Probioticos 3

Food Microbiology 33 (2013) 282e291

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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, Effie 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 Agricultural University of Athens, Dept. of Food Science and Technology, Lab of Dairy Research, Iera Odos 75, 11855 Athens, Greece 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 b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2012 Received in revised form 10 October 2012 Accepted 15 October 2012 Available online 31 October 2012

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from 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, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>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 efficiency 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 benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Probiotic Starters Olive fermentation Lactic acid bacteria

1. Introduction The term probiotic, literally meaning “for life”, was first addressed by Lilly and Stillwell (1965) and was used to describe substances produced by protozoa to stimulate the growth of other organisms. Nowadays, the term refers to viable, nonpathogenic microorganisms (bacteria or yeasts) that, when ingested, are able to reach the intestines in sufficient numbers to confer health benefits to the host (De Vrese and Schrezenmeir, 2008). Commonly used bacterial probiotics include various species of Lactobacillus, Bifidobacterium, and Streptococcus, as well as Lactococcus lactis and some

* Corresponding author. Tel.: þ30 2102845940/1/2; fax: þ30 2102840740.

E-mail address: ctassou@nagref.gr (C.C. Tassou).

0740-0020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2012.10.005

Enterococcus species. Currently, the only probiotic yeast used is the nonpathogenic Saccharomyces boulardii (Morrow et al., 2012).

It is well established that probiotics confer a number of beneficial health effects to humans and animals. Intake of probiotics stimulates the growth of beneficial microorganisms and reduces the amount of pathogens improving thus the intestinal microbial balance of the host and lowering the risk of gastro-intestinal diseases (Fuller, 1989; Cross, 2002; Chiang and Pan, 2012). Their benefits include also the alleviation of certain intolerances (such as lactose intolerance), the enhancement of nutrients bioavailability, 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, anticarcinogenic, hypocholesterolemic, antihypertensive, anti-osteoporosis, and immunomodulatory effects (Chiang and Pan, 2012). They

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relieve the symptoms of inflammatory bowel diseases, irritable bowel syndrome, colitis, alcoholic liver disease, constipation and reduce the risk for colon, liver and breast cancers (Prado et al., 2008).

Probiotic foods receive market interest as health-promoting, functional foods. Probiotic food products according to FAO/WHO (2002) are in general fermented foods containing an amount of viable and active microorganisms large enough to reach the intestine and exert an equilibrating action on the intestinal microflora. To deliver the health benefits, probiotic foods need to contain an adequate amount of live bacteria (at least 106e107 cfu/g) (Oliveira et al., 2001; Boylston et al., 2004), although there are recent convincing data on beneficial immunological effects derived from dead cells (Vinderola and Reinheimer, 2003; Mottet and Michetti, 2005).

Most probiotic bacteria are lactic acid bacteria and, among them, lactobacilli represent one of the fundamental microbial groups.

They have been introduced in a wide range of food products. Many studies have reported that the best matrices to deliver probiotics are dairy fermented products, such as fermented milks and yogurt.

On the other hand, nowadays there is a need for novel and nondairy probiotics and it has been found that traditional fermented foods may constitute a good working base for the development of probiotic-type functional foods (De Vuyst et al., 2008; Ruiz-Moyano et al., 2008, 2011). A window of opportunity for the development of non-dairy probiotic products has arisen from the increasing number of lactose intolerance cases occurring in the world population, coupled with the unfavourable effect of cholesterol contained in fermented dairy products (Granato et al., 2010).

Among the traditional fermented foods, table olives could be a promising probiotic food through the use of functional probiotic starter cultures. Functional starter cultures contribute to microbial safety and offer organoleptic, technological, nutritional or health advantages. In contrast to well-adapted industrial starters, wildtype strains that naturally dominate traditional fermentations tend to have higher metabolic capacities, which can beneficially affect product quality, for instance with regard to aroma formation and/or food safety. Natural selection is likely to have forced such strains to be more competitive by endowing them with ecological advantages (Ayad et al., 2002). The information provided from traditional fermented foods and scientific research could help develop new probiotic products for the food industry (RiveraEspinoza and Gallardo-Navarro, 2010).

Most of the studies published today about physiological properties of strains intended to be used as probiotics are performed on strains from human or animal internal cavities, considering that strains of these origins would be better adapted and colonize the human/animal gastrointestinal tract (Johansson et al., 1993; Prasad et al., 1998; Xanthopoulos et al., 2000; Ouwehand et al., 2002; RuizMoyano et al., 2009; Zacarías et al., 2011). On the other hand, research has started to increase on probiotic functions of lactic acid bacteria isolated from foods like dairy products (Maragkoudakis et al., 2006; Bao et al., 2010; Monteagudo-Mera et al., 2012; Espeche et al., 2012), dry sausages (Papamanoli et al., 2003; Pennacchia et al., 2004; De Vuyst et al., 2008), foods of plant origin (Husmaini et al., 2011), fruits, cereals, meat or fish (Rivera-Espinoza and Gallardo-Navarro, 2010). Traditional fermented foods are a plentiful source of microorganisms and some of them show probiotic characteristics, although the research of these matrices as raw material for probiotic microorganisms is still scarce compared with their dairy counterpart (Rivera-Espinoza and GallardoNavarro, 2010).

The aim of this work was to perform established in vitro tests to evaluate the probiotic potential and safety of 71 LAB strains originating from olive microbiota and especially from naturally

283

fermented olives. The candidate probiotic strains that fulfil the established criteria could therefore be potentially used as novel probiotic strains by the table olive industry and food industry in general.

2. Materials and methods A total of 71 strains, isolated from naturally fermented olives of cv. Conservolea and Halkidiki, as well as 2 reference strains, namely Lb. casei Shirota (ACA-DC 6002) and Lb. rhamnosus GG (ATCC 53103) were screened for their probiotic potential, following a series of in vitro tests according to relevant proposed guidelines (FAO/WHO, 2002). The studied strains included 17 Ln. mesenteroides, 1 Ln. pseudomesenteroides, 13 Lb. plantarum, 1 Lb. paraplantarum, 37 Lb. pentosus and 2 Lb. paracasei subsp. paracasei strains, whilst their isolation, identification and characterization protocols, are given in details in the article Doulgeraki et al., 2013. The GenBank/EMBL/ DDBJ accession numbers for the 16S rRNA gene sequences of the strains are JX129193eJX129206.

2.1. Survival under conditions simulating the human GI tract 2.1.1. Resistance to low pH The methods used and described below are according to Maragkoudakis et al. (2006) and Zoumpopoulou et al. (2008).

Bacterial cells from overnight (18 h) cultures were harvested (10,000 g, 5 min, 4 C), washed twice with PBS buffer (pH 7.2), before being re-suspended in PBS solution, adjusted to pH 2.5.

Resistance was assessed in triplicates in terms of viable colony counts and enumerated on MRS agar (BK089HA, Biokar Diagnostics) after incubation at 37 C for 0, 0.5, 1, 2, and 3 h, reflecting the time spent by food in the stomach.

2.1.2. Resistance to bile salts Bacterial cells from overnight (18 h) cultures were harvested (10,000 g, 5 min, 4 C), washed twice with PBS buffer (pH 7.2), before being re-suspended in PBS solution (pH 8.0), containing 0.5% (w/v) bile salts (LP0055, Oxoid). Resistance was assessed in triplicates in terms of viable colony counts and enumerated after incubation at 37 C for 0, 1, 2, and 4 h reflecting the time spent by food in the small intestine.

2.1.3. Bile salts hydrolysis Fresh bacterial cultures were streaked in triplicates on MRS agar containing 0.5% (w/v) taurodeoxycholic acid (T0875, Sigma). The hydrolysis effect was indicated by different colony morphology (partial hydrolysis) from the control MRS plates, after 48 h of anaerobic incubation at 37 C. 2.2. Antimicrobial activity against pathogens All strains were tested in triplicates for antimicrobial activity against two Escherichia coli strains (ATCC 35150 and NCTC 12079), two Salmonella Enteritidis strains (ATCC 13076 and WT) and two Listeria monocytogenes strains (FMCC B218 and NCTC 10527). Fresh overnight bacterial MRS culture supernatants of the potential probiotic strains were collected by centrifugation (10,000 g, 15 min, 4 C), adjusted to pH 6.5 and filter-sterilised (0.22 mm). The cell-free culture supernatants (CFCS) of the potential probiotic strains were screened for inhibitory activity using the well diffusion assay.

An initial inoculum of approximately 1$106 cfu/ml of the target strain was incorporated into soft agar (1%, w/v) plates of the appropriate for the target strain medium. CFCS (50 mL) were transferred in holes (5 mm diameter) drilled into the agar. The

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plates were incubated at 37 C, depending on the target strain, and the antimicrobial activity was recorded as growth-free inhibition zones (diameter) around the well. The antibiotic kanamycin (30 mg/ ml) was used as positive control, whilst MRS broth, adjusted to pH 6.5 and filtered was the negative control.

2.3. Haemolytic activity Fresh bacterial cultures were streaked in triplicates on Columbia agar plates, containing 5% (w/v) human blood (Michopoulos S.A., Athens, Greece), and incubated for 48 h at 30 C. Blood agar plates were examined for signs of b-haemolysis (clear zones around colonies), a-haemolysis (green-hued zones around colonies) or ghaemolysis (no zones around colonies).

2.4. Antibiotic resistance

3. Results & discussion

For testing antibiotic resistance, bacterial strains were inoculated (1%, v/v) in MRS broth supplemented with antibiotics (vancomycin, chloramphenicol, penicillin, streptomycin, gentamycin, kanamycin, erythromycin, ampicillin and tetracycline) at various final concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 mg/ ml) and examined in triplicate for growth in a microplate reader (OD at 610 nm) following a 24 h incubation period at 30 C. 2.5. Chemometric analysis Data from viable counts of LAB obtained from the resistance to low pH and bile salts assays, after 3 h exposure to pH 2.5 and after 4 h exposure to 0.5% (w/v) bile salts respectively, were used as input variables in hierarchical cluster analysis (HCA) based on Euclidian distances to allocate strains into homogeneous groups according to these two characteristics. Prior to analysis, the data were standardized by calculating the relative survival ratio (RSR) as follows:

RSR ¼

ðlogcfuN0 logcfuNt Þ 100 logcfuN0

approximately 107 cfu were added to each well (technical replicates), with each strain being assessed for adherence in triplicate wells in each experiment. Following co-incubation for 4 h at 37 C cells were washed twice with sterile PBS and lysed with 2 ml Triton 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 (expressed as a percentage) was calculated by using the ratio of the number of bacterial cells that remained attached to the total number of bacterial cells added initially to each well. All the above experiments were repeated 6 times (biological replicates) with all strains. Statistical analysis t-test was performed to assess if a strain showed a different adherence compared to each of the two reference strains Lb. casei Shirota and Lb. rhamnosus GG. P values less than 0.05 were considered statistically significant.

(1)

where N0 represents the total viable count for LAB before treatment and Nt the total viable count after the treatment at low pH or bile salts, respectively. The data were analyzed using the XLStat software (version 2006.06, Addinsoft, Paris, France), a built-in statistical software package of Excel.

2.6. Adherence to Caco-2 cells The strains that were selected according to the above described HCA, as well as the two reference strains, were further examined for their ability to adhere to human colon carcinoma (Caco-2) cells.

Quantitative analysis of bacterial adherence to differentiated Caco2 cells (13 days) was tested as described previously (Anderson et al., 2010) with minor modifications. First, 2$105 Caco-2 cells (European Collection of Cell Cultures number 86010202) were seeded in 24well plates in plates with Dulbecco’s modified Eagle’s medium containing 2 mM glutamine, 1% (w/v) nonessential amino acids, and 20% (v/v) foetal bovine serum supplemented with 100 U/ml penicillin/streptomycin (Sigma). The medium in the wells was replaced with fresh medium every 2e3 days and monolayers were maintained for 13 days in 7% CO2 at 37 C until monolayers formed with no further visible differentiation. One hour before coincubation the medium in each well was replaced with prewarmed fresh medium without antibiotics.

Prior to experiments, all bacterial cultures were grown overnight until stationary phase in MRS at 37 C and washed twice with sterile phosphate-buffered saline (PBS). Subsequently,

3.1. Survival under conditions simulating the human GI tract In order to act as a probiotic in the gastrointestinal tract and to exert their beneficial effect on the host, the bacteria must be able to survive the acidic conditions in the stomach and resist bile acids at the beginning of the small intestine (Holzapfel et al., 1998; 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 secreted into the human digestive tract every day. Thus, it is essential for the bacteria to have protection systems to withstand the low pH in the stomach, digestive enzymes and bile in the small intestine.

3.1.1. Resistance to low pH The viable counts of most Ln. mesenteroides and Ln. pseudomesenteroides strains were found to be<1 log cfu/ml after 3 h of exposure to pH 2.5, indicating no resistance at all. On the contrary, the majority of Lb. plantarum and Lb. pentosus strains showed higher resistance to low pH, whilst variability in the final counts was obtained. Table 1 shows the ranges of the final counts of all the tested strains after exposure to low pH for 3 h, whereas Fig. 1 shows the viable counts of the most resistant LAB strains (final counts > 8 log cfu/ml) after 0, 0.5, 1, 2, and 3 h incubation in pH 2.5, including also the reference strains. Overall, 9 strains showed very high resistance to low pH (Lb. plantarum B282, E10, E69; Lb. pentosus B281, E108, E97, E104; Lb. paracasei subsp. paracasei E93, E94) with final populations exceeding 8 log cfu/ml, whereas 12 strains showed high resistance to low pH (Lb. plantarum E4, E45, E50, E71, E73; Lb. pentosus B285, E96, E106B, E111, E119, E121, E141) that along with the reference strains Lb. casei Shirota and Lb. ramnosus GG showed final populations within 6e8 log cfu/ml.

These results are in agreement with those obtained from previous studies, where Lactobacillus strains of food, human or

Table 1 Ranges of the final viable counts of all the tested strains after exposure to low pH for 3 h.

Species

Total no of strains

Ln. mesenteroides Ln. pseudomesenteroides Lb. plantarum Lb. paraplantarum Lb. pentosus Lb. paracasei subsp. paracasei Lb. casei Shirota Lb. ramnosus GG

17 1 13 1 37 2 1 1

Final counts (log cfu/ml) >8

6e8

3

5

4 2

7 1 1

3e6

5 1 15

1e3

<1

1 1 1

16

8

3

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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 pH values of 2.5e4.0 (Conway et al., 1987; Du Toit et al., 1998; Jacobsen et al., 1999; Dunne et al., 2001; Maragkoudakis et al., 2006; Xanthopoulos et al., 2000).

Several in vitro assays have been described to select acid resistant strains, i.e., exposure to pH-adjusted PBS (Conway et al., 1987; Park et al., 2002), incubation in gastric contents (Conway et al., 1987; Fernández et al., 2003) and use of a dynamic model of the stomach (Marteau et al., 1997). Conway et al. (1987) found survival of lactobacilli to be slightly lower when PBS was used rather than gastric juice, because components in the gastric juice may confer some protective effect on the bacterial cell. They suggested the use of PBS at the desired pH to screen strains for their ability to maintain viability in vivo when exposed to gastric juice. Moreover, the probiotic strains could be buffered by food or other carrier matrix molecules following consumption and are thus not likely to be exposed to the pH of the stomach (Prasad et al., 1998). The pH value (2.5) used in this study for the selection of potential probiotic strains is very selective and even though it is not the most common pH value in the human stomach it assures the isolation of the very acid-tolerant strains (Pennacchia et al., 2004).

3.1.2. Resistance to bile salts and bile salts hydrolysis The majority of tested strains were found to be resistant to bile salts even after 4 h of exposure retaining their viability with negligible reduction in viable counts ( 1 log cycle). Only 3 Lb. pentosus strains (625A, E110 and E182) demonstrated approximately 2 log reduction after 4 h of exposure to bile salts. Regarding bile salt hydrolysis, 12 strains exhibited partial bile salt hydrolase activity, recorded as differentiated colony morphology on TDCAMRS agar in comparison with the control MRS agar plates. These were 5 Lb. plantarum (B282, E45, E10, E73, E79) and 7 Lb. pentosus (B279, B283, B284, E43, E100, E106B, E129) strains.

Bile plays a fundamental role in specific (Marteau et al., 1997) and non-specific (Kalambaheti et al., 1994) defence mechanism of the gut and the magnitude of its inhibitory effect is determined primarily by the bile salts concentration (Charteris et al., 2000).

Therefore, bile tolerance is considered as an important characteristic of Lactobacillus strains, which enables them to survive, grow, and exert their action in gastrointestinal transit. According to Sanders et al. (1996), Lactobacillus strains which could grow and metabolize in normal physical bile concentration could survive in gastrointestinal transit. The resistance to bile salt of some strains

was related also to the activity of bile salt hydrolase which can hydrolyse combined bile salt and thus reduce their toxic and side effects. In addition, Gänzle et al. (1999b) and Du Toit et al. (1998) proposed that some components of food could protect and promote the resistance of strain to bile salt.

Bile salt hydrolysis has been correlated to cholesterol lowering (Begley et al., 2006; Liong and Shah, 2005). One hypothesis proposes that the cholesterol lowering property of Lactobacillus is due to incorporation of cholesterol into the cellular membranes of bacteria from the medium during its growth period (Gilliland et al., 1985). The main mechanism however is linked to the bile salt hydrolase activity of the cells (Taranto et al., 1997). Indeed some strains of LAB secrete bile salt hydrolase enzyme, which hydrolyses conjugated bile acids to release de-conjugated bile acids and amino acids (Sridevi et al., 2009; Begley et al., 2006; Franz et al., 2011).

When these salts are secreted from the gastrointestinal tract, the demand for cholesterol is increased, which in turn lowers cholesterol levels (De Rodas et al., 1996; Driessen and de Boer, 1989).

On the other hand, it is yet not completely clear whether BSH activity is a desirable property for probiotics, since large amounts of de-conjugated bile salts may have undesirable effects for the human host (Berr et al., 1996; Mamianett et al., 1999). However, current research data strongly suggest that microbial BSH function in the detoxification of bile salts increases the intestinal survival and persistence of producing strains, which in turn increases the overall beneficial effects associated with the strain (Begley et al., 2005, 2006). Moreover, the bacterial genera that would most likely be used as probiotics (bifidobacteria and lactobacilli) are not capable of dehydroxylating de-conjugated bile salts (Ahn et al., 2003; Gilliland and Speck, 1977; Takahashi and Morotomi, 1994), and thus the majority of the breakdown products of BSH activity by a probiotic strain may be precipitated and excreted in feces (Thomas et al., 2000; Veysey et al., 2001).

3.2. Antimicrobial activity against pathogens None of the supernatants (adjusted to pH 6.5) obtained from the 71 tested strains as well as the two reference probiotic strains inhibited the growth of the pathogenic target strains, leading to the assumption that no bacteriocin-like action exists. This is in agreement with findings for other probiotics like Lb. fermentum strains (Lin et al., 2007) or Lb. casei Shirota, Lb. plantarum, and Lb. paracasei subsp. tolerans (Maragkoudakis et al., 2006) whose inhibitory

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Table 2 Antibiotic resistance of the tested strains.

Strain

MICsa (mg/ml) A

V

G

K

S

E

P

T

C

Ln. mesenteroides B259 Ln. mesenteroides B260 Ln. mesenteroides B261 Ln. mesenteroides B262 Ln. mesenteroides B263 Ln. mesenteroides B264 Ln. mesenteroides B265 Ln. mesenteroides B266 Ln. mesenteroides B267 Ln. mesenteroides B268 Ln. mesenteroides B269 Ln. mesenteroides B270 Ln. mesenteroides B271 Ln. mesenteroides B273 Ln. mesenteroides B274 Ln. mesenteroides B275 Ln. mesenteroides B276 Ln. pseudomesenteroides B277 Lb. paraplantarum B280 Lb. plantarum B282 Lb. plantarum E1 Lb. plantarum E4 Lb. plantarumE10 Lb. plantarum E45 Lb. plantarum E50 Lb. plantarum E66 Lb. plantarum E68 Lb. plantarum E69 Lb. plantarum E71 Lb. plantarum E73 Lb. plantarum E77 Lb. plantarum E79 Lb. pentosus B278 Lb. pentosus B279 Lb. pentosus B281 Lb. pentosus B283 Lb. pentosus B284 Lb. pentosus B285 Lb. pentosus E43 Lb. pentosus E83 Lb. pentosus E84 Lb. pentosus E89 Lb. pentosus E95 Lb. pentosus E96 Lb. pentosus E97 Lb. pentosus E100 Lb. pentosus E101 Lb. pentosus E104 Lb. pentosus E105 Lb. pentosus E106B Lb. pentosus E108 Lb. pentosus E110 Lb. pentosus E111 Lb. pentosus E119 Lb. pentosus E120 Lb. pentosus E121 Lb. pentosus E128 Lb. pentosus E129 Lb. pentosus E130 Lb. pentosus E139 Lb. pentosus E141 Lb. pentosus E182 Lb. pentosus 390A Lb. pentosus 606 Lb. pentosus 609 Lb. pentosus 612 Lb. pentosus 625A Lb. pentosus 632 Lb. pentosus 637 Lb. paracasei subsp. paracasei E93 Lb. paracasei subsp. paracasei E94

<2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 2 2 2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 4R 4R 2 2 2 2 <2 <2 <2 4R <2 2 2 2 <2 <2 <2 32R 32R 32R 32R 32R 32R 32R <2 1024R 1024R 64R 32R 32R <2 <2 4R <2 <2 <2 4R <2 <2

128 256 64 256 256 256 256 256 256 256 256 256 256 512 512 512 256 512 256 1024 1024 1024 1024 1024 1024 512 1024 1024 1024 1024 1024 1024 1024 1024 256 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024 1024

4 4 4 4 4 4 4 4 2 2 2 2 2 2 <2 <2 4 8 16 32R 8 8 16 16 8 32R 32R 64R 64R 16 32R 16 16 16 16 16 16 8 8 16 4 8 16 4 32R 4 32R 128R 4 4 8 8 8 8 8 16 8 32R 8 256R 4 4 4 4 4 4 8 4 16 32R 32R

32R 8 4 32R 16 16 16 32R 32R 32R 32R 32R 32R 32R 16 32R 32R 64R 1024R 1024R 64 64 128R 256R 128R 128R 128R 512R 256R 256R 128R 256R 128R 128R 256R 128R 256R 128R 64 128R 32 64 64 128R 256R 32 128R 512R 32 64 64 64 128R 64 64 64 64 256R 16 <2 64 32 32 32 32 32 64 32 256R 256R 256R

32 8 16 32 16 16 32 32 32 32 16 32 16 16 8 16 16 32 128 128 64 64 64 128 64 128 128 128 128 128 128 128 64 32 128 64 64 64 32 64 32 32 32 64 128 8 64 64 8 16 32 16 64 16 32 32 32 64 8 256 16 16 8 16 16 8 32 16 64 128 128

<2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 2 2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2

<2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 <2 8 8 16 4 4 2 8 4 4 4 4 4 8 32 32 <2 64 32 16 16 <2 <2 8 <2 16 8 4 8 8 8 16 32 32 32 32 32 32 <2 4 <2 8 <2 <2 16 4 16 32 4 <2 16 4 <2

2 <2 <2 <2 <2 <2 2 <2 <2 <2 <2 <2 2 2 2 2 <2 16R 32 4 4 8 4 4 4 8 4 8 8 8 8 8 16 32 32 16 16 16 4 4 4 4 2 4 4 4 8 8 8 8 8 8 16 4 16 16 4 16 8 16 4 4 4 8 8 8 4 8 8 8R <2

2 2 2 2 2 2 2 2 2 4 2 2 2 4 2 2 2 16R 8 8 2 2 2 4 2 2 2 2 2 2 2 2 2 16R 16R 8 8 8 <2 <2 2 2 <2 <2 2 2 2 2 <2 <2 2 2 2 2 2 2 <2 2 <2 2 <2 <2 <2 <2 2 <2 2 2 2 2 2

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Table 2 (continued ) Strain

Lb. casei Shirota Lb. ramnosus GG

MICsa (mg/ml) A

V

G

K

S

E

P

T

C

<2 32R

1024 1024

16 16

4 256R

128 32

2R <2

8 8

16R 2

8R 4

R Resistant according to the EFSA’s 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 et al. (2007) reported that neutralization of the soluble fraction to pH 6.5 significantly reduced the antimicrobial activity against all the selected pathogens.

The production of antimicrobial compounds such as organic acids, short chain fatty acids and bacteriocins is one of the functional properties used to characterize probiotics (Fuller, 1989). The lowering of pH due to organic acids (especially lactic and acetic acids) produced by these bacteria in the gut has a bactericidal or bacteriostatic effect (Shah, 2007). The capacity to produce different antimicrobial compounds may be one of the critical characteristics for effective competitive exclusion of pathogen survival in the intestine and expression of a probiotic effect for the host (Ouwehand and Salminen, 1998). The acidic conditions in the stomach may even enhance the activity of these antimicrobial compounds (Gänzle et al., 1999a). Furthermore, these probiotic characteristics may partly be based on the production of relevant concentrations of lactic acid in the microenvironment, which, in combination with a detergent such as bile salts, inhibits the growth of Gram-negative pathogenic bacteria (Begley et al., 2005).

3.3. Haemolytic activity Absence of haemolytic activity and antibiotic resistance is considered as a safety prerequisite for the selection of a probiotic strain (FAO/WHO, 2002). None of the examined strains exhibited bhaemolytic activity when grown in Columbia human blood agar.

Most of the strains (69 strains) were g-haemolytic (i.e. no haemolysis), while four strains exhibited a-haemolysis. These were Lb. pentosus B278, B279, B281, and B285 strains. Similar observations were made for all the strains of Lb. paracasei subsp. paracasei, Lactobacillus spp. and Lb. casei isolated from dairy products which showed g-haemolysis except of few that showed a-haemolysis (Maragkoudakis et al., 2006).

3.4. Antibiotic resistance Table 2 shows the minimum inhibitory concentrations (MICs) of the 71 tested LAB strains to antibiotics of different groups: cell wall inhibitors (penicillin G, ampicillin and vancomycin) and protein synthesis inhibitors (kanamycin, streptomycin, tetracycline, gentamicin, erythromycin and chloramphenicol). Strains were considered resistant when they showed MIC values higher than the MIC breakpoints established by the European Food Safety Authority (EFSA, 2008).

The profiles of antibiotic susceptibility of LAB have been documented in many publications (Zoumpopoulou et al., 2008; Ammor et al., 2007; Kastner et al., 2006; D’Aimmo et al., 2007; Charteris et al., 1998). According to the breakpoints set by EFSA (2008) the majority of LAB isolates tested in this study can be characterized as resistant to kanamycin, including 12/18 of Leuconostoc strains, 11/13 of Lb. plantarum strains and Lb. paraplantarum strain, 14/37 of Lb. pentosus strains, 2 Lb. paracasei subs. paracasei strains and Lb. ramnosus GG. Regarding the antibiotic gentamycin, 6/13 of Lb.

plantarum strains, and 5/37 of Lb. pentosus strains were found to be resistant. Moreover, 1/2 of Lb. paracasei subsp. paracasei strains, Ln. pseudomesenteroides and Lb. casei Shirota were resistant to tetracycline. Lb. casei Shirota was found to be resistant to erythromycin.

Finally, 17/37 of Lb. pentosus strains and Lb. ramnosus GG strain showed resistance to ampicillin, and 2/37 of Lb. pentosus strains and Lb. casei Shirota strain were resistant to chloramphenicol.

All LAB strains showed resistance to vancomycin (without a specified breakpoint from EFSA though), supporting the native resistance of Lb. plantarum and Lb. casei species to vancomycin which is consistent with previous reports (Liu et al., 2009; Felten et al., 1999; Temmerman et al., 2002; Danielsen and Wind, 2003).

It has also been reported that strains of Lb. rhamnosus, pediococci, and Leuconostoc spp. are resistant to vancomycin (Zhou et al., 2005), while resistance to kanamycin has been confirmed for most Lactobacillus species (Temmerman et al., 2003). Previous studies also confirm the generally lower resistance of the lactobacilli species studied here towards tetracycline and chloramphenicol (Katla et al., 2001; Temmerman et al., 2002; Choi et al., 2003; Maragkoudakis et al., 2006). It has also been reported that strains of Lb. paracasei subsp. paracasei were resistant to aminoglycosides (gentamicin, kanamycin) and to tetracycline (Charteris et al., 1998; Zhou et al., 2005; Ammor et al., 2008; Ripamonti et al., 2011).

Various opinions exist as to whether it might be desirable that some probiotic strains show resistance to specific antibiotics that are, for instance, involved in antibiotic-induced diarrhoea (Charteris et al., 1998). On the other hand, the commercial introduction of probiotics containing antibiotic resistant strains may also have negative consequences, for example, when resistance is transferred to intestinal pathogens (Curragh and Collins, 1992).

However, according to previous studies (Charteris et al., 2001; Katla et al., 2001; Danielsen and Wind, 2003) the antibiotic resistance observed for Lactobacillus strains in this work, are considered to be intrinsic or natural resistance because it is chromosomally encoded and, therefore, non-transmissible. Resistance to aminoglycoside antibiotics, such as gentamicin, streptomycin, kanamycin, is considered to be intrinsic in the Lactobacillus genus and is attributed to the absence of cytochrome-mediated electron transport, which mediates drug uptake. Also, the resistance to vancomycin by Lactobacillus strains has been attributed to the presence of D-Ala-Dlactate in their peptidoglycan instead of the normal dipeptide DAla-D-Ala, which is the target of the antibiotic (Coppola et al., 2005; Danielsen and Wind, 2003; Andrea Monteagudo-Mera et al., 2012).

The vancomycin-resistant genes of lactobacilli are also chromosomal and, therefore, not readily transferable to other species (Morrow et al., 2012).

3.5. Chemometric analysis Using the viable counts of LAB strains derived from the resistance tests to low pH and bile salts, HCA revealed the presence of three well-differentiated groups (clusters) (Fig. 2). The lower cluster (Group I) contained strains that exhibited the highest resistance to low pH and bile salts, whereas strains with little or no

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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 (Group III). Four strains that showed intermediate performance were located in a small cluster (Group II) between the two major groups. It is characteristic that the strains that presented the highest survival to low pH and bile salts were located in the same cub-cluster in Group I (encircled strains), including 4 strains of Lb. pentosus (E281, E97, E104, E108), 3 strains of Lb. plantarum (B282, E10, E69), 2 strains of Lb. paracasei subsp. paracasei (E93, E94).

The strains that showed the highest survival to low pH and bile salts and were grouped together according to HCA, along with the reference strains, were further examined for their ability to

adhere to Caco-2 cells. All the tested strains showed similar or higher adherence compared to the two reference strains after 4 h.

This suggests that all these strains demonstrate comparable or higher adherence properties to the well-known probiotic strains.

In comparison with the Lb. casei Shirota, the higher adherence was demonstrated from the strains Lb. pentosus E108, Lb. plantarum B282 and Lb. paracasei subsp. paracasei E94 (Fig. 3). Though, the strain Lb. paracasei subsp. paracasei E94 showed high aggregation activity, a fact that may false positively increase the measured adherence. On the other hand only Lb. plantarum B282 exhibited a statistically signiďŹ cant higher adherence in comparison to the Lb. rhamnosus GG (Fig. 4).

The ability to adhere to the mucosal surfaces of the intestine and the subsequent long or short-term colonization has long

Fig. 3. 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) against Lactobacillus casei Shirota as a reference strain (Error bars indicate standard deviation from 6 replications per strain).

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) against Lactobacillus rhamnosus GG as a reference strain (Error bars indicate standard deviation from 6 replications per strain).

3.6. Adherence to Caco-2 cells

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289

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

Lb. pentosus B281 Lb. pentosus E97 Lb. pentosus E104 Lb. pentosus E108 Lb. plantarum B282 Lb. plantarum E10 Lb. plantarum E69 Lb. paracasei subsp. paracasei E93 Lb. paracasei subsp. paracasei E94 Lb. casei Shirota Lb. rhamnosus GG a b c d e

Test Low pH (SR%)a

Bile salts (SR%)b

Bile salts hydrolysis

Haemolytic activityd

Antibiotic resistancee

Caco-2 (Adherence%)

95.64 89.69 92.52 91.08 87.79 89.95 98.36 89.41 82.75 82.83 64.02

94.78 96.79 97.64 100.59 100.09 98.67 100.02 96.55 88.80 100.20 100.61

0c 0 0 0 1 1 0 0 0 0 0

a g g g g g g g g g g

K, C, S K, C, S K, G K, A K, G, E K, G K, G K, G, S K, G, S S, E, P, T, C K, A, P

37.21 39.76 33.72 60.78 68.94 44.75 30.51 41.92 74.02 31.41 34.00

Survival rate after 3 h in low pH. Survival rate after 4 h in bile salts.

0: no hydrolysis; 1: partial hydrolysis. a-haemolysis, g-haemolysis.

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 selection of probiotic strains (Collado et al., 2009; Lebeer et al., 2008; Marco et al., 2006). For beneficial (probiotic) health effects in the large intestine, such as the competitive exclusion of pathogens from the intestinal epithelium or the immuneregulation, an effective probiotic should be able to, at least transiently, colonize on the gut mucosa. Adhesive probiotic lactobacilli have been reported to have beneficial health effects, especially related to the inhibition of pathogen adhesion to intestinal cell lines (Hudault et al., 1997; Lievin-Le Moal et al., 2002). Previous studies have reported adhesive strains, such as Lb. johnsonii La1, Lb. rhamnosus GG, as well as Lb. casei Shirota and Lb. casei Imunitass (Tuomola and Salminen, 1998; Juntunen et al., 2001; Ouwehand et al., 2001).

The adhesion mechanisms are not fully understood, however bacterial cell-surface associated proteins with mucus and intestinal cell binding properties have been identified and characterized in probiotic strains (Sánchez et al., 2008; Veléz et al., 2007). Despite new sophisticated methodologies, bacterial adhesion capacity is most commonly studied in vitro with epithelial cell lines, immobilized intestinal mucus or extracellular matrix molecules (Jensen et al., 2012). Probiotic bacteria compete with invading pathogens for binding sites to epithelial cells and the overlying mucus layer in a strain-specific manner (Morrow et al., 2012).

On the other hand, it is increasingly clear that probiotics can provide beneficial effects even without true colonization of the gastrointestinal tract. Some probiotic entities (Bifidobacterium longum, Bacteroides thetaiotaomicron) become part of the established intestinal microflora, whereas others (Lactobacillus casei, Bifidobacterium animalis) temporarily remodel or influence the microbial community, thereby exerting transient effects only as they pass through (Ohland and MacNaughton, 2010).

4. Conclusion In conclusion, the results of this study showed that 4 strains of Lb. pentosus (E281, E97, E104, E108), 3 strains of Lb. plantarum (B282, E10, E69), and two strains of Lb. paracasei subsp. paracasei (E93, E94) were found to possess desirable in vitro probiotic properties similar or superior to the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. The summarized results obtained from all in vitro tests for these strains are given in Table 3.

These strains are good candidates for further investigation with in vivo studies to elucidate their potential health benefits as well as in fermentation studies to assess their technological characteristics for application as novel probiotic starters. A probiotic potential is

expected to greatly enhance the already important nutritional value of table olives, which are regarded as a source of fiber, organic acids, vitamins and minerals. Development of probiotic olive products may indeed convey a favourable impact in rural economy, especially knowing that such products originate from less developed regions.

Acknowledgements The research leading to these results has received funding from the EU (FP7/2007e2013), under grant agreement no 243471-PROBIOLIVES. The information in this document reflects only the authors’ views and the Community is not liable for any use that may be made of the information contained therein. The authors would like to thank Dr. A.I. Doulgeraki for strain identification and Mrs. A. Damaskinou for technical assistance.

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