stomach. For this reason, the acid tolerance of fatty acidenriched L. johnsonii NCC 533 was analyzed by exposing logand stationary-phase-harvested cells to pH 2.0 for 90 min. Viability during acid stress was monitored using cell integrity and CFU counts. In addition, the membrane fatty acid profile was analyzed before and after the acid shock. Log-phase cells showed an immediate membrane integrity loss of approximately 25% following exposure to acid (Fig. 5). Furthermore, there was no significant difference between L. johnsonii NCC 533 cells grown with or without fatty acids. Cells harvested in stationary phase were more resistant to the acidic conditions than cells harvested in the log phase. Interestingly, supplemen-

FIG. 4. Cell integrity (percent live) after 5 min of heat shock for L. johnsonii grown on MLM⫹ (open blue circles) supplemented with oleic (red squares), linoleic (purple triangle), and linolenic (green star) acids. Cells were harvested in log (a) and stationary (b) phases. Integrity was analyzed using a viability kit in combination with flow cytometry. Values are the average of at least three experiments, and error bars represent ⫾ SD.

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fatty acid-supplemented strain and the unsupplemented strain in log phase following 5 min of heat shock at 58 and 62.5°C (Fig. 4a). However, cells harvested in stationary phase and grown in the presence of oleic, linoleic, and linolenic acids showed a significant decrease in cell integrity by 32, 73, and 75% at 58°C and by 39, 88, and 91% at 62.5°C, respectively (Fig. 4b). The CFU data confirmed the trend that supplementation with linoleic and linolenic acids decreased heat tolerance compared to cells grown without fatty acid supplementation. However, the percentage of decrease in viability measured by CFU counts did not correspond with the cell integrity data obtained by flow cytometry. In general, the viability by CFU counts was lower than indicated by cell integrity results (Table 2). This could indicate that several cells were in a viable but nonculturable condition. Analyzing the mem-

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tation with linoleic and linolenic acids decreased resistance to acid stress, which is similar to the findings of heat-shocked cells. Following 90-min exposure to acid, approximately 80% of the linoleic and linolenic acid-supplemented L. johnsonii NCC 533 cells had a loss of cell integrity, compared to 55% of the oleic acid-supplemented and nonsupplemented L. johnsonii NCC 533 cells. Here, the CFU data showed a similar trend to the flow cytometric data, and there was a linear correlation observed between cell integrity and CFU viability counts (R2 ⫽ 0.95) (data not shown). This correlation would suggest that acid stress directly affects membrane integrity and thus cell viability (Table 3). Analysis of the membrane fatty acid profiles before and after acid shock revealed significant differences for stationaryphase-harvested cells while there was no significant difference in membrane profile for log-phase-harvested cells. Table 4 shows the membrane fatty acid composition before and after acid shock for stationary-phase-harvested L. johnsonii grown

TABLE 3. Percentage of live bacteria measured using CFU counts and flow cytometry after a 90-min exposure to pH 2 Cell survival (%) by growth phase and detection method Supplement

MLM⫹ Oleic acid Linoleic acid Linolenic acid a

FC, flow cytometry.

Log phase

Stationary phase

CFU

0.0 0.0 0.0 0.0

0.3 0.1 0.1 0.1

21.7 31.5 5.5 7.9

42.0 45.9 17.9 21.8

on supplemented medium. During acid shock, the levels of oleic acid increased significantly when medium was supplemented with linoleic or linolenic acid (P ⬍ 0.05). The increase in oleic acid was accompanied by a decrease of the respective supplemented fatty acid, suggesting that the polyunsaturated fatty acid was reduced. A similar saturation was observed from oleic acid to stearic acid in the case where medium was supplemented with oleic acid. However, the conversion to oleic acid did not seem to have a protective effect against acid since L. johnsonii NCC 533 grown in the presence of linoleic or linolenic acid had the lowest viability after acid shock, with only 20% survival upon exposure to pH 2 for 30 min. Storage test. The viability of probiotic bacteria is of paramount importance during the shelf life of the food product since sufficient living cells need to exist at the time of consumption. For this reason, we determined the effect of supplementing the growth medium of L. johnsonii NCC 533 with unsaturated fatty acids on survival during storage in acidified milk over a period of 1 month. The viability of the strain decreased steadily by ⬃1 log per week for the first 3 weeks, while prior supplementation with free fatty acids to the growth medium had no significant effect on the survival (data not shown). These results would suggest that the stress responses occurring during storage in a yoghurt-like product are not correlated to survival mechanisms upon exposure to heat or acid, as described above. Adherence and competitive exclusion. Adherence to gut epithelial cells and competitive exclusion of pathogens are considered important probiotic traits. Therefore, the effects of supplementing the growth medium of L. johnsonii NCC 533 with unsaturated fatty acids on the adherence to HT-29 epi-

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FIG. 5. Cell integrity curve during acid shock for 90 min at pH 2.0 of L. johnsonii grown on MLM⫹ (open blue circles) supplemented with oleic (red squares), linoleic (purple triangle), and linolenic (green star) acids. Cells were harvested in log (a) and stationary (b) phases. Integrity was analyzed using a viability kit in combination with flow cytometry. Values are the average of at least three experiments, and error bars represent ⫾ SD.

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TABLE 4. Percentage of FAMEs before and after acid shock of L. johnsonii stationary-phase cells Percentage of FAMEs in relation to heat shock in:a Fatty acid designation and/or name

SFA/UFA a b c

MLM⫹ and oleic acid

MLM⫹ and linoleic acid

MLM⫹ and linolenic acid

Before

After

Before

After

Before

After

Before

After

2.54 4.91 4.31 0.18 1.97 2.70 4.10 47.07 2.49 0.24 1.16 25.34 0.00 0.49 0.41 2.10 0.00

2.59 6.48 4.44 0.42 1.98 2.77 4.06 47.33 2.46 0.25 1.13 23.66 0.00 0.25 0.26 1.91 0.00

1.39 3.00 2.55 0.58 1.16 1.97 2.37 47.05 1.78 0.00 0.74 35.55 0.00 0.40 0.28 1.18 0.00

1.60 3.53 2.90 0.42 1.32 2.07* 2.40 48.85 1.83 0.00 0.78 32.92 0.00 0.11 0.11 1.15 0.00

1.27 3.14 2.57 0.45 1.24 2.17 2.16 24.18 1.30 0.29 41.87 18.09 0.00 0.13 0.00 1.13 0.00

1.33* 3.34* 2.55 0.00 1.28 2.32 2.24 25.44* 1.37* 0.46 40.5* 17.42 0.00 0.27 0.27 1.20 0.00

1.43 3.48 2.80 0.00 1.13 2.39 2.31 25.70 1.35 0.00 0.92 19.96 36.88 0.00 0.14 1.15 0.37

1.51 3.81 3.96 0.00 2.12 2.43 2.33 26.59* 1.40 0.00 0.91 18.37 34.68* 0.13 0.36 1.09 0.30

0.12

0.14

0.07

0.08

Data are the average of three separate acid shocks. ⴱ, significant difference in FAMEs between before and after acid shock (P ⬍ 0.05). CLA, conjugated linoleic acid. Unknown fatty acid is most likely an isomer of stearidonic acid.

thelial cells and competitive exclusion of Salmonella were investigated. The adherence to HT-29 cells of L. johnsonii NCC 533 grown in MLM⫹ alone was used as a control. Supplementing the growth medium with oleic acid (10 ␮g/ml) showed no significant effect on adherence compared to that of the control, while prior addition of linoleic and linolenic acids (10 ␮g/ ml) reduced adherence significantly by 5-fold compared with that of the unsupplemented control. The adhesion indices of Salmonella UK1-lux and NCC 533 grown in supplemented MLM⫹ are summarized in Table 5. The competitive exclusion properties of L. johnsonii NCC 533 grown in fatty acid-enriched medium were analyzed by adding Salmonella UK1-lux to HT-29 cells, which were preincubated with L. johnsonii NCC 533. HT-29 cells challenged with Salmonella were used as a negative control, and 17.5% and 2.1% of added Salmonella cells were found to adhere and invade, respectively. In contrast, preexposure of the HT-29 cells with L. johnsonii NCC 533, grown in fatty acid-supplemented minimal medium, significantly decreased Salmonella adherence and invasion in all cases (Fig. 6). Preexposure of HT-29 cells with L. johnsonii NCC 533 (grown in MLM⫹) reduced Salmonella adhesion and invasion by 5.3- and 17.5-

TABLE 5. Adhesion index and initial cell numbers of Salmonella UK1-lux and NCC 533 grown in different media Strain (medium type)

UK1-lux NCC 533 NCC 533 NCC 533 NCC 533

(MLM⫹) (oleic acid) (linoleic acid) (linolenic acid)

Bacteria added (log CFU ⫾ SD)

Adhesion index (⫾ SD)

6.3 (0.03) 8.61 (0.01) 8.31 (0.17) 8.42 (0.09) 8.20 (0.10)

15.8 (5.6) 40.34 (21.4) 25.59 (13.9) 8.52 (1.6) 8.46 (1.8)

fold, respectively. Adhesion and invasion were further reduced by 4- and 2.5-fold, respectively, when L. johnsonii NCC 533 was grown in fatty acid-supplemented medium. Interestingly, cells preexposed to L. johnsonii NCC 533 grown in complex MRS

FIG. 6. Salmonella UK1-lux competitive exclusion by NCC 533 grown in MLM⫹ (MLM⫹) and supplemented with oleic (OA), linoleic (LA), and linolenic (LIA) acids. Bars represent the percentage of UK1-lux luminescence that has invaded (black) and adhered (checkered) to HT-29 cells normalized to the negative control (UK1-lux), which has no probiotic added. The last bar (MRS) is used as a reference; here, bacteria were cultured in nutrient-rich MRS medium. Values are the average of at least three experiments, and error bars represent ⫾ SD. *, invasion and adherence were significantly different (P ⬍ 0.05) compared with MLM⫹.

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medium displayed 2-fold increased adherence of Salmonella compared to cells preexposed to L. johnsonii NCC 533 grown in MLM⫹. In all assays (both in MRS medium and MLM⫹), the numbers of CFU of added L. johnsonii were identical. DISCUSSION

in situations where microorganisms were exposed to stress and where the cfa synthase genes were found to be upregulated and cyclopropane fatty acid was incorporated into the membrane. Overexpression of the cfa synthase gene in Clostridium acetobutylicum also increased tolerance to acid and solvent (51). Fatty acid composition and, in particular, the degree of saturation in the bacterial membrane are probably correlated with the fluidity of the membrane, with fluidity increasing with desaturation (2). Membrane fluidity is believed to play a crucial role in stress resistance, and the relationship between fluidity and stress resistance has been used to predict the outcome of cell resistance to stress (15, 24, 39). The membrane composition of yeast during stress has been extensively studied, and it was reported that more unsaturation in the membrane increased the sensitivity to heat (40). In another study, Saccharomyces cerevisiae with increased membrane fluidity also showed decreased heat resistance while ethanol resistance increased (29, 30). It was also reported that when Oenococcus oeni was heat or acid stressed, the membrane fluidity increased in contrast to ethanol stress, which decreased membrane fluidity (44). These reports confirm our results, which showed that L. johnsonii NCC 533 supplemented with unsaturated fatty acids had increased sensitivity to heat and acid stress. We hypothesize that the desaturation acquired from the supplemented medium, in combination with the stress, increased the membrane fluidity to the extent of integrity loss, as can also be derived from the observation that cell death occurred with loss of membrane integrity during acid stress. However, heatshocked bacteria retained cell integrity but were unable to propagate. This would suggest that internal mechanisms, such as enzyme inactivation or denaturation, rendered the cells incapable of cell division while membrane integrity was sustained. Similar results were previously reported when the inactivation of Lactobacillus plantarum during pulsed electric field was investigated (50). In that study, cells were exposed to different heat treatments while the membrane integrity was followed, and no correlation was found between heat inactivation and membrane integrity. Our results also showed that the membrane composition of L. johnsonii NCC 533 did not change following heat shock, and this might be because the duration of the heat shock was too short to record a difference. It has been reported that 20 min was required to observe changes in the membrane composition of Streptococcus mutans in response to environmental changes (14). Following acid stress, however, the concentration of unsaturated fatty acids (oleic, linoleic, and linolenic acids) decreased in favor of stearic or oleic acid, which could be part of a survival mechanism to remove increased proton concentration within the cell during acid stress. This observation was similar to earlier findings, where stearic acid increased significantly in lactobacilli following acid exposure when oleic acid was supplemented in the medium (7). Here, during acid stress, linolenic acid (C18:3) was converted to oleic acid (C18:1) in L. johnsonii NCC 533, suggesting that at pH 2, the proton concentration is so high that the desaturation reaction is shifted completely to oleic acid since the intermediate linoleic acid (C18:2) was undetected. The mechanisms underlying probiotic functionality, such as adhesion and related competitive exclusion of pathogens, are considered important probiotic traits. In this study, we reported

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This study has illustrated that the membrane composition in terms of fatty acid composition and concentration of unsaturated fatty acids can be altered dramatically in L. johnsonii NCC 533 by supplementing the growth medium with free fatty acids. To achieve this, a modified minimal Lactobacillus medium was designed that contained sufficient Tween 80 (0.05%, wt/vol) to support an optimal growth rate. Next, this basal medium was supplemented with oleic, linoleic, or linolenic acid. Little is known about biosynthesis of the cell membrane of L. johnsonii NCC 533 and incorporation of fatty acids. However, a comparative genomics study has shown that it lacks common genes for de novo fatty acid synthesis (4), and as a consequence L. johnsonii NCC 533 has to acquire fatty acids from external/environmental sources. The supplemented fatty acids improved the growth of L. johnsonii NCC 533 compared to growth in MLM⫹ alone, but survival following heat and acid stress decreased with increased unsaturation of the membrane fatty acids. The supplementation of free UFA decreased the SFA/(UFA⫹CFA) ratio significantly compared to the level with MLM⫹ alone. Furthermore, only for NCC 533 grown in pure MLM⫹ did the ratio SFA/(UFA⫹CFA) decrease from log to stationary phase. It could be concluded, since log phase cells are more sensitive to stress than stationary phase cells (10, 27, 42), that reducing the SFA/(UFA⫹CFA) ratio is beneficial for cell survival. However, the UFA-supplemented cells had a lower SFA/(UFA⫹CFA) ratio and were the most sensitive. Here, it is hypothesized that the ratio alone is not sufficient to determine stress sensitivity, and it is important to include the kind of unsaturation. Linoleic and linolenic acids have two and three double bonds, respectively, and thus will cause more sterical hindrance for membrane packing than oleic acid. When external stress is applied, this sterical hindrance could result in membrane integrity loss and cell death. Moreover, in all samples an increase in the concentration of CFA and a decrease of its precursor oleic acid were found in the membranes of stationary versus logarithmically grown cells. Importantly, however, while the conversion of oleic acid to CFA would not change the SFA/(UFA⫹CFA) ratio, the structural difference incurred could have an influence on the fluidity of the membrane. It is evident from the structures of oleic acid and CFA that CFA would provide less stereometric hindrance in the lipid layer than oleic acid. It could be that this mechanism actually decreases the fluidity in L. johnsonii NCC 533 and contributes to the stability of the strain when it is stressed by allowing for closer packing of the membrane fatty acid and thus decreasing the risk of membrane integrity loss. This could also explain why L. johnsonii with the highest CFA concentration showed the highest survival rate following acid shock. Earlier studies reported on the increased cyclopropanation of the membrane of several LAB by the addition of oleic acid to the growth medium (20). Moreover, CFA is believed to play a role in acid and solvent resistance in E. coli and other bacteria (5, 41, 51). This hypothesis could be confirmed

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probiotics and lead to better medium components that will increase the survival of probiotic bacteria during and following stress. Finally, at present, probiotics are added to products based on the concentration of living probiotics, and we expect that the results of our study and work from others (17) will support the discussion to focus research on development of bioassays assessing the probiotic functionality or biomass rather than using only viability assays. ACKNOWLEDGMENTS We thank Nestec for funding of this work and the kind gift of the probiotic strain. J. A. Muller is in receipt of a Teagasc Walsh Fellowship. This work is funded in part by SFI funds and by the Irish Government under National Development Plan 2000-2006. We acknowledge Seamus Aherne for his help with maintaining and running of samples on the gas chromatograph. REFERENCES 1. Alander, M., et al. 1999. Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl. Environ. Microbiol. 65:351–354. 2. Aricha, B., et al. 2004. Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae. J. Bacteriol. 186:4638–4644. 3. Bernet, M. F., D. Brassart, J. R. Neeser, and A. L. Servin. 1994. Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 35:483–489. 4. Boekhorst, J., et al. 2004. The complete genomes of Lactobacillus plantarum and Lactobacillus johnsonii reveal extensive differences in chromosome organization and gene content. Microbiology 150:3601–3611. 5. Chang, Y. Y., and J. E. Cronan, Jr. 1999. Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli. Mol. Microbiol. 33:249–259. 6. Collado, M. C., J. Meriluoto, and S. Salminen. 2007. Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus. Lett. Appl. Microbiol. 45:454–460. 7. Corcoran, B. M., C. Stanton, G. F. Fitzgerald, and R. P. Ross. 2007. Growth of probiotic lactobacilli in the presence of oleic acid enhances subsequent survival in gastric juice. Microbiology 153:291–299. 8. Corcoran, B. M., C. Stanton, G. F. Fitzgerald, and R. P. Ross. 2005. Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl. Environ. Microbiol. 71:3060–3067. 9. Cronan, J. E., Jr. 2002. Phospholipid modifications in bacteria. Curr. Opin. Microbiol. 5:202–205. 10. De Angelis, M., et al. 2004. Heat shock response in Lactobacillus plantarum. Appl. Environ. Microbiol. 70:1336–1346. 11. FAO/WHO. 2001. Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Report from FAO/WHO expert consultation, 1 to 4 October, Cordoba, Argentina. FAO/WHO, Rome, Italy. http://www.who.int/foodsafety/publications/fs_management/en/probiotics .pdf. 12. Finlay, B., and S. Falkow. 1989. Common themes in microbial pathogenicity. Microbiol. Rev. 53:210–230. 13. Fourniat, J., C. Colomban, C. Linxe, and D. Karam. 1992. Heat-killed Lactobacillus acidophilus inhibits adhesion of Escherichia coli B41 to HeLa cells. Ann. Vet. Res. 23:361–370. 14. Fozo, E. M., and R. G. Quivey Jr. 2004. Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments. Appl. Environ. Microbiol. 70:929–936. 15. Giraud, M. N., C. Motta, D. Boucher, and G. Grizard. 2000. Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. Hum. Reprod. 15:2160–2164. 16. Guerzoni, M. E., R. Lanciotti, and P. S. Cocconcelli. 2001. Alteration in cellular fatty acid composition as a response to salt, acid, oxidative and thermal stresses in Lactobacillus helveticus. Microbiology 147:2255–2264. 17. Jankovic, I., W. Sybesma, P. Phothirath, E. Ananta, and A. Mercenier. 2010. Application of probiotics in food products—challenges and new approaches. Curr. Opin. Biotechnol. 21:175–181. 18. Jenkins, J. K., and P. D. Courtney. 2003. Lactobacillus growth and membrane composition in the presence of linoleic or conjugated linoleic acid. Can. J. Microbiol. 49:51–57. 19. Jin, L. Z., Y. W. Ho, N. Abdullah, and S. Jalaludin. 1998. Acid and bile tolerance of Lactobacillus isolated from chicken intestine. Lett. Appl. Microbiol. 27:183–185. 20. Johnsson, T., P. Nikkila, L. Toivonen, H. Rosenqvist, and S. Laakso. 1995.

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that the infection of Salmonella was significantly decreased by preexposing HT-29 cells to fatty acid-modified L. johnsonii NCC 533. Previously, it was reported that Caco2 binding sites were altered by addition of linolenic acid to the growth medium, and the adhesion properties of bacteria were changed (22). This would not, however, explain the results reported here since no additional fatty acids were added to the growth medium of HT-29 cells, and carryover was minimized by washing the bacteria suspension twice with buffer. Other investigators have shown that adhesion was increased for L. casei Shirota when unsaturated fatty acids (5 ␮g/ml) were added to the growth medium while at higher concentrations (10 to 40 ␮g/ ml) adhesion of Lactobacillus GG and L. casei Shirota to Caco2 cells was inhibited (22). These results confirm our findings, i.e., that increased unsaturated fatty acids interfered with the binding of L. johnsonii NCC 533 to HT-29 cells. This could be related to minor changes in the hydrophilic and hydrophobic characteristics of membranes when bacteria are grown in the presence of unsaturated fatty acids, which could change the hydrophobic interactions in microbe-microbe interactions (21). Another aspect of this study was to investigate the effect of fatty acid supplementation on competitive exclusion of S. Typhimurium UK1-lux. L. johnsonii NCC 533 grown in MLM⫹ reduced Salmonella invasion significantly. Surprisingly, even though fewer L. johnsonii NCC 533 bacteria adhered to HT-29 cells when growth medium was supplemented with linoleic and linolenic acids, Salmonella infection was reduced. This may be because the increased unsaturation decreased the hydrophobic interaction between L. johnsonii NCC 533 and HT-29 cells, resulting in a weaker adhesion, and while still blocking adhesion sites on HT-29 cells, the bacteria might have been washed away prior to enumeration. One of the mechanisms proposed for how probiotics prevent pathogens from infecting cells is that they sterically hinder the adherence of pathogens (13), which might explain the increased exclusion properties when L. johnsonii NCC 533 was grown in MLM⫹ compared to growth in nutrient-rich MRS medium. The amount of L. johnsonii NCC 533 added to HT-29 cells was based on CFU counts, and since it was observed that the size of L. johnsonii NCC 533 grown in MLM⫹ increased 10-fold compared to growth in MRS medium, the steric hindrance of L. johnsonii NCC 533 grown in MLM⫹ would be much larger per CFU than that of L. johnsonii NCC 533 grown in MRS medium. Conclusions. The market for probiotic products is still growing in terms of volumes and product varieties while the regulations concerning probiotic health claims continue to become stricter. Since probiotic products are required to contain a certain number of live probiotic bacteria, much research has been devoted to technical properties such as the survivability of strains during shelf life and gastric transit. This study has shown that by supplementing growth medium with unsaturated fatty acids, the responses to heat and acid stress were affected. In addition, the morphology, adherence, and competitive exclusion properties were affected by this supplementation. Based on these results, it can be concluded that medium components can be selected to affect bacterial properties, such as physiology, biomass yield, or stress tolerance. Furthermore, it shows that the membrane composition contributes to stress resistance and competitive exclusion properties. This could be used to further understand the stress response mechanisms of

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