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Food Microbiology 33 (2013) 282e291

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Selection of potential probiotic lactic acid bacteria from fermented olives by


in vitro tests
Anthoula A. Argyri a, Georgia Zoumpopoulou b, Kimon-Andreas G. Karatzas c, Efe Tsakalidou b,
George-John E. Nychas d, Efstathios Z. Panagou d, Chrysoula C. Tassou a, *
a
Hellenic Agricultural Organisation DEMETER, Institute of Technology of Agricultural Products, Sof. Venizelou 1, 14123 Lycovrissi, Attikis, Greece
b
Agricultural University of Athens, Dept. of Food Science and Technology, Lab of Dairy Research, Iera Odos 75, 11855 Athens, Greece
c
University of Reading, Dept. of Food and Nutritional Sciences, Whiteknights, Reading, Berkshire RG6 6AH, UK
d
Agricultural University of Athens, Dept. of Food Science and Technology, Lab of Microbiology and Biotechnology of Foods, Iera Odos 75, 11855 Athens, Greece

a r t i c l e i n f o a b s t r a c t

Article history: The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from
Received 28 July 2012 naturally fermented olives and select candidates to be used as probiotic starters for the improvement of
Received in revised form the traditional fermentation process and the production of newly added value functional foods. Seventy
10 October 2012
one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lacto-
Accepted 15 October 2012
bacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from
Available online 31 October 2012
table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used
as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions,
Keywords:
Probiotic
antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7),
Starters Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb.
Olive fermentation plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest nal population
Lactic acid bacteria (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile
salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt
hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efciency to
adhere to Caco-2 cells was observed. This was the same regarding strains susceptibility towards different
antibiotics. None of the strains exhibited b-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3
strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable
in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota
and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo
studies to elucidate their potential health benets and in olive fermentation processes to assess their
technological performance as novel probiotic starters.
2012 Elsevier Ltd. All rights reserved.

1. Introduction Enterococcus species. Currently, the only probiotic yeast used is the
nonpathogenic Saccharomyces boulardii (Morrow et al., 2012).
The term probiotic, literally meaning for life, was rst It is well established that probiotics confer a number of bene-
addressed by Lilly and Stillwell (1965) and was used to describe cial health effects to humans and animals. Intake of probiotics
substances produced by protozoa to stimulate the growth of other stimulates the growth of benecial microorganisms and reduces
organisms. Nowadays, the term refers to viable, nonpathogenic the amount of pathogens improving thus the intestinal microbial
microorganisms (bacteria or yeasts) that, when ingested, are able to balance of the host and lowering the risk of gastro-intestinal
reach the intestines in sufcient numbers to confer health benets diseases (Fuller, 1989; Cross, 2002; Chiang and Pan, 2012). Their
to the host (De Vrese and Schrezenmeir, 2008). Commonly used benets include also the alleviation of certain intolerances (such as
bacterial probiotics include various species of Lactobacillus, Bido- lactose intolerance), the enhancement of nutrients bioavailability,
bacterium, and Streptococcus, as well as Lactococcus lactis and some and prevention or reduction of the prevalence of allergies in
susceptible individuals (Isolauri, 2001; Chiang and Pan, 2012).
Probiotics are reported to have also antimutagenic, anticarcino-
* Corresponding author. Tel.: 30 2102845940/1/2; fax: 30 2102840740. genic, hypocholesterolemic, antihypertensive, anti-osteoporosis,
E-mail address: ctassou@nagref.gr (C.C. Tassou). and immunomodulatory effects (Chiang and Pan, 2012). They

0740-0020/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fm.2012.10.005
A.A. Argyri et al. / Food Microbiology 33 (2013) 282e291 283

relieve the symptoms of inammatory bowel diseases, irritable fermented olives. The candidate probiotic strains that full the
bowel syndrome, colitis, alcoholic liver disease, constipation and established criteria could therefore be potentially used as novel
reduce the risk for colon, liver and breast cancers (Prado et al., probiotic strains by the table olive industry and food industry in
2008). general.
Probiotic foods receive market interest as health-promoting,
functional foods. Probiotic food products according to FAO/WHO 2. Materials and methods
(2002) are in general fermented foods containing an amount of
viable and active microorganisms large enough to reach the A total of 71 strains, isolated from naturally fermented olives of
intestine and exert an equilibrating action on the intestinal cv. Conservolea and Halkidiki, as well as 2 reference strains, namely
microora. To deliver the health benets, probiotic foods need to Lb. casei Shirota (ACA-DC 6002) and Lb. rhamnosus GG (ATCC 53103)
contain an adequate amount of live bacteria (at least 106e107 cfu/g) were screened for their probiotic potential, following a series of
(Oliveira et al., 2001; Boylston et al., 2004), although there are in vitro tests according to relevant proposed guidelines (FAO/WHO,
recent convincing data on benecial immunological effects derived 2002). The studied strains included 17 Ln. mesenteroides, 1 Ln.
from dead cells (Vinderola and Reinheimer, 2003; Mottet and pseudomesenteroides, 13 Lb. plantarum, 1 Lb. paraplantarum, 37 Lb.
Michetti, 2005). pentosus and 2 Lb. paracasei subsp. paracasei strains, whilst their
Most probiotic bacteria are lactic acid bacteria and, among them, isolation, identication and characterization protocols, are given in
lactobacilli represent one of the fundamental microbial groups. details in the article Doulgeraki et al., 2013. The GenBank/EMBL/
They have been introduced in a wide range of food products. Many DDBJ accession numbers for the 16S rRNA gene sequences of the
studies have reported that the best matrices to deliver probiotics strains are JX129193eJX129206.
are dairy fermented products, such as fermented milks and yogurt.
On the other hand, nowadays there is a need for novel and non- 2.1. Survival under conditions simulating the human GI tract
dairy probiotics and it has been found that traditional fermented
foods may constitute a good working base for the development of 2.1.1. Resistance to low pH
probiotic-type functional foods (De Vuyst et al., 2008; Ruiz-Moyano The methods used and described below are according to
et al., 2008, 2011). A window of opportunity for the development of Maragkoudakis et al. (2006) and Zoumpopoulou et al. (2008).
non-dairy probiotic products has arisen from the increasing Bacterial cells from overnight (18 h) cultures were harvested
number of lactose intolerance cases occurring in the world pop- (10,000  g, 5 min, 4  C), washed twice with PBS buffer (pH 7.2),
ulation, coupled with the unfavourable effect of cholesterol con- before being re-suspended in PBS solution, adjusted to pH 2.5.
tained in fermented dairy products (Granato et al., 2010). Resistance was assessed in triplicates in terms of viable colony
Among the traditional fermented foods, table olives could be counts and enumerated on MRS agar (BK089HA, Biokar Diagnos-
a promising probiotic food through the use of functional probiotic tics) after incubation at 37  C for 0, 0.5, 1, 2, and 3 h, reecting the
starter cultures. Functional starter cultures contribute to microbial time spent by food in the stomach.
safety and offer organoleptic, technological, nutritional or health
advantages. In contrast to well-adapted industrial starters, wild- 2.1.2. Resistance to bile salts
type strains that naturally dominate traditional fermentations Bacterial cells from overnight (18 h) cultures were harvested
tend to have higher metabolic capacities, which can benecially (10,000  g, 5 min, 4  C), washed twice with PBS buffer (pH 7.2),
affect product quality, for instance with regard to aroma formation before being re-suspended in PBS solution (pH 8.0), containing 0.5%
and/or food safety. Natural selection is likely to have forced such (w/v) bile salts (LP0055, Oxoid). Resistance was assessed in tripli-
strains to be more competitive by endowing them with ecological cates in terms of viable colony counts and enumerated after incu-
advantages (Ayad et al., 2002). The information provided from bation at 37  C for 0, 1, 2, and 4 h reecting the time spent by food in
traditional fermented foods and scientic research could help the small intestine.
develop new probiotic products for the food industry (Rivera-
Espinoza and Gallardo-Navarro, 2010). 2.1.3. Bile salts hydrolysis
Most of the studies published today about physiological prop- Fresh bacterial cultures were streaked in triplicates on MRS agar
erties of strains intended to be used as probiotics are performed on containing 0.5% (w/v) taurodeoxycholic acid (T0875, Sigma). The
strains from human or animal internal cavities, considering that hydrolysis effect was indicated by different colony morphology
strains of these origins would be better adapted and colonize the (partial hydrolysis) from the control MRS plates, after 48 h of
human/animal gastrointestinal tract (Johansson et al., 1993; Prasad anaerobic incubation at 37  C.
et al., 1998; Xanthopoulos et al., 2000; Ouwehand et al., 2002; Ruiz-
Moyano et al., 2009; Zacaras et al., 2011). On the other hand, 2.2. Antimicrobial activity against pathogens
research has started to increase on probiotic functions of lactic acid
bacteria isolated from foods like dairy products (Maragkoudakis All strains were tested in triplicates for antimicrobial activity
et al., 2006; Bao et al., 2010; Monteagudo-Mera et al., 2012; against two Escherichia coli strains (ATCC 35150 and NCTC 12079),
Espeche et al., 2012), dry sausages (Papamanoli et al., 2003; two Salmonella Enteritidis strains (ATCC 13076 and WT) and two
Pennacchia et al., 2004; De Vuyst et al., 2008), foods of plant origin Listeria monocytogenes strains (FMCC B218 and NCTC 10527). Fresh
(Husmaini et al., 2011), fruits, cereals, meat or sh (Rivera-Espinoza overnight bacterial MRS culture supernatants of the potential
and Gallardo-Navarro, 2010). Traditional fermented foods are probiotic strains were collected by centrifugation (10,000  g,
a plentiful source of microorganisms and some of them show 15 min, 4  C), adjusted to pH 6.5 and lter-sterilised (0.22 mm). The
probiotic characteristics, although the research of these matrices as cell-free culture supernatants (CFCS) of the potential probiotic
raw material for probiotic microorganisms is still scarce compared strains were screened for inhibitory activity using the well diffusion
with their dairy counterpart (Rivera-Espinoza and Gallardo- assay.
Navarro, 2010). An initial inoculum of approximately 1$106 cfu/ml of the target
The aim of this work was to perform established in vitro tests to strain was incorporated into soft agar (1%, w/v) plates of the
evaluate the probiotic potential and safety of 71 LAB strains origi- appropriate for the target strain medium. CFCS (50 mL) were
nating from olive microbiota and especially from naturally transferred in holes (5 mm diameter) drilled into the agar. The
284 A.A. Argyri et al. / Food Microbiology 33 (2013) 282e291

plates were incubated at 37  C, depending on the target strain, and approximately 107 cfu were added to each well (technical repli-
the antimicrobial activity was recorded as growth-free inhibition cates), with each strain being assessed for adherence in triplicate
zones (diameter) around the well. The antibiotic kanamycin (30 mg/ wells in each experiment. Following co-incubation for 4 h at 37  C
ml) was used as positive control, whilst MRS broth, adjusted to pH cells were washed twice with sterile PBS and lysed with 2 ml Triton
6.5 and ltered was the negative control. X-100 (1% v/v) in PBS. Following incubation for 5 min at 37  C, cell
lysates were serially diluted and plated on MRS agar. The adherence
2.3. Haemolytic activity (expressed as a percentage) was calculated by using the ratio of the
number of bacterial cells that remained attached to the total
Fresh bacterial cultures were streaked in triplicates on Columbia number of bacterial cells added initially to each well. All the above
agar plates, containing 5% (w/v) human blood (Michopoulos S.A., experiments were repeated 6 times (biological replicates) with all
Athens, Greece), and incubated for 48 h at 30  C. Blood agar plates strains. Statistical analysis t-test was performed to assess if a strain
were examined for signs of b-haemolysis (clear zones around showed a different adherence compared to each of the two refer-
colonies), a-haemolysis (green-hued zones around colonies) or g- ence strains Lb. casei Shirota and Lb. rhamnosus GG. P values less
haemolysis (no zones around colonies). than 0.05 were considered statistically signicant.

2.4. Antibiotic resistance


3. Results & discussion
For testing antibiotic resistance, bacterial strains were inocu-
3.1. Survival under conditions simulating the human GI tract
lated (1%, v/v) in MRS broth supplemented with antibiotics (van-
comycin, chloramphenicol, penicillin, streptomycin, gentamycin,
In order to act as a probiotic in the gastrointestinal tract and to
kanamycin, erythromycin, ampicillin and tetracycline) at various
exert their benecial effect on the host, the bacteria must be able to
nal concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 mg/
survive the acidic conditions in the stomach and resist bile acids at
ml) and examined in triplicate for growth in a microplate reader
the beginning of the small intestine (Holzapfel et al., 1998;
(OD at 610 nm) following a 24 h incubation period at 30  C.
Klaenhammer and Kullen, 1999). Approximately 2.5 l of gastric
juice (Cotter and Hill, 2003) and 1 l of bile (Begley et al., 2005) are
2.5. Chemometric analysis
secreted into the human digestive tract every day. Thus, it is
essential for the bacteria to have protection systems to withstand
Data from viable counts of LAB obtained from the resistance to
the low pH in the stomach, digestive enzymes and bile in the small
low pH and bile salts assays, after 3 h exposure to pH 2.5 and after
intestine.
4 h exposure to 0.5% (w/v) bile salts respectively, were used as input
variables in hierarchical cluster analysis (HCA) based on Euclidian
3.1.1. Resistance to low pH
distances to allocate strains into homogeneous groups according to
The viable counts of most Ln. mesenteroides and Ln. pseudome-
these two characteristics. Prior to analysis, the data were stan-
senteroides strains were found to be<1 log cfu/ml after 3 h of
dardized by calculating the relative survival ratio (RSR) as follows:
exposure to pH 2.5, indicating no resistance at all. On the contrary,
logcfuN0  logcfuNt the majority of Lb. plantarum and Lb. pentosus strains showed
RSR  100 (1) higher resistance to low pH, whilst variability in the nal counts
logcfuN0
was obtained. Table 1 shows the ranges of the nal counts of all the
where N0 represents the total viable count for LAB before treatment tested strains after exposure to low pH for 3 h, whereas Fig. 1 shows
and Nt the total viable count after the treatment at low pH or bile the viable counts of the most resistant LAB strains (nal counts > 8
salts, respectively. The data were analyzed using the XLStat soft- log cfu/ml) after 0, 0.5, 1, 2, and 3 h incubation in pH 2.5, including
ware (version 2006.06, Addinsoft, Paris, France), a built-in statis- also the reference strains. Overall, 9 strains showed very high
tical software package of Excel. resistance to low pH (Lb. plantarum B282, E10, E69; Lb. pentosus
B281, E108, E97, E104; Lb. paracasei subsp. paracasei E93, E94) with
nal populations exceeding 8 log cfu/ml, whereas 12 strains
2.6. Adherence to Caco-2 cells
showed high resistance to low pH (Lb. plantarum E4, E45, E50, E71,
E73; Lb. pentosus B285, E96, E106B, E111, E119, E121, E141) that
The strains that were selected according to the above described
along with the reference strains Lb. casei Shirota and Lb. ramnosus
HCA, as well as the two reference strains, were further examined
GG showed nal populations within 6e8 log cfu/ml.
for their ability to adhere to human colon carcinoma (Caco-2) cells.
These results are in agreement with those obtained from
Quantitative analysis of bacterial adherence to differentiated Caco-
previous studies, where Lactobacillus strains of food, human or
2 cells (13 days) was tested as described previously (Anderson et al.,
2010) with minor modications. First, 2$105 Caco-2 cells (European
Collection of Cell Cultures number 86010202) were seeded in 24-
Table 1
well plates in plates with Dulbeccos modied Eagles medium Ranges of the nal viable counts of all the tested strains after exposure to low pH for
containing 2 mM glutamine, 1% (w/v) nonessential amino acids, 3 h.
and 20% (v/v) foetal bovine serum supplemented with 100 U/ml
Species Total no Final counts (log cfu/ml)
penicillin/streptomycin (Sigma). The medium in the wells was of strains
replaced with fresh medium every 2e3 days and monolayers were >8 6e8 3e6 1e3 <1

maintained for 13 days in 7% CO2 at 37  C until monolayers formed Ln. mesenteroides 17 1 16


Ln. pseudomesenteroides 1 1
with no further visible differentiation. One hour before co-
Lb. plantarum 13 3 5 5 1
incubation the medium in each well was replaced with pre- Lb. paraplantarum 1 1
warmed fresh medium without antibiotics. Lb. pentosus 37 4 7 15 8 3
Prior to experiments, all bacterial cultures were grown over- Lb. paracasei subsp. paracasei 2 2
night until stationary phase in MRS at 37  C and washed twice Lb. casei Shirota 1 1
Lb. ramnosus GG 1 1
with sterile phosphate-buffered saline (PBS). Subsequently,
A.A. Argyri et al. / Food Microbiology 33 (2013) 282e291 285

Fig. 1. Resistance to low pH after 0, 0.5, 1, 2, and 3 h of the strains Lb. plantarum B282, E10, and E69, Lb. pentosus B281, E108, E97, and E104, Lb. paracasei subsp. paracasei E93 and E94
and the reference strains Lb. casei Shirota and Lb. ramnosus GG (Error bars indicate standard deviation from three replications).

animal origin, were able to retain their viability when exposed to was related also to the activity of bile salt hydrolase which can
pH values of 2.5e4.0 (Conway et al., 1987; Du Toit et al., 1998; hydrolyse combined bile salt and thus reduce their toxic and side
Jacobsen et al., 1999; Dunne et al., 2001; Maragkoudakis et al., effects. In addition, Gnzle et al. (1999b) and Du Toit et al. (1998)
2006; Xanthopoulos et al., 2000). proposed that some components of food could protect and
Several in vitro assays have been described to select acid resis- promote the resistance of strain to bile salt.
tant strains, i.e., exposure to pH-adjusted PBS (Conway et al., 1987; Bile salt hydrolysis has been correlated to cholesterol lowering
Park et al., 2002), incubation in gastric contents (Conway et al., (Begley et al., 2006; Liong and Shah, 2005). One hypothesis
1987; Fernndez et al., 2003) and use of a dynamic model of the proposes that the cholesterol lowering property of Lactobacillus is
stomach (Marteau et al., 1997). Conway et al. (1987) found survival due to incorporation of cholesterol into the cellular membranes of
of lactobacilli to be slightly lower when PBS was used rather than bacteria from the medium during its growth period (Gilliland et al.,
gastric juice, because components in the gastric juice may confer 1985). The main mechanism however is linked to the bile salt
some protective effect on the bacterial cell. They suggested the use hydrolase activity of the cells (Taranto et al., 1997). Indeed some
of PBS at the desired pH to screen strains for their ability to strains of LAB secrete bile salt hydrolase enzyme, which hydrolyses
maintain viability in vivo when exposed to gastric juice. Moreover, conjugated bile acids to release de-conjugated bile acids and amino
the probiotic strains could be buffered by food or other carrier acids (Sridevi et al., 2009; Begley et al., 2006; Franz et al., 2011).
matrix molecules following consumption and are thus not likely to When these salts are secreted from the gastrointestinal tract, the
be exposed to the pH of the stomach (Prasad et al., 1998). The pH demand for cholesterol is increased, which in turn lowers choles-
value (2.5) used in this study for the selection of potential probiotic terol levels (De Rodas et al., 1996; Driessen and de Boer, 1989).
strains is very selective and even though it is not the most common On the other hand, it is yet not completely clear whether BSH
pH value in the human stomach it assures the isolation of the very activity is a desirable property for probiotics, since large amounts of
acid-tolerant strains (Pennacchia et al., 2004). de-conjugated bile salts may have undesirable effects for the
human host (Berr et al., 1996; Mamianett et al., 1999). However,
3.1.2. Resistance to bile salts and bile salts hydrolysis current research data strongly suggest that microbial BSH function
The majority of tested strains were found to be resistant to bile in the detoxication of bile salts increases the intestinal survival
salts even after 4 h of exposure retaining their viability with and persistence of producing strains, which in turn increases the
negligible reduction in viable counts (1 log cycle). Only 3 Lb. overall benecial effects associated with the strain (Begley et al.,
pentosus strains (625A, E110 and E182) demonstrated approxi- 2005, 2006). Moreover, the bacterial genera that would most
mately 2 log reduction after 4 h of exposure to bile salts. Regarding likely be used as probiotics (bidobacteria and lactobacilli) are not
bile salt hydrolysis, 12 strains exhibited partial bile salt hydrolase capable of dehydroxylating de-conjugated bile salts (Ahn et al.,
activity, recorded as differentiated colony morphology on TDCA- 2003; Gilliland and Speck, 1977; Takahashi and Morotomi, 1994),
MRS agar in comparison with the control MRS agar plates. These and thus the majority of the breakdown products of BSH activity by
were 5 Lb. plantarum (B282, E45, E10, E73, E79) and 7 Lb. pentosus a probiotic strain may be precipitated and excreted in feces
(B279, B283, B284, E43, E100, E106B, E129) strains. (Thomas et al., 2000; Veysey et al., 2001).
Bile plays a fundamental role in specic (Marteau et al., 1997)
and non-specic (Kalambaheti et al., 1994) defence mechanism of 3.2. Antimicrobial activity against pathogens
the gut and the magnitude of its inhibitory effect is determined
primarily by the bile salts concentration (Charteris et al., 2000). None of the supernatants (adjusted to pH 6.5) obtained from the
Therefore, bile tolerance is considered as an important character- 71 tested strains as well as the two reference probiotic strains
istic of Lactobacillus strains, which enables them to survive, grow, inhibited the growth of the pathogenic target strains, leading to the
and exert their action in gastrointestinal transit. According to assumption that no bacteriocin-like action exists. This is in agree-
Sanders et al. (1996), Lactobacillus strains which could grow and ment with ndings for other probiotics like Lb. fermentum strains
metabolize in normal physical bile concentration could survive in (Lin et al., 2007) or Lb. casei Shirota, Lb. plantarum, and Lb. paracasei
gastrointestinal transit. The resistance to bile salt of some strains subsp. tolerans (Maragkoudakis et al., 2006) whose inhibitory
286 A.A. Argyri et al. / Food Microbiology 33 (2013) 282e291

Table 2
Antibiotic resistance of the tested strains.

Strain MICsa (mg/ml)

A V G K S E P T C
Ln. mesenteroides B259 <2 128 4 32R 32 <2 <2 2 2
Ln. mesenteroides B260 <2 256 4 8 8 <2 <2 <2 2
Ln. mesenteroides B261 <2 64 4 4 16 <2 <2 <2 2
Ln. mesenteroides B262 <2 256 4 32R 32 <2 <2 <2 2
Ln. mesenteroides B263 <2 256 4 16 16 <2 <2 <2 2
Ln. mesenteroides B264 <2 256 4 16 16 <2 <2 <2 2
Ln. mesenteroides B265 <2 256 4 16 32 <2 <2 2 2
Ln. mesenteroides B266 <2 256 4 32R 32 <2 <2 <2 2
Ln. mesenteroides B267 <2 256 2 32R 32 <2 <2 <2 2
Ln. mesenteroides B268 <2 256 2 32R 32 <2 <2 <2 4
Ln. mesenteroides B269 <2 256 2 32R 16 <2 <2 <2 2
Ln. mesenteroides B270 <2 256 2 32R 32 <2 <2 <2 2
Ln. mesenteroides B271 <2 256 2 32R 16 <2 <2 2 2
Ln. mesenteroides B273 <2 512 2 32R 16 <2 <2 2 4
Ln. mesenteroides B274 <2 512 <2 16 8 <2 <2 2 2
Ln. mesenteroides B275 <2 512 <2 32R 16 <2 <2 2 2
Ln. mesenteroides B276 2 256 4 32R 16 <2 <2 <2 2
Ln. pseudomesenteroides B277 2 512 8 64R 32 <2 <2 16R 16R
Lb. paraplantarum B280 2 256 16 1024R 128 <2 <2 32 8
Lb. plantarum B282 2 1024 32R 1024R 128 2 8 4 8
Lb. plantarum E1 <2 1024 8 64 64 <2 8 4 2
Lb. plantarum E4 <2 1024 8 64 64 <2 16 8 2
Lb. plantarumE10 <2 1024 16 128R 64 <2 4 4 2
Lb. plantarum E45 <2 1024 16 256R 128 <2 4 4 4
Lb. plantarum E50 <2 1024 8 128R 64 <2 2 4 2
Lb. plantarum E66 <2 512 32R 128R 128 <2 8 8 2
Lb. plantarum E68 <2 1024 32R 128R 128 <2 4 4 2
Lb. plantarum E69 <2 1024 64R 512R 128 <2 4 8 2
Lb. plantarum E71 <2 1024 64R 256R 128 <2 4 8 2
Lb. plantarum E73 <2 1024 16 256R 128 <2 4 8 2
Lb. plantarum E77 <2 1024 32R 128R 128 <2 4 8 2
Lb. plantarum E79 <2 1024 16 256R 128 <2 8 8 2
Lb. pentosus B278 4R 1024 16 128R 64 2 32 16 2
Lb. pentosus B279 4R 1024 16 128R 32 2 32 32 16R
Lb. pentosus B281 2 256 16 256R 128 <2 <2 32 16R
Lb. pentosus B283 2 1024 16 128R 64 <2 64 16 8
Lb. pentosus B284 2 1024 16 256R 64 <2 32 16 8
Lb. pentosus B285 2 1024 8 128R 64 <2 16 16 8
Lb. pentosus E43 <2 1024 8 64 32 <2 16 4 <2
Lb. pentosus E83 <2 1024 16 128R 64 <2 <2 4 <2
Lb. pentosus E84 <2 1024 4 32 32 <2 <2 4 2
Lb. pentosus E89 4R 1024 8 64 32 <2 8 4 2
Lb. pentosus E95 <2 1024 16 64 32 <2 <2 2 <2
Lb. pentosus E96 2 1024 4 128R 64 <2 16 4 <2
Lb. pentosus E97 2 1024 32R 256R 128 <2 8 4 2
Lb. pentosus E100 2 1024 4 32 8 <2 4 4 2
Lb. pentosus E101 <2 1024 32R 128R 64 <2 8 8 2
Lb. pentosus E104 <2 1024 128R 512R 64 <2 8 8 2
Lb. pentosus E105 <2 1024 4 32 8 <2 8 8 <2
Lb. pentosus E106B 32R 1024 4 64 16 <2 16 8 <2
Lb. pentosus E108 32R 1024 8 64 32 <2 32 8 2
Lb. pentosus E110 32R 1024 8 64 16 <2 32 8 2
Lb. pentosus E111 32R 1024 8 128R 64 <2 32 16 2
Lb. pentosus E119 32R 1024 8 64 16 <2 32 4 2
Lb. pentosus E120 32R 1024 8 64 32 <2 32 16 2
Lb. pentosus E121 32R 1024 16 64 32 <2 32 16 2
Lb. pentosus E128 <2 1024 8 64 32 <2 <2 4 <2
Lb. pentosus E129 1024R 1024 32R 256R 64 <2 4 16 2
Lb. pentosus E130 1024R 1024 8 16 8 <2 <2 8 <2
Lb. pentosus E139 64R 1024 256R <2 256 <2 8 16 2
Lb. pentosus E141 32R 1024 4 64 16 <2 <2 4 <2
Lb. pentosus E182 32R 1024 4 32 16 <2 <2 4 <2
Lb. pentosus 390A <2 1024 4 32 8 <2 16 4 <2
Lb. pentosus 606 <2 1024 4 32 16 <2 4 8 <2
Lb. pentosus 609 4R 1024 4 32 16 <2 16 8 2
Lb. pentosus 612 <2 1024 4 32 8 <2 32 8 <2
Lb. pentosus 625A <2 1024 8 64 32 <2 4 4 2
Lb. pentosus 632 <2 1024 4 32 16 <2 <2 8 2
Lb. pentosus 637 4R 1024 16 256R 64 <2 16 8 2
Lb. paracasei subsp. paracasei E93 <2 1024 32R 256R 128 <2 4 8R 2
Lb. paracasei subsp. paracasei E94 <2 1024 32R 256R 128 <2 <2 <2 2
A.A. Argyri et al. / Food Microbiology 33 (2013) 282e291 287

Table 2 (continued )

Strain MICsa (mg/ml)

A V G K S E P T C
Lb. casei Shirota <2 1024 16 4 128 2R 8 16R 8R
Lb. ramnosus GG 32R 1024 16 256R 32 <2 8 2 4
R
Resistant according to the EFSAs breakpoints (EFSA, 2008).
A: ampicillin, V: vancomycin, G: gentamycin, K: kanamycin, S: streptomycin, P: penicillin, E: erythromycin, T: tetracycline, C: chloramphenicol.
a
MIC: minimum inhibitory concentration.

activity became negligible in neutral pH 7.0. Moreover, Millette plantarum strains, and 5/37 of Lb. pentosus strains were found to be
et al. (2007) reported that neutralization of the soluble fraction to resistant. Moreover, 1/2 of Lb. paracasei subsp. paracasei strains, Ln.
pH 6.5 signicantly reduced the antimicrobial activity against all pseudomesenteroides and Lb. casei Shirota were resistant to tetra-
the selected pathogens. cycline. Lb. casei Shirota was found to be resistant to erythromycin.
The production of antimicrobial compounds such as organic Finally, 17/37 of Lb. pentosus strains and Lb. ramnosus GG strain
acids, short chain fatty acids and bacteriocins is one of the func- showed resistance to ampicillin, and 2/37 of Lb. pentosus strains and
tional properties used to characterize probiotics (Fuller, 1989). The Lb. casei Shirota strain were resistant to chloramphenicol.
lowering of pH due to organic acids (especially lactic and acetic All LAB strains showed resistance to vancomycin (without
acids) produced by these bacteria in the gut has a bactericidal or a specied breakpoint from EFSA though), supporting the native
bacteriostatic effect (Shah, 2007). The capacity to produce different resistance of Lb. plantarum and Lb. casei species to vancomycin
antimicrobial compounds may be one of the critical characteristics which is consistent with previous reports (Liu et al., 2009; Felten
for effective competitive exclusion of pathogen survival in the et al., 1999; Temmerman et al., 2002; Danielsen and Wind, 2003).
intestine and expression of a probiotic effect for the host It has also been reported that strains of Lb. rhamnosus, pediococci,
(Ouwehand and Salminen, 1998). The acidic conditions in the and Leuconostoc spp. are resistant to vancomycin (Zhou et al.,
stomach may even enhance the activity of these antimicrobial 2005), while resistance to kanamycin has been conrmed for
compounds (Gnzle et al., 1999a). Furthermore, these probiotic most Lactobacillus species (Temmerman et al., 2003). Previous
characteristics may partly be based on the production of relevant studies also conrm the generally lower resistance of the lactoba-
concentrations of lactic acid in the microenvironment, which, in cilli species studied here towards tetracycline and chloramphenicol
combination with a detergent such as bile salts, inhibits the growth (Katla et al., 2001; Temmerman et al., 2002; Choi et al., 2003;
of Gram-negative pathogenic bacteria (Begley et al., 2005). Maragkoudakis et al., 2006). It has also been reported that strains of
Lb. paracasei subsp. paracasei were resistant to aminoglycosides
3.3. Haemolytic activity (gentamicin, kanamycin) and to tetracycline (Charteris et al., 1998;
Zhou et al., 2005; Ammor et al., 2008; Ripamonti et al., 2011).
Absence of haemolytic activity and antibiotic resistance is Various opinions exist as to whether it might be desirable that
considered as a safety prerequisite for the selection of a probiotic some probiotic strains show resistance to specic antibiotics that
strain (FAO/WHO, 2002). None of the examined strains exhibited b- are, for instance, involved in antibiotic-induced diarrhoea
haemolytic activity when grown in Columbia human blood agar. (Charteris et al., 1998). On the other hand, the commercial intro-
Most of the strains (69 strains) were g-haemolytic (i.e. no hae- duction of probiotics containing antibiotic resistant strains may
molysis), while four strains exhibited a-haemolysis. These were Lb. also have negative consequences, for example, when resistance is
pentosus B278, B279, B281, and B285 strains. Similar observations transferred to intestinal pathogens (Curragh and Collins, 1992).
were made for all the strains of Lb. paracasei subsp. paracasei, However, according to previous studies (Charteris et al., 2001; Katla
Lactobacillus spp. and Lb. casei isolated from dairy products which et al., 2001; Danielsen and Wind, 2003) the antibiotic resistance
showed g-haemolysis except of few that showed a-haemolysis observed for Lactobacillus strains in this work, are considered to be
(Maragkoudakis et al., 2006). intrinsic or natural resistance because it is chromosomally encoded
and, therefore, non-transmissible. Resistance to aminoglycoside
3.4. Antibiotic resistance antibiotics, such as gentamicin, streptomycin, kanamycin, is
considered to be intrinsic in the Lactobacillus genus and is attrib-
Table 2 shows the minimum inhibitory concentrations (MICs) of uted to the absence of cytochrome-mediated electron transport,
the 71 tested LAB strains to antibiotics of different groups: cell wall which mediates drug uptake. Also, the resistance to vancomycin by
inhibitors (penicillin G, ampicillin and vancomycin) and protein Lactobacillus strains has been attributed to the presence of D-Ala-D-
synthesis inhibitors (kanamycin, streptomycin, tetracycline, lactate in their peptidoglycan instead of the normal dipeptide D-
gentamicin, erythromycin and chloramphenicol). Strains were Ala-D-Ala, which is the target of the antibiotic (Coppola et al., 2005;
considered resistant when they showed MIC values higher than the Danielsen and Wind, 2003; Andrea Monteagudo-Mera et al., 2012).
MIC breakpoints established by the European Food Safety Authority The vancomycin-resistant genes of lactobacilli are also chromo-
(EFSA, 2008). somal and, therefore, not readily transferable to other species
The proles of antibiotic susceptibility of LAB have been docu- (Morrow et al., 2012).
mented in many publications (Zoumpopoulou et al., 2008; Ammor
et al., 2007; Kastner et al., 2006; DAimmo et al., 2007; Charteris 3.5. Chemometric analysis
et al., 1998). According to the breakpoints set by EFSA (2008) the
majority of LAB isolates tested in this study can be characterized as Using the viable counts of LAB strains derived from the resis-
resistant to kanamycin, including 12/18 of Leuconostoc strains, 11/13 tance tests to low pH and bile salts, HCA revealed the presence of
of Lb. plantarum strains and Lb. paraplantarum strain, 14/37 of Lb. three well-differentiated groups (clusters) (Fig. 2). The lower
pentosus strains, 2 Lb. paracasei subs. paracasei strains and Lb. cluster (Group I) contained strains that exhibited the highest
ramnosus GG. Regarding the antibiotic gentamycin, 6/13 of Lb. resistance to low pH and bile salts, whereas strains with little or no
288 A.A. Argyri et al. / Food Microbiology 33 (2013) 282e291

Group III

Group II

Group I

0 10 20 30 40 50 60 70

Dissimilarities
Fig. 2. Results of the hierarchical cluster analysis for the grouping of the 71 strains of LAB based on their resistance to low pH and bile salts.

resistance to low pH and bile salts were located in the upper cluster adhere to Caco-2 cells. All the tested strains showed similar or
(Group III). Four strains that showed intermediate performance higher adherence compared to the two reference strains after 4 h.
were located in a small cluster (Group II) between the two major This suggests that all these strains demonstrate comparable or
groups. It is characteristic that the strains that presented the higher adherence properties to the well-known probiotic strains.
highest survival to low pH and bile salts were located in the same In comparison with the Lb. casei Shirota, the higher adherence was
cub-cluster in Group I (encircled strains), including 4 strains of Lb. demonstrated from the strains Lb. pentosus E108, Lb. plantarum
pentosus (E281, E97, E104, E108), 3 strains of Lb. plantarum (B282, B282 and Lb. paracasei subsp. paracasei E94 (Fig. 3). Though, the
E10, E69), 2 strains of Lb. paracasei subsp. paracasei (E93, E94). strain Lb. paracasei subsp. paracasei E94 showed high aggregation
activity, a fact that may false positively increase the measured
3.6. Adherence to Caco-2 cells adherence. On the other hand only Lb. plantarum B282 exhibited
a statistically signicant higher adherence in comparison to the
The strains that showed the highest survival to low pH and bile Lb. rhamnosus GG (Fig. 4).
salts and were grouped together according to HCA, along with the The ability to adhere to the mucosal surfaces of the intestine
reference strains, were further examined for their ability to and the subsequent long or short-term colonization has long

Fig. 3. Comparison of adherence between all strains (Lb. plantarum B282, E10, and E69, Fig. 4. Comparison of adherence between all strains (Lb. plantarum B282, E10, and E69,
Lb. pentosus B281, E108, E97, and E104, Lb. paracasei subsp. paracasei E93 and E94) Lb. pentosus B281, E108, E97, and E104, Lb. paracasei subsp. paracasei E93 and E94)
against Lactobacillus casei Shirota as a reference strain (Error bars indicate standard against Lactobacillus rhamnosus GG as a reference strain (Error bars indicate standard
deviation from 6 replications per strain). deviation from 6 replications per strain).
A.A. Argyri et al. / Food Microbiology 33 (2013) 282e291 289

Table 3
Selected strains with probiotic potential according to in vitro tests in comparison with Lb. casei Shirota, and Lb. rhamnosus GG.

Strains Test

Low pH (SR%)a Bile salts (SR%)b Bile salts hydrolysis Haemolytic activityd Antibiotic resistancee Caco-2 (Adherence%)
Lb. pentosus B281 95.64 94.78 0c a K, C, S 37.21
Lb. pentosus E97 89.69 96.79 0 g K, C, S 39.76
Lb. pentosus E104 92.52 97.64 0 g K, G 33.72
Lb. pentosus E108 91.08 100.59 0 g K, A 60.78
Lb. plantarum B282 87.79 100.09 1 g K, G, E 68.94
Lb. plantarum E10 89.95 98.67 1 g K, G 44.75
Lb. plantarum E69 98.36 100.02 0 g K, G 30.51
Lb. paracasei subsp. paracasei E93 89.41 96.55 0 g K, G, S 41.92
Lb. paracasei subsp. paracasei E94 82.75 88.80 0 g K, G, S 74.02
Lb. casei Shirota 82.83 100.20 0 g S, E, P, T, C 31.41
Lb. rhamnosus GG 64.02 100.61 0 g K, A, P 34.00
a
Survival rate after 3 h in low pH.
b
Survival rate after 4 h in bile salts.
c
0: no hydrolysis; 1: partial hydrolysis.
d
a-haemolysis, g-haemolysis.
e
A: ampicillin, V: vancomycin, G: gentamycin, K: kanamycin, S: streptomycin, P: penicillin, E: erythromycin, T: tetracycline, C: chloramphenicol.

been one of the most commonly encountered criteria for the expected to greatly enhance the already important nutritional
selection of probiotic strains (Collado et al., 2009; Lebeer et al., value of table olives, which are regarded as a source of ber, organic
2008; Marco et al., 2006). For benecial (probiotic) health acids, vitamins and minerals. Development of probiotic olive
effects in the large intestine, such as the competitive exclusion of products may indeed convey a favourable impact in rural economy,
pathogens from the intestinal epithelium or the immuner- especially knowing that such products originate from less devel-
egulation, an effective probiotic should be able to, at least tran- oped regions.
siently, colonize on the gut mucosa. Adhesive probiotic
lactobacilli have been reported to have benecial health effects, Acknowledgements
especially related to the inhibition of pathogen adhesion to
intestinal cell lines (Hudault et al., 1997; Lievin-Le Moal et al., The research leading to these results has received funding from
2002). Previous studies have reported adhesive strains, such as the EU (FP7/2007e2013), under grant agreement no 243471-PRO-
Lb. johnsonii La1, Lb. rhamnosus GG, as well as Lb. casei Shirota and BIOLIVES. The information in this document reects only the
Lb. casei Imunitass (Tuomola and Salminen, 1998; Juntunen et al., authors views and the Community is not liable for any use that may
2001; Ouwehand et al., 2001). be made of the information contained therein. The authors would
The adhesion mechanisms are not fully understood, however like to thank Dr. A.I. Doulgeraki for strain identication and Mrs. A.
bacterial cell-surface associated proteins with mucus and intestinal Damaskinou for technical assistance.
cell binding properties have been identied and characterized in
probiotic strains (Snchez et al., 2008; Velz et al., 2007). Despite References
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