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Probiotic potential of selected lactic acid bacteria strains isolated from Brazilian kefir grains

Open AccessPublished:April 01, 2015DOI:https://doi.org/10.3168/jds.2014-9265

      Abstract

      A total of 34 lactic acid bacteria isolates from 4 different Brazilian kefir grains were identified and characterized among a group of 150 isolates, using the ability to tolerate acidic pH and resistance to bile salts as restrictive criteria for probiotic potential. All isolates were identified by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of representative amplicons. Eighteen isolates belonged to the species Leuconostoc mesenteroides, 11 to Lactococcus lactis (of which 8 belonged to subspecies cremoris and 3 to subspecies lactis), and 5 to Lactobacillus paracasei. To exclude replicates, a molecular typing analysis was performed by combining repetitive extragenic palindromic-PCR and random amplification of polymorphic DNA techniques. Considering a threshold of 90% similarity, 32 different strains were considered. All strains showed some antagonistic activity against 4 model food pathogens. In addition, 3 Lc. lactis strains and 1 Lb. paracasei produced bacteriocin-like inhibitory substances against at least 2 indicator organisms. Moreover, 1 Lc. lactis and 2 Lb. paracasei presented good total antioxidative activity. None of these strains showed undesirable enzymatic or hemolytic activities, while proving susceptible or intrinsically resistant to a series of clinically relevant antibiotics. The Lb. paracasei strain MRS59 showed a level of adhesion to human Caco-2 epithelial cells comparable with that observed for Lactobacillus rhamnosus GG. Taken together, these properties allow the MRS59 strain to be considered a promising probiotic candidate.

      Key words

      Introduction

      Kefir is a fermented milk product originating from the Northern Caucasus. The name kefir is derived from the Turkish language word keyif, meaning “good feeling” for the feelings experienced after drinking it (
      • Leite A.M.O.
      • Miguel M.A.
      • Peixoto R.S.
      • Rosado A.S.
      • Silva J.T.
      • Paschoalin V.M.
      Microbiological, technological and therapeutic properties of kefir: A natural probiotic beverage.
      ). The fermented beverage is acidic, viscous, and slightly carbonated, and it contains small amounts of alcohol (
      • Leite A.M.O.
      • Leite D.C.
      • Del Aguila E.M.
      • Alvares T.S.
      • Peixoto R.S.
      • Miguel M.A.
      • Silva J.T.
      • Paschoalin V.M.
      Microbiological and chemical characteristics of Brazilian kefir during fermentation and storage processes.
      ). Traditionally, kefir is made by using kefir grains as a starter (
      • Leite A.M.O.
      • Leite D.C.
      • Del Aguila E.M.
      • Alvares T.S.
      • Peixoto R.S.
      • Miguel M.A.
      • Silva J.T.
      • Paschoalin V.M.
      Microbiological and chemical characteristics of Brazilian kefir during fermentation and storage processes.
      ,
      • Leite A.M.O.
      • Miguel M.A.
      • Peixoto R.S.
      • Rosado A.S.
      • Silva J.T.
      • Paschoalin V.M.
      Microbiological, technological and therapeutic properties of kefir: A natural probiotic beverage.
      ). Kefir grains are white to yellowish, cauliflower-like grains, 0.3 to 3.5 cm in diameter, with a slimy but firm texture. The grains are composed of an inert matrix made up of polysaccharides and proteins. The matrix is densely populated by lactic acid bacteria (LAB) species, acetic acid bacteria, and yeasts (
      • Leite A.M.O.
      • Mayo B.
      • Rachid C.T.C.C.
      • Peixoto R.S.
      • Silva J.T.
      • Paschoalin V.M.F.
      • Delgado S.
      Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.
      ,
      • Leite A.M.O.
      • Miguel M.A.
      • Peixoto R.S.
      • Rosado A.S.
      • Silva J.T.
      • Paschoalin V.M.
      Microbiological, technological and therapeutic properties of kefir: A natural probiotic beverage.
      ).
      Kefir beverage has a long tradition of consumption in Eastern Europe, and it is now spreading around the world due to its potential health-associated properties. Although some of the reported health benefits do not yet have well-documented scientific evidence or clinical demonstration, several in vitro and animal studies have associated kefir beverage with alleviation of lactose intolerance (
      • Hertzler S.R.
      • Clancy S.M.
      Kefir improves lactose digestion and tolerance in adults with lactose maldigestion.
      ), immunomodulation (
      • Hong W.-S.
      • Chen H.-C.
      • Chen Y.-P.
      • Chen M.-J.
      Effects of kefir supernatant and lactic acid bacteria isolated from kefir grain on cytokine production by macrophage.
      ), antimicrobial activity against pathogenic microorganisms (
      • Chifiriuc M.C.
      • Cioaca A.B.
      • Lazar V.
      In vitro assay of the antimicrobial activity of kephir against bacterial and fungal strains.
      ), and balance of the intestinal microbiota (
      • Urdaneta E.
      • Barrenetxe J.
      • Aranguren P.
      • Irigoyen A.
      • Marzo F.
      • Ibáñez F.C.
      Intestinal beneficial effects of kefir-supplemented diet in rats.
      ). Traditionally, the functional properties of kefir have been attributed mainly to its bioactive peptide content and to kefiran, its main soluble exopolysaccharide (
      • Santos A.
      • San Mauro M.
      • Sanchez A.
      • Torres J.M.
      • Marquina D.
      The antimicrobial properties of different strains of Lactobacillus spp. isolated from kefir.
      ;
      • Rodrigues K.L.
      • Caputo L.R.
      • Carvalho J.C.
      • Evangelista J.
      • Schneedorf J.M.
      Antimicrobial and healing activity of kefir and kefiran extract.
      ). However, the potential beneficial effects might also be mediated by the undefined microbial composition of this fermented milk or by the secondary metabolites (
      • Nielsen B.
      • Gurakan G.C.
      • Unlu G.
      Kefir: A multifaceted fermented dairy product.
      ).
      Although a reasonable number of well-characterized probiotic strains are commercially available around the world, screening for novel strains is still of great interest from an industrial point of view (
      • Vinderola G.
      • Capellini B.
      • Villarreal F.
      • Suárez V.
      • Quiberoni A.
      • Reinheimer J.
      Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use.
      ;
      • Ayeni F.A.
      • Sanchez B.
      • Adeniyi B.A.
      • de Los Reyes-Gavilan C.G.
      • Margolles A.
      • Ruas-Madiedo P.
      Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow’s intestine.
      ). Additionally, strains expressing unique and particular characteristics that may enable health benefits may arise in the characterization of natural fermented dairy products such as kefir. This traditional product might be an interesting source of LAB strain with specific functional properties. Even though many authors advocate the importance of human origin as a selective criterion for the search of probiotic strains, an expert panel proposed by
      FAO/WHO
      suggested that the probiotic activity is more important than the source of the microorganism. In fact, previous reports described the evaluation and selection of kefir LAB isolates for potential use as probiotics (
      • Golowczyc M.A.
      • Gugliada M.J.
      • Hollmann A.
      • Delfederico L.
      • Garrote G.L.
      • Abraham A.G.
      • Semorile L.
      • De Antoni G.
      Characterization of homofermentative lactobacilli isolated from kefir grains: Potential use as probiotic.
      ;
      • Zheng Y.
      • Lu Y.
      • Wang J.
      • Yang L.
      • Pan C.
      • Huang Y.
      Probiotic properties of Lactobacillus strains isolates from Tibetan kefir grains.
      ).
      The aim of the present study was to identify and characterize LAB strains isolated from traditional kefir grains, displaying in vitro properties related to their probiotic potential, according the guidelines recommended by
      FAO/WHO
      . After a complete characterization and the corresponding in vivo trials, these strains could be ultimately included as probiotics in functional foods.

      Materials and Methods

       Isolation of Bacteria from Kefir Grains

      The LAB were isolated by dilution and plating from 4 kefir grains collected in different regions of Brazil. Briefly, 10 g of each kefir sample was homogenized in 90 mL of sodium citrate (2%). Serial decimal dilutions were obtained and plated on lactobacilli de Man, Rogosa, Sharpe (MRS) and M17 agar media (Difco, Sparks, MD) supplemented with 200 μg/mL of cycloheximide (Sigma-Aldrich, St. Louis, MO), and incubated in aerobic and anaerobic (Gaspak EZ, Difco) conditions at 30°C for 72 h. Representative colonies of all morphologies were taken randomly and purified on the same media by subculturing. Gram-positive, catalase-negative isolates were considered as presumptive LAB, which were stored in 15% glycerol at −80°C.
      For all subsequent assays, LAB were activated in the corresponding media at 30°C for 18 to 24 h, and subcultured in the same conditions.

       Tolerance of the Isolates at Low pH

      The ability of the isolates to tolerate low pH was assayed as described by
      • Nishida S.
      • Michinaka A.
      • Nakashima K.
      • Iino H.
      • Fujii T.
      Evaluation of the probiotic potential of Lactobacillus paracasei KW3110 based on in vitro tests and oral administration tests in healthy adults.
      in MRS broth supplemented with 0.2% sodium thioglycolate (MRS-THIO). In short, overnight cultures were harvested by centrifugation and cells were suspended in PBS (pH 6.5) to obtain an optical density at 600 nm (OD600) = 0.5. Cell suspensions (≈107 to 109 cfu/mL) were 10-fold diluted with MRS medium, adjusted to pH 3.0 with HCl, and incubated at 37°C for 3 h. The pH tolerance of the cells was determined by enumerating the viable cells on MRS agar plates. Nontreated cultures used as controls were suspended in conventional, nonacidified MRS (pH 6.5).

       Bile Tolerance of the Isolates

      Tolerance to bovine bile (Oxgall, Difco) was assayed by growing the isolates in agar plates and broth, following the procedure reported by
      • Delgado S.
      • O'Sullivan E.
      • Fitzgerald G.
      • Mayo B.
      Subtractive screening for probiotic properties of Lactobacillus species from the human gastrointestinal tract in the search for new probiotics.
      and
      • Guo Z.
      • Wang J.
      • Yan L.
      • Chen W.
      • Liu X.-m.
      • Zhang H.-p.
      In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a potential new probiotic, with selected probiotic strains.
      , respectively. The growth rate of the strains was estimated in MRS-THIO broth in the absence (control) and in the presence (test) of 0.3% Oxgall. Overnight cultures were inoculated (1%) into the liquid medium, and cultured at 37°C for up to 9 h. Absorbance at 620 nm was measured every hour. The effect of the bile salts was scored as the time difference required to increase by 0.3 units the absorbance of the culture at 620 nm (OD620) in MRS-THIO broth with and without 0.3% bile salts. The growth delay (hours) between the culture media was considered as the lag time (LT).
      Additionally, tolerance of strains to different concentrations of bile salts was assayed by a plate assay. Individual colonies growing in MRS agar plates were suspended in 2 to 5 mL of sterile saline solution 0.85% at a density corresponding to McFarland standard 1. Aliquots of the suspensions (10 µL) were spotted onto bile-containing 0.3, 0.5, 1, and 2% (wt/vol) agar plates. The plates were incubated at 37°C under anaerobic conditions and growth was recorded after 24 to 48 h. A plate without bile was used as positive control. The experiments were performed in duplicate.

       Identification of LAB Isolates

      Total genomic DNA of the isolates was extracted using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich), following the manufacturer’s recommendations. Purified DNA was used as a template to amplify a segment of the 16S rRNA gene by the PCR technique using the universal prokaryotic primers S-D-Bact-0008-a-S-20 (27F; 5′-AGAGTTTGATCCTGGCTCAG-3′) and S-*-Univ-1492R-b-A-21 (1492R; 5′-GGTTACCTTGTTACGACTT-3′). For the amplified ribosomal DNA restriction analysis (ARDRA), amplicons were purified through GenElute PCR Clean-Up columns (Sigma-Aldrich), digested with HaeIII and HhaI restriction enzymes (Invitrogen, Paisley, UK) and electrophoresed in agarose gels. Gels were stained with ethidium bromide (0.5 mg/mL) and photographed under UV light. Representative amplicons of the different ARDRA profiles were sequenced. Sequencing was accomplished in an ABI 373 DNA sequencer (Applied Biosystems, Carlsbad, CA). On average, 850 bp of sequence were obtained, which were compared with those deposited in the GenBank database using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/). Following the criterion applied by
      • Palys T.
      • Nakamura L.K.
      • Cohan F.M.
      Discovery and classification of ecological diversity in the bacterial world: The role of DNA sequence data.
      , sequences with a percentage of identity of 98% or higher to those in databases were allocated to the same species.

       Molecular Typing Analyses

      To exclude replicates, LAB isolates were grouped by both repetitive extragenic palindromic PCR (rep-PCR), using the primer BOXA2R (5′-ACGTGGTTTGAAGAGATTTTCG-3′), as reported by
      • Koeuth T.
      • Versalovic J.
      • Lupski J.R.
      Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria.
      , and random amplification of polymorphic DNA (RAPD) with primer M13 (5′-GAGGGTGGCGGTTCT-3′), as reported by
      • Rossetti L.
      • Giraffa G.
      Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.
      . Banding patterns were examined with the Bionumerics 6.5 software program (Applied Maths, Sint-Martens-Latem, Belgium) using Dice’s coefficient. Cluster analyses of composite data obtained with rep-PCR and RAPD were achieved using the unweighted pair group method with arithmetic averages.

       Antimicrobial Activity

       Pathogen Inhibition

      The capability of the strains to inhibit a group of foodborne pathogens was determined using an agar spot test as described by
      • Ripamonti B.
      • Agazzi A.
      • Bersani C.
      • De Dea P.
      • Pecorini C.
      • Pirani S.
      • Rebucci R.
      • Savoini G.
      • Stella S.
      • Stenico A.
      • Tirloni E.
      • Domeneghini C.
      Screening of species-specific lactic acid bacteria for veal calves multi-strain probiotic adjuncts.
      . Overnight test cultures were spotted (2 µL) on the surface of modified MRS agar (without ammonium citrate and sodium acetate) and incubated anaerobically for 24 h at 30°C. Cells were then inactivated with chloroform for 30 min. Escherichia coli ATCC 25922, Salmonella enterica var. Enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes ATCC 15313 were used as indicators. A 100-µL volume of an overnight culture of each indicator was mixed with 10 mL of brain heart infusion (Difco) soft agar (0.7%), and poured onto MRS agar plates. These were incubated aerobically at 37°C for 24 h. Lactobacillus acidophilus ATCC 4356 was used as a negative control. Inhibition but no clear-cut halo or a halo <1 mm was recorded as ±; a clear zone of growth inhibition around spots >1 mm was scored as + (positive); and an inhibition zone between 2 and 5 mm surrounding the colony was recorded as ++.

       Bacteriocin Production

      The production of bacteriocin-like inhibitory substances (BLIS) was successively examined using an agar spot test and a well-diffusion assay. Lactobacillus sakei CECT 906 and Lactococcus lactis IL 1403, 2 well-recognized bacteriocin-susceptible strains (
      • Alegría A.
      • Delgado S.
      • Roces C.
      • Lopez B.
      • Mayo B.
      Bacteriocins produced by wild Lactococcus lactis strains isolated from traditional, starter-free cheeses made of raw milk.
      ), and Listeria monocytogenes ATCC 15313 were used as indicators. Overnight cultures were spotted on the surface of M17 and MRS agar plates (0.2% glucose), incubated for 24 h at 30°C for lactococci and leuconostoc strains, and at 37°C for lactobacilli, and inactivated as described previously with chloroform. Spots were covered with 10 mL of soft agar (0.75%) inoculated with the indicators and incubated under the required conditions.
      Positive cultures were tested by a well-diffusion assay. Briefly, 20 mL of agar medium at 45°C was vigorously mixed with 200 µL of an overnight culture of each indicator and poured into Petri dishes. Supernatants from overnight cultures of the test strains were neutralized to pH 6.5 to 7.0 with 0.1 M NaOH, centrifuged at 10,000 × g for 5 min at 4°C, and filter-sterilized through a 0.20-µm pore membrane (Millipore, Bedford, MA). Fifty-microliter aliquots of each supernatant were placed in a well excavated in the agar. To allow the diffusion of the supernatant into the agar, plates were maintained at 4°C for 1 h before incubation. Inhibition of the indicators was evaluated after incubation at 37°C for 24 h.
      To investigate the proteinaceous nature of the BLIS, supernatants were tested in the vicinity of wells filled with 50 µL of a solution of either proteinase K or pronase (each at a concentration of 20 mg/mL). After incubation, plates were examined to judge whether the inhibitory substance was sensitive to proteolysis. Lactococcus lactis 1A6, a nisin producer strain (
      • Alegría A.
      • Delgado S.
      • Roces C.
      • Lopez B.
      • Mayo B.
      Bacteriocins produced by wild Lactococcus lactis strains isolated from traditional, starter-free cheeses made of raw milk.
      ), was used as control. Tests were performed in duplicate.

       Antioxidative Activity

       Sample Preparation for the Antioxidative Assays

      Overnight cultures in MRS broth were harvested by centrifugation at 4°C for 10 min, washing with isotonic saline solution (0.85%) at 4°C, and suspended in phosphate buffer with 1 mM EDTA, pH 7.5. The suspension was adjusted to an OD600 = 1.0. To obtain the cell extracts, cells were disrupted in a cell disruptor (Constant Systems, Daventry, UK) and deposited immediately in an ice bath. The extracts were then centrifuged at 10,000 × g at 4°C for 10 min to eliminate cell debris.

       Total Antioxidative Activity

      To evaluate the total antioxidative activity (TAA) of the strains, the linolenic acid (LA) test was used as described by
      • Kullisaar T.
      • Zilmer M.
      • Mikelsaar M.
      • Vihalemm T.
      • Annuk H.
      • Kairane C.
      • Kilk A.
      Two antioxidative lactobacilli strains as promising probiotics.
      , using 45 µL of the samples (lysate or whole bacterial cells). The absorbance at 534 nm was measured on a UV-Vis Spectrophotometer (Hitachi High-Technologies, Tokyo, Japan) and the percentage of TAA of the samples was expressed as [1 − (As/Ac)] × 100], where As is the absorbance in the presence of the sample and Ac is the absorbance of the control without sample. Intact cells and cell lysates were assayed in triplicate.

       Glutathione Assay

      Reduced and oxidized glutathione and the glutathione redox status were evaluated using cell-free extracts and the GSH/GSSG Ratio Assay kit (Millipore, Billerica, MA) following the manufacturer’s instructions. The glutathione content was quantified on the basis of a standard curve generated with known amounts of glutathione. The reduced glutathione (GSH) content was calculated as the difference between the total GSH and the oxidized glutathione (GSSG). The glutathione redox ratio was expressed as GSH/GSSG. Escherichia coli CECT 515 was used as a positive control (
      • Masip L.
      • Veeravalli K.
      • Georgiou G.
      The many faces of glutathione in bacteria.
      ).

       Adhesion Assay

      The epithelial intestinal cell line Caco-2 purchased from the European Collection of Cell Culture (ECACC 86010202) was used to assess the adhesion ability of selected strains. The culture and maintenance of the cell line were carried out following standard procedures (
      • Sánchez B.
      • Fernández-García M.
      • Margolles A.
      • de los Reyes-Gavilán C.G.
      • Ruas-Madiedo P.
      Technological and probiotic selection criteria of a bile-adapted Bifidobacterium animalis ssp. lactis strain.
      ) using DMEM medium supplemented with 20% fetal bovine serum, nonessential aminoacid solution, and a mixture of antibiotics (50 µg/mL of penicillin-streptomycin, 50 µg/mL of gentamicin, and 1.25 µg/mL of amphotericin B; all reagents from Sigma-Aldrich). The cell line was used after reaching the confluent-differentiated monolayer state (13 ± 1 d). The strain Lactobacillus rhamnosus LMG 18243 (also known as Lb. rhamnosus GG) was used as a reference control for adherence.
      Lactobacilli strains, grown overnight in 10 mL of MRS under standard conditions, were harvested by centrifugation, washed twice in Dulbecco’s PBS solution (Sigma-Aldrich), and suspended in supplemented DMEM media without antibiotics, at a concentration of approximately 108 cfu/mL. To remove antibiotics from the cells, monolayers were washed twice in Dulbecco’s PBS. Subsequently, the bacterial suspensions were added at a bacteria:eukaryotic cell ratio of 10:1, and incubated for 1 h at 37°C in a 5% CO2 atmosphere. Afterward, wells were gently washed 3 times with Dulbecco’s PBS buffer to remove nonadhered bacteria. Monolayers were disrupted with an EDTA-trypsin solution (Sigma-Aldrich), and the attached bacteria were counted by plating in MRS agar. Adhesion was expressed as the percentage of bacteria adhered with respect to total number of bacteria added. Experiments were carried out using 2 independent Caco-2 plates (2 consecutive passes), and in each plate bacterial strains were analyzed in duplicate.

       Safety Assessment

       Hemolysin Production

      Hemolysin production was analyzed on Columbia agar plates containing 5% sheep blood (bioMérieux, Montalieu-Vercieu, France). The presence of β- or α-hemolysis is indicated by the formation of clear or greenish zones around the colonies, respectively.

       Antibiotic Resistance

      The MIC of a series of antibiotics was assayed on the selected strains by microdilution in VetMIC plates for LAB (SVA, Uppsala, Sweden), following the manufacturer’s recommendations. Colonies grown on LSM (
      • Klare I.
      • Konstabel C.
      • Muller-Bertling S.
      • Reissbrodt R.
      • Huys G.
      • Vancanneyt M.
      • Swings J.
      • Goossens H.
      • Witte W.
      Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria.
      ) agar plates were suspended in 5 mL of sterile saline solution (0.9%) to obtain a density corresponding to McFarland standard 1. Suspensions were further diluted 1:1,000 in LSM. One hundred microliters of this dilution was added to each well of the VetMIC plate. The plates were incubated at 37°C for 48 h. The MIC were defined as the lowest antibiotic concentration at which no visual growth was observed.

       Enzyme Activities

      Enzyme activities were measured by the commercial, semiquantitative API-ZYM system (bioMérieux) following the manufacturer’s recommendations. In short, 65 µL of a cell suspension corresponding to McFarland standard 5 were inoculated in each well of the API-ZYM strips. Enzyme activities were evaluated after 4 h of incubation in anaerobiosis at 37°C. Enzyme activities were recorded from 0 (no activity) to 5 (≥40 nmol of product released) with the API-ZYM color reaction chart.

       Statistical Analyses

      Statistical comparisons for both the adhesion and antioxidative tests were performed using the Statistica software package for Windows version 7.0 (Statsoft, Tulsa, OK). Significant differences between treatments were tested by the ANOVA test (1-way ANOVA), followed by a comparison between means using Fisher’s least significance difference method, with levels of significance set at P < 0.05.

      Results

       LAB Screening for pH and Bile-Salt Tolerance

      Thirty-seven out of the 150 tested LAB isolates showed tolerance to pH 3.0 during 3 h of incubation. However, the growth in these conditions was not comparable with that in conventional MRS (pH 6.5), with reductions in counts at least 2 logarithmic units compared with controls. These 37 isolates were tested by a plate assay for resistance to different bile concentrations ranging from 0.3 to 2%, and by tolerance in liquid containing 0.3% of Oxgall. In the latter medium, a LT ranging from 0.5 to 4 h was observed for 34 isolates (Table 1); these were considered to be bile tolerant. In contrast, 3 LAB isolates presented an LT >9 h; these were considered bile susceptible.
      Table 1Bile resistance of 32 identified and typed lactic acid bacteria isolated from kefir grains
      StrainIdentificationOrigin
      Region of Brazil: AR=Niterói, RJ; AD=Lavras, MG; AV=Viçosa, RJ; and AF=Alfenas, MG.
      Bile assay
      Lag time (h),

      0.3% bile
      Growth plate
      Growth plate: –=no growth; +=positive growth.
      0.3% bile0.5% bile1% bile2% bile
      MRS17Lactococcus lactis ssp. cremorisAR2++++
      M1711BAR3++
      MRS26AR1.5++
      MRS47AD3+
      M1732AR1+
      M1734AR2++
      M171AR1++
      MR2Lc. lactis ssp. lactisAV1++++
      MRS52AD1++
      MRN3AV4++
      MRN4Lactobacillus paracaseiAR1++++
      MRS59AF2+++
      M1743AF2++++
      MRS55AF3+++
      MRS2Leuconostoc mesenteroidesAR2++
      GYP8AR3+++
      MRS12AR1.5++
      MRS50AD2+
      MRS48AF3+
      MRS53AF2+
      MRS25AR2++++
      GYP7AR1++++
      MRS8AR2++
      MRS14AR1.5++
      GYP5AR2+
      MRS10AR4++
      GYP9AR1++++
      GYP12AR0.5+++
      M1721AR2.5+
      MRS19AR1.5+
      MRS24AR1+
      M1711AAR2++
      1 Region of Brazil: AR = Niterói, RJ; AD = Lavras, MG; AV = Viçosa, RJ; and AF = Alfenas, MG.
      2 Growth plate: – = no growth; + = positive growth.
      As a result of the bile plate assay, 34 isolates grew in 0.3%, 25 grew in 0.5%, 13 in 1%, and 8 in 2% (Table 1).

       LAB Identification

      Among the 34 isolates 4 different representative ARDRA profiles were observed with the HaeIII and HhaI restriction enzymes (data not shown). Representative 16S rDNA amplicons of each of the different profiles were selected for sequencing. Sequence comparisons showed a homology higher than 98% to 4 different LAB species. Therefore, the 34 isolates were identified as follows: Leuconostoc mesenteroides (18), Lactococcus lactis ssp. cremoris (8), Lactobacillus paracasei (5), and Lactococcus lactis ssp. lactis (3).

       LAB Typing

      The LAB isolate typing was performed by a combination of rep-PCR and RAPD techniques. Using a 90% similarity as threshold (Figure 1), 32 different strains (4 Lb. paracasei, 10 Lc. lactis, and 18 Leu. mesenteroides) were considered. These were all subjected to further analyses.
      Figure thumbnail gr1
      Figure 1Combined dendrogram obtained from repetitive extragenic palindromic (rep)-PCR and random amplification of polymorphic DNA (RAPD)-PCR profiles, using BOXA2R and M13 primers corresponding to 5 Lactobacillus paracasei strains (panel A), 11 Lactococcus lactis strains (panel B), and 18 Leuconostoc mesenteroides strains (panel C).

       Antimicrobial Properties

       Antagonistic Activity Against Pathogens

      Pathogens were inhibited by most strains in the agar spot test (Table 2). Listeria monocytogenes, E. coli, and S. enterica were inhibited by 23, 22, and 20 strains, respectively. In contrast, S. aureus was inhibited by only 7 strains. Pathogen inhibition, however, was not confirmed by the agar well-diffusion assay, with the exception of L. monocytogenes, which was inhibited by 4 strains after neutralization of the cell-free supernatants (see below).
      Table 2Antimicrobial activity data of 32 identified and typed lactic acid bacteria isolated from kefir grains
      Strain
      Strains in bold produce bacteriocin-like inhibitory substances.
      IdentificationOriginInhibition profile spot test
      Indicators: I=Escherichia coli ATCC 25922; II=Salmonella enterica serovar Enteritidis ATCC 13076; III=Staphylococcus aureus ATCC 25923; IV=Listeria monocytogenes ATCC 15313; V=Lactobacillus sakei CECT 906; VI=Lactococcus lactis IL1403. Activity: ±=inhibition but no clear halo; +=presence of a clear zone of growth inhibition around spots >1mm; ++=presence of a clearly defined inhibition zone between 2 and 5mm surrounding the colony in the spot test or the wells containing neutralized, cell-free supernatant; –=no inhibition.
      Pathogen inhibitionBacteriocin production
      IIIIIIIVIVVVI
      MRS17Lactococcus lactis ssp. cremorisAR±++
      M1711BAR±±
      MRS26AR++±++++++++
      MRS47AD++
      M1732AR++±±
      M1734AR++±±±
      M171AR++±++++++++
      MR2Lc. lactis ssp. lactisAV++++++
      MRS52AD++++++++
      MRN3AV++±++++
      MRN4Lactobacillus paracaseiAR±++++
      MRS59AF++++++++++
      M1743AF++++±±±±
      MRS55AF++++++++++++
      MRS2Leuconostoc mesenteroidesAR±±±
      GYP8AR±±±
      MRS12AR+±±±±
      MRS50AD±±±±
      MRS48AF+±±±
      MRS53AF
      MRS25AR±±±±
      GYP7AR±
      MRS8AR±±±
      MRS14AR±±±
      GYP5AR+±±±
      MRS10AR±±±±
      GYP9AR±±±±
      GYP12AR
      M1721AR±±±±
      MRS19AR±±±±
      MRS24AR±±±±
      M1711AAR±±
      1 Strains in bold produce bacteriocin-like inhibitory substances.
      2 Indicators: I = Escherichia coli ATCC 25922; II = Salmonella enterica serovar Enteritidis ATCC 13076; III = Staphylococcus aureus ATCC 25923; IV = Listeria monocytogenes ATCC 15313; V = Lactobacillus sakei CECT 906; VI = Lactococcus lactis IL1403. Activity: ± = inhibition but no clear halo; + = presence of a clear zone of growth inhibition around spots >1 mm; ++ = presence of a clearly defined inhibition zone between 2 and 5 mm surrounding the colony in the spot test or the wells containing neutralized, cell-free supernatant; – = no inhibition.

       Bacteriocin-Like Inhibitory Substance Production

      Lactobacillus sakei CECT 906 was inhibited by 4 strains and Lc. lactis IL 1403 by 12 strains (Table 2) in the agar spot assay. The isolates demonstrating antibacterial activity against any of the indicators were subsequently subjected to the well-diffusion assay. None of the Leuconostoc strains showed inhibition against indicators in this assay. Lactococcus lactis IL 1403 was inhibited by 3 lactococci strains (MRS26, M171, and MRS52). Both L. monocytogenes ATCC 15313 and Lb. sakei CECT 906 were inhibited by the 3 above-mentioned lactococci strains and one Lb. paracasei (MRS55) strain. The proteinaceous nature of the BLIS produced by these 4 strains was confirmed by proteinase treatment of the cell-free supernatants.

       Antioxidative Activity

      According to
      • Hütt P.
      • Shchepetova J.
      • Loivukene K.
      • Kullisaar T.
      • Mikelsaar M.
      Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens.
      , LAB strains with a TAA value >20% are considered to have antioxidative activity. Three of the LAB strains from kefir grains (2 Lb. paracasei; MRS 59 and M1743, and 1 lactococcal strain; MRS 52) showed percentages above this value in either intact cells or lysate supernatants (Table 3). All other strains showed mean TAA values below 15% (data not shown), except for 2 other lactobacilli strains (MRS55 and MRN4), which showed a TAA between 15 and 20% (Table 3).
      Table 3Data on total antioxidative activity (TAA) and glutathione determinations (total, reduced, and oxidized glutathione and glutathione redox ratio) of representative lactic acid bacteria strains from kefir grains showing antioxidative activity
      Data are expressed as the means ± SD, based on 3 replicates.
      StrainIdentificationTAA test (%)Glutathione test
      Glutathione test: tGSH=total glutathione; GSSG=oxidized glutathione; GSH=reduced glutathione; ND=not determined.
      M)
      Intact cellsCell lysatetGSHGSSGGSHGSH/GSSG
      MRS52Lactococcus lactis ssp. lactis20.3 ± 3.5
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      11.8 ± 3.9
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.586 ± 0.23
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.062 ± 0.03
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.461 ± 0.14
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      8.75 ± 3.19
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      MRS59Lactobacillus paracasei18.4 ± 4.1
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      34.1 ± 9.9
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.157 ± 0.21
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.024 ± 0.01
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.110 ± 0.19
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      5.72 ± 3.09
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      MRN4Lb. paracasei3.5 ± 2.9
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      14.7 ± 12.7
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      NDNDNDND
      M1743Lb. paracasei22.3 ± 9.0
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      26.0 ± 4.2
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.106 ± 0.13
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      00.106 ± 0.13
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0
      MRS55Lb. paracasei10.4 ± 2.3
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      18.2 ± 2.1
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      NDNDNDND
      CECT515
      E. coli CECT 515 was used as positive control.
      Escherichia coliNDND2.313 ± 0.48
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      0.31 ± 0.04
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      1.686 ± 0.40
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      5.33 ± 0.67
      Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P<0.05).
      a–c Means within columns that do not share a superscript are statistically different according to the least significant differences (LSD) mean comparison test (P < 0.05).
      1 Data are expressed as the means ± SD, based on 3 replicates.
      2 Glutathione test: tGSH = total glutathione; GSSG = oxidized glutathione; GSH = reduced glutathione; ND = not determined.
      3 E. coli CECT 515 was used as positive control.
      The 3 LAB strains that showed higher values in the TAA test (P < 0.05) were further analyzed by the glutathione assay (Table 3). The total gluthatione content values found for the LAB strains were lower than the positive control (E. coli; P < 0.05). However, the calculated glutathione redox ratio (GSSG/GSH), used to investigate oxidative stress, was similar for all the strains, except for the Lb. paracasei M1743 strain, in which the oxidized glutathione (GSSG) content was not detectable and, consequently, its GSH/GSSG redox ratio could not be calculated.
      Based on the results obtained in the antimicrobial and antioxidative tests, 3 Lb. paracasei strains (MRS55, MRS59, and M1743) were considered as presenting probiotic potential and were selected for further analyses.

       Adhesion Capability

      The results of the adhesion assay for the 3 selected strains are depicted in Figure 2. Adhesion percentages ranged from 0.9 to 9%. The adhesion level of the MRS59 strain was similar to that of the reference strain GG, whereas those of M1743 and MRS 55 were lower (P < 0.05).
      Figure thumbnail gr2
      Figure 2Percentage of adhesion measured as the percentage of colony-forming units per milliliter adhered bacteria with respect to colony-forming units per milliliter of added bacteria of the 3 Lactobacillus paracasei strains to the intestinal epithelial cell line Caco-2. Columns that do not share the same letter are statistically different according to the least significant differences (LSD) mean comparison test (P < 0.05). Lactobacillus rhamnosus GG was used as an adherent reference strain.

       Enzymatic Activities and Hemolysin Production

      Undesirable activities, such as trypsin, α-chymotrypsin, and β-glucuronidase activities, were not detected in any of the selected strains. In addition, none of the 3 strains showed hemolytic activity under the assay conditions.

       Antibiotic Resistance

      The selected lactobacilli strains were susceptible to all the analyzed antimicrobial agents (including tetracycline, erythromycin, clindamycin, ampicillin, and aminoglycosides) with the exception of vancomycin, for which an intrinsic resistance was observed. The chloramphenicol MIC for MRS55 and M1743 strains was only one dilution higher (8 µg/mL) than the microbiological breakpoint defined by the EFSA (4 µg/mL), which is within the normal acceptable variation around the means (
      EFSA
      EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP): Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance.
      ).

      Discussion

      Beyond their technological function, demand is currently increasing for new LAB strain probiotic candidates (
      • Ayeni F.A.
      • Sanchez B.
      • Adeniyi B.A.
      • de Los Reyes-Gavilan C.G.
      • Margolles A.
      • Ruas-Madiedo P.
      Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow’s intestine.
      ;
      • Argyri A.A.
      • Zoumpopoulou G.
      • Karatzas K.-A. G.
      • Tsakalidou E.
      • Nychas G.-J. E.
      • Panagou E.Z.
      • Tassou C.C.
      Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests.
      ). The complex microbiota of kefir, a traditional beverage endowed with several health benefits (
      • Leite A.M.O.
      • Miguel M.A.
      • Peixoto R.S.
      • Rosado A.S.
      • Silva J.T.
      • Paschoalin V.M.
      Microbiological, technological and therapeutic properties of kefir: A natural probiotic beverage.
      ), could be a source for obtaining novel probiotic strains (
      • Santos A.
      • San Mauro M.
      • Sanchez A.
      • Torres J.M.
      • Marquina D.
      The antimicrobial properties of different strains of Lactobacillus spp. isolated from kefir.
      ;
      • Kumura H.
      • Tanoue Y.
      • Tsukahara M.
      • Tanaka T.
      • Shimazaki K.
      Screening of dairy yeast strains for probiotic applications.
      ).
      In the present study, LAB were isolated from 4 kefir grain samples, identified and typed by molecular methods, and characterized in vitro for recognized probiosis properties, such as acidity and bile tolerance and antimicrobial and antioxidant activities, key features to consider bacterial strains as probiotics. The secretion of gastric acid and transit through the stomach constitutes a primary defense mechanism that all ingested microorganisms must overcome, including probiotics (
      • Gueimonde M.
      • Salminen S.
      New methods for selecting and evaluating probiotics.
      ). The LAB isolates from kefir grains were screened and selected for their resistance and survival in an acidic environment, as well as for their growth in the presence of 0.3% bile salts, a similar concentration to that present in the small intestine (
      • Vinderola G.
      • Capellini B.
      • Villarreal F.
      • Suárez V.
      • Quiberoni A.
      • Reinheimer J.
      Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use.
      ). Though no scientific consensus exists on the pH and bile concentration to which probiotic strains should be tolerant (
      • Zago M.
      • Fornasari M.E.
      • Carminati D.
      • Burns P.
      • Suàrez V.
      • Vinderola G.
      • Reinheimer J.
      • Giraffa G.
      Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses.
      ), based on the results, the lactobacilli were able to grow in higher bile concentrations (1%). Similar results have been previously reported by other authors analyzing LAB strains from different environments (
      • Delgado S.
      • O'Sullivan E.
      • Fitzgerald G.
      • Mayo B.
      Subtractive screening for probiotic properties of Lactobacillus species from the human gastrointestinal tract in the search for new probiotics.
      ;
      • Vinderola G.
      • Capellini B.
      • Villarreal F.
      • Suárez V.
      • Quiberoni A.
      • Reinheimer J.
      Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use.
      ;
      • Zago M.
      • Fornasari M.E.
      • Carminati D.
      • Burns P.
      • Suàrez V.
      • Vinderola G.
      • Reinheimer J.
      • Giraffa G.
      Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses.
      ;
      • Ramos C.L.
      • Thorsen L.
      • Schwan R.F.
      • Jespersen L.
      Strain-specific probiotics properties of Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus brevis isolates from Brazilian food products.
      ).
      Molecular typing showed a rather high genetic heterogeneity among the LAB isolates from the 4 kefir grains as judged by the large number of different profiles obtained.
      Some of these LAB strains exhibited antimicrobial activity against pathogens, and 4 of them produced BLIS, which might provide advantage in competing either in a food product or in the gut (
      • Servin A.L.
      Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens.
      ;
      • Vinderola G.
      • Capellini B.
      • Villarreal F.
      • Suárez V.
      • Quiberoni A.
      • Reinheimer J.
      Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use.
      ). Moreover, we observed one Lactococcus BLIS-producing strain, as well as 2 Lb. paracasei, that showed an antioxidative activity higher than those of other tested LAB strains (P < 0.05), indicating they may aid in protecting cells from oxidative damage (
      • Lin M.Y.
      • Chang F.J.
      Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356.
      ;
      • Zhang Y.
      • Du R.
      • Wang L.
      • Zhang H.
      The antioxidative effects of probiotic Lactobacillus casei Zhang on the hyperlipidemic rats.
      ). In particular, the lactococci strain showed higher TAA in intact cells; meanwhile, in the Lb. paracasei strain, the antioxidative activity was more relevant in cell extracts indicating that this activity might be also relevant in case of bacterial lysis into the gastrointestinal tract releasing of the intracellular content. Furthermore, GSH, an important component of the cell defense system against oxidative stress (
      • Masip L.
      • Veeravalli K.
      • Georgiou G.
      The many faces of glutathione in bacteria.
      ), was detected for all the LAB strains that showed TAA values >20%. However, the obtained values suggest that the antioxidative effect displayed by some of these strains could be attained by means of other mechanisms different from glutathione protection, such as enzymatic antioxidants. The antioxidative activities of LAB strains have been under investigation in other studies (
      • Kullisaar T.
      • Zilmer M.
      • Mikelsaar M.
      • Vihalemm T.
      • Annuk H.
      • Kairane C.
      • Kilk A.
      Two antioxidative lactobacilli strains as promising probiotics.
      ;
      • Li S.
      • Zhao Y.
      • Zhang L.
      • Zhang X.
      • Huang L.
      • Li D.
      • Niu C.
      • Yang Z.
      • Wang Q.
      Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods.
      ;
      • Chen P.
      • Zhang Q.
      • Dang H.
      • Liu X.
      • Tian F.
      • Zhao J.
      • Chen Y.
      • Zhang H.
      • Chen W.
      Screening for potential new probiotic based on probiotic properties and α-glucosidase inhibitory activity.
      ). This protective property may be useful as a defense mechanism in the intestinal microbial ecosystem.
      Strains belonging to the Lactobacillus genus are commonly used as probiotics (
      FAO/WHO
      ;
      • Gueimonde M.
      • Salminen S.
      New methods for selecting and evaluating probiotics.
      ;
      • Zago M.
      • Fornasari M.E.
      • Carminati D.
      • Burns P.
      • Suàrez V.
      • Vinderola G.
      • Reinheimer J.
      • Giraffa G.
      Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses.
      ). Three Lb. paracasei strains were considered as appropriate probiotic candidates and selected for further characterization of desirable and undesirable probiotic-related properties.
      The capacity to adhere to the intestinal mucosa is an important property for probiotic strains because they should, at least transiently, colonize the host gut (
      FAO/WHO
      ). The human colon adenocarcinoma cell line Caco-2 is widely accepted as a model for assessing the adhesion ability of probiotic candidates (
      • Gueimonde M.
      • Salminen S.
      New methods for selecting and evaluating probiotics.
      ;
      • Ayeni F.A.
      • Sanchez B.
      • Adeniyi B.A.
      • de Los Reyes-Gavilan C.G.
      • Margolles A.
      • Ruas-Madiedo P.
      Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow’s intestine.
      ). One Lb. paracasei strain in this study (MRS59) showed similar adhesion values to the recognized probiotic strain Lb. rhamnosus GG, suggesting it may well be a good in vivo colonizer.
      None of these lactobacilli showed β-glucuronidase activity, which may have negative effects in the colon and has been considered a carcinogenic enzyme (
      • Monteagudo-Mera A.
      • Caro I.
      • Rodriguez-Aparicio L.B.
      • Rua J.
      • Ferrero M.A.
      • Garcia-Armesto M.R.
      Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products.
      ). In contrast, all 3 strains produced β-galactosidase, a beneficial enzyme considering both probiotic and technological aspects, supporting the reduction of lactose intolerance as well as milk acidification (
      • Monteagudo-Mera A.
      • Caro I.
      • Rodriguez-Aparicio L.B.
      • Rua J.
      • Ferrero M.A.
      • Garcia-Armesto M.R.
      Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products.
      ). These strains also presented α-glucosidase and β-glucosidase activity, which could contribute to polysaccharide digestion (
      • Papamanoli E.
      • Tzanetakis N.
      • Litopoulou-Tzanetaki E.
      • Kotzekidou P.
      Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties.
      ). The presence of glycosidase activities in food cultures seems to have an effect on sensory properties because flavor is often linked to sugar metabolism (
      • Papamanoli E.
      • Tzanetakis N.
      • Litopoulou-Tzanetaki E.
      • Kotzekidou P.
      Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties.
      ;
      • Mesas J.M.
      • Rodriguez M.C.
      • Alegre M.T.
      Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra.
      ).
      In summary, 32 different LAB strains isolated from kefir grains showing good survival under normal gastrointestinal conditions were evaluated in vitro for antimicrobial and antioxidative activities in this study. Among the analyzed strains, 3 Lb. paracasei strains were further selected. The presence of harmful enzymatic activities and atypical antibiotic resistances among the selected strains was ruled out. Based on a long history of human consumption, Lb. paracasei, like many other LAB species, is granted a qualified presumption of safety (QPS) status (
      EFSA
      EFSA Panel on Biological Hazards (BIOHAZ). Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2011 update).
      ), allowing unrestricted application in food and feed, provided antibiotic resistance is absent. Among the 3 selected strains, Lb. paracasei MRS59 showed the highest number of in vitro probiosis properties; consequently, it was considered as the most appropriate kefir-derived candidate to be used as a probiotic. Further in vivo studies should be also performed to confirm its potential beneficial effects.

      Acknowledgments

      This study was supported by Fundação Carlos Chagas Filho de de Amparo à Pesquisa do Estado do Rio de Janeiro (Rio de Janeiro, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brasília, Brazil) Foundation (process number PDEE 5019109 ), and funds from the Spanish Ministry of Science and Innovation (MICINN, Spain ; reference AGL2011-24300 and AGL2012-33278 ). S. Delgado was supported by a research contract from MICINN under the Juan de la Cierva Program (reference JCI-2008-02391).

      References

        • Alegría A.
        • Delgado S.
        • Roces C.
        • Lopez B.
        • Mayo B.
        Bacteriocins produced by wild Lactococcus lactis strains isolated from traditional, starter-free cheeses made of raw milk.
        Int. J. Food Microbiol. 2010; 143: 61-66
        • Argyri A.A.
        • Zoumpopoulou G.
        • Karatzas K.-A. G.
        • Tsakalidou E.
        • Nychas G.-J. E.
        • Panagou E.Z.
        • Tassou C.C.
        Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests.
        Food Microbiol. 2013; 33: 282-291
        • Ayeni F.A.
        • Sanchez B.
        • Adeniyi B.A.
        • de Los Reyes-Gavilan C.G.
        • Margolles A.
        • Ruas-Madiedo P.
        Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow’s intestine.
        Int. J. Food Microbiol. 2011; 147: 97-104
        • Chen P.
        • Zhang Q.
        • Dang H.
        • Liu X.
        • Tian F.
        • Zhao J.
        • Chen Y.
        • Zhang H.
        • Chen W.
        Screening for potential new probiotic based on probiotic properties and α-glucosidase inhibitory activity.
        Food Contr. 2014; 35: 65-72
        • Chifiriuc M.C.
        • Cioaca A.B.
        • Lazar V.
        In vitro assay of the antimicrobial activity of kephir against bacterial and fungal strains.
        Anaerobe. 2011; 17: 433-435
        • Delgado S.
        • O'Sullivan E.
        • Fitzgerald G.
        • Mayo B.
        Subtractive screening for probiotic properties of Lactobacillus species from the human gastrointestinal tract in the search for new probiotics.
        J. Food Sci. 2007; 72: M310-M315
        • EFSA
        EFSA Panel on Biological Hazards (BIOHAZ). Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2011 update).
        EFSA J. 2011; 9
        • EFSA
        EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP): Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance.
        EFSA J. 2012; 10
        • FAO/WHO
        Probiotic in foods. Health and nutritional properties and guidelines for evaluation. 85. FAO Food and Nutrition Paper, Rome, Italy2006
        • Golowczyc M.A.
        • Gugliada M.J.
        • Hollmann A.
        • Delfederico L.
        • Garrote G.L.
        • Abraham A.G.
        • Semorile L.
        • De Antoni G.
        Characterization of homofermentative lactobacilli isolated from kefir grains: Potential use as probiotic.
        J. Dairy Res. 2008; 75: 211-217
        • Gueimonde M.
        • Salminen S.
        New methods for selecting and evaluating probiotics.
        Dig. Liver Dis. 2006; 38: S242-S247
        • Guo Z.
        • Wang J.
        • Yan L.
        • Chen W.
        • Liu X.-m.
        • Zhang H.-p.
        In vitro comparison of probiotic properties of Lactobacillus casei Zhang, a potential new probiotic, with selected probiotic strains.
        Lebenson. Wiss. Technol. 2009; 42: 1640-1646
        • Hertzler S.R.
        • Clancy S.M.
        Kefir improves lactose digestion and tolerance in adults with lactose maldigestion.
        J. Am. Diet. Assoc. 2003; 103: 582-587
        • Hong W.-S.
        • Chen H.-C.
        • Chen Y.-P.
        • Chen M.-J.
        Effects of kefir supernatant and lactic acid bacteria isolated from kefir grain on cytokine production by macrophage.
        Int. Dairy J. 2009; 19: 244-251
        • Hütt P.
        • Shchepetova J.
        • Loivukene K.
        • Kullisaar T.
        • Mikelsaar M.
        Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens.
        J. Appl. Microbiol. 2006; 100: 1324-1332
        • Klare I.
        • Konstabel C.
        • Muller-Bertling S.
        • Reissbrodt R.
        • Huys G.
        • Vancanneyt M.
        • Swings J.
        • Goossens H.
        • Witte W.
        Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria.
        Appl. Environ. Microbiol. 2005; 71: 8982-8986
        • Koeuth T.
        • Versalovic J.
        • Lupski J.R.
        Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria.
        Genome Res. 1995; 5: 408-418
        • Kullisaar T.
        • Zilmer M.
        • Mikelsaar M.
        • Vihalemm T.
        • Annuk H.
        • Kairane C.
        • Kilk A.
        Two antioxidative lactobacilli strains as promising probiotics.
        Int. J. Food Microbiol. 2002; 72: 215-224
        • Kumura H.
        • Tanoue Y.
        • Tsukahara M.
        • Tanaka T.
        • Shimazaki K.
        Screening of dairy yeast strains for probiotic applications.
        J. Dairy Sci. 2004; 87: 4050-4056
        • Leite A.M.O.
        • Leite D.C.
        • Del Aguila E.M.
        • Alvares T.S.
        • Peixoto R.S.
        • Miguel M.A.
        • Silva J.T.
        • Paschoalin V.M.
        Microbiological and chemical characteristics of Brazilian kefir during fermentation and storage processes.
        J. Dairy Sci. 2013; 96 (a): 4149-4159
        • Leite A.M.O.
        • Mayo B.
        • Rachid C.T.C.C.
        • Peixoto R.S.
        • Silva J.T.
        • Paschoalin V.M.F.
        • Delgado S.
        Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.
        Food Microbiol. 2012; 31: 215-221
        • Leite A.M.O.
        • Miguel M.A.
        • Peixoto R.S.
        • Rosado A.S.
        • Silva J.T.
        • Paschoalin V.M.
        Microbiological, technological and therapeutic properties of kefir: A natural probiotic beverage.
        Braz. J. Microbiol. 2013; 44 (b): 341-349
        • Li S.
        • Zhao Y.
        • Zhang L.
        • Zhang X.
        • Huang L.
        • Li D.
        • Niu C.
        • Yang Z.
        • Wang Q.
        Antioxidant activity of Lactobacillus plantarum strains isolated from traditional Chinese fermented foods.
        Food Chem. 2012; 135: 1914-1919
        • Lin M.Y.
        • Chang F.J.
        Antioxidative effect of intestinal bacteria Bifidobacterium longum ATCC 15708 and Lactobacillus acidophilus ATCC 4356.
        Dig. Dis. Sci. 2000; 45: 1617-1622
        • Masip L.
        • Veeravalli K.
        • Georgiou G.
        The many faces of glutathione in bacteria.
        Antioxid. Redox Signal. 2006; 8: 753-762
        • Mesas J.M.
        • Rodriguez M.C.
        • Alegre M.T.
        Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra.
        Lett. Appl. Microbiol. 2011; 52: 258-268
        • Monteagudo-Mera A.
        • Caro I.
        • Rodriguez-Aparicio L.B.
        • Rua J.
        • Ferrero M.A.
        • Garcia-Armesto M.R.
        Characterization of certain bacterial strains for potential use as starter or probiotic cultures in dairy products.
        J. Food Prot. 2011; 74: 1379-1386
        • Nielsen B.
        • Gurakan G.C.
        • Unlu G.
        Kefir: A multifaceted fermented dairy product.
        Probiotics Antimicrob. Proteins. 2014; 6: 123-135
        • Nishida S.
        • Michinaka A.
        • Nakashima K.
        • Iino H.
        • Fujii T.
        Evaluation of the probiotic potential of Lactobacillus paracasei KW3110 based on in vitro tests and oral administration tests in healthy adults.
        J. Gen. Appl. Microbiol. 2008; 54: 267-276
        • Palys T.
        • Nakamura L.K.
        • Cohan F.M.
        Discovery and classification of ecological diversity in the bacterial world: The role of DNA sequence data.
        Int. J. Syst. Bacteriol. 1997; 47: 1145-1156
        • Papamanoli E.
        • Tzanetakis N.
        • Litopoulou-Tzanetaki E.
        • Kotzekidou P.
        Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties.
        Meat Sci. 2003; 65: 859-867
        • Ramos C.L.
        • Thorsen L.
        • Schwan R.F.
        • Jespersen L.
        Strain-specific probiotics properties of Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus brevis isolates from Brazilian food products.
        Food Microbiol. 2013; 36: 22-29
        • Ripamonti B.
        • Agazzi A.
        • Bersani C.
        • De Dea P.
        • Pecorini C.
        • Pirani S.
        • Rebucci R.
        • Savoini G.
        • Stella S.
        • Stenico A.
        • Tirloni E.
        • Domeneghini C.
        Screening of species-specific lactic acid bacteria for veal calves multi-strain probiotic adjuncts.
        Anaerobe. 2011; 17: 97-105
        • Rodrigues K.L.
        • Caputo L.R.
        • Carvalho J.C.
        • Evangelista J.
        • Schneedorf J.M.
        Antimicrobial and healing activity of kefir and kefiran extract.
        Int. J. Antimicrob. Agents. 2005; 25: 404-408
        • Rossetti L.
        • Giraffa G.
        Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.
        J. Microbiol. Methods. 2005; 63: 135-144
        • Sánchez B.
        • Fernández-García M.
        • Margolles A.
        • de los Reyes-Gavilán C.G.
        • Ruas-Madiedo P.
        Technological and probiotic selection criteria of a bile-adapted Bifidobacterium animalis ssp. lactis strain.
        Int. Dairy J. 2010; 20: 800-805
        • Santos A.
        • San Mauro M.
        • Sanchez A.
        • Torres J.M.
        • Marquina D.
        The antimicrobial properties of different strains of Lactobacillus spp. isolated from kefir.
        Syst. Appl. Microbiol. 2003; 26: 434-437
        • Servin A.L.
        Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens.
        FEMS Microbiol. Rev. 2004; 28: 405-440
        • Urdaneta E.
        • Barrenetxe J.
        • Aranguren P.
        • Irigoyen A.
        • Marzo F.
        • Ibáñez F.C.
        Intestinal beneficial effects of kefir-supplemented diet in rats.
        Nutr. Res. 2007; 27: 653-658
        • Vinderola G.
        • Capellini B.
        • Villarreal F.
        • Suárez V.
        • Quiberoni A.
        • Reinheimer J.
        Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use.
        Lebenson. Wiss. Technol. 2008; 41: 1678-1688
        • Zago M.
        • Fornasari M.E.
        • Carminati D.
        • Burns P.
        • Suàrez V.
        • Vinderola G.
        • Reinheimer J.
        • Giraffa G.
        Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses.
        Food Microbiol. 2011; 28: 1033-1040
        • Zhang Y.
        • Du R.
        • Wang L.
        • Zhang H.
        The antioxidative effects of probiotic Lactobacillus casei Zhang on the hyperlipidemic rats.
        Eur. Food Res. Technol. 2010; 231: 151-158
        • Zheng Y.
        • Lu Y.
        • Wang J.
        • Yang L.
        • Pan C.
        • Huang Y.
        Probiotic properties of Lactobacillus strains isolates from Tibetan kefir grains.
        PLoS ONE. 2013; 8: e69868