characterization of lactic acid bacteria isolated from italian bella di cerignola table olives:...

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Characterization of Lactic Acid BacteriaIsolated from Italian Bella di CerignolaTable Olives: Selection of PotentialMultifunctional Starter CulturesAntonio Bevilacqua, Clelia Altieri, Maria Rosaria Corbo, Milena Sinigaglia, and Labia Irene Ivette Ouoba

Abstract: Lactic acid bacteria (19 isolates) from Bella di Cerignola Italian table olives were investigated for theirtechnological and probiotic properties for the selection of multifunctional starter cultures for table olives. The bacteriawere first identified by phenotyping and genotyping, then characterized for the production of biogenic amines, growth atdifferent pH, NaCl concentrations, and temperatures. The potentiality of the bacteria to have some probiotic properties(antimicrobial activity against foodborne pathogens, survival in low pH and in the presence of bile salts, ability to adhere tothe mammalian cells model IPEC-J2) was also investigated. Eighteen of the studied isolates were identified as Lactobacillusplantarum and one as Enterococcus faecalis. All bacteria were able to grow at a range of pH between 4.0 and 10.0 as wellas in media supplemented with 2.5 to 7.5% of NaCl and 0.3% bile salts and survived in MRS broth acidified at pH2.5; moreover, they inhibited significantly Escherichia coli O157:H7. The adhesion to IPEC-J2 cells was in general low tomoderate (5.3 to 8.3%); however, 2 isolates of L. plantarum (c16 and c19) showed interesting higher adhesion values (up to16%). Our results suggest that at least 3 isolates could be possible multifunctional starters for Bella di Cerignola olives: L.plantarum 16 and 19 for mainly their probiotic properties and L. plantarum 10 for mainly its technological characteristics.

Keywords: Italian table olives, LAB, probiotic potential, starter cultures, technological properties

Practical Application: A functional starter is a microorganism exerting benefits on human health (probiotic) and able toguide a fermentation (starter). The main goal of this article was to select a functional starter for table olives.

IntroductionBella di Cerignola is a traditional variety of table olives from

the Apulian region in Southern Italy; it is appreciated for the bigdimension of the olives and the green/black color of the skin. Inthe last decade, this variety of olive received the protected de-nomination of origin (PDO) from the European Union (EU),with the full name of Bella della Daunia variety Bella di Cerig-nola. Bella di Cerignola olives are produced as follows: olives aremanually harvested into plastic boxes (20 kg) and transported toproduction sites where they are sorted and graded according totheir size. They are then treated with an alkaline lye (1.3% to 2.6%of NaOH, depending on the size and fruit ripeness) for 12 to 15 hto remove their bitterness, washed twice with water, respectively,for 2 to 3 and 10 to 12 h and finally brined and let to ferment for30 to 60 d in increasing concentrations of NaCl (from 6% to 8%up to 10% at the end of the fermentation).

MS 20100439 Submitted 4/22/2010, Accepted 6/30/2010. Authors Bevilacqua,Altieri, Corbo, and Sinigaglia are with Dept. of Food Science, Faculty of AgriculturalScience, Univ. of Foggia, via Napoli 25, Foggia 71122, Italy. Author Ouoba is withDept. of Food Science, Faculty of Life Sciences, Univ. of Copenhagen, Rolighedsvej30, DK-1958 Frederiksberg C, Denmark and also with Microbiology Research Unit,School of Human Sciences, Faculty of Life Science, London Metropolitan Univ., 166-220 Holloway Road, London N7 8DB, U.K. Direct inquiries to author Bevilacqua(E-mail: [email protected], [email protected]).

A preliminary study of the natural microflora of Bella di Cerig-nola olives, based only on phenotyping, revealed that Lactobacillusplantarum is the predominant species among lactic acid bacteria(LAB), but other LAB such as L. brevis and Leuconostoc mesenteroideswere also recovered (Campaniello and others 2005). For yeasts, thestrains isolated were identified as Candida spp., Rhodotorula mucilagi-nosa, and Cryptococcus laurentii (Campaniello and others 2005).

The largest group of LAB belongs to the genus Lactobacillusthat includes more than 50 species (Tannock 2004; de Vries andothers 2006). In many cases, lactobacilli are used as starters forfood fermentation, as they contribute significantly to the stor-age and improvement of flavor and produce some antimicrobialcompounds (de Vries and others 2006). In addition, lactobacilli,such as L. plantarum, are important members of the healthy hu-man microbiota and exert several beneficial physiological effects,such as antimicrobial and antitumorigenic activities (Salminen andothers 1998; Maragkoudakis and others 2006; Fayol-Messaoudiand others 2007; Nguyen and others 2007; Chiu and others 2008;Mathara and others 2008).

Nowadays, the fermentation of Bella di Cerignola table olivesrelies on a system of small-scale producers, with uncontrolled fer-mentation leading to a variation of the quality, the safety, and thestability of the product. One factor that could contribute to theoptimization of the processing condition is the selection of well-identified (for example, by genotyping) and characterized startercultures for controlled fermentation leading to a better product in

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Functional starter for table olives . . .

terms of quality, safety, and stability. It will also be of great interestif the selected starter cultures possess some probiotic properties assuggested by Lavermicocca and others (2005) and Valerio and oth-ers (2006) for olives and artichokes, respectively. This new frontieris called “ortobiotica” (that means probiotic vegetable gastronomy)and is currently covered by some patents (World Intellectual Prop-erty Organization 2005, 2006). This should be a promising wayfor the valorization of traditional fermented vegetables of South-ern Italy; however, it shows a main limit: the strains proposed andinoculated as probiotics are of dairy origin, not adapted to thestressful conditions of a fermented vegetable, such as table olives.

Therefore, the aim of this study was to select a multifunctionalstarter for Bella di Cerignola olives; this final goal was achievedthrough some intermediate milestones:

(1) identification of LAB from Bella di Cerignola olives bygenotyping;

(2) evaluation of their technological properties (for example,growth at different pH, salt concentrations, and temperatures);

(3) study of their probiotic properties (for example, survival atgastric pH and in media supplemented with bile salts, antimicrobialactivity against some foodborne pathogens, and adhesion to celllines).

Materials and MethodsSources of LAB and reference strains

This study focused on 19 isolates of LAB recovered during thefermentation of Bella di Cerignola table olives, processed accord-ing to Spanish style. Originally 322 bacteria were isolated andscreened for their ability to grow at 15 and 45 ◦C. Bacteria (19isolates) that showed a good and quick grow at the precited tem-perature were further selected for the present study. The selectedisolates were classified with a numeric code, ranging from 1 to 19,and stored at −20 ◦C in MRS broth (Oxoid, Milan, Italy), addedwith 33% of sterile glycerol (J.T. Baker, Milan, Italy). One refer-ence strain of L. plantarum and one of L. pentosus were added in thestudy for the identification of the bacteria. The reference strainswere kindly provided by Dept. of Food Sciences/Food Microbi-ology, Faculty of Life Science, Univ. of Copenhagen, Denmark.

Phenotyping of LABInitial typing was based upon gram staining, microscope exami-

nation, catalase production, glucose metabolism (homo or hetero-fermentative metabolism) (Hayard 1957), growth in MRS broth(Oxoid) at 15 and 45 ◦C for 7 d, production of ammonium fromarginine in a laboratory medium (tryptone, 5 g/L; yeast extract,2.5 g/L; glucose, 0.5 g/L; K2HPO4, 2 g/L; arginine monohy-drochloride, 3 g/L; the pH of the medium was 7), hydrolysis ofesculin (MRS broth containing 2 g/L of esculin, C. Erba, Milan;pH, 6.5), production of biogenic amines (cadaverine, histamine,putrescine, and tyramine) evaluated through the qualitative testby Bover-Cid and Holzapfel (1999). The fermentation of carbo-hydrates was assessed using API 50 CHL galleries (Biomerieux,Marcy L’Etoile, France).

Genotyping of LABDNA extraction. Each strain was grown in MRS Agar (Ox-

oid), incubated at 37 ◦C for 24 h. A single isolated colony wasadded with 1 mL of sterile Milli-Q water (Millipore, purifica-tion PAK, Molsheim, France) in an eppendorf tube, centrifugedat 12000 rpm for 1 min and resuspended in 100 μL of Insta-Gene Matrix (Biorad, Richmond, Calif., U.S.A.). The mixturewas incubated at 56 ◦C for 30 min and at 100 ◦C for 8 min andcentrifuged at 12000 rpm for 3 min. The supernatant was stored

at −20 ◦C and used as DNA template for the polymerase chainreaction (PCR) reactions.

Typing of bacteria by 16S-23S rDNA Internal Tran-scribed Spacer PCR (ITS-PCR). The amplification of the16S-23S ITS internal transcribed region was carried out in 50 μLof reaction mixture containing 1 μL of DNA template, 8 μLof dNTP (1.25 mmol/L) (Amersham Pharmacia Biotech,Uppsala, Sweden), 4 μL of MgCl2 (25 mmol/L) (DNA Tech-nology, Aarhus, Denamark), 5 μL of PCR buffer (10×) (Amer-sham Pharmacia Biotech), 30.25 μL of Milli-Q water, 0.5 μLof the primer cy5-16S-1500 F (50 pmol/μL) (5′-5AA GTCGTA ACA AGG TA-3′) (DNA Technology), 0.5 μL of the“reverse” primer 23S-32R (50 pmol/μL) (5′-GCC ARG GCATCC ACC-3′) (DNA Technology), 0.25 μL of Taq Polymerase(5000 U/mL) (Amersham Pharmacia Biotech), and 0.5 μL offormamide (Biorad). The amplification was performed with a to-tal of 35 cycles in a thermalcycler TrioTermoblock

R©(Biometra,

Gottingen, Germany) as follows: a first denaturation at 94 ◦C for5 min, then 10 cycles at 94 ◦C for 30 s, 48 ◦C for 4 min, and72 ◦C for 30 s, followed by 25 cycles at 94 ◦C for 30 s, 55 ◦C for30 s, and 72 ◦C for 30 s. The final extension was carried out at72 ◦C for 7 min and the product cooled at 4 ◦C.

The DNA fragments were separated by applying 10 μL of eachPCR product and 2 μL of a loading dye solution (Fermentas, Vil-nius, Lithuania) to 2.0% agarose gel (Quantum Appligene, Illrich,France), containing 4 μL of ethidium bromide (Sigma-Aldrich,Milan, Italy) per 100 mL of gel. DNA molecular markers (100and 500 bp) (Fermentas) were included as standards. The gel wasrun in 1.0× TAE buffer (Tris-Acetate-EDTA; pH 8.0) for 1 h at125 V and photographed using an ultraviolet transilluminator. TheDNA profiles obtained were observed and all bacteria showing thesame profile were clustered in the same group.

Typing of bacteria by Repetitive-Sequence-BasedPCR (Rep-PCR). Isolates of each group obtainedby ITS-PCR were further discriminated by rep-PCR.Amplification was carried out in 25 μL of reaction mixture con-taining 2 μL of DNA, 4 μL of dNTP, 2 μL of MgCl2, 2.5 μL ofPCR buffer, 10 μL of Milli-Q water, 4 μL of the primer GTG5(5 pmol/μL) (5′-GTG GTG GTG GTG GTG-3′) (DNATechnology), 0.25 μL of Taq Polymerase, and 0.25 μL of 100%formamide. The amplification was performed with a total of 30PCR cycles. The cycling program was started with an initial denat-uration at 94 ◦C for 4 min, followed by 30 cycles of denaturationat 94 ◦C for 30 s, annealing at 40 ◦C for 4 min, and elongation at65 ◦C for 8 min. The PCR was ended with a final extensionat 65 ◦C for 16 min and the product was cooled at 4 ◦C.

DNA fragments were separated by applying 10 μL of each PCRproduct with 2 μL of loading dye solution to 1.5% agarose gel,containing 4 μL of ethidium bromide per 100 mL of gel. Thegel was run in 0.5× TBE buffer (Tris-borate-EDTA; pH 8.2) for2 h at 125 V. The DNA profiles obtained were observed andall bacteria showing the same profile were clustered in the samegroup.

The DNA profiles obtained by ITS and rep-PCR were clus-tered using the Bionumerics system (Bio-Numerics 4.50, UP-GMA Dice Correlation, Applied Maths, Sint-Martens-Latem,Belgium).

Identification of bacteria by 16S rDNA sequencing. Thevariable region 968-1041 of 16S rDNA was sequenced for the phy-logenetic identification. PCR reaction was carried out in a 50 μLmix, containing 1 μL of DNA, 8 μL of dNTP, 3 μL of MgCl2,5 μL of PCR buffer, 30.75 μL of Milli-Q water, 0.25 μL of Taq

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Polymerase, 1 μL of primer Bact-0011f (192 pmol/μL) (5′-AGAGTT TGA TYM TGG CTC AG-3′) (DNA Technology), and1 μL of the reverse primer 1510r (163 pmol/μL) (5′-ACG GYTACC TTG TTA CGA CTT-3′) (DNA Technology). The amplifi-cation was achieved by 30 PCR cycles in the following conditions:first denaturation at 94 ◦C for 5 min, then 30 cycles at 94 ◦C for90 s, 52 ◦C for 30 s, and 72 ◦C for 90 s. The final extension wascarried out at 72 ◦C for 7 min and the product cooled at 4 ◦C.PCR product was purified using a QIAquick PCR purificationkit (Qiagen, Hilden, Germany). The sequence reaction was per-formed with the primer 338f (1.6 pmol/μL) (5′-ACT CCT ACGGGA GGC AGC AG-3′) (DNA Technology), following the man-ufacture’s instructions. The sequencing was achieved by an expressautomatic sequencer CEQ 2000 (Beckman Coulter Inc., Fuller-ton, Calif., U.S.A.) and the identification at genus and specieslevel was carried out by performing a search in GenBank database,using Basic Local Alignment Search Tool (BLAST) programme(Natl. Center for Biotechnology, Md., U.S.A.).

Growth of LAB at different pHs and NaCl concentrationsThe assay was performed in MRS broth, adjusted to pH 4.0,

5.0, 8.0, 8.5, 9.0, 9.5, 10.0, using HCl, or NaOH 2.0 mol/L, oradded with NaCl (2.5%; 5.0%; 7.5%; 10.0% w/v).

Samples were inoculated with approximately 7 log CFU/mL ofbacteria and incubated at 37 ◦C; aliquots of not modified MRS(pH 6.2), inoculated with the LAB strains, were used as positivecontrols. Microbial growth was evaluated (after 24, 48, and 96 h)as absorbance values at 600 nm, by mean of a spectrophotometerShimadzu UV-visible 1601 model 1642 (Shimadzu Europe Ltd.,Duisburg, Germany). Data were modeled as Growth Index (GI)(Blaszyk and Holley 1998), modified as follows:

GI = (Abss /Absc ) × 100

where Abss is the absorbance of the samples at different pH valuesor NaCl concentrations and Absc the absorbance of the positivecontrol.

In the sample containing 10% of NaCl, LAB survival was eval-uated after 48 and 96 h through the pour plate method on MRSagar (incubated under anaerobic conditions at 37 ◦C for 72 to96 h).

All the experiments were performed on 2 independent batches,labeled A and B.

Survival/growth at pH 2.5 and with 0.3% of bile salt addedSurvival was evaluated on stationary phase cultures (7 log

CFU/mL) by plate count on MRS agar, after 1, 3, 6, 8, and24 h of incubation at 37 ◦C in MRS broth, acidified to pH 2.5with HCl 2.0 mol/L, or added with 0.3% of bile salts (Oxoid).Not modified MRS was used as positive control.

The experiments were performed on 2 different batches; datawere reported as difference between the initial and the final cellnumbers (�logN, log CFU/mL).

Bioactivity toward foodborne pathogensThe antimicrobial activity of LAB strains was performed

against Listeria monocytogenes, Staphylococcus aureus, Escherichia coliO157:H7, and Yersinia enterocolitica, belonging to the Culture Col-lection of Dept. of Food Science (Univ. of Foggia). The pathogenswere grown in Nutrient Broth (Oxoid), incubated at 37 ◦C for24 to 48 h.

Aliquots of 100 μL of each pathogen (7 log CFU/mL) werespread separately on MRS agar or modified MRS agar (mMRS),containing 0.5 g/L of glucose as the only difference. Sterile discs

(9 mm) (Schleicher & Schuell Microscience, Dassel, Germany)were soaked (15 μL) in each of the 4 following culture solutionstested and laid upon the agar surface:

(1) Solution A: not-buffered cells prepared with LAB suspen-sion in a saline solution (0.9% NaCl) (ca. 7 log CFU/mL);

(2) Solution B: not-buffered LAB supernatant prepared by cen-trifuging cell culture at 3000 rpm for 10 min.

(3) Solution C: buffered cells prepared with LAB suspension ina saline solution, buffered to pH 7.0 through NaOH 1 mol/L.

(4) Solution D: buffered LAB supernatant prepared by cen-trifuging cell culture at 3000 rpm for 10 min and adjusted to pH7.0 through NaOH 1 mol/L.

The plates were incubated at 37 ◦C for 24 to 48 h.An inhibition zone around the disc was assumed as a significant

antimicrobial action of LAB against the pathogens. The tests wereperformed on 3 independent batches.

Adhesion assayThe porcine jejunal epithelial cell line IPEC-J2 was furnished

by Pr. Antony Blikslager, North Carolina State Univ., U.S.A., andbelong to the cell collection of the Dept. of Food Science, Facultyof Life Sciences, Univ. of Copenhagen. The cells were grown at37 ◦C in a 5% CO2, 95% air-humidified incubator in a mediumcontaining a 1:1 mixture of Dulbecco’s modified Eagle medium(DMEM; Sigma-Aldrich) and F12 (Sigma-Aldrich) supplementedwith 100 mg/L streptomycin (Fluka Chemie GmbH, Steinheim,Switzerland), 100 mg/L penicillin (Sigma-Aldrich), 2 mmol/L L-glutamine (Sigma-Aldrich), 1 mmol/L pyruvate (Sigma-Aldrich),and 10% fetal bovine serum (Cambrex Bio Science, Verviers, Bel-gium).

IPEC-J2 cells were seeded at a concentration of 5 × 105 cells perwell in 12-well tissue culture plates (Corninc Inc., Corning, N.Y.,U.S.A.) and grown to 100% confluence. The cell culture mediumwas changed every 2 d. MRS broth (10 mL) was inoculated witha loop full of bacterial cells. After 24 h at 37 ◦C, 2 mL of MRSbroth were inoculated with 5 log CFU/mL) in a 5-mL steriletubes, added with 100 μL of metabolic radiolabeling 2 Mq/mLL-[methyl-3H]methionine (Amersham Biosciences) and incubatedat 37 ◦C for 21 h. To remove the excess of radiolabel after growth,cultures were centrifuged (3000 rpm) and the pellet washed 3times with phosphate-buffered saline (PBS) (9.0 g NaCl, 0.144 gKH2PO4 and 0.795 g Na2HPO4, H2O per liter, pH 7) and finallysuspended in the cell culture medium to achieve a concentrationof 7 log CFU/mL. The adhesion assay was performed by adding1 mL of this suspension to each of 3 wells containing IPEC-J2monolayer. After incubation at 37 ◦C for 1 h, IPEC-J2 cells werewashed 3 times with cell culture medium (DMEM + F12) andtreated with 500 μL of NaOH/SDS solution (0.1 mmol/L NaOH,0.1% w/v SDS-Amersham Biosciences) overnight to lyse the cells.The lysed cells were mixed with OptiPhase “Hisafe” 2 scintilla-tion liquid (Fisher Chemicals, Louhghsborough Leisc., U.K.); cellradioactivity was measured through a Scintillation Counting (Wal-lac 1414 WinSpectral, Turku, Finlandia). Adhesion was calculatedas the ratio between the radioactivity of the lysed cells and ra-dioactivity of the labeled bacterial suspension. Lactobacillus reuteri12002 (El-Ziney and others 1999) was used as adhesive type strain(positive control).

Each assay was performed on 4 independent batches; for eachbatch, the experiments were performed in triplicate (n = 12).

Statistical analysesData were submitted to one-way analysis of variance

(ANOVA) and Duncan’s test (P < 0.05), through the software

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Statistica for Windows, version 6.0 (Statsoft, Tulsa, Okla.,U.S.A.).

Results

Identification of bacteria by phenotyping and genotypingAll the 19 isolates were Gram positive, rod-shaped, and cata-

lase negative. The experiments on the influence of the tempera-ture showed that they were able to grow both at 15 and 45 ◦C.Considering the metabolism, they possessed an homofermenta-tive metabolism of glucose; moreover, they produced ammoniumfrom arginine, with the exception of the isolates 5, 6, 10, 15, and16. Otherwise the hydrolysis of esculin was recovered only for theisolates 5, 6, and 10. The assay for the production of biogenicamines (putrescine, cadaverine, histamine, and tyramine) was al-ways negative.

As seen in Figure 1, ITS-PCR classified the isolates in 3 groupsat species level. Eighteen isolates (1 to 10 and 12 to 19) from tableolives Bella di Cerignola showed the same DNA pattern as thereference strain R1 of L. plantarum and were then consequentlyclustered in the same group 1. Group 2 included isolate 11 andgroup 3 the reference strain of L. pentosus. Even though similaritieswere observed between reference strains R1 of L. plantarum and R2of L. pentosus as expected, the 2 species were clearly distinguishedand were clustered in different groups (Figure 1).

Sequencing of 16S rDNA and API 50 CHL fermentation pro-files allowed identification of each group of bacteria (from theITS-PCR) at genus and species level (Table 1). The DNA se-quences of the isolates were compared with sequences present inthe GenBank database; thus, the microorganisms were identifiedwith a similarity of 97% to 99%, with the only exception of theisolate 11. As expected, isolates of group 1 were identified as L.plantarum; isolate 11 constituting group 2 was identified as Ente-rococcus faecalis and the identity of reference strain R2 (group 3)was confirmed as L. pentosus. Isolate 18 of group 1 was identifiedwith the same percentage of similarity as L. plantarum and L. pen-tosus. However, combining the results of API 50 CHL, ITS-PCR,

Figure 1–Cluster analysis of ITS-PCR profiles of lactic acid bacteria isolatedfrom Bella di Cerignola table olives. R1 = reference strain of L. plantarum,R2 = reference strain of L. pentosus.

and DNA sequencing the isolate was identified as L. plantarum. Insome cases, differences were observed between the identificationby phenotyping and genotyping. This was the case for isolates 15and 16, which were identified by API 50 CHL as L. pentosus,but turned out to be L. plantarum using the described genotypicmethods. It was also the case of isolate 11, which was identi-fied phenotypically as L. plantarum but genotypically as E. faecalis(Table 1).

Rep-PCR was more discriminatory than ITS-PCR. It con-firmed the differences between the ITS-PCR groups and alloweddifferentiation of 3 subgroups of L. plantarum isolates included inITS-PCR group 1 using the primer GTG5 (Table 1, Figure 2).Subgroup 1.1 was formed by isolates 1 to 4, 6 to 17, and 19; sub-group 1.2 included isolate 18; and subgroup 1.3 isolate 5. Usingrep-PCR, the very closely related species of L. plantarum and L.pentosus were again clearly distinguished even though similaritiesin DNA pattern were observed especially between L. pentosus R2and L. plantarum 18 (Figure 2).

Effect of pH and salt on growth of isolatesTable 2 shows GI values of LAB at pH values ranging between

4.0 and 10.0 after 48 h of incubation. These data highlight thatpH values of 5.0 to 9.0 did not affect significantly the growth ofthe tested isolates, although some differences among the isolatescould be recovered. However, a reduction of GI was observed atpH 4.0, 9.5, and 10.

In particular at pH 4.0, the isolate 5 appeared to be the mostresistant (GI of 112.45%), whereas isolate 18 was the most sensitive(GI of 72.30%). The other isolates could be divided into 2 groups:the 1st (including the isolates 6, 10, 17, and 19 and labeled withthe capital letter C) characterized by a GI of 80% to 86% and a2nd group (including the isolates 1, 2, 3, 4, 7, 8, 9, 11, 12, 13, 14,15, 16-capital letter A) experiencing a GI of ca. 90%.

Concerning the growth at pH 10.0, all the isolates experienceda GI of ca. 56% to 69% (statistical groups labeled with the capital

Table 1–Identification of lactic acid bacteria isolated from Belladi Cerignola table olives.

Identification SimilarityDNA with

ITS-PCR Rep-PCR sequencing sequencespattern pattern Identification + ITS-PCR in the

Bacteria type type API similarity GenBank

1 1 1.1 L. plantarum L. plantarum 97%2 1 1.1 L. plantarum L. plantarum 98%3 1 1.1 L. plantarum L. plantarum 98%4 1 1.1 L. plantarum L. plantarum 98%5 1 1.3 L. plantarum L. plantarum 97%6 1 1.1 L. plantarum L. plantarum 97%7 1 1.1 L. plantarum L. plantarum 97%8 1 1.1 L. plantarum L. plantarum 98%9 1 1.1 L. plantarum L. plantarum 97%

10 1 1.1 L. plantarum L. plantarum 99%11 2 1.1 L. plantarum E. faecalis 86%12 1 1.1 L. plantarum L. plantarum 97%13 1 1.1 L. plantarum L. plantarum 97%14 1 1.1 L. plantarum L. plantarum 97%15 1 1.1 L. pentosus L. plantarum 99%16 1 1.1 L. pentosus L. plantarum 99%17 1 1.1 L. plantarum L. plantarum 97%18 1 1.2 L. plantarum L. plantarum 97%19 1 1.1 L. plantarum L. plantarum 97%R1 1 1.1 L. plantarum L. plantarum -∗

R2 3 1.1 L. pentosus L. pentosus -∗Not available.


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letters A and B), except isolate 11, which showed the lowest GI(38.41%).

Table 3 reports GI values after 48 h of incubation in MRS brothadded with NaCl. A concentration of salt of 2.5% did not affectsignificantly LAB growth, as evidenced by GI of ca. 100%; in ad-dition, some isolates (10, 11, and 12) appeared slightly stimulated,as they experienced a GI of ca. 120%.

As expected, the increase of salt concentration exerted a negativeinfluence on the growth of the 19 isolates with a subsequentdecrease of GI. In particular, GI was strongly affected by 7.5% ofNaCl (the mean value of salt in the brine of Bella di Cerignolaolives) and the minimum values were recovered for the isolates3, 4, and 9 (ca. 15% to 18%), whereas isolates 10, 11, and 12experienced the highest ones (60% to 68%).

At 10% of NaCl, LAB were not able to grow throughout 96 h,as evidenced by a GI of 1% to 3%. However, cell count, evaluatedthrough the pour plate method, remained constant (ca. 6 to 7 logCFU/mL) for the entire running time (data not shown).

Survival of the isolates to gastric pHand their tolerance to bile salts

Figure 3 shows the survival of LAB in acidified MRS broth (pH2.5): viability loss was not significant after 3 h; otherwise, it variedfrom 0.06 (isolate 1) to 1.43 log CFU/mL (isolate 16) after 24 h.

Figure 4 reports survival/growth in MRS broth, containing0.3% bile salts. Isolates 6 and 10 showed a significant viabilityloss after 24 h: 1.16 and 1.82 log units, respectively; on the otherhand, the remaining 17 isolates showed an increase of cell number.The maximum increase was recovered for the isolate 18 (1.55 logCFU/mL), whereas the lowest �logN values were observed forthe isolates 2, 15, and 17 (0.67 to 0.69 log units). The other strainsshowed an increase of the cell number of approximately 1 logCFU/mL.

Antimicrobial activityLAB did not affect the growth of L. monocytogenes and Y. en-

terocolitica, on both MRS and mMRS; on the other hand, not-buffered cells and supernatant (solution A and solution B) showeda strong antimicrobial action against E. coli O157:H7 on MRS and

mMRS agar, although the inhibition zone on mMRS agar werelower.

By measuring the ray of the inhibition zones of LAB cells (so-lution A) against E. coli O157:H7 on MRS agar, it was possible topoint out the isolates with the lowest and the highest antimicrobialability. Data were submitted to one-way ANOVA and Duncan’stest (Table 4), using the homogeneous-group approach (Altieriand others 2008). The results showed that the data varied in acontinuous way from a minimum (2.5 mm for the isolate 10) to amaximum value (15.5 mm for the isolate 18) and could be dividedinto 8 groups (from A to H). However, the groups were not clearlydefined, as they overlapped each other; thus, the 19 isolates couldbe grouped into 3 classes of antimicrobial effectiveness: weak (in-hibition zone 2.5 to 4.3 mm), moderate (4.3 to 10.3 mm), andstrong (≥11.3 mm). The threshold values of the 3 classes were

Table 2–Growth index at 48 h of lactic acid bacteria from Belladi Cerignola table olives in MRS adjusted at different pH.

Bact- pH pH pH pH pH pH pHeria∗ 4.0 5.0 8.0 8.5 9.0 9.5 10.0

1 94.55Ab 99.95A

a 101.74Aa 99.12A

a 95.15Aa 83.91A

b 67.13Ac

2 90.51Aa 98.38A

a,b 97.45Aa,b 95.77A

a,b 95.00Aa,b 84.44A

c 59.51Ad

3 89.05Aa 95.55A

a 95.35Aa 95.04A

a 95.19Aa 78.30A

b 60.41Ac

4 91.49Ab 98.38A

a 97.45Aa 97.13A

a 95.54Aa 82.23A

c 59.51Ad

5 112.45Bb 127.47B

a 95.07Ac 97.33A

c 97.88Ac 89.99A,B

c,d 60.17Ae

6 80.17Cb 96.31A

a 102.59Aa 99.38A

a 96.96Aa 86.57A

a,b 56.13Bc

7 93.72Aa 102.15A

a 103.48Aa 104.83A

a 101.84Aa 89.66A,B

a,b 63.19Ac

8 91.48Ab 101.17A

a 101.34Aa 102.15A

a 98.09Aa 82.08A

c 69.19Ad

9 90.13Ab 93.45A

a,b 97.45Aa 100.32A

a 95.00Aa 82.23A

c 61.33Ad

10 85.31Cb 97.04A

a 98.30Aa 101.19A

a 95.82Aa 80.73A

b 56.65Bc

11 89.01Ab 98.51A

a 90.36A,Bb 87.20A,B

b 86.46Bb 58.83C

c 38.41Cd

12 87.22Ab 93.99A

a 84.26Bb 80.10B

b 82.19Bb 68.24D

c 54.85Bd

13 89.53Ab 98.79A

a 98.33Aa 96.33A

a 96.94Aa 84.61A

b 64.86Ac

14 89.07Aa 91.59A

a 94.36Aa 94.05A

a 90.29Aa 81.76A

b 61.54Ac

15 89.41Ab 96.26A

a 99.13Aa 95.98A

a 94.10Aa 84.58A

b 63.08Ac

16 90.80Ab 97.00A

a 102.59Aa 102.25A

a 100.00Aa 88.89A,B

b 62.64Ac

17 86.18Ca 93.16A

b 100.33Ab 100.00A

b 100.16Ab 84.66A

a 63.14Ac

18 72.30Da 83.73A

b 97.67Ac 95.24A

c 93.45Ac 83.23A

b 61.24Ad

19 86.12Cb 97.54A

a 100.00Aa 99.39A

a 98.58Aa 83.05A

b 63.64Ac

aValues in a row with the same uppercase are not significantly different (P > 0.05; Duncan’stest).AValues in a column with the same capital letter are not significantly different (P > 0.05;Duncan’s test).

Figure 2–Cluster analysis of Rep-PCR profiles oflactic acid bacteria isolated from Bella diCerignola table olives. R1 = reference strain ofL. plantarum, R2 = reference strain of L.pentosus.


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set to 4.3 mm (overlapping of the group A and B) and 11.3 (firstvalue of the group H). Therefore, the isolates could be classifiedas follows: weak inhibition against E. coli O157:H7: L. plantarum10; moderate inhibition: L. plantarum 1, 2, 4, 5, 7, 9, 14, 15, 16,17; strong inhibition: L. plantarum isolates 3, 6, 8, 11, 12, 13, 18,19.

Concerning S. aureus no inhibition zone was observed; however,the colonies of the pathogen were very little around the disccontaining LAB cells; this means a type of inhibition, although we

Table 3–Growth index at 48 h of lactic acid bacteria from Belladi Cerignola table olives in MRS broth containing different con-centrations of NaCl.

Isolates 2.5% NaCl 5.0% NaCl 7.5% NaCl 10.0% NaCl

1 108.03Aa 95.13A

a 36.84Ab 1.73A

c

2 105.60Aa 91.85A

b 29.49Ac 1.98A

d

3 96.60Aa 83.32B

b 20.03A,Bc 1.69A

d

4 97.12Aa 81.10B

b 15.72Bc 1.87A

d

5 98.72Aa 83.19B

b 37.80Ac 1.91A

d

6 97.50Aa 79.43B

b 24.67Ac 1.59A

d

7 98.01Aa 83.32B

b 43.52Cc 2.06A

d

8 99.95Aa 82.80B

b 45.44Cc 2.28A

d

9 97.03Aa 84.28B

b 17.98Bc 1.83A

d

10 126.96Ba 113.76C

b 68.76Dc 3.29A

d

11 125.39Ba 112.87C

b 67.84Dc 3.05A

d

12 118.04A,Ba 105.84C

b 60.84Dc 2.64A

d

13 96.26Aa 83.74B

b 22.51A,Bc 2.27A

d

14 97.70Aa 83.93B

b 46.25Cc 1.93A

d

15 103.41Aa 85.01Bb 42.39C

c 1.66Ad

16 100.77Aa 84.88B

b 27.18Ac 1.69A

d

17 103.96Aa 86.71B

b 57.21Dc 1.36A

d

18 97.17Aa 85.81B

b 41.38Cc 1.81A

d

19 101.17Aa 83.31B

b 44.46Cc 1.95A

d

aValues in a row with the same capital letter are not significantly different (P > 0.05;Duncan’s test).AValues in a column with the same capital letter are not significantly different (P > 0.05;Duncan’s test).

were not able to measure it through a quantitative index (data notshown).

Adhesion to IPEC-J2 cellsFigure 5 shows the adhesion to IPEC-J2 cells. Interesting results

were recovered for L. plantarum 16 and 19, respectively, an averageof 10.2% and 13.6% of adhesion with a maximum of about 16%

Table 4– One-way ANOVA and Duncan’s test (P < 0.05) per-formed on the inhibition rays of lactic acid bacteria from Belladi Cerignola table olives against E. coli O157:H7.

Inhibition Homogeneous groupszones∗

Strains (ray, mm) A B C D E F G H

10 2.514 4.35 4.89 6.32 6.5

16 6.74 7.3

15 7.51 8.37 8.8

17 10.33 11.3

11 11.512 11.88 12.26 13.0

13 13.319 14.018 15.5∗Data are the average of 3 replicates. Values are referred to not-buffered LAB cells (solutionA).

Figure 3–Viability loss (log CFU/mL) (±SD) oflactic acid bacteria in acidified MRS (pH 2.5).

Figure 4–Survival/growth (log CFU/mL) (±SD) oflactic acid bacteria cultured in MRS + 0.3% ofbile salts.


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for both isolates. According to the results of the statistical analysis,the remaining 17 isolates could be divided into 3 different groups,indicated by the different capital letters: group A including L.plantarum 6, 7, 14, and 17 with very low percentages of adhesion(0.70% to 2.18%) to IPEC-J2; group BC including L. plantarum 3,4, 9, 5, and E. faecalis 11 with low percentage of adhesion (5.3% to6.4%); and group CD that includes L plantarum 1, 2, 5, 8, 10, 11,12, 13, 18 with moderate (6.8% to 8.3%) adhesion properties.

DiscussionIn the present study, characterization of isolates of Italian olives

Bella di Cerignola was initiated by identifying bacteria using phe-notypic and genotypic methods. The 16S ribosome RNA (16SrRNA), a 1500 bp sequence that codes for a portion of the 30S ri-bosome, and comparative analysis of the sequences are commonlyused for bacterial identification (Petty 2007). Strains that gener-ally show a similarity higher than 97% sequence are considered tobe the same species (McCartney 2002); however, the sequencesof 16S rRNA could be not suitable for the identification of thespecies of L. plantarum group (L. plantarum subsp. plantarum, L.plantarum subsp. argentoratensis, L. paraplantarum, L. pentosus, andL. fabifermentans), because the species of this group show a similar-ity of 98.9% to 99.9% (Huang and others 2010). Thus, differentmethods have been proposed and validated, such as a partial dnaKsequences and DNA fingerprinting techniques (Huang and others2010). In the present study, this main drawback was overcome by acombination of some molecular (ITS and Rep-PCR, sequencing)and phenotypic methods.

ITS-PCR was a powerful method for discriminating the bacte-ria at species level (Gurtler and Stanisich 1996; Johnson and others2000; Lei and Jakobsen 2004; Ouoba and others 2008), and L.plantarum and L. pentosus were clearly distinguished. As expected,rep-PCR was more discriminatory and allowed differentiation of3 subgroups within the same group of ITS-PCR using primerGTG5. It has been reported that subgroups of bacteria includingLAB obtained by rep-PCR represent subspecies (Gevers and oth-ers 2001; Cherif and others 2003; Masco and others 2003; Ouobaand others 2008); thus, the studied isolates of L. plantarum wereidentified at subspecies level in some extend. Differences observedin some cases between the phenotypic and genotypic identificationis not surprising. Identification of bacteria based only on pheno-

typing is not reliable for some bacteria; nowadays, a combinationof both phenotypic and genotypic methods is more suitable formore accurate identification (Towner and Cockayne 1993).

The selection of a starter is a complex process, involving dif-ferent steps. A key feature for starter for table olives is the growthover a wide range of temperatures. In Southern Italy, in fact, tableolives are produced from September to December; therefore, thegrow at low and high temperatures is an essential prerequisite. Allthe isolates were able to grow at 15 and 45 ◦C, thus suggesting thatthey could start and control the fermentation both in Septemberand in November to December.

In addition to the ability to grow in a large range of temper-atures, another prerequisite for the selection of starter culturesfor fermented vegetables is the production of biogenic amines,which are undesirable compounds, produced by LAB from someaminoacids (arginine, tyrosine, lysine, histidine). As reported bymany authors (Bover-Cid and Holzapfel 1999; Madera and oth-ers 2003; Nieto-Arribas and others 2009), tyramine is the amineproduced by the largest number of LAB and was recovered alongwith other amines at significant levels in table olives of differentorigins (Garcıa-Garcıa and others 2000, 2001, 2004). None of thestrains recovered from Bella di Cerignola olives and investigatedin the present study produced amines, thus suggesting that theycould be regarded as safe for this kind of foods.

Apart from temperature, NaCl and pH are the most importantvariables, which can influence the fermentation of table olives.The isolates were able to grow in MRS broth containing 7.5% ofNaCl added and survived at 10%, thus confirming their suitabilityas starter. Focusing on the effect of pH, what is important from atechnological point of view is the ability of the isolates to grow atpH 10.0; this pH, in fact, can be found in brines throughout thelye treatment or in the 1st fermentation phase, although it usuallydecreases within few days as a result of the number of washingsand the duration of each of them.

After studying the technological characteristics, attention wasgiven to classical probiotic properties of isolates. The use of pro-biotics in table olives fermentation was proposed by Lavermicoccaand others (2005); however, they inoculated some probiotic strainsof dairy origin, not adapted to the environmental conditions oftable olives; therefore, this article proposes the isolation and char-acterization of probiotic strains of LAB from olives, as suggested

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

L. reu

teri

Strains

Adh

esio

n (%

)

C,DC,D C,D C,D C,DC,D C,D C,D

C,DB,C B,C

B,C

B,C

A

AA,B

A

DE

F

Figure 5–Adhesion (%) (±SD) of lactic acidbacteria on IPEC-J2 cells. Values with differentletters are significantly different (one-wayANOVA and Duncan’s test) (P < 0.05).


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from many other vegetables (Chiu and others 2008; Kos and others2008; Mathara and others 2008).

An important requirement for a probiotic strain is its ability tosurvive into the gastrointestinal tract. The gastric pH in healthyhumans is about 2.0 to 2.5 (Fernandez and others 2003). Stud-ies about probiotic lactobacilli species (L. acidophilus, L. gasseri)demonstrated that these bacteria could survive in these acidicconditions, for at least 90 min (Fernandez and others 2003). Inaddition, Cebeci and Gurakan (2003) recovered a significant sur-vival of 3 strains of L. plantarum in a laboratory medium con-taining bile salts. In contrast, Jacobsen and others (1999) found astrain-dependant survival when they analyzed strains of LAB fromAfrican traditional fermented foods (Gari and maize dough fromGhana). The isolates investigated in this article survived at pH 2.5,showing only a moderate viability loss and were able to grow inmedium supplemented with bile salts, thus suggesting that theycould transit and persist into the gastrointestinal tract.

Another issue of great concern for the selection of pro-biotic strains is the antimicrobial activity toward foodbornepathogens. Lactobacilli have been reported to inhibit some in-testinal pathogens and commensals (Drago and others 1997). Thisinhibitory activity of lactobacilli against Gram-positive and Gram-negative bacteria has been found to be very variable and strain spe-cific (Jacobsen and others 1999). Generally, lactobacilli are highlycompetitive, largely due to the production of several antimicro-bial compounds (organic acids, hydrogen peroxide, bacteriocins,and other minor compounds). Our results showed that the 19strains were able to inhibit E. coli O157:H7 and compete withS. aureus. In particular, the antagonistic effect against E. coli wasstrain-dependant and greatly variable and this is due probably tolactic acid production, as inhibition was observed only for the cellsand the supernatant not-buffered, in agreement with the data ofOgawa and others (2001).

Experiments of adhesion of the isolates to an intestinal cell linewere done as an indirect measure of the ability to colonize the in-testinal tract. The adhesion to the gut by the studied bacteria couldbe the result of many synergistic mechanisms. According to Sav-age (1987), lactobacilli seem to adhere with extracellular substancescontaining polysaccharides, proteins, lipids, and lipoteichoic acids.Lipoteichoic acids are glycerolphospahte polymers of the cell wall,covalently linked with glycolipids, containing both hydrophilicand hydrophobic regions. Sherman and Savage (1986) detectedmacromolecular protein complexes, rich in lipotheicoic acids inmany Lactobacillus spp. Strains, and some of them are known tohave good adhesive properties. Wadstrom and others (1987) ob-served that some Lactobacillus spp. strains from the small intestine ofpigs contained carbohydrate capsule polymers and possessed highhydrophobicity; these authors suggested that the capsule was themost important determinant for the intestinal colonization of pigby lactobacilli. Considering the fact that a L. casei strain was con-sidered “good,” as it showed mean adhesion percentage of 0.38%(Bertazzoni Minelli and others 2004), the isolates studied in thecurrent work, in particular the isolates 16 and 19 that showed upto 16% of adhesion may be considered potential probiotic cultures,from the adhesive point of view.

ConclusionsTo our knowledge, this article is the 1st research aimed to the

technological and probiotic characterization of LAB microlfora oftable olives Bella di Cerignola. Moreover, our results suggest thatat least 3 isolates could be possible multifunctional starters for Belladi Cerignola olives: L. plantarum 16 and 19 for mainly their pro-

biotic properties and L. plantarum 10 for mainly its technologicalcharacteristics.

Further investigations are required to validate these resultsthrough the formulation of a multistrain starter, able to perform,and/or control the fermentation of this traditional food of South-ern Italy.

AcknowledgmentsThis work was supported by the Apulian Region under grant

No. PE-003 “Selection and characterization of lactic acid bacteriawith probiotic characteristics as starters for table olives.”

Authors would like to thank Dr. Dennis Sandris Nielsen andprof. Mogens Jakobsen (Dept. of Food Science, Faculty of LifeSciences, Univ. of Copenhagen) for their technical support andassistance, when authors visited their dept. to perform some ex-periments reported in the paper.

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