Probiotic potential of Lactobacillus strains isolated from dairy products

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International Dairy Journal 16 (2006) 189–199 www.elsevier.com/locate/idairyj

Probiotic potential of Lactobacillus strains isolated from dairy products Petros A. Maragkoudakisa, Georgia Zoumpopouloua, Christos Miarisa, George Kalantzopoulosa, Bruno Potb, Effie Tsakalidoua, a Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece Institute Pasteur de Lille; Laboratoire Bacte´riologie des Ecosyste`mes, 1 rue de Prof. Calmette BP245, 59019 Lille Cedex, Lille, France

b

Received 27 October 2004; accepted 7 February 2005

Abstract Twenty-nine Lactobacillus strains of dairy origin were examined in vitro for their probiotic potential. Only a few strains were able to survive at pH 1 or in the presence of pepsin, while all were unaffected by pH 3, pancreatin and bile salts. Strains exhibited variable bile salt hydrolase activity. None was haemolytic. The majority of strains were resistant to vancomycin and teicoplanin, but sensitive to chloramphenicol and tetracycline. A few strains were able to adhere to Caco-2 cells. Although no bacteriocin activity was detected in vitro, strains L. casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were able to inhibit the adhesion of Escherichia coli and Salmonella typhimurium to Caco-2 cells. They also induced the secretion of proand anti-inflammatory cytokines by human peripheral blood mononuclear cells. These three strains were therefore found, in vitro, to possess desirable probiotic properties. r 2005 Elsevier Ltd. All rights reserved. Keywords: Lactobacillus; Probiotic; Survival; GI tract; Adhesion; Cytokines

1. Introduction The genus Lactobacillus has a long history of safe use, especially in the dairy industry, and plays a major role in the production of fermented milk products. Over the past few decades, an increased drive has existed for the isolation of novel Lactobacillus strains that exert a beneficial health effect when ingested by humans. Such strains are termed probiotic. According to Guarner and Shaafsma (1998) probiotics are ‘‘living micro-organisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition’’. Beneficial effects conferred by lactobacilli include inhibition of pathogenic organisms, such as Salmonella, Shigella and Helicobacter (Bernet-Camard et al., 1997; Corresponding author. Tel.: +30 210 5294661; fax: +30 210 5294672. E-mail address: [email protected] (E. Tsakalidou).

0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.02.009

Hudault, Lievin, Bernet-Camard, & Servin, 1997; Aiba, Suzuki, Kabir, Tagaki, & Koga, 1998; HammiltonMiller, 2003; Sgouras et al., 2004). Furthermore, lactobacilli have been associated with numerous other health benefits, such as reduction of lactose intolerance (Gilliland & Kim, 1984) and increased immune response (Matsuzaki, Yamazaki, Hashimoto, & Yokokura, 1998). A beneficial role for lactobacilli has also been implied in cancer (Hirayama & Rafter, 2000; KoppHoolithan, 2001), and especially in the case of colon cancer (Mercennier, Pavan, & Pot, 2002; Guarner & Malagelada, 2003). In order for a probiotic strain to exert its beneficial effect on the host, it has to be able to survive passage through the host’s digestive tract. So far, research has mainly focused on strains sensitivity towards low pH, proteolytic enzymes and bile salts (Conway, Gorbach, & Goldin, 1987; Charteris, Kelly, Morelli, & Collins, 1998; Du Toit et al., 1998; Jacobsen et al., 1999). Another

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relevant property is the ability of probiotic bacteria to assimilate cholesterol (Dashkevicz & Feighner, 1989; Buck & Gilliland, 1994; Du Toit et al., 1998; Franz, Specht, Haberer, & Holzapfel, 2001). This has been linked to the bile salt deconjugation activity of some strains because of the enzyme bile salt hydrolase (BSH). The ability of Lactobacillus strains to adhere to the mucosal surfaces of the intestine and the subsequent long or short-term colonization has long been one of the most commonly encountered criteria for the selection of probiotic strains. 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, Amsellem, Servin, & Coconnier, 2002), although the underlying mechanism has not been clearly elucidated yet. The immunostimulatory effect of probiotic lactobacilli has also been examined in the past. Lactobacillus strains are generally good inducers of the pro-inflammatory IL-12, TNF-a and IFN-g, with fewer strains able to induce IL-10 production by immune system cells, such as peripheral blood mononuclear cells (PBMC) (Miettinen, Vuopio-Varkila, & Varkila, 1996; Miettinen et al., 1998; Hessle, Hanson, & Wold, 1999; Maassen et al., 2000). The aim of this study was to apply established in vitro tests to evaluate the probiotic potential of Lactobacillus strains isolated from dairy sources, and to select candidate probiotic strains that fulfill the established criteria and could therefore be potentially used as novel probiotic strains in the food industry.

2. Materials and methods 2.1. Bacterial strains and growth conditions A total of 29 Lactobacillus strains isolated from dairy products (and held at the ACA-DC Collection at the Agricultural University of Athens, Athens, Greece) were included in the present study (Table 1). Strains were stored at 80 1C in MRS broth (Biokar Diagnostics, Beauvais, France), supplemented with 20% (v/v) glycerol. For routine analysis, strains were subcultured twice in MRS broth (Biokar Diagnostics), for 18 h at 30 1C. For the antimicrobial production screening and the inhibition of pathogen adhesion to Caco-2 cells, pathogenic strains of clinical origin were included in this study. These were two Escherichia coli strains (CFA1 and C1845), one Salmonella typhimurium strain (SL1344) and four Helicobacter pylori strains (SS1, 069A, CCUG 38771, HPI137). They were kindly provided by Dr. Alain Servin (U510 INSERM, Paris, France) and Dr. Andreas Mentis (Hellenic Pasteur Institute, Athens, Greece). Enteric bacteria were routinely grown in BHI broth (Biokar Diagnostics) at 37 1C,

Table 1 Species and origin of the lactobacilli strains Strain

Origin

L. acidophilus ACA-DC 295 L. casei Shirota ACA-DC 6002 L. casei Imunitass ACA-DC 6003 L. paracasei subsp. paracasei ACA-DC 116 L. paracasei subsp. paracasei ACA-DC 117 L. paracasei subsp. paracasei ACA-DC 118 L. paracasei subsp. paracasei ACA-DC 119 L. paracasei subsp. paracasei ACA-DC 120 L. paracasei subsp. paracasei ACA-DC 126 L. paracasei subsp. paracasei ACA-DC 129 L. paracasei subsp. paracasei ACA-DC 130 L. paracasei subsp. paracasei ACA-DC 221 L. paracasei subsp. paracasei ACA-DC 233 L. paracasei subsp. paracasei ACA-DC 3304 L. paracasei subsp. paracasei ACA-DC 3334 L. paracasei subsp. paracasei ACA-DC 3335 L. paracasei subsp. paracasei ACA-DC 3343 L. paracasei subsp. paracasei ACA-DC 3344 L. paracasei subsp. paracasei ACA-DC 3345 L. paracasei subsp. tolerans ACA-DC 177 L. paracasei subsp. tolerans ACA-DC 196 L. paracasei subsp. tolerans ACA-DC 4037 L. paracasei subsp. tolerans ACA-DC 4038 L. plantarum ACA-DC 146 L. rhamnosus ACA-DC 112 Lactobacillus sp. ACA-DC 107 Lactobacillus sp. ACA-DC 108 Lactobacillus sp. ACA-DC 109 Lactobacillus sp. ACA-DC 6001

Raw cows’ milk Yakult Actimel (Danone) Feta brine Feta cheese Feta cheese Feta brine Raw cows’ milk Feta brine Feta brine Raw cows’milk Feta brine Feta cheese Raw cows’ milk Cheddar cheese Cheddar cheese Cheddar cheese Cheddar cheese Cheddar cheese Kaseri cheese Kaseri cheese Kaseri cheese Kaseri cheese Feta brine Galotyri cheese Raw cows’ milk Raw cows’ milk Raw cows’ milk Sour milk

while Helicobacter strains were cultured in Brucella broth (Beckton Dickinson, Sparks, USA), supplemented with 10% (v/v) inactivated horse serum (Oxoid, Basingstoke, UK), under anaerobic conditions (GasPak kit; Beckton Dickinson). Total viable counts of Lactobacillus strains were determined using MRS agar (Biokar Diagnostics) at 30 1C. E. coli and S. typhimurium strains were enumerated after 18 h on MacConkey agar (Becton Dickinson) at 37 1C. H. pylori strains were enumerated in Tryptic Soya Agar (Becton Dickinson), under anaerobic conditions (GasPak kit; Beckton Dickinson), at 37 1C for 48 h. 2.2. Survival under conditions simulating the human GI tract The resistance of the examined lactobacilli in a low pH environment was tested as previously described (Conway et al., 1987). Briefly, bacterial cells from overnight (18 h) lactobacilli cultures were harvested (10 000 g, 5 min, 4 1C), washed twice with PBS buffer, pH 7.2, before being resuspended in PBS solution, adjusted to pH 1 and pH 3. Initial populations ranged from 7.0 to 9.0 log cfu mL 1. Resistance was assessed in

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terms of viable colony counts and enumerated after incubation at 37 1C for 0, 1 and 3 h, reflecting the time spent by food in the stomach. Resistance of the lactobacilli to pepsin and pancreatin was tested as described previously (Charteris et al., 1998). Briefly, bacterial cells from overnight (18 h) lactobacilli cultures were harvested (10 000 g, 5 min, 4 1C), washed twice with PBS buffer, pH 7.2, before being resuspended either in PBS solution, pH 2, containing pepsin (3 mg mL 1; Sigma, St. Louis, USA), or in PBS solution, pH 8, containing pancreatin USP (1 mg mL 1; Sigma). Initial populations ranged from 8.2 to 9.0 log cfu mL 1. Resistance was assessed in terms of viable colony counts and enumerated after incubation at 37 1C for 0, 1 and 3 h with pepsin, and 0 and 4 h with pancreatin, reflecting the time spent by food in the stomach and small intestine, respectively. Tolerance to bile salts was tested at 37 1C by inoculation of fresh cultures in MRS broth (Biokar Diagnostics), enriched with 0.3% (w/v) Oxgall (Ox-Bile LP0055, Oxoid). Resistance was assessed in terms of viable colony counts, enumerated after incubation for 0 and 4 h, reflecting the time spent by food in the small intestine. For bile salt hydrolysis testing, fresh cultures were streaked on MRS agar (Biokar), enriched with 0.5% (w/v) taurodeoxycholic acid (TDCA, Sigma), as described previously (Dashkevicz & Feighner, 1989). The hydrolysis effect was indicated by different colony morphology (partial hydrolysis recorded as 1) from the control MRS plates, after 48 h of anaerobic incubation (GasPak kit; Beckton Dickinson) at 37 1C.

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H. pylori strains. Fresh overnight lactobacilli MRS culture supernatants (Biokar Diagnostics) were collected by centrifugation (15 000 g, 15 min, 4 1C), and adjusted to pH 6.5 or 4.5. The antimicrobial activity was screened using well diffusion, spot-on-lawn and microtitre plate assays. In the well diffusion and spot-on-lawn assays, 105 cfu of the pathogenic strains were incorporated into soft agar (1%, v/v) plates of BHI (Biokar Diagnostics) for the enteric pathogens and of TS agar (Oxoid) for the Helicobacter strains. Lactobacilli supernatant samples (50 mL) were pipetted into holes drilled into the agar (well diffusion assay) or spotted (10 mL) on the surface of the agar (spot-on-lawn assay). The plates were then incubated at 37 1C. Antimicrobial activity was recorded as growth free inhibition zones around the well or the spotted area. In the microtitre plate assay the reaction mixture comprised of 100 mL of the appropriate growth medium for the pathogen, 100 mL of the lactobacilli supernatant and 50 mL of pathogenic culture inoculum, diluted in quarter strength Ringer solution (Oxoid) to achieve a final population of 105 cfu in the well. A layer of 20 mL of sterile mineral oil was added on the surface of the wells in the Helicobacter plates to achieve anaerobic conditions. The plates were incubated at 37 1C and their OD was measured at 620 nm at regular time intervals for a period of 24 h. Antimicrobial activity was recorded as growth delay or a complete inhibition of growth of the pathogenic organisms. Appropriate controls were incorporated in each case, i.e. 100 mL of MRS (pH 6.5 and 4.5) or 100 mL of BHI or Brucella broth, instead of the supernatant.

2.3. Haemolytic activity and antibiotic resistance For testing haemolytic activity, fresh lactobacilli cultures were streaked on Columbia agar plates, containing 5% (w/v) human blood (Michopoulos S.A., Athens, Greece), and incubated for 48 h at 30 1C. Blood agar plates were examined for signs of b-haemolysis (clear zones around colonies), a-haemolysis (green-hued zones around colonies) or g-haemolysis (no zones around colonies). For testing antibiotic resistance, lactobacilli strains were inoculated (1%, v/v) in MRS broth supplemented with antibiotics (vancomycin, teicoplanin, chloramphenicol and tetracycline; Sigma) at various concentrations (2, 4, 8, 16, 32, 64 and 128 mg mL 1) and examined for growth (OD at 610 nm) following a 24 h incubation period at 30 1C. 2.4. Antimicrobial activity against Gram-negative pathogens All strains were tested for antimicrobial activity against two E. coli, one S. typhimurium and four

2.5. Stimulation of peripheral blood mononuclear cells (PBMCs) and cytokine determination Fresh human blood, obtained from eight healthy donors from the blood bank of the General Hospital of Lille (France) was diluted at 1:1 ratio with PBS-Ca (GIBCO, Paisley, UK), added over a layer of Ficoll (GIBCO) and centrifuged (400 g, 30 min, 20 1C). PBMCs formed an interphase ring layer in the serum and were aspirated carefully, resuspended to a final volume of 50 mL using PBS-Ca and washed three times (750 g, 10 min, 20 1C). PBMCs were subsequently resuspended using complete RPMI medium, supplemented with 10% (w/v) foetal calf serum inactivated at 56 1C for 30 min, 1% (w/v) L-glutamine, and gentamycin (30 mg mL 1) (all purchased from GIBCO). PBMCs were counted under the microscope, adjusted at a concentration of 2  106 cells mL 1 and distributed (1 mL) in 24-well tissue culture plates (Corning Inc., Fisher Scientific, Loughborough, Leicestershire, UK). Overnight (18 h) lactobacilli cultures were re-inoculated (5%, v/v) in fresh MRS broth (Biokar Diagnostics)

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and grown for 3 h. Bacterial cells were then harvested (3500 g, 10 min, 4 1C) and washed twice with PBS buffer, pH 7.2, before being resuspended in simple RPMI medium at a concentration of 2  109 cfu mL 1. From these suspensions 10 mL were transferred into the wells of the PBMC plates, which were incubated at 37 1C in a 5% CO2/95% air atmosphere. As negative controls, 10 mL of PBS buffer, pH 7.2, or 10 mL of RPMI medium were used in place of the lactobacilli suspension. L. salivarius Ls33 was used as a positive control for IL-10 induction, while L. plantarum 8826 as a positive control in the IL-12, TNF-a and IFN-g assays. After a 24 h incubation the well mixture was aspirated, centrifuged (4000 g, 10 min, 4 1C) and the supernatant was removed and stored at 20 1C. PBMC stimulation experiments were performed twice per blood donor. Quantitative determination of cytokines produced by the stimulation of PBMCs by lactic acid bacteria was effected using an ELISA kit (Pharmingen, BD Biosciences, Cowley, Oxford, UK) for IL-10, IL-12, IFN-g and TNF-a. The ELISA reaction was performed according to the instructions of the manufacturer. Basal cytokine levels in donors were also determined. Cytokine determination was performed in duplicate. 2.6. Adhesion to Caco-2 cells Caco-2 cells were routinely grown in DMEM, supplemented with 10% (v/v) foetal calf serum inactivated at 56 1C for 30 min, 1% (v/v) non-essential amino acids, 1% (v/v) L-glutamine and 20 mg mL 1 of streptomycin and penicillin (all purchased from GIBCO). Cells were incubated at 37 1C in a 10% CO2/90% air atmosphere. For the adhesion assay, monolayers of Caco-2 cells were prepared in six-well tissue culture plates (Corning Inc.), seeded (1 mL) at a concentration of 1.2  105 cells mL 1 inside the wells, and incubated for 15 days, with the culture medium changed daily. Lactobacilli MRS broth cultures (18 h, 10 mL) were harvested (3500 g, 10 min, 4 1C) and washed twice with 5 mL PBS buffer, pH 7.2. Cells were resuspended in 1 mL of PBS and then properly diluted in nonsupplemented DMEM (GIBCO) to achieve a concentration of 108 cfu mL 1. The growth medium in the six-well tissue culture plates of Caco-2 monolayers (15 days old) was aspirated and the cells washed twice with PBS. Subsequently, 1 mL of bacterial DMEM suspension was transferred onto the Caco-2 monolayers. The plates were incubated at 37 1C in a 10% CO2/90% air atmosphere for 90 min, and then the bacterial suspension was aspirated and the Caco-2 monolayers were washed twice with PBS, before 1 mL of Tween 80 (0.04%, w/v; Research Organics, Cleveland, USA) was added to detach the adhered bacterial cells. The bacterial suspension was then enumerated as described before. The adhesion of the

lactobacilli strains to Caco-2 cells was expressed as a percentage of the viable bacteria compared to their initial population in the DMEM suspension. E. coli TG-1 strain was used as a positive control, while L. plantarum V299 adh non-adhesive strain was used as a negative control. Adhesion experiments were performed in quadruplicate. The enumeration of the adhered lactobacilli cells was performed in duplicate. 2.7. Inhibition of pathogen adhesion to Caco-2 cells E. coli CFA1 and S. typhimurium SL1344 strains were grown overnight in BHI broth (Becton Dickinson) at 37 1C, harvested and washed with PBS buffer, pH 7.2 (3500 g, 10 min, 4 1C), and then resuspended in simple DMEM medium at a population of 108 cfu mL 1. Caco2 cells were challenged with lactobacilli strains as described earlier, and washed four times with PBS, after the completion of the lactobacilli adhesion. Caco-2 cells were then challenged with 1 mL of E. coli CFA1 and S. typhimurium SL1344 suspension, at 37 1C in a 10% CO2/ 90% air atmosphere for 60 and 90 min, respectively. Caco-2 cells were solubilised and the adhered pathogen populations were enumerated as described earlier. Adhesion experiments were performed in quadruplicate. The enumeration of the adhered pathogen populations was performed in duplicate.

3. Results and discussion The in vitro criteria used in our study for the selection of candidate probiotics have been described in previous studies and are referred to as selection guidelines by the FAO/WHO committee (Joint FAO/WHO Working Report, 2002). The in vitro screening of the survival of lactobacilli in simulated GI tract conditions may only have value in predicting the actual in vivo survival of a strain when consumed in a non-protected way. Strains embedded in a food matrix, for example, or consumed in an encapsulated form, may behave differently. Consequently, the in vitro test conditions for survival need to be adapted, according to the desired application for a particular candidate probiotic. Also, very little or no information is available on the in vivo behaviour of consumed bacteria in the GI tract. However, the survival tests proposed in the literature still have the potential to elucidate possible bacterial behaviour under adverse GI tract conditions, revealing whether high numbers of bacteria may remain stable, be reduced, or increase during passage through the GI tract. These observations could help to suggest minimal numbers of intake or point toward the need for protection measures (e.g., embedding in milk or other food matrices) and lead to better designed in vivo studies.

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For the selection of highly potent probiotic strains, safety and functionality properties such as antibiotic resistance, adhesion to intestinal cell lines, antimicrobial activity and inhibition of pathogenic adhesion, as well as immunomodulation potential, are highly important and should be studied using reliable in vitro screening methods. Performance of the strains, selected by the above protocols, should be confirmed through in vivo studies and/or clinical studies. The type of the in vivo study as well as the experimental parameters (e.g., mode of administration) can be determined most effectively by the outcome of the above in vitro test results. 3.1. Survival under conditions simulating the human GI tract All strains retained their viability even after 3 h of exposure to pH 3 (data not shown). On the contrary, after 1 h at pH 1, only six out of the 29 strains maintained some viability. These were L. paracasei subsp. paracasei ACA-DC 119 and 3345, L. paracasei subsp. tolerans ACA-DC 4037, L. plantarum ACA-DC

146, and Lactobacillus sp. ACA-DC 108 and 109 (Table 2). After 3 h exposure to pH 1, viable counts were determined only for Lactobacillus sp. ACA-DC 108 (4.0 log cfu mL 1; 0.04% survival) and ACA-DC 109 (3.6 log cfu mL 1; 0.008% survival). A wide variation in survival was observed when the strains were subjected to the pepsin solution at pH 2 (Table 2). After 1 h, highest survival was observed with L. casei Shirota ACA-DC 6002, L. casei Imunitass ACA-DC 6003 (o1.0 log cycle reduction). Strains L. paracasei subsp. paracasei ACA-DC 130, L. paracasei subsp. tolerans ACA-DC 196, L. rhamnosus ACA-DC 112, and Lactobacillus sp. ACA-DC 108 and 109 displayed reductions ranging between 1.0 and 2.3 log cycles, while the remaining strains displayed a loss of viability 42.5 log cycles. Eight strains did not survive at all. After 3 h of exposure to pepsin, the best survival was obtained with strains L. rhamnosus ACA-DC 112 and L. paracasei subsp. paracasei ACA-DC 130 (o2.0 log cycles reduction). The rest of the strains displayed loss of viability of 43 log cycles. Thirteen strains were completely inhibited (Table 2).

Table 2 Survivala of the lactobacilli strains at pH 1 and in the presence of pepsin at pH 2.0, expressed in log cfu mL Strain

L. acidophilus ACA-DC 295 L. casei Shirota ACA-DC 6002 L. casei Imunitass ACA-DC 6003 L. paracasei subsp. paracasei ACA-DC 116 L. paracasei subsp. paracasei ACA-DC 117 L. paracasei subsp. paracasei ACA-DC 118 L. paracasei subsp. paracasei ACA-DC 119 L. paracasei subsp. paracasei ACA-DC 120 L. paracasei subsp. paracasei ACA-DC 126 L. paracasei subsp. paracasei ACA-DC 129 L. paracasei subsp. paracasei ACA-DC 130 L. paracasei subsp. paracasei ACA-DC 221 L. paracasei subsp. paracasei ACA-DC 233 L. paracasei subsp. paracasei ACA-DC 3304 L. paracasei subsp. paracasei ACA-DC 3334 L. paracasei subsp. paracasei ACA-DC 3335 L. paracasei subsp. paracasei ACA-DC 3343 L. paracasei subsp. paracasei ACA-DC 3344 L. paracasei subsp. paracasei ACA-DC 3345 L. paracasei subsp. tolerans ACA-DC 177 L. paracasei subsp. tolerans ACA-DC 196 L. paracasei subsp. tolerans ACA-DC 4037 L. paracasei subsp. tolerans ACA-DC 4038 L. plantarum ACA-DC 146 L. rhamnosus ACA-DC 112 Lactobacillus sp. ACA-DC 107 Lactobacillus sp. ACA-DC 108 Lactobacillus sp. ACA-DC 109 Lactobacillus sp. ACA-DC 6001 a

193

pH 1

1

Pepsin at pH 2

0h

1h

0h

1h

3h

7.4 7.6 7.7 9.0 8.4 8.4 9.0 8.4 7.8 8.2 7.1 7.4 8.2 7.0 7.0 7.7 7.6 7.7 9.0 7.2 7.2 8.0 7.9 7.6 8.0 7.6 7.4 7.7 9.0

0 0 0 0 0 0 4.8 0 0 0 0 0 0 0 0 0 0 0 3.0 0 0 3.3 0 3.0 0 0 5.2 4.5 0

8.9 9.0 9.0 9.0 8.3 8.6 8.4 8.3 8.7 8.7 8.5 8.8 8.9 8.3 8.3 8.7 9.0 8.9 8.6 8.8 8.9 8.7 9.0 8.6 8.6 8.2 8.4 8.2 8.5

5.6 8.4 8.1 5.8 0 0 4.5 4.2 0 3.3 7.2 0 0 3.9 4.2 3.5 0 0 4.1 4.5 6.8 5.0 0 5.7 7.2 5.6 6.6 5.9 4.8

4.0 0 0 5.0 0 0 4.0 3.0 0 0 6.8 0 0 0 3.3 3.1 0 0 0 3.9 4.2 4.8 0 5.7 7.1 3.3 3.0 3.3 3.8

Values are means from three replicates, with standard deviation ranging from 0 to 0.8.

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their viability when exposed to pH values of 2.5–4.0, but displayed loss of viability at lower pH values (Conway et al., 1987; Du Toit et al., 1998; Jacobsen et al., 1999; Dunne & Mahony, 2001). Our findings on the viability of the lactobacilli in the presence of pepsin at pH 2 are also in agreement with existing literature data (Charteris et al., 1998; Fernandez, Boris, & Barbes, 2003). The combined effect of a pepsin-pH solution aims at simulating the gastric juice, although it is not clear whether the decrease of viability conferred by the pepsin solution at pH 2 was due to the enzyme alone, or in synergy with low acidity. It should be mentioned, however, that probiotic bacteria are mainly consumed in the presence of milk proteins. Milk proteins have a protective effect on the starters and thus support bacterial survival in the acidic environment of the stomach (Conway et al., 1987; Charteris et al., 1998; Fernandez et al., 2003). Also, the gastric juice itself may offer some degree of protection, when compared with low pH buffers (Conway et al., 1987). In this context, even strains not able to survive at pH 1 in vitro could

In contrast, all strains were resistant to pancreatin, as even after 4 h of exposure retained their viability with little (o1 log cycle) or no loss at all (data not shown). This was also the case when strains were incubated for 4 h in MRS broth in the presence of 0.3% (w/v) bile salts (data not shown). Regarding the bile salt hydrolase activity, 13 strains exhibited partial bile salt hydrolase activity, recorded as differentiated colony morphology on TDCA-MRS agar in comparison with the control MRS agar plates (Table 3). The pH in human stomach ranges from 1, during fasting, to 4.5, after a meal, and food ingestion can take up to 3 h. Since Lactobacillus strains are known to survive at pH 4.6, which is the common final acidity of many fermented dairy products, lower pH values (1 and 3) were examined. Although all of the examined strains were completely resistant to pH 3 even after 3 h of exposure, most of the strains displayed loss of viability when exposed to pH 1 for 1 h. These results are in agreement with those obtained from previous similar studies, where Lactobacillus strains were able to retain

Table 3 BSH activity, haemolytic activity, tetracycline resistance (MIC, mg mL 1) and % adhesion (7SEM) on Caco-2 cells of the lactobacilli strains Strain

BSHa

Haemolysisa

Tetracyclinea MIC

% Adhesionb on Caco-2

L. acidophilus ACA-DC 295 L. casei Shirota ACA-DC 6002 L. casei Imunitass ACA-DC 6003 L. paracasei subsp. paracasei ACA-DC 116 L. paracasei subsp. paracasei ACA-DC 117 L. paracasei subsp. paracasei ACA-DC 118 L. paracasei subsp. paracasei ACA-DC 119 L. paracasei subsp. paracasei ACA-DC 120 L. paracasei subsp. paracasei ACA-DC 126 L. paracasei subsp. paracasei ACA-DC 129 L. paracasei subsp. paracasei ACA-DC 130 L. paracasei subsp. paracasei ACA-DC 221 L. paracasei subsp. paracasei ACA-DC 233 L. paracasei subsp. paracasei ACA-DC 3304 L. paracasei subsp. paracasei ACA-DC 3334 L. paracasei subsp. paracasei ACA-DC 3335 L. paracasei subsp. paracasei ACA-DC 3343 L. paracasei subsp. paracasei ACA-DC 3344 L. paracasei subsp. paracasei ACA-DC 3345 L. paracasei subsp. tolerans ACA-DC 177 L. paracasei subsp. tolerans ACA-DC 196 L. paracasei subsp. tolerans ACA-DC 4037 L. paracasei subsp. tolerans ACA-DC 4038 L. plantarum ACA-DC 146 L. rhamnosus ACA-DC 112 Lactobacillus sp. ACA-DC 107 Lactobacillus sp. ACA-DC 108 Lactobacillus sp. ACA-DC 109 Lactobacillus sp. ACA-DC 6001

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

a g g g g g g g a g g g g g g g g g g g g g g g a g a g g

4 8 8 o2 o2 o2 o2 o2 42 o2 o2 o2 o2 64 32 32 64 64 16 4 4 32 8 o2 o2 8 8 8 o2

0.270.03 2.470.4 1.770.3 6.670.7 0.470.1 0.970.1 2.470.3 0.170.02 0.270.01 1.970.3 2.170.3 13.171.5 4.670.6 0.970.2 13.872.4 11.870.9 6.570.5 2.070.3 2.870.4 1.170.1 9.471.2 0.870.1 0.970.1 25.573.1 0.270.03 0.970.1 4.470.3 1.370.4 0.470.1

a

Experiments were performed in triplicate. Adhesion to Caco-2 cells was done in quadruplicate and enumeration of the adhered lactobacilli cells in duplicate; adhesion of E. coli TG-1 (positive control) was 48.3% (73.8); adhesion of the non-adhering L. plantarum V299 adh strain (negative control) was 0.6% (70.1). c 0, no hydrolysis; 1, partial hydrolysis. b

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exhibit substantial viability when consumed as starters or adjuncts in a matrix of fermented milk. In contrast to pepsin, most strains examined in this study could survive well in a pancreatin solution at pH 8.0 or in the presence of bile salts (0.3%, w/v), simulating the near neutral small intestine environment. Most studies so far have shown that the majority of the strains survived well under such conditions, suggesting a potential recuperation of the initial levels during the passage of the small intestine (Charteris et al., 1998; Du Toit et al., 1998; Jacobsen et al., 1999; Fernandez et al., 2003). These considerations could explain why, for instance, L. casei Shirota ACA-DC 6002 is completely inhibited after 1 h exposure at pH 1 and 3 h exposure to pepsin, but, in another study by our group, survived the passage of the mouse GI tract in very high numbers (Sgouras et al., 2004). In the present study, only some strains exhibited partial bile salt hydrolysis. Bile salt hydrolase (BSH) activity deconjugates bile salts, which are then readily excreted from the GI tract. Hypothetically, this increases the demand on cholesterol for the de novo synthesis of bile salts, and thus leads to lower blood serum cholesterol levels (Dashkevicz & Feighner, 1989; Tanaka, Doesburg, Iwasaki, & Mierau, 1999; Franz et al., 2001; Pereira, McCartney, & Gibson, 2003). However, it should be mentioned that the presence of BSH activity in lactic acid bacteria is controversial, as BSH activity might be detrimental and undesirable (Brody, 1999).

resistant to vancomycin and teicoplanin. This is confirmed by many other studies (Felten, Barreau, Bizet, Lagrange, & Philippon, 1999; Temmerman, Pot, Huys, & Swings, 2002; Danielsen & Wind, 2003). Previous studies also confirm the generally lower resistance of the lactobacilli species studied here towards tetracycline and chloramphenicol (Kattla, Kruse, Johnsen, & Herikstad, 2001; Temmerman et al., 2002; Choi, Chang, Kim, & So, 2003).

3.2. Haemolytic activity and antibiotic resistance

Most of the strains tested (20 out of 29) had low adhesion to Caco-2 cells (o4%, Table 3). Nine strains were found to adhere to Caco-2 cells with percentages ranging from 4.4% to 25.5%, with highest values obtained for L. plantarum ACA-DC 146 (25.5%), and L. paracasei subsp. paracasei strains ACA-DC 221, 3334 and 3335 (13.1, 13.8 and 11.8%, respectively). Almost 50% of the E. coli TG-1 cells adhered to Caco-2 cells (48.3%), while the non-adhering L. plantarum V299 adh strain resulted in an adhesion percentage of only 0.6%. Adhesion of lactobacilli has been claimed to be essential for the exertion of a beneficial (probiotic) effect in the large intestine. Previous studies have reported adhesive strains such as L. johnsonii La1, L. rhamnosus GG, as well as L. casei Shirota and L. casei Imunitass (Tuomola & Salminen, 1998; Juntunen, Kirjavainen, Ouwenhand, Salminen, & Isolauri, 2001; Ouwenhand, Tuomola, Tolkko, & Salminen, 2001). In our study, where viable adherent bacteria were measured by enumeration, a few strains displayed high adhesion to Caco-2 cells. L. casei Shirota ACA-DC 6002 adhesion was measured at 2.4%, confirming the adhesion observed for that particular strain in other studies (Tuomola & Salminen, 1998; Juntunen et al., 2001; Ouwenhand et al., 2001).

None of the strains examined exhibited b-haemolytic activity when grown in Columbia human blood agar. Most of the strains (25 strains) were g-haemolytic (i.e. no haemolysis), while four strains exhibited a-haemolysis (Table 3). Concerning antibiotic resistance, the majority of the strains (25 strains) were found to be resistant to vancomycin and teicoplanin, with MICs higher than 128 mg mL 1. Only four strains, namely L. paracasei subsp. paracasei ACA-DC 130, L. rhamnosus ACA-DC 112, and Lactobacillus sp. ACA-DC 107 and ACA-DC 109 were sensitive (MICsp4 mg mL 1 ). In the case of chloramphenicol, MICs of 8 mg mL 1 were determined for all strains, apart from L. paracasei subsp. paracasei strains ACA-DC 3304, 3334 and 3335 with MIC of 16 mg mL 1, and L. paracasei subsp. paracasei strains ACA-DC 3343 and 3344 with MIC of 64 mg mL 1. A broader variation was observed in the case of tetracycline, with the last group of strains again exhibiting the highest resistance (Table 3). It is generally known that some Lactobacillus species display resistance to glycopeptide antibiotics (Swenson, Facklam, & Thornsberry, 1990). In our study, the majority of the lactobacilli examined were found to be

3.3. Antimicrobial activity against Gram-negative pathogens None of the supernatants of the lactobacilli strains, whether at pH 6.5 or 4.5, inhibited the growth of the pathogenic strains tested, using either the spot-on-lawn or the well diffusion assay. In the microtitre plate assay, the growth of all pathogens was inhibited by the supernatants at pH 4.5, as well as by the respective control (MRS at pH 4.5). Since no inhibition at all was observed when the pathogens were grown in the presence of near-neutral supernatants (pH 6.5) nor with the appropriate control medium (MRS at pH 6.5), inhibition effects cannot be explained by bacteriocin action and are most probably due to the production of organic acids along with the low pH. 3.4. Adhesion of lactobacilli to Caco-2 cells

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3.5. Stimulation of peripheral blood mononuclear cells (PBMCs) and cytokine production Eight lactobacilli strains, selected on the basis of their performance in the in vitro tests described above were examined for cytokine production by stimulated PBMCs (Table 4). The selection included strains that displayed favourable performance in the pH 1 and pepsin at pH 2 survival assays, as well as in the bile salt hydrolysis and Caco-2 attachment assays. The haemolytic activity and antibiotic resistance assays were not considered, since no strains exhibiting b-haemolysis or unexpected resistance to antibiotics were found. In addition, survival at pH 3, in the presence of pancreatin or bile salts did not differentiate between bacteria in the in vitro survival assays. Strain L. plantarum ACA-DC 146 was the highest inducer of IL-12 (1538 pg mL 1), TNF-a (163208 pg mL 1) and IFN-g (72937 pg mL 1). Additionally, L. paracasei subsp. tolerans ACA-DC 4037 also induced considerable levels of all three pro-inflammatory cytokines IL-12, IFN-g and TNF-a (1018, 41004 and 142742 pg mL 1, respectively), with elevated levels of IL-10 as well (723 pg mL 1). However, the highest secretion of IL-10 (1225 pg mL 1) was displayed when PBMCs were incubated with strain L. casei Shirota ACA-DC 6002, which also induced notable levels of TNF-a secretion (105971 pg mL 1). In the case of IL-12, strains Lactobacillus sp. ACA-DC 108 and L. paracasei subsp. tolerans ACA-DC 196 were noteworthy (653 and 922 pg mL 1, respectively). For IFN-g, high secretion levels were also observed with L. paracasei subsp. paracasei ACA-DC 3334 (53231 pg mL 1). Finally, high secretion levels for TNF-a were obtained with strain L. paracasei subsp. paracasei ACA-DC 221 (141244 pg mL 1). Immune modulation by probiotics is presumed to be one of the main mechanisms of probiotic action in human health, explaining reported effects on inflammatory bowel diseases and allergies (Kopp-Hoolithan, 2001; Cross, 2002; Mercennier et al., 2002; Guarner & Malagelada, 2003). The Th-1 group cytokine IL-12

Table 4 Cytokine secretion levelsa (pg mL

1

reduces the specific immunological response of the Th-2 pathway, as well as the IgE secretion in mice (Matsuzaki et al., 1998). Also, secretion of Th-2 cytokines plays a major role in the perpetuation of the immunological responses in allergic diseases (Pochard et al., 2002). IL12 can also induce secretion of IFN-g, which can activate specific immune responses, and has reported anti-tumour activity (Murosaki, Muroyama, Yamamoto, & Yoshikai, 2000). Specific Lactobacillus strains have been described as good inducers of IL-12, IFN-g and TNF-a (Miettinen et al., 1996, 1998; Hessle et al., 1999; Maassen et al., 2000). In our study, L. plantarum ACADC 146 and L. paracasei subsp. tolerans ACA-DC 4037 induced high levels of the Th-1 cytokines IL-12, TNF-a and IFN-g, suggesting a pro-inflammatory reaction. IL-10 is an anti-inflammatory regulatory cytokine of the Th-2 pathway and IL-10 inducing strains or genetically engineered strains excreting murine IL-10 have been applied with great success in mice colitis models (Madsen, Doyle, Jewell, Tavernini, & Fedorak, 1999; Steidler, 2002). The lack of IL-10, on the other hand, in IL-10 knockout mice leads to the development of colitis (Berg et al., 2002). Lactobacillus strains inducing strong IL-10 secretion from PBMCs are rare, but have been reported previously and included mainly strains of L. casei (Miettinen et al., 1996; Maassen et al., 2000). In our study, L. casei Shirota ACA-DC 6002 was found to induce considerable levels of IL-10 from PBMCs. This may suggest a possible anti-inflammatory regulation of immune system components for the particular probiotic. The relevance of the in vitro measured immunomodulatory activity of these lactic acid bacteria in an in vivo system needs to be established. L. casei Shirota ACA-DC 6002 may be applied in a murine colitis model, while L. plantarum ACA-DC 146 or L. paracasei subsp. tolerans ACA-DC 4037 are good candidates for application in models of allergy, as they induce high level of IL-12, which plays a pivotal role in the control of allergic disease development.

7SEM) from PBMCs stimulated with lactobacilli

Strain

IL 10

IL 12

TNFa

IFN g

L. casei Shirota ACA-DC 6002 L. paracasei subsp. paracasei ACA-DC 116 L. paracasei subsp. paracasei ACA-DC 221 L. paracasei subsp. paracasei ACA-DC 3334 L. paracasei subsp. tolerans ACA-DC 196 L. paracasei subsp. tolerans ACA-DC 4037 L. plantarum ACA-DC 146 Lactobacillus sp. ACA-DC 108

12257132 971 157712 147713 252729 723756 416757 3676

448756 1072 5374 448756 922781 10187234 15387167 653744

105971712459 2320273205 141244715850 7967079023 3250074238 142742714340 163208717340 7932479486

2157974280 396971200 1937773265 5323177356 16537234 4100475600 7293779860 2410373256

a

PBMC stimulation experiments were performed twice per blood donor (eight donors). Cytokine determination was performed in duplicate. Mean basal cytokine levels (pg mL 17SEM) in donors were 1075 for IL-10 and IL-12, 23507860 for TNF-a and 8907360 for IFN-g.

ARTICLE IN PRESS P.A. Maragkoudakis et al. / International Dairy Journal 16 (2006) 189–199 Table 5 Inhibition of pathogen adhesiona to Caco-2 cells pre-treated with lactobacilli, expressed as % (7SEM) decrease of pathogen adhesion Strain

E. coli CFA1

S. typhimurium SL1344

L. casei Shirota ACA-DC 6002 L. paracasei subsp. paracasei ACA-DC 221 L. paracasei subsp. tolerans ACA-DC 4037 L. plantarum ACA-DC 146

371 4873

4075 2573

3573

2873

4174

1472

a

Adhesion experiments were performed in quadruples and enumeration of the adhered pathogen cells was performed in duplicate.

3.6. Inhibition of pathogen adhesion to Caco-2 cells Four lactobacilli strains (Table 5) were used in the adhesion inhibition experiment. Strains were chosen on the basis of their adhesion ability to Caco-2 cells (L. plantarum ACA-DC 146, L. paracasei subsp. paracasei ACA-DC 221) and their immunostimulation of PBMC cells (L. plantarum ACA-DC 146, L. paracasei subsp. tolerans ACA-DC 4037 and L. casei Shirota ACA-DC 6002). The adhesion of E. coli CFA1 and S. typhimurium SL1344 to Caco-2 cells was found to be 13 and 8%, respectively. Adhesion of E. coli CFA1 was reduced by 35%, 41% and 48%, when the Caco-2 cells were previously challenged with strains L. paracasei subsp. tolerans ACA-DC 4037, L. plantarum ACA-DC 146 and L. paracasei subsp. paracasei ACA-DC 221, respectively (Table 5). In contrast, L. casei Shirota ACA-DC 6002 did not affect the binding of E. coli CFA1, resulting in only 3% inhibition of adhesion. As far as S. typhimurium SL1344 is concerned, L. paracasei subsp. paracasei ACA-DC 221 and L. paracasei subsp. tolerans ACA-DC 4037 reduced adhesion to Caco-2 cells by 25% and 28%, respectively, while L. casei Shirota ACA-DC 6002 was more effective, as it reduced adhesion of the pathogen by 40% (Table 5). L. plantarum ACA-DC 146 only reduced pathogen adhesion by 14%. The in vitro inhibition of Gram-negative pathogens by probiotic strains, but also the inhibition of pathogen adhesion to eukaryotic cell lines, has already been reported for strains such as L. johnsonii La1, Bifidobacterium CA1 and F9, and L. acidophilus LB (BernetCamard et al., 1997; Cocconier, Lievin, Lorrot, & Servin, 2000; Lievin et al., 2000; Lievin-Le Moal et al., 2002). In many cases, direct inhibitory activity has been linked to possible bacteriocin production by the probiotic strains and/or to their competitive adherence to epithelial cells, resulting in the inability of the pathogen to bind to its normal attachment site (Ouwenhand et al., 2001). As no bacteriocin activity

197

was observed in vitro, the latter mechanism would be probable in our study, where the adhesion of both pathogenic E. coli and S. typhimurium strains to Caco-2 cells was reduced even up to 50%. Strains with higher adhesive ability might well occupy common adhesion sites and thus prevent the further adhesion of the pathogenic bacterium. On the other hand, even the weak binding of a Lactobacillus strain on a Caco-2 cell could stimulate cellular mechanisms, which may lead to increased colonisation resistance. Further studies are required to elucidate the adhesive mechanism and the inhibition of pathogen adhesion observed in this study. To conclude, in this study the strains L. casei Shirota ACA-DC 6002, L. plantarum ACA-DC 146 and L. paracasei subsp. tolerans ACA-DC 4037 were found in vitro to possess desirable probiotic properties. These strains are good candidates for further investigation in in vivo studies to elucidate their potential health benefits and their application as novel probiotic strains in the food industry.

Acknowledgements The present work was financially supported by the Greek General Secretariat of Research and Technology (EPET II, Project 97-DIATRO-26). Petros Maragkoudakis would like to express his thanks to the Marie Curie Programme (Project type PHD20, HPMT-GH01-00275-02) of the Research-Directorate General of the European Commission for financial support.

References Aiba, Y., Suzuki, N., Kabir, A. M. A., Tagaki, A., & Koga, Y. (1998). Lactic acid mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarious as a probiotic in a gnotobiotic murine model. American Journal of Gastroenterology, 93, 2097–2101. Berg, D. J., Zhang, J., Weinstock, J. V., Ismail, H. F., Earle, K. A., Alila, H., Pamukcu, R., Moore, S., & Lynch, G. (2002). Rapid development of colitis in NSAID-treated IL-10 deficient mice. Gastroenterology, 123, 1527–1542. Bernet-Camard, M. F., Lievin, V., Brassart, D., Neeser, J. R., Servin, A. L., & Hudault, S. (1997). The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo. Applied Environmental Microbiology, 63, 2747–2753. Brody, T. (1999). Digestion and absorption. In Nutritional Biochemistry (pp. 57–131). San Diego: Academic Press. Buck, L. M., & Gilliland, S. E. (1994). Comparison of freshly isolated strains of Lactobacillus acidophilus of human intestinal origin for ability to assimilate cholesterol during growth. Journal of Dairy Science, 77, 2925–2933. Charteris, W. P., Kelly, P. M., Morelli, L., & Collins, J. K. (1998). Development of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. Journal of Applied Microbiology, 84, 759–768.

ARTICLE IN PRESS 198

P.A. Maragkoudakis et al. / International Dairy Journal 16 (2006) 189–199

Choi, Y. S., Chang, C. E., Kim, S. C., & So, J. S. (2003). Antimicrobial susceptibility and strain prevalence of Korean vaginal Lactobacillus sp. Anaerobe, 9, 277–280. Cocconier, M. H., Lievin, V., Lorrot, M., & Servin, A. L. (2000). Antagonistic activity of Lactobacillus acidophilus LB against intracellular Salmonella enterica serovar typhimurium infecting human enterocyte-like Caco-2/TC-7 cells. Applied and Environmental Microbiology, 66, 1152–1157. Conway, P. L., Gorbach, S. L., & Goldin, B. R. (1987). Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. Journal of Dairy Science, 70, 1–12. Cross, M. L. (2002). Immunoregulation by probiotic lactobacilli: pro Th-1 signals and their relevance to human health. Clinical and Applied Immunology Reviews, 3, 115–125. Danielsen, M., & Wind, A. (2003). Susceptibility of Lactobacillus sp. to antimicrobial agents. International Journal of Food Microbiology, 82, 1–11. Dashkevicz, M. P., & Feighner, S. D. (1989). Development of a differential medium for bile salt hydrolase active Lactobacillus sp. Applied and Environmental Microbiology, 55, 11–16. Du Toit, M., Franz, C. M. A. P., Dicks, L. M. T., Shillinger, U., Haberer, P., Warlies, B., Ahrens, F., & Holzapfel, W. H. (1998). Characterization and selection of probiotic lactobacilli for a preliminary mini pig feeding trial and their effect on serum cholesterol levels, feces pH and feces moisture content. International Journal of Food Microbiology, 40, 93–104. Dunne, C. O., & Mahony, L. (2001). In vitro selection criteria for probiotic bacteria of human origin: correlation in vivo findings. American Journal of Clinical Nutrition, 73, 386–392. Felten, A., Barreau, C., Bizet, C., Lagrange, P. H., & Philippon, A. (1999). Lactobacillus species identification, H2O2 production, and antibiotic resistance and correlation with human clinical status. Journal of Applied Microbiology, 37, 729–733. Fernandez, M. F., Boris, S., & Barbes, C. (2003). Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. Journal of Applied Microbiology, 94, 449–455. Franz, C. M. A. P., Specht, I., Haberer, P., & Holzapfel, W. H. (2001). Bile salt hydrolase activity of enterococci isolated from food screening and quantitative determination. Journal of Food Protection, 64, 725–729. Gilliland, S. E., & Kim, H. S. (1984). Effect of viable starter culture bacteria in yogurt on lactose utilization in humans. Journal of Dairy Science, 67, 1–6. Guarner, F., & Shaafsma, G. J. (1998). Probiotics. International Journal Food Microbiology, 39, 237–238. Guarner, F., & Malagelada, J. R. (2003). Gut flora in health and disease. Lancet, 361, 512–519. Hammilton-Miller, J. M. T. (2003). The role of probiotics in the treatment and prevention of H. pylori infection. International Journal of Antimicrobial Agents, 22, 360–366. Hessle, C., Hanson, A., & Wold, A. E. (1999). Lactobacilli from human gastrointestinal mucosa are srong stimulators of IL-12 production. Clinical Experimental Immunology, 116, 276–282. Hirayama, K., & Rafter, J. (2000). The role of probiotic bacteria in cancer prevention. Microbes and Infection, 2, 681–686. Hudault, S., Lievin, V., Bernet-Camard, M. F., & Servin, A. L. (1997). Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (Strain GG) against Salmonella typhimurium C5 infection. Applied and Environmental Microbiology, 63, 513–518. Jacobsen, C. N., Roesnfeldt Nielsen, A. E., Moller, P. L., Michaelsen, K. F., Paerregaard, A., Sandstrom, B., Tvede, M., & Jacobsen, M. (1999). Screening of probiotic activities of forty-seven strains of Lactobacillus sp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Applied and Environmental Microbiology, 65, 4949–4956.

Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food, London, Ontario, Canada, April 30 and May 1, 2002. Juntunen, M., Kirjavainen, P. V., Ouwenhand, A. C., Salminen, S. J., & Isolauri, E. (2001). Adherence of probiotic bacteria to human intestinal mucus in healthy infants during rotavirus infection. Clinical and Diagnostic Laboratory Immunology, 8, 293–296. Kattla, A. K., Kruse, H., Johnsen, G., & Herikstad, H. (2001). Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. International Journal of Food Microbiology, 67, 147–152. Kopp-Hoolithan, L. (2001). Prophylactic and therapeutic uses of probiotics: A review. Journal of the American Dietetic Association, 101, 229–238. Lievin, V., Peiffer, I., Hudault, S., Rochart, D., Brassart, D., Neeser, J. R., & Servin, A. L. (2000). Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity. Gut, 47, 646–652. Lievin-Le Moal, V., Amsellem, R., Servin, A. L., & Coconnier, M. H. (2002). Lactobacillus acidophilus (strain LB) from resident human adult gastrointestinal microflora exerts activity against brush border damage promoted by a diarrhoeagenic Escherichia coli in human enterocyte-like cells. Gut, 50, 803–811. Maassen, C. B. M., van Holten-Neelen, C., Balk, F., den BakGlashouwer, H. M., Leer, R. J., Laman, J. D., Boersma, W. J. A., & Claassen, E. (2000). Strain-dependent induction of cytokine profiles in the gut by orally administered Lactobacillus strains. Vaccine, 18, 2613–2623. Madsen, K. L., Doyle, J. S., Jewell, L. D., Tavernini, M. M., & Fedorak, R. N. (1999). Lactobacillus species prevents colitis in Interleukin 10 gene-deficient mice. Gastroenterology, 116, 1107–1114. Matsuzaki, T., Yamazaki, R., Hashimoto, S., & Yokokura, T. (1998). The effect of oral feeding of Lactobacillus casei strain Shirota on immunoglobulin E production in mice. Journal of Dairy Science, 81, 48–53. Mercennier, A., Pavan, S., & Pot, B. (2002). Probiotics as biotherapeutic agents: present knowledge and future prospects. Current Pharmaceutical Design, 8, 99–110. Miettinen, M., Matikainen, S., Vuopio-Varkila, J., Pirhonen, J., Varkila, K., Kurimoto, M., & Julkunen, I. (1998). Lactobacilli and Streptococci induce Interleukin-12 (IL-12), IL-18 and Gamma Interferon production in human peripheral blood mononuclear cells. Infection and Immunity, 66, 6058–6062. Miettinen, M., Vuopio-Varkila, J., & Varkila, K. (1996). Production of human tumor necrosis factor alpha, Interleukin-6, and Interleukin 10 is induced by lactic acid bacteria. Infection and Immunity, 64, 5403–5405. Murosaki, S., Muroyama, K., Yamamoto, Y., & Yoshikai, Y. (2000). Antitumor effect of heat killed Lactobacillus plantarum L-137 through restoration of impaired interleukin-12 production in tumor-bearing mice. Cancer Immunology and Immunotherapy, 49, 157–164. Ouwenhand, A. C., Tuomola, E. M., Tolkko, S., & Salminen, S. (2001). Assessment of adhesion properties of novel probiotic strains to human intestinal mucus. International Journal of Food Microbiology, 64, 119–126. Pereira, D. I., McCartney, A. L., & Gibson, G. R. (2003). An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties. Applied Environmental Microbiology, 69, 4743–5472. Pochard, P., Gosset, P., Grangette, C., Andre, C., Tonnel, A. B., Pestel, J., & Mercennier, A. (2002). Lactic acid bacteria inhibit Th2 cytokine production by mononuclear cells in allergic patients. Journal of Allergy and Clinical Immunology, 110, 617–623. Sgouras, D., Maragkoudakis, P. A., Petraki, K., Martinez-Gonzalez, B., Eriotou, E., Michopoulos, S., Kalantzopoulos, G., Tsakalidou, E.,

ARTICLE IN PRESS P.A. Maragkoudakis et al. / International Dairy Journal 16 (2006) 189–199 & Mentis, A. (2004). In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota. Applied and Environmental Microbiology, 70, 518–526. Steidler, L. (2002). In situ delivery of cytokines by genetically engineered Lactococcus lactis. Antonie van Leewenhoek, 82, 323–331. Swenson, J. M., Facklam, R. R., & Thornsberry, C. (1990). Antimicrobial susceptibility of vancomycin resistant Leuconostoc, Pediococcus and Lactobacillus species. Antimicrobial Agents and Chemotherapy, 34, 543–547.

199

Tanaka, H., Doesburg, K., Iwasaki, T., & Mierau, I. (1999). Screening of lactic acid bacteria for bile salt hydrolase activity. Journal of Dairy Science, 82, 2530–2535. Temmerman, R., Pot, B., Huys, G., & Swings, J. (2002). Identification and antibiotic susceptibility of bacterial isolates from probiotic products. International Journal of Food Microbiology, 81, 1–10. Tuomola, E. M., & Salminen, S. J. (1998). Adhesion of some probiotic and dairy Lactobacillus strains to Caco-2 cell cultures. International Journal of Food Microbiology, 41, 45–51.