In vitro and in vivo gastrointestinal survival, antibiotic susceptibility and genetic identification of Propionibacterium freudenreichii ssp. shermanii JS

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International Dairy Journal 18 (2008) 271–278 www.elsevier.com/locate/idairyj

In vitro and in vivo gastrointestinal survival, antibiotic susceptibility and genetic identification of Propionibacterium freudenreichii ssp. shermanii JS Tarja Suomalainena,b,, Pia Sigvart-Mattilaa, Jaana Ma¨tto¨b,1, Soile Tynkkynena a

Valio Ltd, R&D, Helsinki, Finland VTT Technical Research Centre of Finland, Espoo, Finland

b

Received 26 March 2007; accepted 2 September 2007

Abstract The in vitro tolerance of Propionibacterium freudenreichii strains to low pH and high bile concentration, their antibiotic susceptibility and enzyme activity profiles were assessed. The gastrointestinal survival of the P. freudenreichii ssp. shermanii JS strain was also examined when administered to healthy adult volunteers in whey-based fruit juice. For tracking the strain JS in faecal samples, a genetic identification method for the strain was developed. No loss of viability was observed in any of the P. freudenreichii strains during the three-hour treatment at pH 4 or 3, whereas at pH 2 the cell counts of all strains decreased remarkably. Bile salt at 0.5% concentration had no apparent effect on the viability of the strains. No atypical antibiotic resistance was detected. P. freudenreichii ssp. shermanii JS strain was recovered in high numbers in faecal samples during the consumption of whey-based fruit juice enriched with the JS strain, and with few exceptions, it was not detected after the 2-week follow-up period. r 2007 Elsevier Ltd. All rights reserved. Keywords: Dairy propionibacteria; Probiotics; Gastric and intestinal tolerance

1. Introduction Several species of Lactobacillus and Bifidobacterium are used as probiotics with a number of beneficial health promoting effects documented for them, such as prophylactic and therapeutic uses in allergy and gastrointestinal disorders (Andersson et al., 2001 Andersson, Asp, Bruce, Roos, Wadstro¨m, & Wold, 2001; Ouwehand, Salminen, & Isolauri, 2002a). The growing use of probiotics in functional foods has led to numerous research programs and a search for novel probiotic candidates including representatives from other genera than Lactobacillus and Bifidobacterium. The beneficial health promoting probiotic effects are strain specific (Fooks & Gibson, 2002). Therefore, while Corresponding author. Present address: Verso Finland Ltd, Viikinkaari 6, FIN-00790 Helsinki, Finland. Tel.: +358 40 5497117; fax: +358 9 3193 6322. E-mail address: tarja.suomalainen@versofinland.fi (T. Suomalainen). 1 Present address: Finnish Red Cross, Blood Service, Helsinki, Finland.

0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.09.004

selecting new candidates for probiotic applications, functionality and technological characteristics should be tested separately for each strain. The Food and Agricultural Organization/World Health Organization (FAO/WHO) working group has provided guidelines and recommendations for the evaluation of probiotics in food (FAO/WHO, 2002). The guidelines include specifications for identification methods and require the assessment of safety characteristics such as the origin and lack of atypical antibiotic resistances in the strain. The functionality of a probiotic is ascertained then by in vivo tests including tolerance to gastric conditions, adherence to mucus or human epithelial cells and inhibition of harmful pathogenic bacteria. Finally, the physiological efficiency should be proven in well designed human studies. Dairy propionibacteria have traditionally been used as starters in Swiss-type cheeses (Kerjean et al., 2000). Besides their technological application, propionibacteria have been introduced as potential probiotics (Perez-Chaia, Zarate, & Oliver, 1999). Propionibacteria have several metabolic

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functions that may contribute to their possible role as probiotics, such as production of bacteriocins (Holo et al., 2002) and vitamin B12 (Hugenholtz, Hunik, Santos, & Smid, 2002). In addition, propionate produced by propionibacteria may enhance absorption of calcium (Trinidad, Wolever, & Thompson, 1996) and iron (Bougle et al., 2002) in the gut. Propionate together with acetate has also been shown to induce in vitro apoptosis in human adenocarsinoma cells, which might help against colon cancer development (Jan et al., 2002). Interestingly, propionibacteria are able to modulate the intestinal microbiota by enhancing growth of bifidobacteria (Bougle, Roland, Lebeurrier, & Arhan, 1999; Hojo et al., 2002; Kaneko, Mori, Iwata, & Meguro, 1994; Satomi, Kurihara, Isawa, Mori, & Kaneko, 1999). Kaneko et al. (1994) have isolated and characterized an aminocarboxynaphthoquinone (ACNQ) compound produced by Propionibacterium freudenreichii ET-3 which stimulated the growth of selected bifidobacteria strains in vitro. The bifidogenic effect of the compound was verified in vivo in healthy adult volunteers (Hojo et al., 2002; Satomi et al., 1999). Similarly, Bougle et al. (1999) have shown that some dairy P. freudenreichii strains increase bifidobacteria counts in adults. In addition to the bifidogenic effect, propionibacteria have also been reported to reduce mutagen-producing faecal enzyme activities (Perez-Chaia et al., 1999), and to stimulate the immune system (Alvarez et al., 1996 Alvarez, Medici, Vintini, Oliver, de Ruiz Holgado, & Perdigon, 1996). P. freudenreichii ssp. shermanii JS was originally isolated from Swiss-type cheese and has been used for decades as a starter culture for Jarlsberg cheese in Finland. Besides the starter use, the strain has been combined with Lactobacillus rhamnosus LC705 in a protective culture, BioprofitTM, which is used in biopreservation of fermented foods (Suomalainen & Ma¨yra¨-Ma¨kinen, 1999). Several interesting probiotic characteristics have been reported for P. freudenreichii ssp. shermanii JS. The strain adheres well both on mucus and Caco-2-cells (Lehto & Salminen, 1997; Ouwehand, Suomalainen, To¨lkko¨, & Salminen, 2002b) and has an excellent mycotoxin binding capacity (El-Nezami et al., 2000 El-Nezami, Mykka¨nen, Kankaanpa¨a¨, Suomalainen, Salminen, & Ahokas, 2000). Kirjavainen, El-Nezami, Salminen, Ahokas, and Wright (1999) found that P. freudenreichii ssp. shermanii JS enhanced proliferation of T- and B-cells in mouse lymphocytes. The aims of the present study were to evaluate the in vitro tolerance of P. freudenreichii ssp. shermanii JS to low pH and high bile concentration as encountered in the upper parts of the gastrointestinal tract and to assess the in vivo gastrointestinal survival of this strain when administered in wheybased fruit juice by healthy adult volunteers. In order to track the JS strain in faecal samples, a genetic identification method for the strain was developed. In addition, the safety related characteristics, i.e., antibiotic susceptibility defined as a minimal inhibitory concentration of different antibiotics and an enzyme activity profile of the strain were also examined.

2. Materials and methods 2.1. Bacterial strains and culture conditions Bacterial strains used in the study were P. freudenreichii ssp. shermanii JS (DSMZ 7076; Deutsche Sammlung von Mikroorganismen und Zellkulturen; DSMZ GmbH, Braunschweig, Germany) and P. freudenreichii ssp. freudenreichii P131 (Valio Ltd., Helsinki, Finland), P. freudenreichii ssp. shermanii NCIMB 10585 and P. freudenreichii ssp. freudenreichii NCIMB 5959T (National Collection of Industrial, Food and Marine Bacteria; NCIMB Ltd., Aberdeen, UK). In addition P. freudenreichii ssp. freudenreichii ITG P20 and P. freudenreichii ssp. freudenreichii ITG P23 (Institut Technique Francais des Fromages (ITFF), Cedex 09, France), P. thoenii ATCC 4874T, P. jensenii ATCC 4868T (American Type Culture Collection; ATCC, Manassas, USA) and P. acidipropionici DSMZ 20272 were used during validation of the genotyping methods. The strains were maintained at 80 1C and subcultured in propioni medium (PA, pH 7.3) consisting of 0.5% (w/v) tryptone (LabM, Bury, UK), 1% (w/v) yeast extract (LabM), 1.0% (w/v) sodium lactate (Merck, Darmstadt, Germany). PA-medium was used as a broth (PA-broth) or supplemented with 1% (w/v) of b-glycerolphospate (Merck) and 1.5% agar (PPA-agar) for the cell count enumerations. Alternatively, whey permeate medium (WPM) consisting of 5%(w/v) whey permeate (Valio Ltd), 2%(w/v) casein hydrolysate (Valio Ltd.), 1%(w/v) yeast extract (LabM), pH 6.8, was used for the cultivation. All strains were incubated anaerobically (Anaerocult; Merck) for 3 d at 30 1C. 2.2. Bile salt and acid tolerance The strains were cultivated anaerobically in PA-broth (pH 7.3) for 3 d at 301C (stationary growth phase). The survival of the propionibacteria strains during bile exposure was studied in PA-broth supplemented with 0.3% or 0.5% (w/v) oxgall powder (Bile bovine, Sigma B-3883, Sigma-Aldrich, St. Louis, MO, USA) by inoculating the broths with 1% of stationary growth phase culture and by enumerating the viable cell counts after 0 and 3 h of incubation at 30 1C. The acid tolerance test was performed in whey permeate medium (WPM-broth) adjusted with 37% HCl to pH of 6.5, 4.0, 3.0 or 2.0. WPM-broth was inoculated with 1% of stationary growth phase culture and incubated for 1, 2 and 3 hours at 301C, after which viable cell counts were enumerated. Viable cell counts from both experiments were determined on PPA-agar by incubating the plates anaerobically for 7 d at 301C. 2.3. Enzyme activity profiles Propionibacteria strains were grown for 3 d at 30 1C in PPA-broth. Enzyme activity profiles of the strains were detected by the chromogenic, semiquantitative API ZYM

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system (bioMe´rieux sa, Marcy-l’Etoile, France) according to the manufacturer’s instructions. Briefly, strains were harvested by centrifugation at 8000  g for 10 min at 4 1C. The cells were washed and suspended into API ZYM medium provided by the manufacturer and adjusted with the same medium to the McFarland standard value of 6 (equivalent to a cell count of 8 log10 cfu mL1), and a 65 mL of this suspension was added to each well and then incubated for 4 h at 30 1C. The reaction was stopped by adding one drop of the reagent Zym A and the colour development was obtained within 5 min by adding one drop of the reagent Zym B to each wells. Reagents Zym A and Zym B were provided by the manufacturer. Enzyme activity was detected through colour reaction by comparing the colour intensity to the colour reading chart provided by the manufacturer and expressed as nM of substrate hydrolysed per 65 mL of culture on a scale of 0 (no activity) to X40 nM (high activity). The cell numbers in the wells were enumerated by plate counting on PPA-agar for 7 d at 30 1C and they were 8.070.1 log10 cfu mL1 for the JS strain and 7.970 log10 cfu mL1 for the P131 and NCIMB 5959T strains. The test was performed twice for all strains. 2.4. Minimal inhibitory concentration (MIC) of antibiotics Determination of the minimal inhibitory concentration (MIC) of antibiotics was performed on VetMICTM E-cocci microdilution panel (National Veterinary Institute, Uppsala, Sweden) by using the recently developed lactic acid bacteria susceptibility test medium (LSM; Klare et al., 2005) consisting of isosensitest broth (IST, Oxoid Ltd., Cambridge, UK) supplemented with 10% of MRS medium (LabM). The assay included the following antibiotics: ampicillin, erythromycin, virginiamycin, gentamicin, streptomycin, kanamycin, tetracycline, chloramphenicol, vancomycin, narasin, bacitracin and linezolid in a concentration range of 0.125–1024 mg mL1. Briefly, P. freudenreichii ssp. shermanii JS, P131, NCIMB 5959T and NCIMB 10583 were cultivated in LSM broth for 48 h at 30 1C anaerobically. For the final inoculum, 1 mL of propionibacteria culture grown for 3 d at 30 1C was suspended aseptically to sterile saline until a density corresponding to a spectrophotometric OD625 of 0.16–0.2 was reached. This saline suspension was then diluted 1:1000 in LSM broth to obtain a final concentration of 5.5–5.8 log10 cfu mL1. For each well in VetMICTM E-cocci microdilution plate, 100 mL of this inoculum was added within 30 min after the preparation of the final inoculum. The wells were sealed with a transparent covering tape and the panel was incubated for 3 d at 30 1C, anaerobically. The growth of the culture in the wells was assessed visually. The MIC was defined as the lowest concentration of antibiotic giving a complete inhibition of visible growth out of three repeated experiments. The antibiotic susceptibility of P. freudenreichii ssp. shermanii JS was assessed additionally using the Sensititre

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microdilution panel (Trek Diagnostics System Ltd., East Grinstead, West Sussex, UK). The propionibacteria cells were diluted in sterile water to the value of 0.5 on the MacFarland scale, and the mixture was further diluted 1:200 in buffered PA-broth supplemented with 0.5% glucose. The inoculum was transferred into the wells of Sensititre standard veterinary MIC susceptibility plates CMV1ECOF, CMV1ABPF and CMV1BURF (Urinary plate) (Trek Diagnostics System Ltd.) plates according to the manufacturer’s instructions. The plates were incubated anaerobically at 30 1C for 4 d. The growth of the culture in the wells was studied visually and recorded daily. 2.5. Identification and genotyping of propionibacteria Genomic DNA was isolated as described previously (Varmanen, Rantanen, Palva, & Tynkkynen, 1998). Identification of Propionibacterium species was performed according to Fessler, Casey, and Puhan (1998). Briefly, part of the 23S rDNA was amplified followed by restriction analysis with MspI (New England Biolabs, Ipswich, MA, USA). Randomly amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) analyses were used for typing of the strains. In RAPD oligonucleotide primers 50 CGAGCCGTC30 and 50 ACG CGCCCT30 were used in combination. PCR-amplification was performed with a DyNAzyme DNA Polymerase kit (Finnzyme, Espoo, Finland) according to the instructions of the manufacturer. The PCR reaction contained 10 mM Tris–HCl, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100 (pH 8.8), primers were used at 1 mM and deoxynucleotides at 200 mM concentration. Initial denaturation was at 94 1C for 2 min and the thermocycling programme of 40 cycles used was 94 1C for 15 s, 30 1C for 30 s and 72 1C for 2 min. The PFGE analysis was performed as previously described (Tynkkynen, Satokari, Saarela, Mattila-Sandholm, & Saxelin, 1999 Tynkkynen et al., 1999). Restriction enzymes XbaI and SspI (New England Biolabs) were used to cut the genomic DNA. 2.6. Survival during gastrointestinal transit The in vivo trial was performed with commercially available calcium-enriched whey-based orange juice (pH 3.870.2), Hedelma¨tarhas (Valio Ltd.). During the test period the juice was supplemented with P. freudenreichii ssp. shermanii JS in combination with L. rhamnosus LC705 or with L. rhamnosus E800 (VTT E-97800) alone. For preparation of the juice, a concentrated culture, Bioprofits (Valio Ltd.) (Suomalainen & Ma¨yra¨-Ma¨kinen, 1999), containing P. freudenreichii ssp. shermanii JS and L. rhamnosus LC705 strains was added (1%, v/v) or L.rhamnosus E800 (VTT E-97800) culture was added (1%, v/v). After preparation, the juice was stored at 4 1C until consumption. The cell number of P. freudenreichii ssp. shermanii JS at the juice was enumerated in the beginning

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and during the shelf life of 3 weeks and it remained constant at the level of 2–4  108 cfu mL1 as determined by viable cell counts on PPA agar for 7 d at 30 1C. Healthy adult volunteers (n ¼ 22) were recruited to study the survival of P. freudenreichii ssp. shermanii JS in the gastrointestinal (GI)-tract. The participants were students and staff members (19 women, three men, aged 20–44 years) from the Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland. Before participation in the study, all subjects were examined by a physician for their health status. The exclusion criteria included known gastrointestinal tract disease or antibiotic consumption within the last 4 weeks. The subjects were advised to maintain their lifestyle habits unchanged and not to consume preparations containing known probiotics during the study. The consumption of fermented dairy products was recorded. The study was approved by the Ethical Committee of the Hospital District of VarsinaisSuomi, and a signed informed consent was obtained from every subject. The study was conducted as a single blind parallel study with three 2-week periods (baseline, consumption and follow-up). Volunteers (n ¼ 22) were randomized into two groups receiving 100 mL of the juice drink twice a day throughout the study. During the consumption period, the subjects were administering the juice which was supplemented either with P. freudenreichii ssp. shermanii JS and L. rhamnosus LC705 (JS group, n ¼ 10) or with L. rhamnosus E800 (VTT E-97800) alone (non-JS group, n ¼ 12 of which one person interrupted the study). Faecal samples were collected for microbiological analysis at the beginning and after each study period. Immediately after the collection, the sample tubes for microbial analysis were sealed into anaerobic bags (Anaerocult Mini A, Merck) and frozen at 20 1C. The sample was transported frozen to the laboratory and was kept frozen at 75 1C until analysis. Frozen faecal samples were thawed, mixed at a ratio of 1:10 with peptone-saline buffer, homogenized and serially diluted in the same buffer and plated on PPA-agar. The plates were grown anaerobically at 30 1C for 7 d. Presumptive colonies of propionibacteria were purified on PPA-agar, transferred into PPA-broth and incubated for 3 d at 30 1C. For identification of P. freudenreichii ssp. shermanii isolates, Gram-staining, catalase activity, fermentation of lactose and sucrose, nitrate reduction (Nitrate test, Merck) and carbohydrate fermentation by API 50 CH (bioMe´rieux sa) were studied. In order to confirm the strain identification, one to three presumable P. freudenreichii ssp. shermanii JS colonies from each sample were studied by RAPD analysis as described in Section 2.5. 2.7. Statistical analysis Bacterial cell numbers in tests for exposure to bile salts or low pH were transformed into log10 values from three repetitions. The statistical significance of differences in the

survival of P. freudenreichii strains during exposure to bile salts or low pH was analysed within each strain by comparing initial cell counts separately with cell counts of 1-, 2-, and 3-hour treatments by using the t-test (paired, twotailed) (Windows XP, Microsofts Excel 2002). The statistical significance of differences in the survival between strains was analysed pair wise by comparing differences in changes of cell counts at different pH and time points separately by using the t-test (paired, two-tailed). Bacterial cell numbers in a single blind parallel human study were transformed into log10 values and presented separately for each subject. The statistical significance of differences in the cell counts of the strain JS at baseline versus after consumption and after follow-up within each individual was analysed by using the ttest (paired, two-tailed). A p-value below 0.05 was considered to be statistically significant. 3. Results 3.1. Acid and bile tolerance, minimal inhibitory concentrations of antibiotics, and enzyme activity profiles of the strains P. freudenreichii ssp. freudenreichii P131 and NCIMB 5959T and P. freudenreichii ssp. shermanii JS strains survived well during the 3-hour treatment at pH 3 but weakly at pH 2, the cell counts of all strains decreasing progressively to the level of 2 log10 cfu mL1 after 3 hours. At pH 2, the change in cell count of P. freudenreichii ssp. shermanii JS was higher than the change in cell count of P. freudenreichii ssp. freudenreichii P131, but only after 1 h of treatment (po0.05). All propionibacteria strains survived well and similarly to each other in the presence of 0.5%(w/v) bile without any decrease in viable cell numbers (Table 1). Antibiotic susceptibilities of P. freudenreichii ssp. shermanii JS and NCIMB 10585, and P. freudenreichii ssp. freudenreichii 131 and NCIMB 5959T as assessed with the VetMICTM broth microdilution assay are presented in Table 2. All strains showed low minimal inhibitory concentration (MIC o4 mg mL1) for ampicillin, erythromycin, virginiamycin, tetracycline, chloramphenicol, vancomycin, narasin, bacitracin, and linezolid, and high MICs (44 mg mL1) for aminoglycosides (streptomycin, gentamicin and kanamycin). P. freudenreichii ssp. shermanii JS displayed similar MICs compared with the other tested P. freudenreichii strains. The antibiotic susceptibilities of P. freudenreichii ssp. shermanii JS revealed by VetMIC and Sensititre analysis were parallel (data not shown). As determined with the semiquantitative API ZYM system, the b-galactosidase activity of P. freudenreichii ssp. shermanii JS was high, which was indicated by liberation of over 40 nM of 2-naphthyl derivatives, whereas no b-galactosidase activity was detected in the other P. freudenreichii strains (Table 3). None of the strains indicated b-glucuronidase activity or activity of alkaline phosphatase, lipase, valine arylamidase, trypsin, chymotrypsin, N-acetyl-b-glucosaminidase or a-mannosidase.

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Table 1 Acid and bile tolerance of P. freudenreichii ssp. shermanii JS, P. freudenreichii ssp. freudenreichii P131 and P. freudenreichii ssp. freudenreichii NCIMB 5959T cells after 0, 1, 2, and 3-hour treatments in WPM-broths adjusted to pH2, pH3 and pH4 and after 0 and 3-hour treatments in PA-broth supplemented with 0.3% and 0.5% of bile salt Strain

Cell numbers (log10 cfu mL1) after 0, 1, 2, and 3-hour acid treatment and after 0, and 3-hour bile salt treatmenta

pH and bile concentrations

JS

pH 4 pH 3 pH 2 Bile 0.3% Bile 0.5% pH 4 pH 3 pH 2 Bile 0.3% Bile 0.5% pH 4 pH 3 pH 2 Bile 0.3% Bile 0.5%

P131

NCIMB5959T

0h

1h

2h

3h

7.870.06 7.870.06 7.870.06 7.870.09 7.870.09 7.570.05 7.570.05 7.570.05 7.670.11 7.670.11 7.870.07 7.870.07 7.870.07 7.770.05 7.770.05

7.870.02 7.870.05 6.770.30e*

7.970.02 7.870.03 3.670.55bc**

7.570.14 7.470.08 4.271.33b*

7.570.10 7.170.10 2.771.22c**

7.870.04 7.870.01 6.070.42b*

7.670.04 7.770.07 2.970.83c***

7.870.07 7.770.12 o3.070.0d*** 7.770.05 7.870.05 7.570.08 6.870.30 2.470.64d*** 7.670.06 7.670.09 7.770.01 7.670.09 2.370.55d*** 7.570.08 7.670.12

a

Results are shown as mean values and standard deviations (sd) of three repetitions. Statistical significant difference in cell counts within the strain between initial count (0 h)and 1 h; t-test *po0.05, **po0.01, ***po0.001. c Statistical significant difference in cell counts within the strain between initial count (0 h) and 2 h; t-test *po0.05, **po0.01, ***po0.001. d Statistical significant difference in cell counts within the strain between initial count (0 h) and 3 h; t-test *po0.05, **po0.01, ***po0.001. e Statistical significant difference in cell counts after one hour at pH 2 between the JS strain and P131; t-test *po0.05, **po0.01, ***po0.001. b

Table 2 Antibiotic susceptibilities expressed as minimal inhibitory concentration (MIC) of P. freudenreichii ssp. shermanii JS, P. freudenreichii ssp. freudenreichii 131, P. freudenreichii ssp. freudenreichii NCIMB 5959T and P. freudenreichii ssp. shermanii NCIMB 10585 on VetMICTM broth microdilution assay (three repetitions) Antibiotic

Table 3 API ZYM system profiles of P. freudenreichii ssp.shermanii JS, P. freudenreichii ssp. freudenreichii P131 and P. freudenreichii ssp. freudenreichii NCIMB 5959T strains Enzyme

Quantity of substrate hydrolyzed by each strain (nM)

Minimal inhibitory concentration (mg mL1)a JS JS

P131

NCIMB 5959T

Ampicillin 0.25 2.0 o0.125 Erythromycin o0.25 o0.25 o0.25 Virginiamycin o0.25 1.0 0.50 Gentamicin 8 4 8 Streptomycin 8 16 16 Kanamycin 32 64 64 Tetracycline 1.0 1.0 0.25 Chloramphenicol 2.0 1.0 0.50 Vancomycin 1.0 1.0 1.0 Narasin 0.125 0.50 1.0 Bacitracin, U mL1 o1 o1 o1 Linezolid 1.0 2.0 1.0

P131

NCIMB5959

0 0 5 40 5 5 5 0 0 10 10

0 0 0 40 5 5 5 0 0 10 10

NCIMB 10585 0.25 o0.25 0.50 16 32 128 0.50 1.0 1.0 0.50 o1 1.0

a

The minimal inhibitory concentration (MIC) was defined as the lowest antibiotic concentration of three repetition all inhibiting the visible bacterial growth.

3.2. Species- and strain-specific identification of P. freudenreichii ssp. shermanii JS Biochemical identification of propionibacteria strains was confirmed by 23S rDNA restriction analysis. The amplified 23S rDNA of strain JS gave a corresponding

Alkaline phosphatase 0 Esterase (C4) 10 Esterase lipase 5 Leucine arylamidase 10 Cystine arylamidase 0 Phosphatase acid 5 Naphthol-AS-phosphohydrolase 5 a-Galactosidase 20 b-Galactosidase 440 a-Glucosidase 10 b-Glucosidase 0

The results are expressed as nM of substrate hydrolysed by 65 mL of each cell culture after anaerobic growth in PA-broth for 3 d at 30 1C. The colour intensity was scaled from 0 (no activity) to X40 nM (high activity).

restriction pattern to the P. freudenreichii-type strain NCIMB 5959T, as did all the studied P. freudenreichii ssp. freudenreichii and ssp. shermanii strains, while the type strains P. jensenii ATCC 4868, P. thoenii ATCC 4874 and P. acidipropionici ATCC 4875 gave distinct restriction profiles (data not shown). Strain JS did not reduce nitrate, which further classified the strain to ssp. shermanii. RAPD was used to differentiate P. freudenreichii ssp. shermanii JS

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Viable count of JS log10 cfu g-1 faeces

9.0

10/10∗∗∗

8.0 7.0 6.0 5.0 4.0 3.0

2/10

1/10

2.0 Baseline

Fig. 1. Pulsed field gel electrophoresis (PFGE) profiles of Propionibacterium strains, the genomic DNA was cut with XbaI enzyme. Lanes 1–10: P. acidipropionici DSM 20272 (1), P. jensenii ATCC 4868T (2), P. thoenii ATCC 4874T (3), P. freudenreichii ssp. freudenreichii strains NCIMB 5959T (4), P131 (5), ITG P20 (6), ITG P23 (7), P. freudenreichii ssp. shermanii strains NCIMB 10585 (8) and JS (9).

from other strains representing the same (sub) species. Strain JS showed a unique RAPD-profile consisting of approx. 900-, 340- and 250-bp fragments (data not shown). Though the used random primers amplified the 340 and 250-bp fragments from all P. freudenreichii-strains studied, the profile with 900-bp amplification product was strainspecific. P. thoenii ATCC 4874, P. jensenii ATCC 4868 and P. acidipropionici DSM 20272 strains did not display the 250-bp fragment. To evaluate the specificity of the method, RAPD analysis was used for typing of 91 isolates collected from faecal samples of persons consuming the strain JS. Of the isolates, which were primarily selected as strain JS according to their biochemical characteristics, 94.5% were identified as strain JS by RAPD-analysis. Identification of 12 random isolates, determined as strain JS by RAPD, was further confirmed with PFGE analysis. All the 12 isolates gave identical profiles to strain JS in PFGE (data not shown), which confirmed that strain-specific identification was achieved by RAPD. All the studied propionibacteria reference strains could be distinguished from strain JS and from each other by PFGE using restriction enzymes XbaI (Fig. 1) and SspI. 3.3. Recovery of P. freudenreichii ssp. shermanii JS during gastrointestinal transit In the group consuming the whey-based fruit juice supplemented with P. freudenreichii ssp. shermanii JS (JS group), the strain JS was identified in 1 out of 10 subjects (detection limit 3 log10 cfu g1) at the baseline. During the administration period the strain was detected in high numbers 7.6 log10 cfu g1 (range 6.1–8.3 log10 cfu g1) (po0.001) in all subjects (Fig. 2). After the 2-week followup period, the counts decreased below the detection limit in 8 out of 10 subjects and were at the levels of 5.6 and 6.3 log10 cfu g1 in the two remaining subjects. In the group

Consumption

Follow-up

Fig. 2. Cell numbers and the detection frequency of P. freudenreichii ssp. shermanii JS (JS) in faecal samples obtained at the end of baseline, consumption of fruit juice containing the JS strain and follow-up periods (2 weeks each). The bold line shows the mean count of the JS strain in the faecal samples of 10 subjects and the thinner lines show the cell numbers of individual samples from each person.

consuming whey-based fruit juice without P. freudenreichii ssp. shermanii JS (non-JS), no isolates representing the JS strain were detected throughout the study. Other propionibacteria than P. freudenreichii ssp. shermanii JS were detected at the baseline in two and after the consumption period in three out of 11 subjects with relatively high cell numbers ranging between 5.6 and 6.7 log10 cfu g1 (data not shown). 4. Discussion This single blinded parallel study with adult human volunteers demonstrated that P. freudenreichii ssp. shermanii JS may survive through the gastrointestinal tract when administered in whey-based fruit juice. During the consumption period, the JS strain was detected in high numbers (7.670.7 log10 cfu g1) in faeces of all volunteers, and it comprised the predominant Propionibacterium population in the faeces (data not shown). The in vitro assessment carried out at different pH levels and high bile concentration supported the finding. A probiotic strain should survive the passage through the upper gastrointestinal tract and arrive alive and active to its site of action where it should transiently colonize the gut (Lee, Nomoto, Salminen, & Gorbach, 1999). During gastrointestinal transit, the strain has to tolerate low pH of the stomach which generally ranges from pH 2 to 3.5. In our in vitro study, the P. freudenreichii strains showed excellent survival during bile exposure, and although the cells did not survive the treatment at pH 2 for 3 h, the acid survival was good during exposure to pH 3. Our findings are comparable with those of Zarate, Gonzalez, Perez-Chaia, and Oliver (2000) who showed that some P. acidipropionici and P. freudenreichii strains tolerated bile concentrations up to 0.3% well and survived in artificial gastric fluids at pH 3, but not at pH 2. Huang and Adams (2004) have reported similar limiting pH ranges for propionibacteria isolated from milk and cheese, and when

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the cells were administered in a food matrix such as soybased medium, even pH 2 was well tolerated. To the best of our knowledge, no studies on antibiotic susceptibility of dairy propionibacteria have been published. In this study, antibiotic susceptibility of the P. freudenreichii strains JS, P131, NCIMB 5959T and NCIMB 10585 did not differ remarkably from each other. There are no MIC distribution data available for dairy propionibacteria. In our study we were not able to define the breakpoints differentiating the susceptible and resistant strain population due to the insufficient number of strains available for testing. Based on the MIC distributions reported for lactobacilli (Danielsen & Wind, 2003) and bifidobacteria (Ma¨tto¨ et al., 2007) and described by the panel on additives and products or substances used in animal feed (FEEDAP Panel, 2005) the P. freudenreichii strains could be regarded susceptible to all other antibiotics included in the VetMICTM E-cocci panel, except for aminoglycosides (streptomycin, gentamicin and kanamycin). The resistance to aminoglycosides is a common characteristic of anaerobic bacteria as they lack a cytochrome-mediated drug transport system (Bryan & Kwan, 1981). For tracking the JS strain from faeces, genotyping methods (RAPD and PFGE) were applied for the strainlevel identification of faecal isolates. The RAPD method is widely used for strain-level identification of bacteria (Olive & Bean, 1999) since it is non-laborious and does not need any strain-specific sequence information. However, setting up a reliable, discriminative and reproducible RAPDmethod needs careful optimization. We used the PFGEmethod, known to be one of the most discriminative but laborious typing methods (Olive & Bean, 1999; Tynkkynen et al., 1999), to confirm the strain specificity of P. freudenreichii ssp. shermanii JS RAPD-pattern. Same isolates were identified as the JS strain by both methods, which confirmed that the developed RAPD analysis was reliable and could be used alone for identification of the strain JS in future clinical studies. Only a few studies reported the cell number of propionibacteria in faeces of human volunteers. Bougle et al. (1999) reported that the propionibacteria counts increased in 15 out of 18 volunteers from the initial level of o5 log10 cfu g1 to 6.37 (70.89) log10 cfu g1 after administration of P. freudenreichii SI 26 and SI 41 for 2 weeks. Our results with P. freudenreichii ssp. shermanii JS showed that the strain was detectable in high numbers (7.670.7 log10 cfu g1) in faeces of all volunteers during administration. The colonization was transient with the cell counts declining to the baseline level within 2 weeks after the administration period. Recently, in a study of Myllyluoma et al. (2005) P. freudenreichii ssp. shermanii JS levels increased from baseline (detection) level of 4 log10 cfu g1 to the level of 7–8 log10 cfu g1 during the 1-month intervention period with milk-based fruit drink containing L. rhamnosus GG, L. rhamnosus LC705, Bifidobacterium breve Bb99 and JS in subjects receiving

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intense antimicrobial treatment for Helicobacter pylori eradication. Our results on viable cell counts of strain JS in faeces are in accordance with the numbers achieved by Myllyluoma et al. (2005) in which the cell counts were analysed by using a strain-specific DNA-based quantitative PCR method. At the baseline of our study, the individual variation of propionibacteria counts was high while the detection frequency was low. Both in our study and in the study conducted by Bougle et al. (1999) a few people were carriers of propionibacteria at the baseline. Upon consumption of Swiss-type cheeses, live propionibacteria may be taken into the intestine. In our study, the consumption of Jarlsberg-type cheese was not included in the exclusion criteria. Therefore, the study volunteers who were consumers of Jarlsberg-type cheese were found to carry strain JS in their faeces at the baseline. Probiotic properties are strain specific and should be demonstrated for each candidate strain separately prior to the strain being labelled and marketed as a probiotic to consumers. The FAO/WHO working group has generated guidelines and recommendations for the evaluation of probiotics in food (FAO/WHO, 2002). This study and earlier studies (Kajander, Hatakka, Poussa, Fa¨rkkila¨, & Korpela, 2005; Myllyluoma et al., 2005; Ouwehand, Lagstro¨m, Suomalainen, & Salminen, 2002c) showed that P. freudenreichii ssp. shermanii JS is a promising probiotic strain. When the JS strain was administered in fruit juice with L. rhamnosus LC705, the combination showed a weak positive effect on defecation frequency in elderly (Ouwehand et al., 2002c). In a study with irritable bowel syndrome (IBS) patients, the administration of capsules containing L. rhamnosus GG, L. rhamnosus LC705, B. breve Bb99 and JS reduced the IBS symptoms by 42% compared to reduction of 6% in the placebo group (Kajander et al., 2005). Interestingly, the same probiotic mixture with the JS strain improved the tolerance to H. pylori eradication therapy (Myllyluoma et al., 2005). 5. Conclusions The present study with adult human volunteers showed excellent in vitro and in vivo gastrointestinal survival of the P. freudenreichii ssp. shermanii JS strain. The strain did not show antibiotic resistance deviating from other studied dairy P. freudenreichii strains and the strain JS did not produce potential procarcinogenic enzymes, such as betaglucoronidase, in vitro. In addition, in the present study, DNA fingerprinting tools for strain level identification of P. freudenreichii ssp. shermanii were developed. These findings combined with a long history of safe use as a starter culture and with other beneficial features published for the JS strain, such as mycotoxin binding capacity and alleviation of symptoms in IBS and H. pylori treatments, support that the JS strain is an interesting candidate as a probiotic and thus the findings form a basis for studies on the therapeutic potential of this strain.

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