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Food Research International 50 (2013) 135–142

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Screening of lactic acid bacteria isolated from fermented table olives with
probiotic potential
J. Bautista-Gallego a, b,⁎, F.N. Arroyo-López a, K. Rantsiou b, R. Jiménez-Díaz a,
A. Garrido-Fernández a, L. Cocolin b
Food Biotechnology Department, Instituto de la Grasa (CSIC), Avda. Padre García Tejero, 4, 41012, Sevilla, Spain
DIVAPRA, Agricultural Microbiology and Food Technology Sector, Faculty of Agriculture, University of Turin, Via Leonardo da Vinci 44, 10095 Grugliasco, Torino, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this work was to study the potential probiotic properties of lactobacilli associated with table
Received 9 February 2012 olives. From a total of 111 isolates from spontaneously fermented green olive brines, 109 were identified
Accepted 10 October 2012 at species level by multiplex PCR amplifications of the recA gene. One hundred and seven of these were iden-
Available online 18 October 2012
tified as Lactobacillus pentosus, one as Lactobacillus plantarum, and another as Lactobacillus paraplantarum.
Repetitive bacterial DNA element fingerprinting (rep-PCR) with GTG5 primer revealed a higher variability
Table olives
within the L. pentosus isolates and nine different clusters were obtained. Most of them showed high
Lactic acid bacteria autoaggregation ability, low hydrophobicity properties, and lower survival to gastric than to pancreatic
Molecular identification digestion; however, no isolate showed bacteriocin, haemolytic or bile salt hydrolase activities. A multivariate
Starter analysis based on results from phenotypic tests led to the segregation of some L. pentosus isolates with prom-
Probiotic potential ising potential probiotic characteristics, which are even better than probiotic reference strains. Due to the
autochthonous origin of the strains, their use as starter cultures may contribute to improving natural fermen-
tation and the nutritional characteristics of table olives.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction benefit to the host”. They “should be resistant to gastric juices and be
able to grow in the presence of bile”. There is a series of in vitro tests
According to the latest consolidated statistics, worldwide table olive such as acid and bile tolerance, antimicrobial production, etc., that, al-
production for the 2007/2008 season was 2,153,500 tonnes (IOOC, though requiring further refinement, are usually applied as a first ap-
2008). Most table olives are prepared as Spanish-style or alkali treated proach for the selection of potential probiotic microorganisms (FAO/
green olives (IOOC, 2004), which represent about 60% of production, WHO, 2001).
but there is also a general increasing interest for traditional and home- Reviews on the importance of foods in the efficacy of probiotics have
made products such as “seasoned” olives, usually prepared from direct- recently been published (Champagne & Gadner, 2005; Ranadheera,
ly brined fruits (Arroyo López et al., 2005). Baines, & Adams, 2010). New sources of probiotic microorganisms such
Among the microorganisms involved in the Spanish style green as Greek dry-fermented sausage (Papamanoli, Tzanetakis, Litopoulou-
table olive fermentation, lactic acid bacteria (LAB) is the most relevant Tzanetaki, & Kotzekidou, 2003) cereal-based substrates (malt, barley
group (Garrido-Fernández, Fernández Díaz, & Adams, 1997). The devel- and wheat media) (Charalampopoulos, Pandiella, & Webb, 2002) or
opment of LAB in this process leads to a rapid acidification of brines and kimchi (Chang, Shim, Cha, & Chee, 2010) are currently being investigat-
a drop in pH to below 4.5 (Delgado et al., 2007; Garrido-Fernández et al., ed. The regular intake of probiotic preparations is considered useful to
1997; Sánchez, Rejano, Montaño, & De Castro, 2001). However, the increase the bioavailability of trace minerals and proteins in the vegetar-
growth of LAB in directly brined olives is more difficult due to the ian diet (Famularo, De Simone, Pandey, Ranjan Sahu, & Minisola, 2005).
high presence of phenolic compounds in the storage solutions. For this Potentially probiotic strains of Lactobacillus plantarum and Lactobacillus
reason, LAB from non alkali-treated fruits have been scarcely studied paracasei survived in artichokes, which improved their survival in simu-
(Garrido-Fernández et al., 1997). lated gastrointestinal digestion (Valerio et al., 2006). L. paracasei IMPC2.1
According to the FAO/WHO (2001), probiotics are “live microorgan- (isolated from the human intestine) incorporated into an artichoke fed
isms which, when administered in adequate amounts, confer a health to a human was recovered from stools and also showed high viability
for 3 months in ripe and green olive fermentations (Lavermicocca,
Lonigro, et al., 2005). The use of table olives as a reliable food vehicle
⁎ Corresponding author at: Food Biotechnology Department, Instituto de la Grasa
(CSIC), Avda. Padre García Tejero, 4, 41012, Sevilla, Spain. Tel.: + 34 954 690850;
for this bacterium into the human gastrointestinal tract has been
fax: + 34 954 691262. subjected to patents (Hurtado, Reguant, Bordons, & Rozès, 2012;
E-mail address: (J. Bautista-Gallego). Lavermicocca et al., 2005). Recently, L. paracasei IMPC2.1 has been used

0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.

The resulting pellet was washed twice in PBS.9 ml of in 110 g ml −1. Gopal. One milliliter of analyses of the pH. where at ambient temperature. Fermentation control were centrifuged (10. and Lombardi (2001). After solidifica- 0. Both factories used plastic drum containers with temperature for 5 h. 0. Gordal ol. Then. was sampled. LTD Tokyo. 40. the addition of lysozyme (50 mg ml −1. At 1 h intervals. Autoaggregation assays were performed according to the methodol- tion was 25 g ml −1. St. 5 min). paraplantarum. mogenized by vigorous vortexing for 10 s and then incubated at room Spain) were visited. At represents the absorbance at any time (1. For directly brined olives. the most common 2. 4 or 5 h). For the detection of bacteriocin production. two factories which ogy described by Kos et al. Molecular identification and characterization of the lactobacilli lactic acid fermented vegetable in western countries. Belgium). L. ives were first treated with a 20 g ml −1 sodium hydroxide solution until reaching 2/3 of the flesh. follow- ing the protocol described by Gevers. milliliters of an overnight culture containing ~10 8 CFU ml−1 were ho- villa. Germany). located in Alcalá de Guadaira. which process. A lawn of BHI soft agar (10 g l−1. None of the three sampled factories had pre. at least Bacterial cell surface hydrophobicity was assessed by measuring mi- two fermentation containers were analyzed.5. respectively. Spain) and Aloreña cultivar (plant C. transferred to a new tube containing 3.1 Gel Software (Eppendorf. Previously. and Dellaglio (2001). The similarity among digi- es both Gordal and Manzanilla cultivars in underground. NaCl concentration (g ml−1). The autoaggregation percentage was expressed as a function of were maintained in a cold room (~8 °C) and also in a covered yard time until it was constant. namely Salmonella enteritidis saline solution (9 g l −1 NaCl) and then plated using a Spiral System ATCC13076 and the strain named as WT. Four ferment Gordal and Manzanilla fruits (plant B. the hole and allowed to diffuse at 4 °C for 30 min. USA) as selective medium tion. Then. Briefly. In vitro phenotypic tests related to probiotic potential then immersed in 110 g ml −1 of an NaCl brine. following the protocol described by isolated from two types of table olive elaborations: lye-treated (Spanish Torriani. Briefly. package (Applied Maths. 108 CFU ml−1) and the A600 was measured (A0). Manzanilla and Gordal (A600) was measured in a SmartSpec™3000 (Bio-Rad Laboratories. In this case. Sigma) for cell lysis. re-suspended in 3 ml of 0. 10. Lonigro. fiberglass talized profiles was calculated using the Pearson correlation and an av- containers with a 10-tonne capacity. 3. Materials and methods characterization with the single oligonucleotide primer (GTG)5. could have a con. isolates siderable value for developing starter cultures with the double role of being technologically relevant and able to enhance the health aspect of The different isolates grown on MRS agar supplemented with so- table olives. rep-PCR fingerprinting was performed for molecular strain 2. except that the sodium hydroxide solu. respectively..1 mol l−1 KNO3 (final concentra- Physicochemical control of the fermentation was achieved through tion ca.2. Brine samples were diluted in sterile containing each indicator microorganism. a hole was made in the center of the plate and 100 μl of a 36–48 h for LAB isolation. and Swings (2001). olives were washed and directly brined was carefully removed. cubation at 37 °C. LAB isolation ity (H%) was calculated using the formula H%=(1−A1 /A0)×100. a food isolate. Hamburg. These parameters were analyzed according and xylene phases were separated by incubation for 20 min at room tem- to the methodology described by Garrido-Fernández et al. with microorganisms. 2. Seville. perature (~25 °C). Barcelona. crobial adhesion to hydrocarbons using the procedure described by Crow. and b) the study of certain in vitro phe. pro platinum 1. DNA was extracted style) and directly brined olives. cultures were purified by subsequent re-streaking through molecular identification (De Bellis.3. 2010).3. Prod- 2.2 g l −1 sodium azide (Sigma. Rogosa and Sharpe (MRS) agar (Oxoid) supplemented with 35150 and NCTC 12079. and A0 the viously used LAB starter cultures. expressed system. Bautista-Gallego et al. 60 and two-phase system was mixed by vortexing for 2 min. Listeria model dwScientific (Dow Whitley Scientific Limited. titratable (lactic xylene was then added to the cell suspension to form a two-phase acid) and combined (undissociated organic salts) acidities. erage linkage (UPGMA) dendrogram was derived from the profiles. the water 105 days of fermentation. Valerio. Alhaurín el Grande. After an overnight in- Spain) image analysis system and expressed as log10 CFU ml −1.136 J. Sisto. using the formula 1 − (At / A0) × 100. Felis. from 1 ml of an overnight culture of each isolate according to the pro- notypic characteristics related to the probiotic potential of these cedure describe by Andrigetto. Manzanilla olives followed the same treatment. Se. pentosus. cells at the stationary phase 2.4. Huys. 20. washed with tap water for 18 h and 2. containers were kept in an open yard whereas Aloreña containers USA). England) on de monocytogenes FMCC B-128 and NCTC 10527. a single factory resulting fingerprints were analyzed with the BioNumerics 4. Sanlucar la Mayor. 80 from Spanish style green olives. & on MRS agar and observed under a phase contrast microscope Lavermicocca. molecular characterization at strain level of the lactobacilli microbiota plantarum and L. 28 from streaked onto Columbia agar plates containing 50 g l−1 of horse blood . 80 g l −1 and 70 g l −1 of NaCl solutions for Aloreña. Japan) to differentiate cell morphology. After a 10 min pre-incubation at room temperature. (2003) with slight modifications. The percentage of the cell surface hydrophobic- 2. A total of 111 isolates were randomly selected during the different To test for haemolytic activity. evolve spontaneously.000×g. dium azide were identified at species level using multiplex PCR anal- Therefore.1. Luis. The availability of LAB strains isolated from this product. phosphate buffered saline (PBS). / Food Research International 50 (2013) 135–142 as starter culture for processing green table olives and its survival and Gordal and Manzanilla directly brined olives and 3 from Aloreña directly dominance on the olive surface during fermentation was studied brined olives. and Escherichia coli ATCC Man. Spain). fresh lactobacilli broth cultures were stages of fermentation. the agar well diffusion Samples for microbiological purposes were taken at the same assay test was used. the present study focused on a) the identification and ysis of the recA gene with species-specific primers for L. However. Plates were incubated at 30 °C for 48 h and then cell-free MRS broth of the different lactobacilli strains were placed in the colonies were counted using a CounterMat v. and then the absorbance at 600 nm Gordal and Manzanilla cultivars. the as g ml−1 and mEq l −1.6 software (plant A. was poured onto Petri dishes. Málaga. the probiotic potential of LAB naturally (Olympus Optical Co. Then. and Wicken (1995).10 (IUL. at 0 (initial). All fermentations were allowed to absorbance at time t =0 h. The aqueous phase was carefully removed and the A600 was measured (A1). The In the case of Spanish style green olive fermentations. Kortrijk. For each factory and type of process.1 ml of the upper suspension 500-kg capacity. Olive cultivars and processing ucts of rep-PCR were electrophoresed in a 20 g l−1 agarose gel and the profiles obtained were visualized under ultraviolet light using UVI Samples were directly obtained from three Spanish olive factories. Zampese. (1997). occurring in table olive fermentations still remains unexplored. the plates were examined for halos around the hole.) medium sampling times described above.

nating variables were hydrophobicity. Multivariate analysis er (Gevers et al. USA).1. using a varimax rotation.81 g l−1 of sodium phosphate dibasic cold room. Oxoid) and pancreatin (0. The cases introduced in the the first half of fermentation. Overall. pancreatic digestion survival and overall digestion 3. paraplantarum. / Food Research International 50 (2013) 135–142 137 (Oxoid.5 in directly brined olives (because Simulated gastric digestion (SGD) was studied using the protocol of the initial brine correction with acetic acid) while it was higher described by Corcoran. The results obtained indi- quently plated onto MRS agar. KH2PO4 (0. and Ross (2007) with slight (~ 5. & Sedlacek. Molecular identification and characterization of the lactobacilli initial counts in the freshly prepared simulated pancreatic juice. As shown in Fig. Italy). the NaCl concentration was kept con- plates containing 5 g l−1 of Oxgall (Sigma-B3883) were inoculated with stant at around 64. Initial counts were around 6 log10 CFU ml −1.9%) was identified as L. the fermentation produced modifications. auto-aggregation. to know the 3.0 consisting of 50. acidities.6. Serial dilutions were done. L. pentosus. PCR analysis of the recA gene with species-specific primers for ryover of the buffers to the agar. from 9 of cluster VIII to 2 of clus- number of principal components or factors.4%) were identi- Simone Guglielmetti from the University of Milan. The lactobacilli cultures to The changes in the LAB population in Spanish style green table be tested were grown to early stationary phase. ters II and V. HM2T0-63. and subse. 2005). obtained from the second half and end of fermentation. showed the highest auto-aggregation values (more The initial and final NaCl concentrations. the populations were kept constant until (3 g l−1. the plates studied in this work are shown in Table 1. representing 66% of the L. tion. plantarum and comparison of the survival rates after simulated gastric and pancreatic another strain (0. 107 strains (96.27 g l−1. GM2FT1-129 and HG2T1-138.4. was isolates performed by serial dilutions of the cultures. The pH was adjusted with a solu. Statistical analyses were performed with Statistic 7. characterization to define their intra-specific variability.9%) as L. incubated at 37 °C for 72 h. lactobacilli counts at the start of the simulated gastric digestion and The 107 L. LAB were not detected during the period of study while heptahydrate and 8. a decrease in pH and an increase in titratable acidity at the end of fer- er solution at pH 2. J. serial dilutions ly brined Gordal and Manzanilla olives. Milan.00 were retained. centrifuged olive fermentations showed the typical evolution for this type of pro- (10. and then observed was significantly higher in Spanish style compared to directly green for colonies with precipitated bile salts (BSH activity). Simulated.2. one strain (0. Among them. cipal component analysis (PCA) was used to discriminate among pentosus strains considered in this study. while GM2FT4-288 showed values below 10%. Briefly. clusters VI and VII check their suitability to be analyzed by chemometric techniques. LAB changes during fermentation (0. CaCl2 (0. to any of these three bacterial species. Stanton. Fitzgerald. constituted mainly by isolates of plant A (42 out of 46) obtained in values higher than 1. a total Lactobacilli activities were subjected to multivariate analysis to of 9 clusters could be identified. Finally.0 g l −1 throughout the process. In direct- (~200 rpm) to simulate peristaltic movements. which was successfully accomplished by means of rep-PCR using the GTG5 prim- 2..05 g l−1). Drab.. and cluster VIII contained isolates analysis were the 109 identified lactobacilli isolates while the discrimi. and only factors with eigen.3. they grew tion of potassium phosphate monobasic anhydrous at 1. Prior to use. Physicochemical changes 80% (Fig. the initial LAB counts were of the cultures were plated onto MRS agar medium and counted after much lower (below 2 log10 CFU ml −1) and reached their maximum incubation at 30 °C for 3–5 days.0133 g l−1) and lysozyme (0. 3a). 3. in the drums placed in the yard at ambient temperature.0 with 1 M HCl and autoclaved at 121 °C for 15 min. synthetic gastric juice was prepared in a buff. mentation. slowly after a very large lag phase and reached a population of around Harvested cells from the previous gastric digestion step were washed 5 log10 CFU ml −1 at the 20th week.0) mentioned above. 1). It is worth noting that the composition of cluster VI was tioned by Jolliffe (1986) was followed. pentosus isolates were then subjected to molecular those remaining at the end of the simulated pancreatic digestion.000 ×g.0 containing NaCl (2. reaching (pH 2. and incubated for 48 h at 30 °C. gastric diges- tion survival. LAB counts decreased slowly but a pop- 1010 CFU ml−1 and incubated for 3 h at 37 °C in an orbital shaker ulation level of above 6 log10 CFU ml −1 was always present.0 software package (StatSoft Inc. Results This phenotype exhibited a normal distribution. If present. In Aloreña directly brined olives stored in a pH 8. the Kaiser criterion men. which was more pronounced in Spanish style olives.1 g l−1. with the largest number of isolates being included in the interval between 50% and 3. the brined olives. the pellet was washed and In the present survey. Then.6) in the Spanish style. pepsin 3. All the compo- nents were obtained from Sigma-Aldrich. grouped 46 and 25 isolates.37 g l−1). and pH values for the different table olive elaborations the hydrophobicity distribution pattern was strongly skewed and . the cells were re-suspended in a maximum population level of ~ 8 log10 CFU ml −1 at the second the synthetic gastric juice to a final concentration between 107 and week of fermentation. By contrast. population levels (6 log10 CFU ml −1) between the 3rd and the 4th Simulated pancreatic digestion (SPD) was formulated using bile week of processing. The initial pH was around 4. Reference probiotic strains Lactobacillus cated that a total of 2 isolates (1. Four isolates. Phenotypic characteristics related to probiotic potential survival. A colony count on MRS plates at time zero (T0). Combined acidity an overnight MRS culture. a total of 111 LAB isolates were subjected to then re-suspended in a volume of isotonic solution in order to avoid car. pentosus.5 g l−1 of NaCl. Tulsa. Prin. The initial NaCl concentra- were examined for signs of α. Then. respectively. The other 7 clusters lactobacilli isolates.0 g l −1 at the end of the described by Dashkevicz and Feighner (1989). These last two species were digestion. Then.0 g l −1 (Aloreña) and decreased to ~ 20. After shaking at 200 rpm in an orbital shaker overnight at 37 °C. bile salt–MRS agar process. β or γ-haemolysis. It was adjusted to pH 2. The results obtained from the sedimentation experiments showed that lactobacilli isolates had a strong auto-aggregating phenotype.11 g l−1) and KCl (0. 2. combined and titratable than 90%).01 g l−1) were added.60 g l −1). by arbitrarily selecting a similarity coefficient of 80%. OK. in which it increased markedly at the end of fermenta- diameters of the precipitate halos around the colonies were measured. Bautista-Gallego et al. GG1ST0-4.5 g l −1 (Manzanilla) to The ability to deconjugate bile salts was tested by using the plate assay 91. For the selection of the contained lower amounts of isolates.8%) could not be identified as belonging casei Shirota and Lactobacillus rhamnosus GG (kindly provided by Dr. 2001. L. Sigma) in a buffer at the end of fermentation. paraplantarum. in saline and re-suspended in the same volume of the simulated pancre- atic juice. plantarum and L. Svec. 10 min) and the pellet was washed with the buffer cesses (Fig. Italy) were used for fied as L. survival was obtained by comparison of the initial both isolated from Spanish style green olive fermentations. tions in directly brined olives ranged from 59. In Spanish style olives. Then. As expected.

250% for GG3F-T3-238.6) 11. a few isolates showed high probiotic potential survival rates among those tested: 0. HG1T5-330. GM2S-T0-36. The overall rates were. GM2S-T0-36 at 36.6(±0. creatic digestions) which also had the highest proportions of the according to processing system and cultivar. The overall survival proportions of the probiotic control spe- BSH tests also revealed the absence of these activities in all isolates. 0.1%.. the values found in this work were higher. but its auto-aggregation and hy- ture.041% for GM1S-PRE-41. and 0. The study of the contribution of and 0. directly brined Manzanilla olives. cies were even lower.4) 45. Bautista-Gallego et al. In general. GG2F-T0-13.7) 117(±1) 122(±1) 5.3 (±0.8) 69. also disclosed to the partial survival in the simulated pancreatic digestion was clearly the link between pancreatic digestion and overall digestion as well as skewed (tail) to the right (data not shown).040% for HM2-T3-256.00% for L.7) 63.4%.00 (±0.25 (±0. However. 3b).5. Spanish style green Manzanilla olives.004%.00% and 0. second assay with proteinase K revealed that the inhibition was in low and showed that most of the strains had percentages below fact due to the production of lactic acid (data not shown).048% for GG2F-T1-96. although formed by the first two factors. with a final population of ~3 log10 CFU ml−1 at the end of digestive and pan- Fig. Iso- GMV. Therefore.055% for gestion survival.5 (±3.91 (±0.48) 3. However.2) 49.7(±0.092% for JOLCA A1-71.61%) to auto-aggregation and gastric After SGD. and Factor 3 (20.12) 3. genvalues higher than 1 were obtained.001%. PCA also allowed for the segregation of an- other group characterized by high values of auto-aggregation and gastric digestion (GM1F-T2-195 and GG3F-T3-238) and an average pancreatic and overall digestion survival. Spanish style green Gordal olives.08) 3. All of them showed greater survival rates than the refer.5(±0. In comparison with the survival rate of the ref- (GM2FT2-206. Standard errors in parentheses.31 (±0.1 (±0. Most of the isolates showed a low survival rate (%) after the SGD.2) 15 (±2) 48(±2) 4. rhamnosus GG. Table 3 shows the L. and GG3F-T3-237) but discrimination criteria were more difficult to establish in this case. variables to PCA.45 (±0.05%: 0.9(±0. and overall digestion HG1T4-291). in general. 0. A group of strains showed survival variables to factors (Table 2) showed that Factor 1 (which explained rates close to 0.3) 7. GM2FT3-246.06) 1.05%.007% and 0. all isolates were sequentially subjected to the simulated digestion survival. and HG2T4-297).54%) to hydrophobicity. Factor 2 (21.2 (±0.045% for GG2S-T2-168. 0.043% for L.3) Manzanilla 59. and 0.1(±0. GG1FT3-230.g. population of ~4. Their position was related to the negative side of Factor 1 and the average value for Factor 2.4) 29(±2) 75(±2) 4.3) Gordal 69. 0. directly brined Aloreña olives stored in covered yard at ambient tempera. Regarding bacteriocin production. and overall survival. with a final room.5 (±1. GG4S-T1-111.00738%. only three ei- ence probiotic strains used for comparison: 0. 29. HM2T2-222. auto-aggregation phenotype (75. as a result.1) 11.55 (±0.84 (±0. casei Shirota. 1. erence probiotic strains.5 (±1. rhamnosus GG).70 (±0. time (at selected interval levels).89 (±0. 0.15) 4.1) showed that most of the isolates had values between 0% and 5% there were some particularly resistant strains. The histogram corresponding (Fig.7(0. NaCl Combined acidity pH Titratable acidity (g l−1) (mEq l−1) (g l−1) Elaboration Cultivar Initial Final Initial Final Initial Final Initial Final Directly brined green olives Aloreña 91. When subjecting the auto-aggregation.03) 2.5 (±1.67 (±0. pancreatic digestion survival.7%. The histogram was pathogen microorganisms in the diffusion agar assays.8) 25 (±2) 72(±2) 4. Changes in lactic acid bacteria counts vs. they are characterized by high values of pancreatic digestion survival and overall digestion (due to their negative relationship with Factor 1). 4.5(±0.004% for L.102% for GG1S-T1-84. e.61%) and hydrophobicity (8.075% for GG2S-T1-91. using the isolates as grouping variables. There was a cloud of isolates around the center of the new axes (their identities have been omitted to im- prove readability).00804%. 2. Vertical bars denote 95% confidence intervals. 5b shows the projection of the isolates (cases) onto the plane rate for the pancreatic digestion was generally below 25%.07) 1. 0. There were only two strains with survival values between none of the lactobacilli isolates had the capacity to produce bacterio.3) Spanish style green olives Gordal 65. some strains with relatively high survival rates.076% for GM2S-T1-127. a higher survival rate to the pancreatic tion of the variables onto the plane formed by the first two factors than to the gastric digestion was noticed. HJG. with a high number of lactobacilli included in an interval of survival 3.3) 5. This could indicate that most of the lactobacilli were weakly ing 56. pentosus isolates with the most promising po- tential probiotic characteristics based on PCA analysis.0 (±3. indicating the presence of that between auto-aggregation and gastric digestion survival. However. The partial survival Fig. GG2F-T0-13 at 39.5 (±1.3) 5. Isolates . in addition. directly brined Aloreña olives stored in cold late GM2FT5-327 had outstanding survival (0. all of them can be considered to be very close to each other. GGAS-T1-111 show- (Fig. Multivariate analysis of phenotypic characteristics related to rate between 0. AMC.52%).5) 105(±6) 126(±4) 5. although some of them showed levels higher than 25% GM2F-T5-327 at 36.06) 4.6(±0. hydrophobicity. gastric di- 0. 0.6) Manzanilla 64. a strongly skewed (tail) to the right. the first screening indicated The overall lactobacilli survival in the digestive process with re- that some strains were able to inhibit the growth of the indicator spect to the initial population is shown in Fig. HM2T4-308.3 (±1. GG2S-T2-168 and GMSF-T5-327 were clearly distinguished. / Food Research International 50 (2013) 135–142 Table 1 Physicochemical conditions of the brines obtained from the different table olive elaborations studied in this work.23 log10 CFU ml −1). There were also other isolates with a slight segregation from the cloud of points (such as HM2-T2- 222.05) 1. and finally hydrophobic. drophobicity values were lower than those of GG2S-T2-168. casei Shirota and 2.5) 64.14% for L. The highest over- all survival rate was observed in isolate GG2S-T2-168 (0.09) 1.009% while a few more were between 0. GGV. there were also a few isolates separated from the rest. 5a) clearly depicted this relationship but.138 J. The results of the haemolytic and 0. AMP.2 (±1.150% for GG1S-T3-229. A projec- pancreatic digestion.002% and cins under the assayed conditions.3(0.7%).54% variance) was mainly related to pancreatic digestion survival 0. GM2ST3-245. directly brined Gordal olives. HJM.

Histogram of the overall simulated gastric and pancreatic digestion survival. Discussion LAB starter cultures are widely used in combination with probiotic bacteria to produce fermented dairy derivatives (Vinderola. & Reinheimer. table olives. However. . Bautista-Gallego et al. are especially relevant due to their worldwide production. Among others. which are indicated with roman numerals. there are an increasing number of con- sumers who are allergic to milk. however. or simply. 4. expressed as percentage of the initial population. 4. Bailo. both showed high auto-aggregation and hydrophobicity values. 2. distribution. Dendrogram generated after cluster analysis of the digitized rep-PCR finger- prints of lactobacilli. J. GG3F-T3-238 had survival rates comparable to those of the reference while isolates GM1F-T2-195 did not survive at all. the most common fermented vegetable product in western countries. / Food Research International 50 (2013) 135–142 139 Fig. 3. prefer more variation with respect to probiotic sources. Histograms showing the distribution of the auto-aggregation (a) and hydropho- bicity (b) characters in lactobacilli isolates. An 80% coefficient of similarity was arbitrarily selected to identify the clusters. 2003). Fig. and progressively increasing consumption Fig. Products traditionally subjected to lactic acid fermentation during their processing are good candidates.

and most of the isolated strains belonged to this last species. the BSH activity has also been mooted to be potentially detrimental to the human host and thus it is not yet completely clear that it could be a desirable trait in a probiotic bacterium. 2008. Catulo. 2008). BSH activity was not found in any of the isolates. Brito. pentosus and L.. these two traits may be used for pre- liminary screening to identify potentially adherent isolates (Del Re. belonged to typical Spanish and directly brined green olive processes Variable Factor 1 Factor 2 Factor 3 and the strains could a priori have a wide application spectrum. 1984). with some isolates show- ing values above 25%. However.. et al. and the isolates characterized in this study formed 9 different clusters. 2000). Esteve-Zarzoso. & Barbés.6076 0. those phenotypic characteristics considered relevant for a first criterion of selection based on their probiotic potential were evaluated. from different spontaneous industrial green table olive fermentations 2008.. 2010. 1997). It has been shown that those strains able to adhere to cell monolayers also show auto-aggregation and hydrophobicity characteristics as determined by microbial adhe- sion to hydrocarbons. By contrast. green olives. pentosus were identified in sociated with the Mediterranean diet and enjoy a healthy and natural Gallega fermentations (Oliveira et al.0817 to their processing. Isolate HM2-T2-222 had a good auto-aggregation phenotype (nearly 58%) and an important resistance to gastric and pan- creatic digestions. Bautista-Gallego et al. is gaining prominence (Panagou. casei and L. Boris. In fact. with a reduced lactobacilli inter-specific biodiversity al- though the strains originated from different factories and elaboration processes. L. olives could be well accepted by consumers. L. olive cultivar or day of isolation (Fig. it is impor- tant to select probiotic strains with high resistance to bile because they could develop better in the upper intestine (Gilliland. & Caggia. 2003. However.. Shortt. Tassou. Valerio et al. 2007). 2004). Romano. were isolated from Arbequina directly brined olives (Hurtado. 2007b). In this work. L. while L. Selection criteria for LAB to be used as functional starter cultures have been recently updated (Ammor & Mayo. However. García García. Adhesion ability to the intestinal epithelium is another important criterion for selecting potentially probiotic strains (Ouwehand. recently. Sgorbati. we have selected lactobacilli isolates Panagou. & Rozès. 2005.140 J.0001 0. results should be interpreted with caution because this adherence does not necessarily mean an in vivo adhesion. Pancreatic digestión survival 0. no definitive differ- entiation could be observed based on the processing plant of origin. Because of the unfavorable environmental conditions for microor- ganism survival in the stomach. table olives are also as. & with promising in vitro phenotypic characteristics related to their Garrido Fernández. Reguant. Schillinger. 1999). 2005. However.0662 0. 2007a.3051 0. Franz. & Fig. Furthermore.0315 numerous (Garrido-Fernández et al. hydro- phobicity had a strongly skewed distribution. & Palenzona. Therefore. & Nychas. paraplantarum ives have already been proposed. Segovia-Bravo. although two main groups (VI and VII) accounted for the majority of the microorganisms.0 (Fernández. 2006). & Pintado. inter- pretation of this result is not straightforward because. Restuccia. / Food Research International 50 (2013) 135–142 Table 2 probiotic potential.. The probiotic products produced with them will thus have the invaluable Auto-aggregation 0. an intra-specific diversity was found within the L. Miglioli. although the ability of probiotic strains to hydrolyze bile salts is usually included among the criteria for probiotic strain selection. pentosus Lavermicocca. where the pH is around 2. & Salminen. 2004) and L. In our study. however.4445 b0. In addition. 1997). The results of this work are in agree- ment with this trend. 2003). Durán Quintana. 2006) was predominant in naturally fermented Algerian (Garrido-Fernández et al. In this study.0147 0.8667 advantage of their origin without the introduction of any strange factor Gastric digestion survival 0. Projection of the variables (a) and cases/isolates (b) onto the plane formed by Bush. In this work. Bordons. Peres. & Katsaboxakis. 5. 2). pentosus species. plantarum and L. plantarum (Mourad & Nour-Eddine..0655 0. intestinal tract has been considered as one of the most critical factors . Valerio. Lavermicocca.0040 0. probiotic table ol. the LAB species mentioned were Overall digestión 0.0011 In traditional olive fermentations. mentation process were isolated from humans (De Bellis et al. plantarum reputation.0190 Hydrophobicity 0. but in recent studies the species isolated have been more limited. Similarly. Arroyo López. it is essential to select probiotic strains with high tolerance to acidic conditions. survival during transit through the gastro the first two factors deduced from PCA.4706 0. These results prevent any speculation regarding a plant-specific population or strain evolution during the fer- mentation process. Staley. it is highly probable that presumptive probiotic table were detected in naturally fermented Sicilian olives (Randazzo. the bacteria used for the fer. Thus. Kirjavainen. et al. The fermentations from which they were selected Contribution of variables to the factors in the PCA based on correlations.0218 0. Lonigro. the auto-aggregation phenotype was fairly widespread and had an almost normal distribution among most of the strains showing values higher than 50%..

. AGL2009-07436/ALI. C. & Garrido Fernández. K. Journal of Dairy Science. Medical Hypotheses.. Valerio.. C. rhamnosus GG. V. V. L. Projection of the cases onto the International Dairy Journal. D. properties as well as on other technological characteristics like survival 449–455. 26–30. 43–52. 9. limited application as potential olives: Microbial populations adhering on olive surface in fermentations sets inoc- probiotics because their differences were linked mainly to character. C. Growth of probiotic probiotic strains used in dairy products (reference strains).. L.0 76. Arroyo López. K. Critical Review in Food Science and Nutrition. L. (2001). Delgado. (2003). Grasas y Aceites. P.. N. A. 5. Gilliland. (2007). C.51 8. J. A. P.30 GM1F-T2-195 L. Simone Guglielmetti. International Journal of Food Microbiology. Bautista-Gallego et al. (2000).9 39. F. Madrid. 851–859. A. Meat Science.00091 2. 205.. Other strains (GM1F-T2-195 and GG3F-T3-238) also far apart from 55. Applied and References Environmental Microbiology.12. A. F. S. T.. S. Jolliffe.. pentosus isolates showed promising probiotic and differ. F. partially financed by European regional development funds. FEMS Microbiology Letters.. Lavermicocca. P. Del Re. S. bile salt hydrolase-active Lactobacillus spp. A... We are also thankful for the financial International. S. Garrido-Fernández.. Y. Champagne. D. Letters in Applied Microbiology. Fitzgerald. 53. F. Isolate reference Species Overall survival rate % Residual populationa CFUml−1 Auto-aggregation (%) Hydrophobicity (%) GG2S-T2-168 L. P. V. Kirjavainen. casei Shirota 0.00007 3. B. pentosus 0. J. ERDF). 57. Grasas y Aceites.. 31. P. M.. & Ross. A. pentosus 0.55 Control L. (2005b). P. Priority date of Milan. 193–201. Staley. Brito. & Rozès.. F. S. & Barbés. 31–36. (2005). et al.. 140. 1. & Nour-Eddine. M. A. Esteve-Zarzoso. J. 6–13.2b requires Certain L. Trade standard applying to table olives. Famularo. & Ouwehand. 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IOOC (International Olive Oil Council) (2004).00000 No survival 51. 94. absence of NaCl and apparently follows a mixed metabolite kinetics. S. among others. 130. L.00007 2.2 27. N.23 5. & Rozès.. F. in agreement with the overall survival rate criterion. B. 94. 109.. Document COI/OT/NC no. Biotechnol- lactobacilli isolated from artisanal meat plants and traditional fermented sausages ogy of olive fermentation of “Galega” Portuguese variety. Journal of entiated characteristics as was proven by PCA. Kos. S. Vukovic. themselves and with the new factors. Word patent: WO 2005/053430 A1. Hurtado. Journal of Applied Microbiology. (1999). K. (2010). Journal of Applied Microbiology. Leitäo. G. Romero. GG2S-T2-168 showed Biotechnology.N. Lonigro. Microbiology. A.. N. G. Food Microbiology. Brito. L. N.. . / Food Research International 50 (2013) 135–142 141 Table 3 In vitro phenotypic characteristics of the selected lactobacilli isolates selected by PCA. K. pentosus 0. De Bellis. population dynamics during the processing of Arbequina table olives. M. Importance of bile tolerance of Lactobacillus Acknowledgments acidophilus used as dietary adjunct.. could have.... J. 1132–1135. Pandiella. Probiotic not only high survival rates but also high values for auto-aggregation lactobacilli: An innovative tool to correct the malabsorption syndrome of vegetarians? and hydrophobicity. Fernández. Probiotic characteristics of lactic acid bacteria isolated from kimchi. Reguant. (2005).. D.. from the University Lavermicocca.. A.00738 4. N. Study of the adhesion and survival of lactobacilli and bifidobacteria on table olives with the aim of formulating a new probiotic food.. pentosus 0. The GM2F-T5-327 isolate had a moderate auto. & Mayo.. C. Sisto. Conclusion Microbiology. (2001). final selection would also depend on their real in vivo probiotic strains to be used in the gastrointestinal tract. Gallegari. Principal component analysis. M.. L. L. Peres. C. F.. 138–146. S. Valerio. L.. The research leading to these results has received funding from the Hurtado. 61–84. and Junta de Andalucía (through financial support to group AGR-125). olive surface. & Minisola. Amerian Córdoba Park Hotel. Lonigro. Bordons.. Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria. with the effects of brine solutions obtained at the end of the olive fermentation. & Feighner.. October–November. Adhesion. first two factors clearly differentiated strains GG2S-T2-168 and Dashkevicz. & Salminen. L. D.. J. (1986).. (2002). 71. Microbial EU's Seventh Framework Program ([FP7/2007-2013] under grant agree. T. production of lactic acid during fermentation or adhesion to Production and processing. Mourad. because the survival rate is extremely low in the gastric digestion and changes in seasoned olives during the shelf period. Bordons. Cordoba (Argentina). Gomes. 981–987. contract. 292–300. 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