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In vitro evaluation of the fermentation properties and potential probiotic activity of L.plantarum C4 in batch culture systems
Triana Bergillos-Meca, Adele Costabile, Gemma Walton, Miriam Moreno-Montoro,Alfonso Ruiz-Bravo, María Dolores Ruiz-López
PII: S0023-6438(14)00494-0
DOI: 10.1016/j.lwt.2014.08.006
Reference: YFSTL 4090
To appear in: LWT - Food Science and Technology
Received Date: 11 January 2014
Revised Date: 29 July 2014
Accepted Date: 10 August 2014
Please cite this article as: Bergillos-Meca, T., Costabile, A., Walton, G., Moreno-Montoro, M., Ruiz-Bravo, A., Ruiz-López, M.D., In vitro evaluation of the fermentation properties and potential probioticactivity of L. plantarum C4 in batch culture systems, LWT - Food Science and Technology (2014), doi:10.1016/j.lwt.2014.08.006.
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In vitro evaluation of the fermentation properties and potential probiotic activity of 1
L. plantarum C4 in batch culture systems 2
3
Triana Bergillos-Mecaa*, Adele Costabileb, Gemma Waltonb, Miriam Moreno-Montoroa, Alfonso 4
Ruiz-Bravoa, María Dolores Ruiz-Lópeza. 5
6
aDepartamento de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Granada, 7
Campus Cartuja, 18012 Granada, Spain 8
bDepartment of Food and Nutritional Sciences, The University of Reading, RG6 6AP, Reading, 9
UK 10
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∗Corresponding author: Triana Bergillos-Meca 13
Telephone: +34 639217021 14
Email: [email protected] 15
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Abstract 17
Lactobacillus plantarum C4 has been tested in in vitro pH-controlled anaerobic faecal batch 18
cultures as compared to Lactobacillus rhamnosus GG to determine changes caused to the 19
composition of faecal bacteria. Effects upon major groups of the microbiota and levels of short-20
chain fatty acids (SCFA) were assessed over 24 h. Concomitantly, hydrophobic character and 21
ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. Quantitative 22
analysis of bacterial populations revealed that there was a significant increase in 23
Lactobacillus/Enterococcus numbers in vessels with probiotic supplemented with 24
fructooligosaccharides (FOS), compared to the negative control. L. plantarum C4 showed to 25
have more hydrophilic behaviour and fulfilled better adhesive properties, compared to L. 26
rhamnosus GG. Thus, L. plantarum C4 can modulate the intestinal microbiota in vitro, promoting 27
changes in some numerically and metabolically relevant microbial populations and shifts in the 28
production of SCFA. 29
30
Keywords 31
Probiotics; Prebiotics; Batch cultures system; Faecal microbiota 32
33
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1. Introduction 34
35
The human colonic microbiota is a complex ecosystem harbouring a vast range of bacteria and 36
dominated by obligate anaerobes that promote normal intestinal function and offers the host 37
protection against infections (Bäckhed, Ley, Sonnenburg, Peterson, & Gordon, 2005). A 38
disturbance in the composition of this complex population of microorganisms can however 39
predispose towards gastrointestinal disorders and intestinal dysfunction (Turnbaugh et al., 2006; 40
Tilg, 2010; Frank et al., 2011). Probiotics have a number of beneficial health effects in humans 41
and animals, leading to improved gut health and wellbeing (Hickson, 2011; Chian & Pan, 2012; 42
Bergillos-Meca et al., 2013a). The genera Lactobacillus have a long and safe history in the 43
manufacture of dairy products (Vaughan & Mollet, 1999; Masco, Huys, De Brandt, Temmerman, 44
& Swings, 2005). In this context, the putative probiotic strain L. plantarum C4, isolated from a 45
commercial kefir, is being tested. This strain could be of high significance for the dairy industry 46
and for the healthcare, since it fulfils the in vitro criteria for the selection of potentially effective 47
probiotic bacteria, has antimicrobial and immuno-modulating properties (Fuentes et al., 2008; 48
Puertollano et al., 2008). This bacterium is being tested as a possible probiotic to be added in a 49
functional fermented goat’s skimmed milk, as reported by Bergillos-Meca et al. (2013b). 50
Skimmed milk has previously been found to be an appropriate vehicle for the intragastric 51
administration of lactobacilli to mice (Bujalance, Moreno, Jiménez-Valera, & Ruiz-Bravo, 2007). 52
53
Short-chain fatty acids (SCFA), the main products arising from the microbial fermentation of 54
carbohydrates, can provide energy to the colonic epithelium, modulate cholesterol and lipid 55
metabolism, suppress pathogenic intestinal bacteria and modulate the immune system (Salazar 56
et al., 2009). pH-controlled faecal batch cultures allow determination of the fermentability of 57
various substrates in the intestinal lumen, simulating the conditions in the human distal colon. 58
However, marked differences can exist due to probiotic adherence to the epithelial cells and the 59
intestinal barrier, which could influence their interaction with the host and the microorganisms 60
present therein (Ouwehand, Kirjavainen, Gronlund, Isolauri, & Salminen, 1999; Deepika, Green, 61
Frazier, & Charalampopoulos, 2009). This process cannot currently be simulated in a vessel 62
system. In contrast, in vitro assays with the Caco-2 cell line have been used to study the 63
adherence of the bacterial cells to the intestinal monolayer (Deepika et al., 2009; Deepika, 64
Rastall, & Charalampopoulos, 2011; Ren et al., 2012). Furthermore, it has been postulated that 65
several physicochemical properties of probiotic cells, such as hydrophobicity, could be a good 66
indicator to preselect strains with a positive adhesive character. Therefore, these models could 67
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give us a more realistic idea of the complex conditions of the colon, while addressing not only 68
for intestinal lumen but also for epithelial barrier. 69
70
pH-controlled faecal batch cultures were carried out to investigate the influence of L. plantarum 71
C4 on the human colonic microbiota, in comparison with L. rhamnosus GG, a species widely 72
used as adjunct culture in functional dairy products, and known to have good adhesive 73
properties (Jacobsen et al., 1999). We quantified modifications in the levels of selected 74
microbial groups by fluorescent in situ hybridisation (FISH), assessed the effect of the probiotic 75
administration on SCFA production and established possible relationships between metabolic 76
changes and variations in microbial populations. Concomitantly, hydrophobic character and 77
ability of both bacterial cells to adhere in vitro to Caco-2 cells were investigated. 78
Both strains were considered in order to ascertain if L. plantarum C4 and its products could 79
influence the microbiota dynamics to give similar or higher benefits to those observed by L. 80
rhamnosus GG. 81
82
2. Materials and methods 83
2.1. Bacterial strains and culture conditions 84
The strain was identified as L. plantarum C4, an internal nomenclature of the Official 85
Microbiology Collection of the University of Granada (Spain), in previous studies (Bujalance, 86
Jiménez-Valera, Moreno, & Ruiz-Bravo, 2006). Its origin and characterization has been 87
previously described by Bujalance et al. (2007). 88
L. plantarum C4 and L. rhamnosus GG (ATCC 53103) were stored at -70oC in 15% (w/w) 89
glycerol Cryobank cryogenic beads (Prolab Diagnostics, UK). Plates of de Man-Rogosa-Sharpe 90
(MRS) agar (Oxoid Ltd, Basingstoke, Hampshire, UK) were inoculated from stock culture 91
collections and were incubated at 37oC in an anaerobic chamber (10% CO2, 10% H2 and 80% 92
N2, Don Whitley Scientific LTD, Shipley, West Yorkshire, UK). 93
After incubation, Bijou bottles containing 10 mL of MRS broth were then inoculated with one 94
colony from each plate. The cultured broths of probiotics were incubated for 24 h under the 95
same conditions mentioned above. 96
2.2. Physicochemical assays: bacterial adhesion to hydrophobic solvent 97
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The hydrophobic characteristics of the cell surface of both strains were evaluated by the 98
microbial adhesion to hexadecane (MATH) assay, previously described by Deepika et al. 99
(2009). Probiotic strains were cultured in MRS for 24 h at 37oC. The culture broths were 100
centrifuged (5000 g for 10 min, 4oC), and cells washed twice in Phosphate Buffer Saline (PBS) 101
before suspending in 10 mmol L-1 KH2PO4 (Sigma). The pH of the bacterial suspension was 102
adjusted to 3 with 1 mol L-1 HCl to minimise the electrostatic interactions between bacterial cells 103
and hexadecane and the initial absorbance (A0) at 600 nm adjusted to 0.8. The absorbance was 104
measured using a Spectrophotometer BioMate 3 (Thermo Electron Corporation, Madison, WI, 105
USA). Two millilitres of bacterial suspension was then mixed with the same volume of 106
hexadecane (Sigma) in a 10 mL syringe. The mixture was vortexed for 1 min and then left 107
undisturbed for 20 min to allow complete phase separation. After equilibration, the lower 108
aqueous phase was removed carefully, in order not to disturb the interfacial equilibrium, into 109
plastic spectrophotometry cuvettes and absorbance at 600 nm (A1) measured. Strains adhering 110
well (>80%) to the hydrocarbons are considered to be hydrophobic and strains adhering poorly 111
(<40%) are considered to be hydrophilic. The percentage of adhesion (% adhesion) was 112
calculated using the following equation: 113
% Adhesion to hexadecane = (1-A1/A0) x 100 114
This experiment was carried out in triplicate and for each biological replicate, two technical 115
reports were used. 116
2.3. Tissue culture assays 117
2.3.1. Caco-2 cell culture 118
The human adeno-carcinoma Caco-2 ECASS 86010202 cell line was obtained from ECACC 119
(Salisbury, UK). The routine culture was performed according to Deepika et al. (2009) to ensure 120
full differentiation. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-121
Aldrich, Dorset, UK), supplemented with 10% heat inactivated foetal bovine serum (Lonza, 122
Slough, UK), 1% mixture of penicillin-streptomycin solution (Lonza), and 1% non-essential 123
amino acid solution (Lonza), at 37oC, in an atmosphere of 5% CO2 and 95% air. The culture 124
medium was changed every other day. 125
2.3.2. Probiotic adhesion to Caco-2 cells 126
Twenty-one day old, fully differentiated cells, cultured in 12-well tissue culture plates (Corning, 127
Kennebunk, ME, USA), were used for the cell adhesion experiments (Deepika et al., 2009). One 128
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day before adhesion assays, the spent medium was replaced with DMEM supplemented with 129
non-essential amino acids and foetal bovine serum, without antibiotics. On the day of the 130
experiment, the monolayer was washed twice with Dulbecco’s Phosphate Buffered Saline 131
(DPBS) (pH 7.2, without Ca and Mg; Sigma-Aldrich, Dorset, UK), in order to remove all traces of 132
the medium. The cells, at around 4 x 105 cells mL-1, were counted using a Nikon microscope 133
(Kingston Upon Thames, UK). L. plantarum C4 and L. rhamnosus GG were cultured in MRS 134
broth for 24 h at 37oC under anaerobic conditions. Cultured broths were centrifuged (5000 g for 135
10 min, 4oC), and cells washed and re-suspended in DPBS (108 CFU mL-1). One millilitre of 136
bacterial suspension was added to each well and the plates were incubated for 60 min at 37oC 137
in 5% CO2 and 95% air. After incubation, the DPBS containing unbound bacteria was removed 138
from the wells; the wells were further washed with 1 mL of DPBS. These two fractions were 139
pooled together. Consequently, the bacteria attached to the Caco-2 cells were detached by 140
trypsinisation. One millilitre of 0.25% trypsin-(ethylenediaminetetraacetic acid) EDTA solution 141
(Sigma, USA) was added to each well and the plates incubated for 15 min at room temperature. 142
The cells were detached by mechanical stirring and repeatedly, but gently, aspirated to make a 143
homogeneous suspension. Numbers of probiotic cells (bound and unbound) were determined 144
by plating serial dilutions on MRS agar. Bacterial cells added to each well of the plates were 145
also quantified. To check the consistency of the results, bound bacteria numbers were also 146
determined by subtracting the unbound bacteria from the total number of bacteria added to 147
wells. This experiment was carried out in triplicate and for each biological replicate, two 148
technical reports were used. 149
2.4. Batch cultures 150
2.4.1. Probiotics culture preparation 151
L. plantarum C4 and L. rhamnosus GG were cultured in MRS broth for 24 h at 37oC under 152
anaerobic conditions. Cells were prepared for addition to the fermenter vessels by centrifuging 153
at 5000 g for 10 min. The supernatant was removed and cells washed and re-suspended in 154
PBS (Oxoid Ltd, Basingstoke, Hampshire, UK) and adjusted to an optical density (OD600) 155
corresponding to 108 CFU mL-1. 156
157
2.4.2. Faecal sample preparation 158
The faecal samples were obtained fresh at the premises of the department from three healthy 159
human donors (one man, two women; average 27 ± 3.3 years of age, omnivores) who were free 160
from known metabolic and gastrointestinal disorders. None of the volunteers had taken 161
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antibiotics during the 6 months leading to the study. Samples were collected, kept in anaerobic 162
cabinet and used within 15 min of collection. A 1/10 w/w dilution in PBS was prepared and 163
homogenised using a stomacher (Seward, Worthing, UK) for 2 min at 460 paddle-beats per min. 164
165
2.4.3. In vitro batch cultures studies 166
Sterile stirred batch culture fermentation vessels (100 mL working volume) were prepared and 167
aseptically filled with 45 mL of sterile basal colonic growth medium, prepared as reported by 168
Martín-Peláez et al. (2008). All media and chemicals were purchased from Oxoid and Sigma. 169
Once in the fermentation vessels, sterile medium was maintained under anaerobic conditions by 170
sparging the vessels with O2-free N2 (15 mL min-1) overnight. Temperature was held at 37oC 171
using a circulating water bath and pH values controlled between 6.7 and 6.9 using an 172
automated pH controller (Fermac 260; Electrolab, Tewkesbury, UK) which added acid or alkali 173
as required (0.25 M HCl and 0.25 M NaOH). 174
175
Seven gently stirred pH-controlled batch fermenters were run in parallel. Two vessels were 176
inoculated with 1 mL of a suspension of L. plantarum C4 (108 CFU mL-1), in which 0.5 g of the 177
following carbohydrates was added: FOS (95% oligosaccharide, β(2-1)-fructan; of which 60% 178
w/w glucose-fructose, 40% fructose w/w, degree of polymerization, 3-10) (BENEO GmbH, 179
Germany) or α-cellulose (Sigma Aldrich, UK) (1% w/v). Another set of two vessels were 180
inoculated with 1 mL of L. rhamnosus GG (108 CFU mL-1), and FOS or cellulose (1% w/v) was 181
added to each one. Two extra vessels were also included as positive and negative controls, one 182
of them containing only FOS and the other one containing only cellulose, respectively. A control 183
with neither probiotics nor carbohydrates added (control) was also included. The experiment 184
was performed in triplicate, using one faecal sample given by a different donor for each run of 185
seven batch fermenters. The probiotics and carbohydrates were added to each vessel just 186
before the addition of 5 mL (10% w/w) of fresh faecal slurry prepared as described above, 187
whose average concentration was 4.07 x 1010 cells mL-1. Batch cultures were conducted for 24 188
h, and 4 mL samples obtained from each vessel at 0, 5, 10 and 24 h for analysis of bacterial 189
populations by FISH and for SCFA analyses using gas chromatography (GC). 190
191
2.4.4. SCFA analysis 192
Samples were taken from the batch culture vessels at each time point and cell-free culture 193
supernatants obtained by centrifugation of 1 mL at 13,000 g for 10 min followed by filter 194
sterilisation (0.22 µm; Millipore) to remove all particulate matter. 195
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196
SCFA was measured by GC (Fernandes, Vogt, & Wolever, 2011; Vogt, Pencharz, & Wolever, 197
2004). 1µL of each sample was injected into a 5890 Series II GC system (HP, Crawley, West 198
Sussex, UK) fitted with a NukolTM Capilllary Column (30 m × 0.53 mm × 1.0 µm, SUPELCOTM 199
Analytical, UK) and flame ionisation detector. The carrier gas, Helium, was delivered at a flow 200
rate of 14 mL min-1. The head pressure was set at 10 psi with split injection. Run conditions 201
were: initial temperature 60°C, 1 min; + 20°C/min t o 145°C; + 4°C/min to 200°C, hold 25 min. 202
Peaks were integrated using Agilent ChemStation software (Agilent Technologies, Oxford, UK) 203
and SCFA content quantified by single point internal standard method. Peak identity and 204
internal response factors were determined using a range from 0.32 to 50 mM calibration cocktail 205
including acetic, propionic, iso-butyric, butyric, iso-valeric, valeric and caproic acids. 206
207
2.4.5. Enumeration of bacterial populations by FISH analysis 208
FISH analysis was performed as described by Martín-Peláez et al. (2008). Briefly, aliquots (375 209
µL) of batch culture samples were fixed in three volumes of ice-cold 4% (w/v) paraformaldehyde 210
for 4 h at 4oC. They were then centrifuged at 13,000 g for 5 min and washed twice in 1 mL of 211
sterile PBS. The cells were again pelleted by centrifugation and re-suspended in 150 µL of 212
sterile PBS, to which 150 µL of ethanol was added. Samples were then vortexed and stored at -213
20oC until used in hybridisations. 214
215
For the hybridisations, all probes were commercially synthesised and 5’-labelled with the 216
fluorescent dye (Sigma-Aldrich, St Louis, MO, USA). The probes used in this study were: Bif164 217
(Langendijk et al., 1995), Erec482 (Franks et al., 1998), Lab158 (Harmsen et al., 1999), 218
Chis150 (Franks et al., 1998), Bac303 (Manz, Amann, Ludwig, Vancanneyt, & Schleifer, 1996), 219
Eub338 I-II-III (Daims, Brüls, Amann, Schleifer, & Wagner, 1999). Eub338 I-II-III probes were 220
used in equimolar concentrations (50 ng mL-1). Formamide (35%) was included in the 221
hybridisation buffer. 222
223
2.5. Statistical analysis 224
Bacterial counts and SCFA were analysed using 2-way ANOVA with Bonferroni post-tests plus 225
least significant difference (P<0.05). 226
Adhesion counts and hydrophobicity data were analysed by ANOVA with two-tailed distribution. 227
Paired t-tests were applied to assess the same treatment at different time points and non-paired 228
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to compare different vessels at the same time points. Significant differences were defined at 229
P<0.05. 230
All analyses were performed using GraphPad Prism 5.0 (GraphPad Software, LaJolla, CA, 231
USA). 232
233
3. Results 234
3.1. Bacterial adhesion to hydrophobic solvent 235
Figure 1 shows the results from the MATH assay for L. plantarum C4 and L. rhamnosus GG 236
cells. Adhesion to hexadecane was low for L. plantarum C4 (16.08 ± 6.73%), whereas the 237
values for L. rhamnosus GG were significantly higher (P<0.001), reaching a mean value of 238
74.29 ± 13.59%. These results showed a hydrophilic and hydrophobic character for L. 239
plantarum C4 and L. rhamnosus GG, respectively. 240
241
3.2. Probiotic adhesion to Caco-2 cells 242
Figure 2 presents the results from the adhesion abilities of L. plantarum C4 and L. rhamnosus 243
GG cells to a Caco-2 intestinal epithelial cell line model. For L. plantarum C4 the adhesion 244
reached a mean value of 266.67 ± 85.50 bacterial cells per Caco-2 cell. This microorganism 245
was seen to be significantly more adhesive than L. rhamnosus GG (151.25 ± 20.50 cells/Caco-246
2) (P<0.05). 247
248
3.3. Modulation of bacterial populations by FISH analysis 249
Figure 3 shows bacterial counts in control and probiotic-supplemented cultures. Trends to 250
increases in all incubations were observed for bifidobacteria, but no significant changes were 251
found, the highest number was found in the fermentations with L. plantarum C4 + FOS at 24 h 252
(Log10 8.96 ± 0.36 cells mL-1). Regarding Lactobacillus-Enterococcus group, a decrease in 253
fermentations with FOS was found at 24 h (P<0.05), compared to 0 h. Comparing all treatments 254
to the negative control, it was observed that Lab158 counts were significantly higher at time 5 h 255
and 10 h (P<0.05) in presence of L. plantarum C4 + FOS and at 5 h, 10 h and 24 h (P<0.01) in 256
the case of L. rhamnosus GG + FOS. Bacteroides-Prevotella group showed an increase in the 257
control at 24 h (P<0.05). Clostridium histolyticum group increased significantly in vessels with L. 258
plantarum C4 + FOS and L. rhamnosus GG + cellulose (P<0.05). This group displayed a similar 259
behaviour in fermentations with probiotic + FOS: higher counts were found in both of them at 10 260
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h (P<0.01) when compared to the positive control, and at 10 h and 24 h comparing to the 261
negative control (P<0.05, P<0.001). Besides, significantly higher values were observed in 262
vessels containing L. plantarum C4 + FOS (10 h) and L. rhamnosus GG + FOS (10 h and 24 h), 263
with respect to L. plantarum C4 + cellulose (P<0.01). Relating the Clostridium cluster XIVa+b 264
group, no significant differences were observed. Total bacteria showed a similar behaviour with 265
all probiotics and carbohydrates tested, they increased during batch culture fermentations. 266
However, a decrease for the control at 24 h (P<0.05) was the only significant difference 267
detected. 268
269
3.4. SCFA production in batch cultures 270
Table 1 illustrates the SCFA concentrations in presence of L. plantarum C4 and L. rhamnosus 271
GG with FOS or cellulose. Marked differences were found between donors with respect to levels 272
of SCFA attained in faecal cultures. Because of this, no significant differences were found 273
between time points and only few differences were observed when comparing treatments. A 274
significantly higher value of iso-butyric acid was found in vessels containing L. plantarum C4 + 275
FOS at 10 h, compared to fermentations with L. rhamnosus GG + cellulose (P<0.05). 276
277
4. Discussion 278
The capacity of lactobacilli to adhere in vitro to epithelial cells is considered one of the main 279
criteria in the selection of new probiotic microorganisms (Collado, Surono, Meriluoto, & 280
Salminen, 2007). Although it is controversially discussed in the scientific community the use of 281
cancers cells for investigation of probiotics, the adhesion of probiotic bacteria to the 282
gastrointestinal tract is commonly tested using Caco-2 cells, as they have morphological and 283
functional properties similar to mature enterocyte (Deepika, Karunakaran, Hurley, Biggs, & 284
Charalampopoulos, 2012). In the present study, the adhesion level of L. plantarum C4 was 285
significantly higher compared to L. rhamnosus GG (P<0.05), with L. plantarum C4 adhering to 286
nearly 270 bacteria per Caco-2 cell, whereas L. rhamnosus GG adhered to around 150 287
cells/Caco-2 (Fig. 2). Many different values for lactobacilli adhesion have been reported, ranging 288
between 10 and 160 bacterial cells per Caco-2 cell (Lee et al., 2000; Gopal, Prasad, Smart, & 289
Gill, 2001; Delgado, O’Sullivan, Fitzgerald, & Mayo, 2007). The differences may reflect bacterial 290
cells with different physiological states, such as cells grown in different growth media and 291
conditions, or taken at different time points. 292
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A correlation between hydrophobicity and adhesion to intestinal cells has been previously 294
suggested (Del Re, Sgorbati, Miglioli, & Palenzona, 2000; Ehrmann, Kurzak, Bauer, & Vogel, 295
2002; Kos et al., 2003). However, some controversy exists in this field and they cannot be 296
always correlated (Zárate, De Ambrosini, Chaia, & González, 2002; Vinderola, Medici, & 297
Perdigón, 2004). We did not find a positive correlation between hydrophobicity and bacterial 298
adhesion, as the most hydrophilic probiotic (L. plantarum C4) adhered to Caco-2 significantly 299
better than the most hydrophobic one (L. rhamnosus GG) (Fig. 1 and 2). The lack of correlation 300
between the capacity for adhesion and hydrophobicity has already been observed, indicating 301
that this property does not play an important role in the mechanism of immunostimulation 302
(Vinderola et al., 2004; Deepika et al., 2009). 303
304
To date, not many studies have investigated the effects of L. plantarum using in vitro batch 305
culture studies. These models have been largely used to study the prebiotic effects of different 306
substrates and also potential synbiotic combinations (Martín-Peláez et al., 2008; Saulnier, 307
Gibson, & Kolida, 2008; Rammani et al., 2012). 308
Although higher values of C. histolyticum were found in vessels with probiotic + FOS, an 309
increase in this group occurred in all the fermentations. It has been reported that this fact could 310
be attributed to factors such as culture conditions rather than to a specific effect mediated by the 311
tested probiotics (Salazar et al., 2009). 312
Amongst the other groups analysed by FISH, our results showed that L. plantarum C4 + FOS 313
and L. rhamnosus GG + FOS clearly stimulated the growth of Lactobacillus/Enterococcus, 314
which was not seen with FOS alone. This fact might have been expected, as it had been 315
previously observed higher levels of beneficial members of the microbiota due to the effect of 316
synbiotics, in comparison with prebiotics alone, although it should be taken into account that 317
enhancement of probiotic growth by the prebiotic in mixed culture has been reported to be strain 318
specific (Saulnier et al., 2008; Ramette, 2007). From these results, it appears that fermentation 319
of the synbiotics could be selective and affect Lactobacillus/Prevotella, one of the major 320
members of the microbiota considered as beneficial. 321
Some probiotics do not increase in mixed culture studies possibly because they do not compete 322
well with the rest of the gut microbiota or the numbers added are small to be detected by FISH, 323
whereas with the prebiotic further enhancement was enabled. That could be why an increase in 324
Lactobacillus/Enterococcus was not found in vessels with L. plantarum C4 + cellulose. 325
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Bacteroides are among the predominant genera in the gut of mammals and produce variable 327
amounts of propionate (Macfarlane et al., 1997; Hooper, Midtvedt, & Gordon, 2002). The 328
significant increase in the control could provide a probable rationale for the trend to increase of 329
propionic acid in these fermentations. 330
5. Conclusions 331
Higher levels of lactobacilli, health-promoting bacteria, can result from the presence of L. 332
plantarum C4 + FOS. This synbiotic may have superior effects compared to FOS alone to 333
modulate the faecal microbiota. L. plantarum C4 showed hydrophilic character and fulfilled 334
desirable adhesive properties. Supplementation of fermented goat’s milk with this strain seems 335
to be a good approach for the regulation of the indigenous microbiota and could have a 336
beneficial effect on the health of the consumer host. 337
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Figure 1. Hydrophobicity of L. plantarum C4 and L. rhamnosus GG cells expressed as 521
percentage of bacteria adsorbed by hexadecane as measured by the MATH assay. Error bars 522
represent SD (n=3). 523
524
Figure 2. Adhesion of L. plantarum C4 and L. rhamnosus GG cells to Caco-2 cells, expressed 525
as the number of adhered bacterial cells per Caco-2 cell. Error bars represent SD (n=3). 526
527
Figure 3. Bacterial populations analysed by fluorescence in situ hybridisation in batch cultures 528
containing different probiotics. Error bars indicate SD (n=3). 529
530
Table 1. SCFA concentrations (mM) in pH-controlled batch cultures at 0, 5, 10 and 24 h (n=3). 531
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Mean SCFA concentration (mM) in treatment (±SD):
Treatment Time point Lb C4 + FOS Lb C4 + cellulose Lb GG + FOS Lb GG +
cellulose Control FOS cellulose
Acetic
0h 5h
10h 24h
7.05 ± 9.21 12.67±11.61 3.26 ± 4.53 0.32 ± 0.07
1.99 ±3.11 5.42 ± 5.40 2.04 ± 3.08 0.40 ± 0.08
6.51 ± 8.54 3.62 ± 5.08 2.44 ± 3.58 0.36 ± 0.18
3.40 ± 2.92 1.72 ± 2.58 1.51 ± 1.88 0.60 ± 0.63
3.21 ± 4.42 8.42 ± 6.71 3.19 ±4.39 0.35 ± 0.08
2.10 ± 3.20 6.85 ± 6.43 6.51 ± 5.92 4.29 ± 6.52
3.70 ± 3.14 3.87 ± 4.05 1.57 ± 2.00 0.35 ± 0.23
Propionic
0h 5h
10h 24h
4.68 ± 4.23 2.20 ± 1.87 1.31 ± 0.69 1.08 ± 0.42
1.45 ± 1.61 1.71 ± 1.73 1.62 ± 1.29 2.15 ± 1.20
2.98 ± 3.07 2.21 ± 0.39 1.51 ± 0.61 1.06 ± 0.81
0.98 ± 0.94 0.94 ± 0.54 1.33 ± 0.80 1.15 ± 0.94
4.44 ± 3.62 1.86 ± 0.94 2.91 ± 3.50 1.12 ± 0.83
1.59 ± 1.68 1.84 ± 1.73 1.44 ± 0.90 1.98 ± 0.47
1.39 ± 1.59 1.27 ± 1.49 0.96 ± 0.62 0.83 ± 0.49
Butyric
0h 5h
10h 24h
3.47 ± 3.83 1.26 ± 1.41 0.27 ± 0.21 0.22 ± 0.30
1.15 ± 1.43 0.86 ± 0.96 0.92 ± 1.01 1.03 ± 1.37
2.04 ± 2.56 0.75 ± 0.55 0.30 ± 0.22 0.31 ± 0.39
0.72 ± 0.78 0.43 ± 0.32 0.34 ± 0.31 0.41 ± 0.47
3.38 ± 3.41 0.86 ± 0.67 0.24 ± 0.17 0.07 ± 0.03
1.24 ± 1.51 0.94 ± 0.88 0.79 ± 0.62 1.13 ± 0.96
1.11 ± 1.40 0.73 ± 0.85 0.51 ± 0.43 0.29 ± 0.36
Valeric
0h 5h
10h 24h
2.70 ± 3.94 0.81 ± 1.09 0.11 ± 0.10 0.07 ± 0.08
1.01 ± 1.58 0.46 ± 0.69 0.29 ± 0.35 0.50 ± 0.79
1.74 ± 2.63 0.41 ± 0.46 0.11 ± 0.10 0.09 ± 0.14
0.61 ± 0.94 0.18 ± 0.24 0.10 ± 0.11 0.11 ± 0.15
2.39 ± 3.33 0.57 ± 0.64 0.12 ± 0.14 0.02 ± 0.02
1.06 ± 1.65 0.52 ± 0.75 0.29 ± 0.27 0.56 ± 0.59
0.97 ± 1.53 0.42 ± 0.64 0.16 ± 0.17 0.13 ± 0.20
Caproic
0h 5h
10h 24h
2.59 ± 3.85 0.88 ± 1.20 0.20 ± 0.12 0.08 ± 0.08
1.03 ± 1.63 0.46 ± 0.70 0.23 ± 0.32 0.23 ± 0.36
1.83 ± 2.78 0.50 ± 0.59 0.14 ± 0.11 0.04 ± 0.03
0.67 ± 1.03 0.23 ± 0.32 0.04 ± 0.02 0.09 ± 0.10
2.21 ± 3.21 0.67 ± 0.72 0.18 ± 0.17 0.06 ± 0.05
1.09 ± 1.71 0.50 ± 0.75 0.22 ± 0.29 0.19 ± 0.19
1.01 ± 1.62 0.47 ± 0.73 0.15 ± 0.20 0.03 ± 0.02
Iso-Butyric
0h 5h
10h 24h
1.72 ± 2.56 0.37 ± 0.37 0.09 ± 0.02a 0.07 ± 0.04
0.48 ± 0.78 0.14 ± 0.19 0.08 ± 0.07 0.13 ± 0.15
0.75 ± 1.09 0.21 ± 0.15 0.08 ± 0.03 0.07 ± 0.06
0.23 ± 0.35 0.06 ± 0.06 0.04 ± 0.01 0.07 ± 0.05
0.26 ± 0.32 0.29 ± 0.22 0.11 ± 0.05 0.06 ± 0.04
0.57 ± 0.94 0.15 ± 0.21 0.07 ± 0.06 0.09 ± 0.04
0.42 ± 0.69 0.12 ± 0.17 0.04 ± 0.04 0.04 ± 0.03
Iso-Valeric
0h 5h
10h 24h
2.06 ± 3.07 0.44 ± 0.54 0.09 ± 0.03 0.12 ± 0.12
0.61 ± 0.95 0.21 ± 0.25 0.15 ± 0.14 0.33 ± 0.41
1.02 ± 1.46 0.23 ± 0.19 0.08 ± 0.04 0.11 ± 0.14
0.31 ± 0.43 0.10 ± 0.09 0.07 ± 0.04 0.10 ± 0.10
2.34 ± 3.41 0.37 ± 0.27 0.09 ± 0.03 0.05 ± 0.01
0.71 ± 1.10 0.25 ± 0.30 0.12 ± 0.09 0.17 ± 0.11
0.54 ± 0.83 0.19 ± 0.24 0.09 ± 0.06 0.10 ± 0.09
aSignificantly different from Lb GG + cellulose. Statistical significance was taken as P<0.05 for the same time point (using 2-way ANOVA with
Bonferroni post-tests plus least significant difference) (n=3).
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*Significantly different compared to Lactobacillus rhamnosus GG (t-test, P<0.001).
0
10
20
30
40
50
60
70
80
90
100A
dh
esio
n t
o h
exad
ecan
e(%
)
C4
GG
*
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*Significantly different compared to Lactobacillus rhamnosus GG (t-test, P<0.05).
0
50
100
150
200
250
300
350
400B
acte
rial
cel
ls p
er C
aco
-2 c
ell
C4
GG
*
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7
7.5
8
8.5
9
9.5
10
10.5
11
Lb C4 +FOS
Lb C4 +cellulose
Lb GG +FOS
Lb GG +cellulose
Control FOS cellulose
Lo
g c
ells
mL
-1Bif164
0 h
5 h
10 h
24 h
7
7.5
8
8.5
9
9.5
10
10.5
11
Lb C4 +FOS
Lb C4 +cellulose
Lb GG +FOS
Lb GG +cellulose
Control FOS cellulose
Lo
g c
ells
mL
-1
Erec482
0 h
5 h
10 h
24 h
7
7.5
8
8.5
9
9.5
10
10.5
11
Lb C4 +FOS
Lb C4 +cellulose
Lb GG +FOS
Lb GG +cellulose
Control FOS cellulose
Lo
g c
ells
mL
-1
Lab158
0 h
5 h
10 h
24 h
a† *
‡ † ‡
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7
7.5
8
8.5
9
9.5
10
10.5
11
Lb C4 +FOS
Lb C4 +cellulose
Lb GG +FOS
Lb GG +cellulose
Control FOS cellulose
Lo
g c
ells
mL
-1
Chis150
0 h
5 h
10 h
24 h
¥
§ a
† ‡
a
¥ ¥
§
* ‡
7
7.5
8
8.5
9
9.5
10
10.5
11
Lb C4 +FOS
Lb C4 +cellulose
Lb GG +FOS
Lb GG +cellulose
Control FOS cellulose
Lo
g c
ells
mL
-1
Bac303
0 h
5 h
10 h
24 h
a
7
7.5
8
8.5
9
9.5
10
10.5
11
Lb C4 +FOS
Lb C4 +cellulose
Lb GG +FOS
Lb GG +cellulose
Control FOS cellulose
Lo
g c
ells
mL
-1
Eub338 I-II-III
0 h
5 h
10 h
24 h
a
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compare replicate means by row).
Significant differences for the same vessels between time points are indicated with letters, aSignificantly different compared to 0 h within the same substrate (using t-test, P<0.05) (n=3).
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In vitro effect of a novel probiotic on the human microbiota composition was studied
Higher levels of lactobacilli can result from the presence of L. plantarum C4 + FOS
Synbiotic may have superior effects than FOS alone to modulate the faecal microbiota
L. plantarum C4 showed hydrophilic character and desirable adhesive properties
