Accepted Manuscript Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano cheeses C. Caggia, M. De Angelis, I. Pitino, A. Pino, C.L. Randazzo PII:

S0740-0020(15)00071-4

DOI:

10.1016/j.fm.2015.03.010

Reference:

YFMIC 2375

To appear in:

Food Microbiology

Received Date: 3 December 2014 Revised Date:

4 March 2015

Accepted Date: 26 March 2015

Please cite this article as: Caggia, C., De Angelis, M., Pitino, I., Pino, A., Randazzo, C.L., Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano cheeses, Food Microbiology (2015), doi: 10.1016/j.fm.2015.03.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Probiotic features of Lactobacillus strains isolated from Ragusano and Pecorino Siciliano

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cheeses

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C. Caggiaa, M. De Angelisb, I. Pitinoa, A. Pinoa, C.L. Randazzoa*

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Department of Agriculture, Food and Environment, University of Catania, Catania, Italy Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Bari, Italy

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Keywords: lactobacilli; traditional cheese; resistance to low pH; tolerance to bile salts; adhesion

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ability; transit to GI tract

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12 Abstract

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In the present study 177 Lactobacillus spp. strains, isolated from Ragusano and Pecorino Siciliano

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cheeses, were in vitro screened for probiotic traits, and their characteristics were compared to those

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of Lactobacillus rhamnosus GG, commercial strain. Based on acidic and bile salt resistance,

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thirteen Lactobacillus strains were selected. The multiplex-PCR application revealed that nine

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strains belonged to Lactobacillus rhamnosus species and four to Lactobacillus paracasei species.

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All selected strains were further investigated for transit tolerance in simulated upper gastrointestinal

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tract (GI), for adhesion capacity to human intestinal cell lines, for hydrophobicity, for co-

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aggregation and auto-aggregation and for antimicrobial activities. Moreover, antibiotic resistance,

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haemolytic and bile salt hydrolase activities were investigated for safety assessment.

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*corresponding author: Tel: +39 095 7580218; fax: +39 095 7141960.

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E-mail address: [email protected] (C.L. Randazzo) 1

ACCEPTED MANUSCRIPT Viable counts after simulated gastric and duodenal transit revealed that overall the selected

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lactobacilli tolerated better pancreatic juice and bile salts than acidic juice. In particular, three L.

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rhamnosus strains (FS10, FS2, and PS11) and one L. paracasei strain (PM8) increased their cell

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density after the simulated GI transit. The same strains showed also high percentage of auto-

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aggregation and co-aggregation with Escherichia coli. All strains were effective against both

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Staphylococcus aureus and E. coli and variability was achieved versus Listeria monocytogenes and

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Enterococcus faecalis used as pathogenic indicator strains.

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Different behavior was revealed by strains for adhesion ability and hydrophobicity, which are not

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always linked each other and are strongly strain-dependent. From the safety point of view, no

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isolate showed hemolytic and bile salt hydrolase activities, except one, and most of the strains were

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sensitive to a broad range of clinical antibiotics.

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This work showed that the L. rhamnosus FS10 and the L. paracasei PM8 are good promising

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probiotic candidates for further in vivo investigations.

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Keywords: probiotics; simulated GI transit tolerance; safety assessment; acidic and bile resistances;

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antibiotic resistance; principal component analysis.

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1. Introduction

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Probiotics are defined as “live microorganisms which, when administered in adequate amounts,

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confer a health benefit on the host” (FAO/WHO, 2002). Probiotics may beneficially affect the host

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by modulating immune responses, influencing its metabolic activities (e.g. cholesterol assimilation)

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and performing useful metabolic activities (e.g., lactase activity, vitamin production, antimicrobial

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activity).

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The most studied probiotics belong to Lactobacillus and Bifidobacterium genera and, among them,

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Lactobacillus species are commensal and non-pathogenic inhabitants of human and animal

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intestine, of oral cavity and vagina (Walter 2008; Chen et al., 2010) and have been considered as

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ACCEPTED MANUSCRIPT valuable probiotic organisms since they contribute to the inhibition of harmful intestinal bacteria

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(Walter 2008; van Baarlen et al., 2013). Lactobacilli are food-grade organisms witnessing a long

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history of safe use being widely consumed with fermented foods and beverages (Salminen et al.,

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1998; Leroy and De Vuyst, 2004; Bernardeau et al., 2008). Most of them received the Qualified

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Presumption of Safety (QPS) status (EFSA, 2012). Lactobacilli display a naturally wide range of

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antibiotic resistance and in most cases it is not of the transmissible type (Charteris et al., 1998).

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Lactobacilli possess inhibitory properties against enteropathogens thanks to production of several

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antimicrobial compounds (Collado et al., 2007). The latter is a key criterion which should be taken

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into account for selection of probiotic strains since it is considered an important ecological factor

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determining the dominant bacteria in the intestinal ecosystem. Nevertheless, characteristics ascribed

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to probiotics are strain specific and should be individually tested. For selection of a promising

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probiotic strain, several aspects of functionality should be considered and the first tool is

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represented by in vitro methods aiming to ascertain the ability to survive through the upper GI

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(Saarela et al., 2000). Tolerance to gastric juice and bile salts become a crucial factor for selection

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of new probiotic strains as well as the ability to adhere to epithelial surfaces and to persist in the

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human GI-tract. The testing of their in vivo efficacy is expensive and time-consuming. For this

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reason, reliable in vitro methods are required for selecting new strains of probiotic bacteria to obtain

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profitable in vivo effects.

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Together with the GI tract, dairy products are considered the main sources for isolation of novel

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promising probiotic organisms (Ambadoyiannis et al., 2005) and several studies have demonstrated

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their potentiality as good vehicle for probiotics delivery to human (Bergamini et al., 2010; Oberg et

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al., 2011; Pitino et al., 2012). In particular, traditional dairy products, such as cheeses, represent a

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plentiful source of lactobacilli. Among traditional cheeses, Ragusano and Pecorino types are

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Protected Denomination of Origin (PDO) cheeses made in Sicily from raw milk through a

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traditional cheese-making process, without addition of commercial starter cultures. During the

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manufacture and the cheese ripening, the microbiota naturally evolves, increasing the diversity of

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ACCEPTED MANUSCRIPT Lactobacillus species (Randazzo et al., 2002; 2006; Licitra et al., 2007), with potential probiotic

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functions.

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Therefore, the aims of the present study were (i) to establish in vitro the functional and safety

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characteristics of potential probiotic Lactobacillus strains, isolated from Ragusano and Pecorino

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Siciliano cheeses; (ii) to compare their features to those of commercial Lactobacillus rhamnosus

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ATCC 53103 (strain GG); and (iii) to identify the promising strains throughout genotypic methods.

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2.1. Bacterial strains and culture conditions

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In the present study one hundred seventy-seven (177) wild strains of Lactobacillus spp., belonging

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to the DiGeSA microbial collection, previously isolated during the manufacture of artisanal Sicilian

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cheeses, were investigated for probiotic in vitro characteristics. All strains, including the L.

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rhamnosus ATCC 53103 (strain GG), used as control, were anaerobically grown using Anaerogen

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kit (Oxoid, Milan, Italy) at 37° C in MRS medium (Oxoid) plus cycloheximide (Fluka) (100 mg/l).

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The type strains Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Listeria

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monocytogenes DSM 12464, E. coli 555 (gently provided by Prof. F. Gardini from the University of

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Bologna, Italy) were routinely cultured on Tryptone Soya Broth (Oxoid) at 37° C under aerobic

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conditions. Lactobacillus casei DSMZ 20011, Lactobacillus paracasei DSMZ 5622, Lactobacillus

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plantarum DSMZ 20174, Lactobacillus acidophilus DRU (gently provided by Dr J. Reinheimer

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from INLAIN, UNL-CONICET, Santa Fe, Argentina) and Enterococcus faecalis CCR300, from

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DiGeSA collection (University of Catania, Italy) were cultured on MRS medium at 37° C under

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anaerobic conditions. Strains were maintained at -80° C in the presence of glycerol 20% (v/v), as

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cryoprotective agent. For inocula preparation, individual colonies of 24 h-old cultures, were

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resuspended in phosphate buffered saline (PBS) solution (80 mM Na2HPO4, 20 mM NaH2PO4, 100

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mM NaCl, pH 7.5). Cell suspensions were diluted until a final cell density of 108 cfu/ml by

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measuring the optical density at 600 nm. When necessary, a final density of 109 cfu/ml was used.

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Acidic resistance was examined in MRS broth added of hydrochloric acid (1 M), to obtain a final

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pH value of 2.5. Strains, grown in MRS broth at 37° C overnight, were inoculated (cell density of

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108 cfu/ml) into acidified MRS broth. The cell survival was monitored up to 6 h, at different

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intervals. The survival rates were determined as number of viable cells enumerated on MRS agar,

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incubated under anaerobic conditions at 37° C for 48 h. Moreover, the optical density (OD) of cell

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growth into acidified MRS broth was evaluated at 600 nm using Microplate Manager software

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(Version 5.2.1, Biorad, Milan, Italy).

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One hundred and five strains, selected on the basis of their resistance to low pH, were evaluated for

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bile salt tolerance in MRS broth containing a standardized bile extract consisting mainly of sodium

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glycocholate (Oxoid) and sodium taurocholate (Oxoid) at different concentrations (0.4 %, 1.0 %

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and 2.0 %, w/v). The strains were inoculated into the medium at cell density of 108 cfu/ml and

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anaerobically incubated at 37° C up to 6 h. The survival rates were determined as described above.

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Strains inoculated onto MRS without addition of bile salts were used as control.

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2.3. Molecular identification lactobacilli strains

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Thirteen strains, selected for the characteristics described above, were subjected to genetic

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identification throughout multiplex-PCR, using species-specific primers targeting a conserved

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region of the tuf gene. DNA was extracted as previously described (Coudeyras et al., 2008) and

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PCR amplification was conducted as reported by Ventura and co-workers (2003).

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2.4. Transit tolerance in simulated upper gastrointestinal (GI) tract

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ACCEPTED MANUSCRIPT Transit tolerance in the simulated upper human gastrointestinal (GI) tract was assessed using an in

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vitro model by adding gastric and pancreatic juices, as reported by Charteris et al. (1998), with

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slight modifications. Simulated gastric juice (SGJ) was prepared by dissolving pepsin from porcine

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gastric mucosa in 0.5% w/v sterile saline to a final concentration of 2 g/l and adjusting the pH to

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2.5. Simulated small intestinal juice (SSIJ) was prepared by dissolving pancreatin from porcine

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pancreas, 250 mg/l, and porcine bile extract, 0.45%, in 0.5% w/v sterile saline and adjusting the pH

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to 7.5. All chemicals were obtained from Sigma Aldrich (St. Louis, MO). The solutions were

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prepared fresh the same day as the experiment. Bacterial tolerance to SGJ and to SSIJ were tested

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by mixing 15 µl of bacterial suspension with 1000 µl of gastric or small intestinal juice and 485 µl

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of 0.5% w/v sterile saline, obtaining a bacterial cell density around 109 cfu/ml. The average final pH

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of the simulated mixtures was 2.62 and 7.46 for the pH 2.5 and pH 7.5 for SGJ and for SSIJ,

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respectively. The mixture was vortexed at maximum settings for 10 s and incubated at 37° C. For

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testing gastric tolerance, 100 µl was removed after 90 and after 120 min for determination of viable

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counts. Tolerance to SSIJ was determined by removing 100 µl after 120 and after 240 min. To

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determine the number of viable counts, serial dilutions were placed on MRS agar and aerobically

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incubated at 30° or 37° C for 48 h before enumeration of colony forming units. The number of

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colony forming units was expressed as log values, and tolerance over time was compared for the

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investigated strains.

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2. 5. Bacterial adhesion capacity

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All the thirteen selected strains were examined for the ability to adhere to human colon carcinoma

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(Caco-2) cells. Caco-2 cell line was purchased from the European Collection of Cell Cultures

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(ECACC). The cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium

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(DMEM; HyClone Laboratories Inc., Logan, UT, USA) supplemented with 10% (v/v) of heat

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inactivated fetal calf serum (Sigma), 1 x non-essential amino acid solution (Sigma Aldrich, Milan

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Italy), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma) at 37° C and 5% of CO2. For 6

ACCEPTED MANUSCRIPT adhesion assays, monolayers of Caco-2 cells were prepared on glass cover slips in 24- well tissue-

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culture plates (Corning, Costar, Sigma). Cells were seeded at a cell density of 5x105 cells per cm2

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and grown to confluence. The monolayers were maintained for 2 weeks prior to use in adhesion

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assays. The cell culture medium was changed every day and replaced by fresh non supplemented

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DMEM at least 1 h before the adhesion assay. The adherence of bacterial strains to Caco-2 cell

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cultures was investigated by adding 1 ml of bacterial suspension (in DMEM, containing 107 cfu/ml)

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to wells containing Caco-2 monolayer. After incubation at 37° C for 3 h, the Caco-2 cell cultures

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were washed three times with 1 ml of Hanks buffered saline solution (Sigma) pre-warmed to 37° C,

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and then treated with 1 ml of 1% Triton X100. Magnetic fleas were added to the wells and the

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plates were then placed on a magnetic stirrer for 10 min to lyse the Caco-2 cells and to release the

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adhered bacteria. The adhesion ratio was calculated by count on MRS agar plates. L. rhamnosus

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ATCC 53103 (strain GG) was used as positive control for both cell line experiments. Each adhesion

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assay was carried out in triplicate.

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2.6. Hydrophobicity, co-aggregation and auto-aggregation activities

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The cell surface hydrophobicity (H%) of the thirteen selected strains was performed as described by

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Solieri et al. (2014) and calculated as percentage of decrease (∆Abs x 100) in the absorbance of the

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aqueous phase after mixing and phase separations (Abst5) relative to that of original suspension

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(Abst0) as follows:

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H% = ∆Abs/Abst0 X 100

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Auto-aggregation (Auto-A%) ability of the thirteen selected strains was performed according to

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Solieri et al. (2014) and was expressed as percent decrease in the absorbance after 5 h (∆Abs x 100)

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relative to that of original suspension (Abst0) as follows:

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Auto-A% = ∆Abs/Abs t0 x 100

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In the co-aggregation test bacterial suspensions were prepared and calculated as described by

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Solieri et al., 2014. The pathogen strain used was E. coli 555.

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ACCEPTED MANUSCRIPT 182 2.7. Antimicrobial activity

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The antagonism towards various bacterial pathogens and common bacteria present in the GI tract

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was evaluated according to Argyri et al. (2013). All the thirteen selected strains were tested in

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duplicate against Escherichia coli ATCC 25922, Staphilococcus aureus ATCC 6538 and Listeria

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monocytogenes DSM 12464. MRS broth adjusted to pH 6.5 was used as negative control.

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188 2.8. Antibiotic resistance

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The following 7 antimicrobial agents (Sigma) were used: ampicillin, penicillin G, and vancomycin

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as inhibitors of cell wall synthesis; kanamycin, chloramphenicol, tetracycline, streptomycin as

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inhibitors of protein synthesis; and rifampicin as inhibitors of nucleic acid synthesis. All the

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antibiotic powders were dissolved in appropriate diluents and filter sterilized prior to addition to

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MRS medium. Serial dilutions of antibiotics, ranging from 0.25 to 256 µg/ml, were prepared, and

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vancomycin concentrations of 514 and 1028 µg/ml were also tested. MRS broth containing

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antibiotics at different concentrations was used to prepare each well of a micro-well plate. The

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inoculum was adjusted to a turbidity equivalent to 0.5 McFarland standard (ca. 105 cfu/ml) and was

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derived from a broth culture which was incubated at 37° C for 18 h. One ml of fresh culture was

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used to inoculate each well, incubated anaerobically at 37° C. Minimal inhibitory concentration

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(MIC) was determined on MRS broth, since it supported the best growth of all bacteria used in this

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study. The MIC was defined as the lowest concentration of antibiotic giving a complete inhibition

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of visible growth in comparison to an antibiotic-free control well. At the same time, the optical

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density (OD) of cell cultures was evaluated using Microplate Manager software (Version 5.2.1,

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Biorad) at 600 nm. The OD ≤ 0.020 was considered an indicator of transparency.

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2.9. Haemolytic and bile salt hydrolase activities

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ACCEPTED MANUSCRIPT The selected strains (13) were tested for haemolytic activity following protocol proposed by Argyri

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et al. (2013). Bile salt hydrolase (BSH) activity was performed using MRS agar plates

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supplemented with 0.5% (w/v) of taurodeoxycholic acid (TDCA, Sigma). Overnight MRS broth

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cultures (15 µl) were inoculated onto the agar medium, which was then incubated anaerobically for

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48 h at 37° C. BSH activity was present when taurodeoxycholic acid precipitated in the agar

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medium below and around a colony. The strain L. acidophilus DRU was used as positive control.

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213 2.10. Statistical analysis

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Results are expressed as the mean and standard deviation error mean (SEM) of three independent

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experiments with duplicate determinations. Differences were considered statistically significant at

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p< 0.05. In order to correlate the origin of the 177 strains with their ability to resist to low pH and to

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tolerate different bile salt concentrations, data obtained were subjected to principal component

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analysis (PCA) using the statistical software Statistica for Windows (Statistica 6.0 per Windows

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1998, StatSoft, Vigonza, Italia).

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3. Results

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The lactobacilli strains

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3.1. Acidic resistance and bile salt tolerance

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In the present study tolerance to pH 2.5 and to bile salts were chosen as criteria to select strains to

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be included in the further experiments. Results are reported in Table 1. Among the 177 lactobacilli

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investigated, sixteen strains did not survive at pH 2.5 and a total of 161 strains were able to survive

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after 2 h at pH 2.5 and 105 strains also after 4 h.

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In detail, 95 and 46 strains maintained the value of about 7-8 log cfu/ml after 4 and 6 h of

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incubation, respectively, as well as the commercial LGG strain; 9 strains decreased of 2 log cfu/ml

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after 6 h.

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ACCEPTED MANUSCRIPT Overall, bile salts at concentration differently affected the survival of the tested strains. Among the

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105 strains selected for their good resistance to low pH, 48 strains exhibited fairly good bile

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tolerance after 6 h of incubation, maintaining their concentration higher than 6 log cfu/ml after

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exposure to 0.4%, while 39 strains were strongly affected by bile salt presence. Only 4 strains were

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able to maintain the value of 8 log cfu/ml after 6 h of exposure to 2.0% bile.

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The data obtained after acidic or bile stress of each strain were subjected to principal component

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analysis (PCA) using a covariance matrix. Only strains showing resistance at least at 0.4% of bile

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salt for 2 h at 37° C were showed. Avoiding speculation, no statistical correlation (P>0.05) was

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found between the origin of the strains and the acidic or bile resistance (data not shown). Fig. 1

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shows the distribution of the strains based on acidic and bile resistance. The first two principal

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components explained ca. 88.19% of the total variance. The strains FS10, PS11, FS2 PM8, CM1,

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MF5 and GG were characterized by the highest stress resistance and were located in different zones

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of the plane delimited by the two principal components. All these strains together with other seven

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strains (SF6, FM13, FM22, SP13, PS2, FM14 and CM2), showing final cell density higher than 6

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log after acidic and bile stresses, were selected for further analyses.

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3.2. Molecular identification of lactobacilli strains

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The thirteen strains, all isolated from Pecorino Siciliano cheese, were selected on the basis of the

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properties described above, and were indentified through multiplex analysis, using tuf gene PCR-

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based assay. Primers used allowed species-specific identification of closely related species, such as

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Lactobacillus casei group. Results demonstrated that the strains FS2, FS10, PS2, SP13, PS11, SF6,

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FM13, FM14, FM22 belong to L. rhamnosus species, generating only one amplicon of 520 bp,

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while the strains CM1, CM2, MF5 and PM8 were ascribed to the Lactobacillus paracasei species,

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which generated two amplicons (240 and 520 bp) (Supplementary Fig. S1). The origin of the 13

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selected strains and their species affiliation are reported in Table 2.

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ACCEPTED MANUSCRIPT 3.3. Transit tolerance in simulated upper gastrointestinal (GI) tract

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The same thirteen lactobacilli strains, which exhibited the highest acidic and bile resistance, were

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selected for the in vitro transit tolerance in the GI tract. L. rhamnosus GG was used as positive

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control (Table 3). Except for the FS10 strain, all tested strains showed a decrease of cell viability

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during the treatment at pH 2.4. However, the loss of viability ranged from less than 1 log (FS2,

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PM8 and GG strains) to more than 4 log (CM2, FM13, FM14, FM22, PS2, SF6 and SP13 strains).

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The viability of the strains CM1, FS2, FS10, PM8 and PS11 was not affected by the exposure to the

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small intestinal juice with pancreatin and bile salt (Table 3). A good survival rate was found for

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MF5 and GG strains.

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3.4. Bacterial adhesion capacity

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In the present study the adhesion to Caco2 intestinal cells was determined for the thirteen tested

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lactobacilli strains and results are illustrated in Fig. 2. Cell adhesion capacity to Caco2 is calculated

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as the percentage of adhering bacteria in relation with the number of bacteria initially present in the

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well. The adhesion capacity of the 13 strains was highly variable (from 58% to 95%), depending on

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the bacterial strain. The highest adherence capacity (more than 90%) was demonstrated from strains

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CM1, MF5, PM8, and GG while adhesion ability ranging from 90 to 70% was revealed by the

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strain CM2, FM13, FM22, FS10 and FS2. The strains FM14, PS2, SF6 and SP13 showed adhesion

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ability between 70% and 50%).

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3.5. Hydrophobicity, co-aggregation and auto-aggregation activities

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Cell surface hydrophobicity was evaluated by the partitioning ratio of cells between aqueous and

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aromatic (xylene) layers and was measured in a two-phase system. Results are illustrated in the Fig.

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3. The cell adherence of the thirteen selected strains ranged from 8 to 93%. Four strains (FS10,

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MF5, PM8 and PS11) as well as the L. rhamnosus GG, exhibited good hydrophobic cell surface,

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with a percentage higher than 70%. CM1, CM2, FM13, FM14, FS2 and SF6 showed 11

ACCEPTED MANUSCRIPT hydrophobicity ranging from 65 to 25%, while the strains FM22, PS2 and SP13, belonging to L.

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rhamnosus species had the most hydrophilic characteristics (Fig. 3).

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Results of co-aggregation ability of the thirteen selected strains with the pathogen E. coli 555 is

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illustrated in Fig. 3. Out of 13 strains, 5 (CM1, FS10, MF5, PM8, and PS11) showed high

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percentage of co-aggregation ranging from 47 to 65%, after incubation at room temperature for 5 h.

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The highest percentages were displayed by the L. rhamnosus FS10 and by the L. paracasei PM8.

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Only the strains FM22 and SP13 showed weak co-aggregation properties (no higher than 5%) with

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E. coli 555.

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In this study the calculated value of auto-aggregation, revealed a variable distribution with

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percentage ranging from 5 to 68% (Fig. 3). Compared to GG, only L. rhamnosus FS10 strain

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displayed similar auto-aggregation value (68%). Five strains (CM1, FS2, MF5, PM8, and PS11)

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showed good auto-aggregation percentage (more than 40%), whereas the lowest values were found

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for CM2, FM13, FM14, PS2, SF6, and SP13.

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The inhibitory abilities of the lactobacilli strains, considered in the present study, in the form of cell

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free spent broth against commensal bacteria of GI tract and against common food pathogens, are

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shown in Table 4. The strains were also tested against L. casei, L. paracasei and L. plantarum, as

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philogenetically related species. None of the 13 selected strains, as well as the L. rhamnosus GG,

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inhibited the growth of the related strains (data not shown), while variability was revealed among

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strains against pathogens and commensal bacteria of GI tract. In particular, all strains indicated

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forceful antimicrobial activity against E. coli (with an inhibition zone higher than 8.0 mm in

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diameter) as L. rhamnosus GG, and variable activity versus L. monocytogenes and E. faecalis

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strains (Table 4). While 5 lactobacilli strains (FS2, FM13, FM4, FM22, and MF5) partially

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inhibited (2.0-4.0 mm) the growth of L. monocytogenes and E. faecalis, the other strains exhibited

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an inhibition zone between 5.0 and 8.0 mm of diameter.

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ACCEPTED MANUSCRIPT 310 3.7. Antibiotic resistance

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In table 4 are shown the MIC values of the 13 selected strains to antibiotics. Strains were considered

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resistant when they showed MIC value higher that the MIC breakpoints established by the European

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Food Safety Authority (EFSA, 2008). None of the strains exhibited resistance to penicillin G and

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rifampicin. On the contrary, all tested strains were resistant to vancomycin (without a specific

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breakpoint from EFSA). Regarding the antibiotics ampicillin, streptomycin and kanamycin, 5

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strains were resistant to ampicillin, 6 strains to streptomycin, and 6 strains to kanamycin as GG.

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Most of the strains, as well as GG, revealed the sensitive to tetracycline and to chloramphenicol.

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3.8. Haemolytic and bile salt hydrolase activities

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None of the examined strains exhibited β-haemolytic activity when grown in Columbia horse blood

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agar; only the strains CM1 and CM2 exhibited α-haemolysis (data not shown). The absence of

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haemolytic activity is considered as a safety prerequisite for the selection of a probiotic strain

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(FAO/WHO, 2002).

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Results of bile salt hydrolase (BSH) activity of lactobacilli strains are reported in Table 4. All the

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strains, excepted the strain MF5, did not exhibit BSH activity, as the GG in TDCA-MRS agar.

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Discussion

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Among fermented dairy products, traditional cheeses represent an alternative and readily source of

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lactobacilli with promising functional properties, which could be impact positively on human

332

health. Recently, Solieri and co-workers (2014) demonstrated that Parmigiano Reggiano cheese is

333

an interesting source of potential probiotic non-starter lactobacilli, as well Hashemi and co-workers

334

(2014) revealed the potential functional features of isolates from Kudish cheese, a traditional raw

335

goat cheese from Iran. In the present study, 177 lactobacilli isolated from traditional Sicilian 13

ACCEPTED MANUSCRIPT cheeses were screened for potential probiotic use. It’s noteworthy that the resistance to gastric

337

acidity and to bile salts, the antimicrobial activity, cell surface adhesion, and antibiotic

338

susceptibility are considered fundamental properties for selection of novel probiotic candidates. The

339

initial pH of the stomach and its decrease during gastric digestion significantly affect probiotic

340

strains viability. In this contest, the exposure to pH 2.5 is considered a discriminating factor for

341

selecting very-acidic tolerant strains even if never are directly exposed to gastric fluid (Prosad et al.,

342

1998). Our results revealed a wide range of heterogeneity among tested strains in relation to low pH

343

resistance, and most of the strains exhibited survival to pH 2.5, as previously reported

344

(Xanthopoulos et al., 2000; Mathara et al., 2008).

345

The presence of bile salts and pancreatic enzymes constitute another barrier to the survival of the

346

ingested bacteria during digestion. In the present study, out of 105 tested strains, 48 were resistance

347

to bile salts after 2 h of exposure at all tested percentages. The results are in agreement with those

348

acquired from previous studies, where L. rhamnosus and L. paracasei strains exhibited high in vitro

349

tolerance to bile salts. Moreover, our data revealed high ability of the L. rhamnosus GG to tolerate

350

both high acidity and bile salts, in contrast to results reported by Goldin et al. (1992). Bile tolerance

351

by probiotic has been revealed to be dependent on bile type and on the strain, with tolerance levels

352

of bile salts ranging from 0.125 to 2.0% (Lion et al., 2005). Among the 13 selected strains from

353

Pecorino cheese, which survived under the simulated in vitro static GI conditions, results

354

demonstrate that the low value of pH in the gastric compartment has more influence on the strains

355

survival than the presence of bile and pancreatic juice in duodenum compartment. This findings are

356

in accordance to previous studies (Succi et al., 2005; Vinderola et al., 2009; Pitino et al., 2010;

357

Solieri et al., 2014) and confirmed that the resistance to low pH and bile salts is a strain specific

358

trait (Papamanoli et al., 2003; Pitino et al., 2012). In the human gut, food matrix certainly protect

359

the bacteria from the deleterious effect of gastric and small intestinal secretions (Pitino et al., 2012),

360

improving the viability of microorganisms during gastric transit (Huang and Adams, 2004; Pitino et

361

al., 2012).

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14

ACCEPTED MANUSCRIPT Up to now is not yet completely clear if bile salt hydrolase (BSH) activity is a desirable trait for

363

promising probiotic strains selection. Current data suggest that this activity could maximize the

364

intestinal survival and persistence of probiotic strains increasing the overall beneficial effects

365

associated with the strain (Begley et al., 2006). Several f bile salt hydrolase enzymes have been

366

characterized and correlated to cholesterol lowering (Liong and Shah, 2005). . Moreover, Our

367

results indicated that all tested strains, except the L. paracasei MF5, were not able to deconjugate

368

bile salts and thus the majority of the breakdown products of the dehydroxylating activity by strains

369

may be precipitated and excreted in feces (Veysey et al., 2001). Moreover, the strains which did not

370

exhibit BSH activity were able to survive at different bile salts concentrations, confirming that the

371

two activities are not correlated each other, in accordance with previous reported data (Moser and

372

Savage, 2001).

373

The ability of the strains to adhere mucosal surface is another commonly encountered criterion for

374

probiotic strains selection, since it is directly related to their colonization and persistence in the GI

375

tract (Morelli, 2007). . Probiotic bacteria compete with invading pathogens for binding site to

376

epithelial cells and the overlying mucus layer in a strain-specific manner (Morrow et al., 2012). Our

377

results for bacterial adherence capacity to Caco-2 cells revealed adhesion ability of the 13 selected

378

strains, confirming that this property for Lactobacillus are strain and matrix dependent (Tallon et

379

al., 2007; Laparra and Sanz, 2009). Moreover, data demonstrated the high adhesion to cells of L.

380

rhamnosus GG strain, in contrast to previous study (Jensen et al., 2012). It is interesting to note that

381

the adhesion ability of lactobacilli has been correlated to cells hydrophobicity, which represents a

382

benefit for bacterial maintenance in the gastrointestinal tract (Kos et al., 2003). This work revealed

383

that strains with high adhesion capacity and hydrofobicity, including L. rhamnosus GG, showed

384

high auto-aggregation and co-aggregation abilities, in accordance with previous data (Pithva et al.,

385

2014). Nevertheless, conflicting evidences have been previously reported (Solieri et al., 2014; Botta

386

et al., 2014), indicating that adhesive and aggregative properties are strain dependent as well as

387

hydrophobic trait. Aggregation and co-aggregation for instance with pathogens, are important

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ACCEPTED MANUSCRIPT properties of probiotic strains in term of colonization, long-term persistence and anti-infection

389

mechanism against pathogen species in the GI tract. Data obtained through in vitro assay are

390

difficult to extrapolate to the situation in the human GI tract, where the host defense systems,

391

competition with the resident microbiota, mucosal shedding, and peristaltic flow are likely to

392

modify the bacterial adhesion (Lebeer et al., 2008). However, in vitro experiments provide

393

important information regarding species and strain differences (Jensen et al., 2012).

394

The ability to produce antimicrobial compounds may be one of the key characteristics for

395

competitive exclusion of pathogen survival in the intestine and expression of a probiotic effect for

396

the host (Salminen et al., 1998; Collado et al., 2005). All 13 selected strains tested showed forceful

397

activity against E. coli, in agreement with previous work (Tulumoglu et al., 2013). However, as

398

previous reported (Ambalam, 2009; Pithva et al., 2014), our results also confirmed the strain-

399

specific nature of the antimicrobial activity, in particular against L. monocytogenes and E. faecalis

400

strains.

401

Evaluating safety aspects, the 13 promising probiotic strains were, therefore, tested for antibiotic

402

resistance and for hemolytic activity. None of the strains exhibited resistance to penicillin G and

403

rifampicin, while all tested strains were resistant to vancomycin, supporting the native resistance of

404

L. casei, L. paracasei, and L. rhamnosus species to this antibiotic (Liu et al., 2009), which is not

405

transferable to other species because it is chromosomally encoded (Morrow et al., 2012). The

406

verification of transferable resistance is recommended prior to consider them safe for human

407

consumption (Reid et al., 2002; Huys et al., 2008). The resistance to vancomycin has been

408

attributed to the presence of D-Ala-D-lactate in their peptidoglycan instead of the normal D-Ala-D-

409

Ala, which is the target of the antibiotic (Monteagudo-Mera et al., 2011). Most of the strains, as the

410

GG strain, revealed the sensitive to tetracycline and chloramphenicol, confirming the generally

411

lower resistance of the lactobacilli species towards these antibiotics (Maragkoudakis et al., 2006)

412

and variable resistance among strains, including the GG, was achieved for the antibiotics ampicillin,

413

streptomycin and kanamycin, in accordance with other previous studies (Kirtzalidou et al. 2011;

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16

ACCEPTED MANUSCRIPT Argyri et al., 2013; Botta et al., 2014). The resistance to kanamycin has already been reported in

415

several Lactobacillus species and it could be attributable to the absence of cytochrome-mediated

416

electron transport, which mediate drug uptake, and to the membrane impermeability (Elkins et al.,

417

2004). The low resistance to vancomycin, ampicillin, streptomycin and kanamycin, could not

418

represent a safety concern, since they exhibited high susceptibility to clinically relevant antibiotics

419

and for their QPS status (EFSA, 2012) they could be totally free of transferable antibiotic resistance

420

genes. Moreover, the safety properties of the promising probiotic strains were also confirmed by the

421

absence of β-haemolytic α-haemolysis and activity, which was revealed only in two L. paracasei

422

strains, in accordance with Maragkoudakis et al. (2006) and Argyri et al. (2013).

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425

Traditional cheeses represent an excellent source of lactic acid bacteria biodiversity, which support

426

the selection of promising strains with functional properties.s. Results of the present study reveal

427

that the L. rhamnosus FS10 and L. paracasei PM8 both isolated from Pecorino Siciliano cheese,

428

exhibited in vitro favorable strain-specific probiotic properties higher than the reference

429

rhamnosus GG. Moreover, these strains were confirmed to be safe for the absence of resistance to

430

most clinical antibiotics. Further in vivo investigation will aim to elucidate both their potential

431

health benefits to human and their technological properties before allowing their utilization as

432

probiotics in functional foods.

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Acknowledgment

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This work was partially founded by AG Pharma srl, Italy. Authors thank Prof. F. Gardini from the

436

University of Bologna, Italy, and Dr J. Reinheimer from INLAIN, UNL-CONICET, Santa Fe,

437

Argentina for gently providing E coli 555 and L. acidophilus DRU strains, respectively.

438 17

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ACCEPTED MANUSCRIPT Table and Figure legend Table 1 Resistance to low pH and tolerance at different bile salt concentrations of Lactobacillus spp. strains.

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Table 2

Origin of selected strains and species affiliation through tuf gene PCR-based assay.

Table 3

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Transit tolerance in simulated upper gastrointestinal (GI) tract expressed as viability of lactobacilli.

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Antimicrobial activity of cell free spent broth of lactobacilli strains against common pathogens, antibiotic resistance and bile salt hydrolase activity of the strains.

Figure 1

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Survival of lactobacilli strains in relation to resistance at pH 2.5 and to tolerance at 1% of bile salts. In the x-and y-axes the OD values of each strain grown onto MRS medium after 4 h of incubation at 37°C at 1% of bile salt and at pH 2.5, respectively, are reported. Figure 2

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Adhesion ability to the Caco 2 cell lines of the 13 selected lactobacilli strains and LGG strain. The capacity is calculated as the percentage of adhered lactobacilli in relation to the total number of strains added (log 8+0.3). (Error bars indicate standard deviation from two replications).

Hydrophobicity, auto-aggregation and co-aggregation with E. coli percentage of lactobacilli strains. (Error bars indicate standard deviation from two replication). Figure S1

Profile of multiplex- PCR using tuf gene of the thirteen selected strains. M: 1kb plus marker; lines 1, 5, 6 and 10: strains identified as L. paracasei species (bands at 540 bp and 240 bp) and lines 2, 3, 4, 7, 8, 9, 11, 12, and 13: strains identified as L. rhamnosus species (band at 540 bp).

ACCEPTED MANUSCRIPT Table 2 Origin of selected strains and species affiliation through tuf gene PCR-based assay Strains

Origin

Species affiliation

PM8

Pecorino cheese 60 days old

L. paracasei

Pecorino cheese 120 days old L. paracasei

CM1, CM2

Pecorino cheese 180 days old L. paracasei

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MF5

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PS11, SP13, PS2 Pecorino cheese 60 days old L. rhamnosus SF6, FM13, FM14, Pecorino cheese 120 days old L. rhamnosus FM22, FS10, FS2

ACCEPTED MANUSCRIPT

Table 1 Resistance to low pH and tolerance at different bile salt concentrations of Lactobacillus spp. strains Strainsa 2h

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CS1, PM1, PM2, PM3, PM5, PM6, PM9, PM13, PM18, PM19, PM20, PM24, PM25, PM26, MP1, MP2, MP3, MP4, MP8, MP9, MP10, MP11, MP12, MP14, MP16, MP17, MP19, MP20, MP21, MP22, MP24, MP15, FM12 CS2, CS3, SP16, SP17, SP18, SP15, SP19, SP21, MC1, PM16, FM2, FM6, FM8, FM9, FM20, FM21

SC1, SF1, SF2, SF3, SF4, SF5, SF7, SF8, SF9, SF10, SF11, SF13, SF14, SF15, SF16, SF17, MF4, MF8, MP7,

SC

MP5, MP6, MP13, FM18 PS2, PS1, PS3, PS8, PS9, PS12, PS13, PS15, PS17, SP4,SP11, SP26, SP29 PS4, PS5, PS6, PS7, SP1, SP2, SP6, SP7, SP8, SP9, SP10, SP20, SP22, SP23

M AN U

PS10, PS14, SP4, SP3, SP14, SP28, SP24, FS18, PM4, PM7, PM10, PM11, PM12, PM14, PM15, PM17, PM21, PM22, PM23, PM27, PM28, MP18, MP23, FM1, FM3, FM4, FM5, FM7, FM10, FM11, FM16, FM19 PS11, FS10, FS14, FS15, FS19 PS16 PS18, PS19, FS1 SP13

CM1

-

ND

ND

ND

-

-

-

ND

ND

ND

+

-

-

ND

ND

ND

+++

+++

++

***

*

*

+++

+++

++

*

-

-

+++

+++

+++

-

-

-

+++

+++

+++

***

**

**

+++

+++

++

-

-

-

+++

+++

+++

*

-

-

+++

++

++

***

-

-

++

+

+

-

-

-

+++

++

+

***

***

***

+

***

***

***

+++

++

***

**

**

SF6, SF12, SF18, SF19

+++

+++

+++

***

*

*

+++

+++

+++

***

**

**

+++

+++

++

***

*

*

+++

++

+

***

**

**

+++

++

++

*

-

-

+++

+++

+++

***

**

*

+++

+++

++

***

***

***

+++

+++

+++

***

**

**

FM17 FM22 MF5, MF7 GG

EP

+++

FM14

c)

-

+++

FM13, FM15

b)

++

+++

PM8

a)

6h

Bile salt tolerance atc: 0.4% 1.0% 2.0%

FS2, FS3, FS4, FS5, FS6, FS7, FS8, FS9, FS11, FS12, FS13, FS16, FS17, FS20, FS21, SF20

AC C

CM2

TE D

SP25, MF1, MF2, MF3, MF6, MF9

pH 2.5b 4h

Strain code +++: range of the final viable counts of 7-8 log cfu/ml; ++: range of the final viable counts of 6-7 log cfu/ml; +: range of the final viable counts of 5-6 log cfu/ml; -: the final viable counts