FEMS Microbiology Letters, 363, 2016, fnv234
doi: 10.1093/femsle/fnv234
Advance Access Publication Date: 10 December 2015
Research Letter
R E S E A R C H L E T T E R – Food Microbiology
In vitro screening of mare’s milk antimicrobial effect
and antiproliverative activity
Anilda Guri1,2 , Michele Paligot3 , Sebastien Crèvecoeur3 , Benoit Piedboeuf3 ,
Jonathan Claes3 , Georges Daube3 , Milena Corredig1 , M. W. Griffiths1,2
and Veronique Delcenserie3,∗
1
Department of Food Science, University of Guelph, Guelph, ON N1G2W1, Canada, 2 Canadian Research
Institute for Food Safety, University of Guelph, Guelph, ON N1G 2W1, Canada and 3 Fundamental and Applied
Research for Animal and Health (FARAH), Food Sciences Department, Faculty of Veterinary Medicine,
University of Liège, Quartier Vallée 2, Avenue de Cureghem 10, B-4000 Liege, Belgium
∗
Corresponding author: Food Sciences Department, Faculty of Veterinary Medicine, University of Liège, Sart-Tilman B43b, B-4000 Liege, Belgium.
Tel: +32-4-366-51-24; Fax: +32-4-366-40-44; E-mail: [email protected]
One sentence summary: This study reports that mare’s milk was able to modulate virulence gene expression of Salmonella Typhimurium and exerts
antiproliferative effects on Caco-2 cells. These results may offer new approaches for promoting gastrointestinal health.
Editor: Abelardo Margolles
ABSTRACT
The aims of this study were to examine the effect of mare’s milk on virulence gene expression of Salmonella Typhimurium
and observe its potential activity on proliferation of adenocarcinoma Caco-2 cells. Different supernatants of mare’s milk,
raw or heat-treated at 65◦ C for 15 s or 30 min, were studied. The changes in hilA gene expression of Salmonella Typhimurium
in presence of mare’s milk supernatants were assessed using a reporter luminescent strain. A significant decrease in hilA
gene expression was observed with all tested supernatants. Virulence gene expression was then assessed using qPCR on a
wild-type strain of Salmonella Typhimurium. A significant decrease of hilA and ssrB2 gene expression was observed with raw
milk supernatants but not with heat-treated supernatants. The same supernatants were administered to Caco-2 cells to
measure their proliferation rate. A significant reduction of proliferative effect was observed only with raw milk
supernatants. This study reports that raw mare’s milk was able to modulate virulence gene expression of Salmonella
Typhimurium and exerts antiproliferative effects on Caco-2 cells. These results may offer new approaches for promoting
gastrointestinal health.
Keywords: mare’s milk; antimicrobial and antiproliferative effects; Salmonella Typhimurium; virulence expression
INTRODUCTION
Mare’s milk has been gaining attention as an alternative to cow’s
milk for its nutritional values and also for its unique properties
as a formula for children allergic to cow’s milk (Businco et al.
2000). For that reason its consumption has increased beyond its
traditional market, mostly Asian countries, into the European
market as well. The chemical profile of mare’s milk has been well
studied and remarkable quantitative differences occur between
mare’s milk and bovine or human milk (Malacarne et al. 2002).
These differences include a higher fat and lactose content, but
less protein and mineral salt in mare’s milk when compared to
that from cows (Csapó-Kiss et al. 1995; Marconi and Panfili 1998;
Received: 4 August 2015; Accepted: 3 December 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2016, Vol. 363, No. 2
Malacarne et al. 2002). However, its protein, sugar and salt mineral content seems to be similar to human milk (Malacarne et al.
2002), which makes its application as an infant formula more appealing than cow’s milk (Marconi and Panfili 1998). Mare’s milk
has a lower casein concentration than bovine milk but it contains twice as much whey. Although information is available on
the chemical composition of mare’s milk, little is known regarding the bioactivity of compounds contained in this milk.
The major antimicrobial activity is associated with the whey
fraction of mare’s milk (Hancock et al. 2002; Palmer 2011). Indeed, some components such as lysozyme (Wulijideligen et al.
2012), immunoglobulins, lactoperoxidase and lactoferrine are
well known for their strong antimicrobial effects. Other components such as complex oligosaccharides are able to prevent adhesion of pathogens to intestinal mucosa (Uniacke-Lowe 2011)
and are digested only by specific bacteria such as lactobacilli
or bifidobacteria, giving them prebiotic properties (Zivkovic and
Barile 2011).
Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a major foodborne pathogen and is the most
commonly isolated serovar of Salmonella in Europe along with
serovar Enteritidis. The pathogenesis of this bacterium requires
the invasion of intestinal epithelial cells and its multiplication
inside phagocytic leukocytes. Those steps are controlled by type
III secretion systems (TTSS) 1 and 2 (Altier et al. 2000; Zhou and
Galan 2001). The components involved in TTSS 1 or 2 are encoded by Salmonella pathogenicity island (SPI) 1 and 2, respectively (Bayoumi and Griffiths 2010). All the genes of SPI1 are
directly controlled and activated by the hilA gene, which is described as the general transcriptional regulator of SPI1 (Bajaj,
Hwang and Lee 1995; Lostroh, Bajaj and Lee 2000). SPI2 encodes
the TTSS2 secretion system responsible for the internalization
of effector proteins inside the macrophages, allowing the survival and the replication of Salmonella Typhimurium inside the
macrophage and then the spread of bacterial cells to different
susceptible organs. The SPI2 central response regulator is the
gene ssrB (Shea et al. 1996; Worley, Ching and Heffron 2000; Feng
et al. 2004; Coombes et al. 2007).
LeBlanc (2003) has shown that hydrolysates of bovine milk fat
globule membrane (MFGM) exhibit antibacterial activity against
Escherichia coli O157:H7 and to a lower extent the antimicrobial
activity against Listeria monocytogenes, Salmonella Typhimurium
and Pseudomonas fluorescens.
A previous study showed that bovine MFGM was able to induce a decrease in virulence gene expression of E. coli O157:H7
using quantitative real-time PCR (qPCR) (Tellez et al. 2012). To
date, no study has examined the antimicrobial effect of mare’s
milk on virulence gene expression of foodborne pathogens using
qPCR.
Several researchers have studied the anti-cancer activities
exerted by molecules present in human or in bovine milk or fermented milk (Xu, Lee and Ahn 2010; Cousin et al. 2012; Zanabria
et al. 2013; Jiang, Du and Lonnerdal 2014). However, there is scant
information on the antiproliferative activity of mare’s milk.
Thus, the objectives of this study were to analyze the potential antimicrobial effect of mare’s milk supernatants on virulence gene expression of Salmonella Typhimurium and to investigate whether raw or heat-treated mare’s milk supernatants
exhibiting antimicrobial effects would have an effect on colon
cancer cell proliferation.
MATERIALS AND METHODS
Mare’s milk supernatants preparation
Three different mare’s raw milk samples (R1, R2 and R3) were
bought frozen at a farm in Wallonia, Belgium. After defrosting
at 4◦ C, 40 ml samples of milk were heated (65◦ C for 15 s in heart
and 65◦ C for 30 min) in sterile containers in a thermostatically
controlled water bath, and immediately cooled to about 20◦ C in
an ice bath. Raw or heat-treated milk was then centrifuged at
10 000 g for 30 min at 4◦ C (Eppendorf 5810; Rotselaar, Belgium)
to obtain the fat and cell-free fraction as described previously
(Nissen et al. 2012; Tellez et al. 2012) but with some modifications. Following centrifugation the cream was removed and the
liquid serum was centrifuged again at the same speed for 30 min
to remove residual cream. Next, the serum fraction was filtersterilized using a 0.22 μm syringe filter (Millipore). This fraction
was used for the next part of the experiments. The total protein concentration assessed using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA) was estimated
to be between 10 and 30 mg mL−1 .
Cell culture
Caco-2 cells obtained from the CRIFS Culture Collection (Food
Science, University of Guelph, ON, Canada) were regularly
maintained in Dulbecco’s Modified Eagle Medium in a humidified 5% CO2 atmosphere, at 37◦ C (Forma Series II Waterjacketed CO2 Incubator, Model No: 3110, Forma Scientific,
California, USA). Medium was supplemented with 25 mM glucose (Sigma-Aldrich, Oakville, ON, Canada), 10% fetal bovine
serum heat inactivated, 2 mM L-glutamine, 1% NEAA and
1% antibiotic solution of penicillin-streptomycin (Invitrogen
Canada Inc., Burlington, ON, Canada) and 25 mM HEPES buffer
(Sigma-Aldrich, Oakville, ON, Canada). Cells were cultured
in T-75 cm2 tissue culture flasks (Fisher Scientific, Mississauga, ON, Canada) and harvested with 0.25% trypsin-1 mM
EDTA (1×), prior to seeding. Cells between passages 20–28
were used in order to maintain relatively constant cellular
phenotypes.
Cell proliferation assay
The effect of mare’s milk on human colon cancer cell line Caco2 was evaluated by the sulforhodamine B (SRB) (Vichai and
Kirtikara 2006). Briefly, Caco-2 cells were seeded at a concentration of 4 × 103 cells per well in clear 96-well plates (Fisher
Scientific, Mississauga, ON, Canada) and allowed to adhere for
24 h. Mare’s milk supernatants were thawed at room temperature and subsequently added to the cells in a final dilution
of 1:5.6 (sample:media) (v/v) and incubated for another 24 h at
37◦ C and 5% CO2 in humidified atmosphere. Control samples
(cells with medium only) along with blank samples (medium
only) were also tested. The optical density of plates was measured using an automated 96-well plate reader (Multi detetctor
Microplate Reader, Biotek Synergy HT Model, Vermont, USA) at
a wavelength of 570 nm. Results were expressed as percentage
proliferation with respect to control wells grown under regular
conditions.
Guri et al.
Effect of milk supernatants on Salmonella Typhimurium
reporter constructs
One reporter plasmid construct, (HilA) Salmonella Typhimurium
HilA::luxCDABE, was used to investigate whether the supernatants played a role in the regulation of the gene expression
of the hilA gene controlling the genes of SPI 1. The construct
was designed in a previous study (Bayoumi and Griffiths 2010).
The reporter strain was grown for 18 h at 37◦ C in Luria-Bertani
(LB) (Becton Dickinson, Mississauga, ON, Canada) broth supplemented with 50 μg mL−1 of ampicillin. The resulting cell suspension was diluted 1:100 in fresh medium with and without
supplementation with 10% (v/v) non-treated and heat-treated
sterilized milk supernatants. Two hundred microliters of each
sample were distributed in triplicate into wells of a sterile,
opaque 96-well plate (Corning No. 3610; Fisher Scientific Canada,
Ottawa, ON, Canada) and incubated at 37◦ C. Luminescence and
cell density were measured every hour for a period of 24 h with a
Wallac 1420 Victor Multilabel Counter (Wallac, PerkinElmer Life
Sciences Canada, Woodbridge, ON, Canada). Luminescence was
measured as relative light units (RLU, counts/min).
Effect of milk supernatants on transcription of
virulence genes of Salmonella Typhimurium
To study the effects of heat-treated milk supernatants or raw
milk supernatants on gene expression of the reference strain
Salmonella Typhimurium ATCC 14028, the expression of the
genes hilA (invasion protein regulator), ssrB2 (TTSS regulator,
transcriptional activator), invA (invasion gene associated with
the penetration into the intestinal epithelium), stn (Salmonella
enterotoxin-stn-gene) and sopD (secreted effector protein) were
analyzed using reverse transcription quantitative PCR (RT-qPCR).
The housekeeping genes 16S rRNA, rpoD and gmk were used as
reference genes.
RNA extraction
Salmonella Typhimurium strain ATCC 14028 was grown in brain
heart infusion (BHI) medium alone (control) or supplemented
with 100 μl mL−1 of raw milk or heat-treated milk supernatants
(tests). As described previously (Tellez et al. 2012), 4 mL culture
samples were withdrawn and mixed with one equivalent volume of RNA protect stabilization reagent (Qiagen, Venlo, the
Netherlands) after 4 h incubation. Cells were collected after centrifugation at 5000 × g for 10 min in a centrifuge (Eppendorf
5810, Rotselaar, Belgium) at room temperature. Lysis of the cells
was realized using reagents from the RNeasy Mini kit (Qiagen,
Venlo, the Netherlands) and samples were stored overnight at
−80◦ C according to the manufacturer’s instructions. RNA was
extracted the following day using the same kit as described previously (Tellez et al. 2012).
After RNA extraction, the DNA contamination was eliminated from each sample using DNase I recombinant RNase-free
(Roche Applied Sciences, Vilvoorde, Belgium). Briefly, 20 μL of
total RNA was incubated for 30 min at 37◦ C with 10 U DNase
I, 10 U RNase inhibitor and 5 μL incubation buffer in a total
volume of 50 μL. The RNA was then purified using the cleanup kit (Qiagen, Venlo, the Netherlands) and solubilized in 30 μL
molecular-grade water. The quantity of RNA present was determined by measuring the absorbance at 260 nm using a NanoDrop 2000 spectrophotometer. The RNA quality was verified by
measuring the ratio of the absorbance at 260 nm/280 nm and by
gel electrophoresis. The purified RNA was used immediately for
Reverse-Transcriptase PCR using a cDNA high capacity reverse
3
transcription kit (Life technologies Europe BV, Gent, Belgium).
Briefly, 1 μg of RNA was reverse transcribed with 0.8 μL of dNTP
(100 mM), 1 μL of Multiscribe Reverse Transcriptase (50 U μL−1 ),
2 μL of 10 × random hexamer primers and 2 μL of 10 × RT buffer
in an adjusted total volume of 20 μL using molecular-grade water. For each sample, a no RT control was included to confirm the
absence of contaminating DNA. cDNA synthesis was performed
in a Mastercycler gradient thermocycler (Eppendorf, Nijmegen,
Nederlands) with the following conditions: 25◦ C for 10 min, 37◦ C
for 120 min, 85◦ C for 5 min and a cooling step to 4◦ C. The cDNA
was stored at −20◦ C.
RT-qPCR
Quantitative PCR amplification was performed using an ABI
7000 thermocycler (Applied Biosystems, Singapore) using
R
GoTaqqPCR
Master Mix (Promega, Leiden, the Netherlands)
according to the manufacturer’s instructions. The primers were
synthesized by Eurogentec (Liège, Belgium). Stock solutions of
40 mM were prepared and stored at −20◦ C. The sequences of the
primers are shown in Table 1. All the primers were checked to
confirm their sensitivity for the tested genome sequences using
a nucleotide BLAST analysis (http://blast.ncbi.nlm.nih.gov).
The annealing temperature was determined experimentally
(54◦ C). The PCR was performed in a total volume of 25 μL, which
contained 12.5 μL Power Sybr Green PCR master mix, 0.5 μL
forward primer (800 pmoles reaction−1 ), 0.5 μL reverse primer
(800 pmoles/reaction), 1 μL of 10× diluted cDNA and 10.5 μL of
molecular-grade water. Each PCR was performed in triplicate.
PCR conditions were as follows: denaturation: 95◦ C for 10 min;
amplification and quantification repeated 40 times: 95◦ C for 30 s,
54◦ C for 30 s and 72◦ C for 30 s; melting curve program: 60◦ C–
95◦ C with a heating rate of 0.1◦ C per second and finally a cooling
step to 40◦ C. Each specific amplicon was verified by the presence of both a single melting-temperature peak and a single
band of expected size on a 1% agarose gel after electrophoresis. Ct values were determined with the ABI software provided
with the instrument. Three housekeeping genes (16S rRNA, rpoD:
sigma factor and gmk: guanylate kinase) were tested. The transcript levels were normalized to the geometric average of expression for all housekeeping genes for each sample (Vandesompele et al. 2002). The relative changes in gene expression were
calculated as described by Pfaffl (2001) using the formula: Ratio
= (E target) delta CT target (control-sample) /(E ref) delta CT ref (control-sample) .
The controls corresponded to the expression of E. coli and
were not exposed to milk supernatants. The samples were analyzed in triplicate and the experiment was replicated three
times.
Statistical analysis
All results in this study are the means ± the standard deviations of three independent trials between test and control groups. Student’s t-test was used when necessary to assess the statistical significance of the differences between
test and control groups, with a P-value of ≤0.05 considered
significant.
RESULTS AND DISCUSSION
Antiproliferative capacity of Mare’s milk supernatants
in Caco-2 cells
A screening test of the antiproliferative activity of mare’s milk
supernatants from three different raw milk samples (Raw1,
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FEMS Microbiology Letters, 2016, Vol. 363, No. 2
Table 1. Sequences of primers used in this study and their annealing temperatures.
Target
genes
Function
hilA
Invasion protein regulator
sopD
Secreted effector protein
ssrB2
TTSS regulator
(transcriptional activator)
16S rRNA
Housekeeping
rpoD
Housekeeping
Gmk
Housekeeping
Primer
sequences
Annealing
temperature
Reference
Forward: 5 -T G T C G G A A G A T A A A G A G C A T-3
Reverse: 5 -A A G G A A G T A T C G C C A A T G T A-3
Forward: 5 -A T T A A T G C C G G T A A C T T T G A-3
Reverse: 5 -C T C T G A A A A C G G T G A A T A G C-3
Forward: 5 -T G G T T T A C A C A G C A T A C C A A-3
58◦ C
This study
58◦ C
This study
58◦ C
This study
58◦ C
Xu et al. (2010)
58◦ C
Botteldoorn et al. (2006)
58◦ C
Botteldoorn et al. (2006)
Reverse: 5 -G G T C A A T G T A A C G C T T G T T T-3
Forward: 5 -AGGCCTTCGGGTTGTAAAGT-3
Reverse: 5 -GTTAGCCGGTGCTTCTTCTG-3
Forward: 5 -ACA TGG GTA TTC AGG TAA TGG AAG A-3
Reverse: 5 -CGG TGC TGG TGG TAT TTT CA-3
Forward: 5 -TTG GCA GGG AGG CGT TT-3
Reverse: 5 -GCG CGA AGT GCC GTA GTA AT-3
Figure 1. Viability of Caco-2 cells after 24 h exposure to raw (R1–R3) and heattreated (15 s: R1-15 to R3-15; and 30 min: R1-30 to R3-30) mare’s milk samples.
All samples were diluted 1:5.6 (sample: medium). The control represents cells
growing in medium only. Values are expressed as the average of at least three
replicates, bars representing standard deviations. ∗ P ≤ 0.05.
Raw2 and Raw3) and their respective heat-treated samples HT15
and HT30 (65◦ C for 15 s and 30 min) was performed on adenocarcinoma Caco-2 cells. After 24 h, growth reached 70% confluence
and the cells were then stimulated with the milk supernatants
as described above for 24 h at 37◦ C. The results (Fig. 1) indicated
that the raw milk supernatants potentially decreased the proliferation rate of cancer cells; however, this decrease was significant only for the R1 fraction (up to 40%). This phenomenon was
not observed for the heat-treated supernatants (15 s or 30 min)
as they all seemed to increase the proliferative activity of the
cells compared to control samples. Similar findings were also reported in other studies (Xu et al. 2015; Zanabria et al. 2013) where
bioactivity of bovine MFGM extracted from raw milk and heat
treated was studied on Ht-29 and Caco-2 colon cancer cells. In
both cases, MFGM fraction obtained from raw milk prompted an
extensive reduction of proliferation of cancer cells than the one
obtained from heat treated. This effect has been explained by
protein–protein interactions that are enhanced during the heat
treatment of the milk (Zanabria et al. 2013) along with structural
changes, which in turn may affect the bioactivity of the milk
components itself. Even though pasteurization has been shown
to affect the bioactivity of raw milk, new processing methods,
such as pulsed electric field treatment (Xu et al. 2015), are being explored to maintain the desired bioactivity of milk components. The samples with the most distinct biological activity,
Figure 2. Effect of heated and raw mare’s milk fractions on Salmonella Typhimurium HilA::luxCDABE after 6 h. Results were expressed as relative light
units (RLU), defined as counts per minute and adjusted to OD600 (RLU/OD600).
Values are the means and standard deviations of luminescence from three independent experiments. RF: raw fraction; HT15: heat-treated fraction during 15
s at 65◦ C; HT30: heat-treated fraction during 30 min at 65◦ C. ∗ P ≤ 0.05.
R1 and its respective heat-treated samples HT15 and HT30, and
we will refer them as Raw, HT15 and HT30, were chosen to further test them for any antimicrobial activity on Salmonella Typhimurium and its effect on transcription of different virulence
genes.
Effect of raw and heat treated mare’s milk supernatants
on hilA reporter construct
Supernatants of Raw, HT15 and HT30 as above mentioned, were
tested against the reporter plasmid constructs: (HilA) Salmonella
Typhimurium HilA::luxCDABE and luminescence was quantified.
Both the raw and heat-treated supernatants (15 s and 30 min at
65◦ C) reduced significantly (P ≤ 0.05) the gene promoter activity (Fig. 2). Even if this test constitutes a valuable tool for a first
screening of activity, previous observations using luminescent
constructs suggest the occurrence of false positives (MedellinPena et al. 2007; Delcenserie et al. 2012; Zeinhom et al. 2012).
Therefore, the samples were further analyzed using RT-qPCR to
confirm the results and have the opportunity to analyze the virulence expression of more genes.
Guri et al.
5
Table 2. Effect of mare’s milk fractions on virulence expression of Salmonella Typhimurium after 4 h.
Cycle threshold (Ct) values and standard deviation
a
Genes
PCR efficiency
No treatmentb
Raw fractionc
HT15d
HT30e
gmk
16S
rpoD
hilA
ssrB2
sopD
102%
98.5%
94.5%
99.5%
94%
108%
24.92 ± 1.97
10.57 ± 1.52
24.77 ± 2.77
29.21 ± 1.56
26.61 ± 2.20
27.18 ± 1.64
24.02 ± 2.21
9.58 ± 1.37
24.44 ± 2.27
29.90 ± 2.06
26.23 ± 2.16
26.66 ± 1.64
23.91 ± 2.55
10.50 ± 2.18
24.35 ± 2.90
27.26 ± 2.02
25.43 ± 1.99
25.49 ± 2.07
25.20 ± 2.94
12.82 ± 4.14
25.87 ± 3.17
28.90 ± 2.36
27.28 ± 2.46
26.74 ± 2.65
PCR efficiency %: E = ((10(−1/slope) )/2) × 1.
No treatment: Salmonella Typhimurium grown in BHI for 4 h.
c
Raw fraction: Salmonella Typhimurium grown for 4 h in BHI supplemented with untreated milk fraction.
d
HT15: Salmonella Typhimurium grown for 4 h in BHI supplemented with heat-treated fraction during 15 s at 65◦ C.
e
Heat-treated fraction: Salmonella Typhimurium grown for 4 h in BHI supplemented with heat-treated fraction during 30 min at 65◦ C.
a
b
Effect of raw and heat-treated mare’s milk
supernatants on virulence expression
All the results were normalized to the geometric average expression of all genes and adjusted accordingly to the efficiency of
each pair of primers (Pfaffl 2001; Vandesompele et al. 2002). The
PCR efficiency, Ct values and standard deviations are presented
in Table 2.
The results were analyzed at the end of exponential
Salmonella growth phase (4 h). As described previously, SPI-1
gene expression is optimal at the end of exponential phase
(Clark et al. 2011) while SPI-2 is maximal at the stationary phase
(Beuzón et al. 1999). At 4 h (Table 2 and Fig. 3), the mare’s milk
fraction from heat-treated milk did not induce any significant
changes in Salmonella virulence gene expression except for an increase in gene expression of sopD (ratio +2.44; P ≤ 0.05), whereas
that from raw milk produced a significant decrease in expression
of hil (ratio −3.80; P ≤ 0.05) and ssrB2 (ratio −0.75; ≤0.05), and an
increase of sopD expression (ratio +1.32; P ≤ 0.01).
Only a few studies have focused on the antimicrobial activity
of mare’s milk. Most of them were performed on fermented mare
milk such as Airag or Koumiss. A heat stable bacteriocin produced by Leuconostoc mesenteroides and having an activity against
L. monocytogenes and Clostridium botulinum was semipurified from
Airag (Wulijideligen et al. 2012). Active peptides (angiotensin
I-converting enzyme inhibitory peptides) were semipurified
from Koumiss (Chen et al. 2010). Our results showed that raw
mare milk was able to induce a decrease in expression of genes
involved in regulation of virulence and TTSSs of Salmonella
Typhimurium. The heat-treated supernatants didn’t show the
same activity, probably due to the denaturation of proteins during the heating process. As described previously for human milk,
heat treatment is able to reduce the concentration of immunological proteins in human milk and consequently decrease their
bacteriostatic effects (Christen et al. 2013). In addition, milk was
previously frozen, which might also decrease the enzymatic activity, especially the production of virulence factors, by the bacteria present in the milk (Lopes Mde et al. 2006). Interestingly, a
study of the effect of isolated bovine milk fractions on another
pathogenic bacteria showed that heat-treated milk samples in
that case presented more activity (Tellez et al. 2012). The fact
the previous study focused on bovine MFGMs only and not on
milk supernatant could explain the observed differences. The
effect of the heat-induced binding of whey proteins to MFGMs
in whole milk or pasteurized milk has been widely discussed,
and the protein– protein interaction or protein–milk fat globules
have been detailed (Corredig and Dalgleish 1996; Ye et al. 2002).
These changes in the protein molecular structure and conformation can lead to changes on the protein functionality itself (Fox
et al. 2015). On the other hand, protein changes and aggregation
caused during heating and processing have lead to an increased
effort from research community on their utilization as encapsulation vehicles for increased functionality (Guri and Corredig
2014).
CONCLUSION
Figure 3. Effect of mare’s milk fractions obtained from heated (HT15 = 15 s at
65◦ C or HT30 = 30 min at 65◦ C) and raw mare’s milk fractions on the gene virulence expression of Salmonella Typhimurium after 4 h incubation time. Expression ratios of the different genes of Salmonella Typhimurium were normalized to
the expression of the geometric average of regulator genes 16S rRNA, rpoD, gmk
and compared with no treatment. Negative values represent downregulation of
the genes in presence of raw or heat-treated fractions while positive values are
upregulation. ∗ P ≤ 0.05; ∗ ∗ P ≤ 0.01.
Raw mare milk supernatant exerted antiproliferative effect on
Caco-2 cells and was able to slightly modulate virulence gene
expression of Salmonella Typhimurium. Those effects were not
observed with heat-treated supernatants suggesting that the effect is probably linked to protein or inhibited by some protein–
protein interactions. Further studies are needed to understand
the mechanisms behind and isolate the biomolecules responsible for the observed activity. If those effects can be confirmed in
vivo, mare’s milk may offer new approaches for promoting gastrointestinal health and the results illustrate the importance of
identifying alternative techniques to pasteurization to preserve
the bioactivity of milk components while maintaining safety.
6
FEMS Microbiology Letters, 2016, Vol. 363, No. 2
FUNDING
Financial support for this project was provided by Dairy Farmers
of Ontario and Special Research Funds from University of Liège.
Conflict of interest. None declared.
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