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Molecular characterization of lactic acid bacteria isolated from industrially fermented Greek table olives
LWT - Food Science and Technology 50 (2013) 353e356
Contents lists available at SciVerse ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Research note
Molecular characterization of lactic acid bacteria isolated from industrially fermented Greek table olives Agapi I. Doulgeraki a, Paraskevi Pramateftaki b, Anthoula A. Argyri b, George-John E. Nychas a, Chrysoula C. Tassou b, Efstathios Z. Panagou a, * a b
Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece Hellenic Agricultural Organisation ‘Demeter’, Institute of Technology of Agricultural Products, Sofokli Venizelou 1, Lycovrissi 14123, Attica, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2012 Received in revised form 28 June 2012 Accepted 5 July 2012
A total of 145 lactic acid bacteria (LAB) isolates have been recovered from fermented table olives and brine and characterized at strain level with molecular tools. Pulsed-Field Gel Electrophoresis (PFGE) of ApaI macrorestriction fragments was applied for strain differentiation. Species differentiation was based either on Denaturing Gradient Gel Electrophoresis (PCR-DGGE) (black olives) or on restriction analysis of the amplified 16S rRNA gene (PCR-ARDRA) (brine and green olives). Species identification was based on sequence analysis of 16S rRNA gene. When the data were insufficient to resolve the species level of the isolates, specific multiplex PCR assays targeting the recA or tuf genes were employed. From 145 LAB isolates, 71 different strains were recovered from fermented olive and brine samples; 17 strains were assigned to Leuconostoc mesenteroides, 51 were grouped in Lactobacillus plantarum group (including 13 L. plantarum, 37 Lactobacillus pentosus, 1 Lactobacillus paraplantarum), 2 Lactobacillus paracasei subsp. paracasei and 1 Leuconostoc pseudomesenteroides. L. plantarum was recovered mainly from green olive fermentation, whereas in black olives the main species identified were L. pentosus and Ln. mesenteroides). These observations reveal that olives are a highly diverse ecosystem regarding the presence of LAB, which may affect the quality of the final fermented product. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Table olives Fermentation Denaturing Gradient Gel Electrophoresis Pulsed-Field Gel Electrophoresis Multiplex PCR
1. Introduction The cultivation of table olives is widespread throughout the Mediterranean basin and holds a major role not only through its contribution to rural economy but also from the cultural and environmental point of view. The estimated world production of table olives for the period 2011/12 amounted to 2.6 million tonnes approximately and has shown an increasing trend over the last years (IOC, 2012). Today, the market offers a wide variety of table olive elaborations that are able to satisfy all consumer tastes, even the most sophisticated ones. However, there are three basic commercial preparations of table olives that have attracted the interest of the scientific community, namely Spanish style green olives, naturally black olives (Greek style) and black ripe olives (Californian style) for which processing conditions are well established in the literature (Sánchez Gómez, García García, & Rejano Navarro, 2006). A basic step in table olive processing, especially for Spanish style green olives and natural black olives, is
* Corresponding author. Tel.: þ30 210 5294693. E-mail address: [email protected] (E.Z. Panagou). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.07.003
fermentation. This process is carried out primarily by the spontaneous microbiota of lactic acid bacteria encountered on olive drupes and in the brine environment aiming at the biotransformation of fermentable substrates (mainly reducing sugars) in olives to organic acids (primarily lactic acid and acetic acid) that are released into the fermentation brine. The net effect is an accumulation of acids and a lowering of brine pH which in combination with the salt content of brine ensures the microbiological safety of the product and at the same time provides the desirable sensory attributes of the final product (Garrido Fernández, Fernández Díez, & Adams, 1997). Moreover, LAB produce small amounts of ethanol and other volatile compounds that make a significant contribution to the final flavor of table olives (for a review on lactic acid bacteria and table olive fermentation see Hurtado, Reguant, Bordons, & Rozès, 2012). It is generally accepted that the table olive industry is highly traditional and even in our days table olive processing remains craft and empirical despite its economic importance. However, new identification and typing techniques for LAB have been continuously developed and implemented to identify the ecological diversity of this microbial group in table olive processing using both culture dependent and independent techniques (Abriouel, Benomar, Lucas, & Gálvez, 2011; Ercolini, Villani, Aponte,
354
A.I. Doulgeraki et al. / LWT - Food Science and Technology 50 (2013) 353e356
& Mauriello, 2006; Hurtado et al., 2011; Hurtado, Reguant, Esteve Zarzoso, Bordons, & Rozès, 2008; Panagou, Schillinger, Franz, & Nychas, 2008; Randazzo, Restuccia, Daniele Romano, & Caggia, 2004). The aim of the present research note is to report on the characterization of LAB isolates from industrially fermented Spanishstyle green olives and natural black olives in Greece using molecular identification techniques. Until now such information came only from small-scale experimental fermentations and there was a lack of LAB characterization from industrial installations. The results obtained in this work will be employed in further research focusing on the investigation of the technological properties of the isolated strains (e.g. probiotic potential, bacteriocin production) for the selection of potential starter cultures with improved characteristics in table olive fermentation. 2. Materials and methods 2.1. Isolation of LAB Brine and olive samples from Spanish-style green olives of cv. Conservolea and Halkidiki, as well as natural black olives from cv. Conservolea and Kalamata were collected from six processing plants in different regions of Greece (Peloponnese, Sterea Ellada). Olive samples were taken directly from fermentation vessels at the end of fermentation process and transported to the laboratory with minimum delay for further analysis. Olive samples (25 g) and brine (1 ml) were aseptically transferred to 225 ml or 9 ml of sterile quarter strength Ringer’s solution, respectively. Decimal dilutions were prepared in the same Ringer’s solution and duplicate 1 ml samples of the appropriate dilutions were mixed on de ManeRogosaeSharp medium (MRS; Merck 1.10660, Darmstadt, Germany), overlaid with 10 ml of the same medium and incubated at 25 C for 48e72 h, to quantify the population of LAB. All plates were examined visually for typical colony types and morphological characteristics that were associated with the growth medium. Moreover, the selectivity of the MRS medium was routinely checked by Gram staining and microscopic examination of smears prepared from randomly selected colonies. LAB were isolated from the highest dilution of MRS growth medium. From each of the aforementioned samplings 10% of the colonies (i.e., 10e15 colonies) were randomly selected and purified. Pure cultures were stored at 80 C in MRS broth medium supplemented with 20% (v/v) glycerol (Serva, Heidelberg, Germany). Before experimental use each isolate was subcultured twice, while the purity of the culture was always checked. A total of 145 pure cultures were finally picked from both olives and brine and subjected to molecular analysis. 2.2. Pulsed Field Gel Electrophoresis (PFGE) Pulsed Field Gel Electrophoresis was performed in order to determine the LAB heterogeneity at strain level. In brief, genomic DNA extraction was performed from all isolates as reported elsewhere (Doulgeraki, Paramithiotis, Kagkli, & Nychas, 2010). The restriction enzyme ApaI (10U) (New England Biolabs, Ipswich, MA, USA) was applied according to the recommendations of the manufacturer. Restriction fragments were separated in 1% PFGE grade agarose gel in 0.5 mM TriseBorate buffer on CHEF-DRII (Bio-Rad, Hercules, CA, USA) equipment with the following running parameters: 6 V cm1, 1 s initial switching time, 10 s final switching time, and 16 h of total run at 14 C. Gels were then stained with ethidium bromide (0.5 mg mL1) in water for 1 h and distained for 2 h before being photographed using a GelDoc system. Conversion, normalization and further analysis were performed using the Pearson
coefficient and UPGMA cluster analysis with Bionumerics software, version 6.1 (Applied Maths, Sint-Martens-Latem, Belgium). 2.3. Identification and characterization of strains Following PFGE differentiation, the different isolates were subjected to PCR-DGGE e(33 isolates from black olives) (Cocolin et al., 2004) and PCR-ARDRA (38 isolates from brine and green olives) (Rodas, Ferrer, & Pardo, 2003), respectively for species differentiation. The identification of a representative number of the different patterns detected in the later methods was achieved by sequence analysis of V1eV6 region of 16S rRNA gene (Doulgeraki et al., 2010). In the case where the acquired data were insufficient to resolve the species level of the bacteria isolates, for the differentiation of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum or Lactobacillus casei and Lactobacillus paracasei, specific multiplex PCR assays targeting the recA gene (Torriani, Felis, & Dellaglio, 2001) or tuf gene (Ventura, Canchaya, Meylan, Klaenhammer, & Zink, 2003) were employed. In all cases, DNA was extracted according to Doulgeraki, Paramithiotis, and Nychas (2011). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences are JX129193 to JX129206. 3. Results and discussion The microbiological analysis of olive samples resulted in average LAB populations of 6.03 (1.03) and 5.73 (0.35) log cfu g1 for black and green olives, respectively, whereas in the brines the respective average values were 6.15 (0.57) and 6.48 (0.83) log cfu ml1. The application of PFGE-ApaI macrorestriction analysis to a total of 145 isolates resulted in 71 different fingerprints (Fig. 1). In total 33 (black olives) and 38 (brine and green olives) different fingerprints were subjected to PCR-DGGE and PCR-ARDRA which resulted in 3 and 2 different patterns, respectively. The results from sequence analysis of a representative number of isolates revealed that the three different patterns detected in PCR-DGGE were assigned to Leuconostoc mesenteroides (17 fingerprints), Leuconostoc pseudomesenteroides (1 fingerprint) and L. plantarum group (15 fingerprints). Additionally, the two patterns detected in PCRARDRA were assigned to L. plantarum group (36 fingerprints) and L. casei group (2 fingerprints). For the differentiation of isolates assigned to L. plantarum group and L. casei group, multiplex PCR assays targeting the recA and tuf genes, respectively were employed, resulting in 13 L. plantarum, 37 L. pentosus, 1 L. paraplantarum and 2 L. paracasei subsp. paracasei. Table 1 summarizes the prevalence of the different genera detected in the different samples. The aforementioned species have been previously associated with the microbiota of spontaneous fermentation of table olives. More accurately, L. plantarum and L. pentosus have been reported as the predominant species in the fermentation of table olives (Abriouel et al., 2011; Asehraou, Peres, Faid, & Brito, 2002; Chamkha, Sayadia, Brub, & Godon, 2008; De Bellis, Valerio, Sisto, Lonigro, & Lavermicocca, 2010; Hurtado et al., 2008; Mourad & Nour-Eddine, 2006; Ruiz-Barba & JiménezDíaz, 1995). It has to be noted that L. pentosus was the most common species detected with a great diversity at strain level. Similarly to these results, L. pentosus has been reported as the most frequently isolated species and found to show a high strain diversity throughout fermentation of green olives (De Bellis et al., 2010), black cv. Conservolea olives (Doulgeraki, Hondrodimou, Iliopoulos, & Panagou, 2012) and brine of green olives (Hurtado et al., 2008). It needs to be noted that L. plantarum was considered to be the dominant LAB species in table olive fermentation (Garrido Fernández et al., 1997). However, when L. pentosus was first described as a new species (Zanoni, Farrow, Phillipps, & Collins,
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355
Fig. 1. Cluster analysis of PFGE ApaI digestion fragments of the lactic acid bacteria isolates recovered from different samples of olives (green and black) and brine calculated by the unweighted average pair grouping method. The distance between the pattern of each strain is indicated by the mean correlation coefficient (r%).
1987), several L. plantarum isolated from the table olive environment have been re-identified as L. pentosus. Additionally, Ln. mesenteroides was detected on green (De Bellis et al., 2010) and black olive fermentation (Ercolini et al., 2006). In
the same studies Ln. pseudomesenteroides was also recovered, while it was the most frequent species detected in the latter study. Furthermore, L. paracasei (De Bellis et al., 2010; Mourad & NourEddine, 2006; Oliveira et al., 2004; Van den Berg et al., 1993) and
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Table 1 Lactic acid bacteria isolated from different olive and brine samples. Species
Source Green olives
Leuconostoc mesenteroides L. pseudomesenteroides Lactobacillus plantarum L. pentosus L. paraplantarum L. paracasei subsp. paracasei
22 1
Total
23
Black olives 20 1 1 21 1 44
Brine
Total
5
20 1 46 72 1 5
78
145
23 50
L. paraplantarum have been previously recovered from olive fermentation (Doulgeraki et al., 2012; Hurtado, Reguant, Bordons, & Rozès, 2009; Hurtado, Reguant, Bordons, & Rozès, 2010; Hurtado et al., 2008). L. casei was found to be the most common species recovered from naturally fermented green Sicilian olives (Randazzo et al., 2004). From Table 1 it can also be inferred that there is greater diversity of LAB in black olives compared to green olives as black olive processing does not include a lye treatment step as in the case of Spanish-style green olives. This observation is in good agreement with a recently published report on the role of lactic acid bacteria in table olive fermentation (Hurtado et al., 2012). The cluster analysis of PFGE ApaI digestion fragments of the LAB isolates showed two major clusters (Fig. 1). The upper cluster contains L. mesenteroides that was recovered almost entirely from black olives. However, no specific information could be provided from the clustering in the second branch which included isolates recovered from brine, green and black olives. The macrorestriction analysis of DNA used for the differentiation of microbiota at strain level was found to be able to distinguish even the L. plantarum from L. pentosus and L. paraplantarum. Similar results have been reported previously (Doulgeraki et al., 2012). PCR-DGGE and PCR-ARDRA were unable to differentiate these species as reported previously for PCR-DGGE (Abriouel et al., 2011; Ercolini et al., 2006), as well as RFLP (Hurtado et al., 2008; Randazzo et al., 2004) and REP-PCR (De Bellis et al., 2010). In conclusion, the above observations reveal that olives are a high diverse ecosystem concerning the biodiversity of LAB, which may affect the quality and/or safety of the final fermented product. To extend the results obtained in this work, future research is necessary to define the technological properties of these strains, such as probiotic potential and bacteriocin production for the selection of certain strains as potential starter cultures in order to enhance the fermentation process and produce consistent and high quality final products. Acknowledgments The research leading to these results has received funding from the EU (FP7/2007e2013), under grant agreement n 243471PROBIOLIVES (www.probiolives.eu). The information in this document reflects only the authors’ views and the Community is not liable for any use that may be made of the information contained therein. References Abriouel, H., Benomar, N., Lucas, R., & Gálvez, A. (2011). Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Alorena green table olives. International Journal of Food Microbiology, 144, 487e496. Asehraou, A., Peres, C., Faid, M., & Brito, D. (2002). Reducing the bloater spoilage incidence in fermented green olives during storage. Grasas y Aceites, 53, 330e334.
Chamkha, M., Sayadia, S., Brub, V., & Godon, J. J. (2008). Microbial diversity in Tunisian olive fermentation brine as evaluated by small subunit rRNA-single strand conformation polymorphism analysis. International Journal of Food Microbiology, 122, 211e215. Cocolin, L., Rantsiou, K., Iacumin, L., Urso, R., Cantoni, C., & Comi, G. (2004). Study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods. Applied and Environmental Microbiology, 70, 1883e1894. De Bellis, P., Valerio, F., Sisto, A., Lonigro, S. L., & Lavermicocca, P. (2010). Probiotic table olives: microbial populations adhering on olives surface in fermentation sets inoculated with the probiotic strain Lactobacillus paracasei IMPC2.1 in an industrial plant. International Journal of Food Microbiology, 140, 6e13. Doulgeraki, A. I., Hondrodimou, O., Iliopoulos, V., & Panagou, E. Z. (2012). Lactic acid bacteria and yeast heterogeneity during aerobic and modified atmosphere packaging storage of natural black Conservolea olives in polyethylene pouches. Food Control, 26, 49e57. Doulgeraki, A. I., Paramithiotis, S., Kagkli, D. M., & Nychas, G.-J. E. (2010). Lactic acid bacteria population dynamics during minced beef storage under aerobic or modified atmosphere packaging conditions. Food Microbiology, 27, 1028e1034. Doulgeraki, A. I., Paramithiotis, S., & Nychas, G.-J. E. (2011). Characterization of the Enterobacteriaceae community that developed during storage of minced beef under aerobic or modified atmosphere packaging conditions. International Journal of Food Microbiology, 145, 77e83. Ercolini, D., Villani, F., Aponte, M., & Mauriello, G. (2006). Fluorescence in situ hybridisation detection of Lactobacillus plantarum group on olives to be used in natural fermentations. International Journal of Food Microbiology, 112, 291e296. Garrido Fernández, A., Fernández Díez, M. J., & Adams, M. R. (1997). Table olives: Production and processing. London: Chapman & Hall. Hurtado, A., Othman, N. B., Hamdi, M., Ferrer, S., Reguant, C., Bordons, A., et al. (2011). Characterization of lactobacillus isolates from fermented olives and their bacteriocin gene profiles. Food Microbiology, 28, 1514e1518. Hurtado, A., Reguant, C., Bordons, A., & Rozès, N. (2009). Influence of fruit ripeness and salt concentration on the microbial processing of Arbequina table olives. Food Microbiology, 26, 827e833. Hurtado, A., Reguant, C., Bordons, A., & Rozès, N. (2010). Evaluation of a single and combined inoculation of a Lactobacillus pentosus starter for processing cv. Arbequina natural green olives. Food Microbiology, 27, 731e740. Hurtado, A., Reguant, C., Bordons, A., & Rozès, N. (2012). Lactic acid bacteria from fermented table olives. Food Microbiology. http://dx.doi.org/10.1016/j.fm.2012. 01.006. Hurtado, A., Reguant, C., Esteve Zarzoso, B., Bordons, A., & Rozès, N. (2008). Microbial population dynamics during the processing of Arbequina table olives. Food Research International, 41, 738e744. International Olive Council (IOC). (2012). World Table Olive Figures. http://www. internationaloliveoil.org/estaticos/view/132-world-table-olive-figures Assessed 24.03.12. Mourad, K., & Nour-Eddine, K. (2006). Microbiological study of naturally fermented Algerian green olives: isolation and identification of lactic acid bacteria and yeasts along with the effects of brine solutions obtained at the end of olive fermentation on Lactobacillus plantarum growth. Grasas y Aceites, 57, 292e300. Oliveira, M., Brito, D., Catulo, L., Leitao, F., Gomes, L., Silva, S., et al. (2004). Biotechnology of olive fermentation of ‘Galega’ Portuguese variety. Grasas y Aceites, 55, 219e226. Panagou, E. Z., Schillinger, U., Franz, C. M. A. P., & Nychas, G.-J. E. (2008). Microbiological and biochemical profile of cv. Conservolea naturally black olives during controlled fermentation with selected strains of lactic acid bacteria. Food Microbiology, 25, 348e358. Randazzo, C. L., Restuccia, C., Daniele Romano, A., & Caggia, C. (2004). Lactobacillus casei, dominant species in naturally fermented Sicilian green olives. International Journal of Food Microbiology, 90, 9e14. Rodas, A. M., Ferrer, S., & Pardo, I. (2003). 16S-ARDRA, a tool for identification of lactic acid bacteria isolated from grape must and wine. Systematic and Applied Microbiology, 26, 412e422. Ruiz-Barba, J. L., & Jiménez-Díaz, R. (1995). Availability of essential B-group vitamins to Lactobacillus plantarum in green olive fermentation brines. Applied and Environmental Microbiology, 61, 1294e1297. Sánchez Gómez, A. H., García García, P., & Rejano Navarro, L. (2006). Elaboration of table olives. Grasas y Aceites, 57, 86e94. Torriani, S., Felis, G. E., & Dellaglio, F. (2001). Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. Applied and Environmental Microbiology, 67, 3450e3454. Van den Berg, D. J. C., Smits, A., Pot, B., Ledeboer, A. M., Kesters, K., Verbakel, J. M. A., et al. (1993). Isolation, screening and identification of lactic acid bacteria from traditional food fermentation processes and culture collections. Food Biotechnology, 7, 189e205. Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T. R., & Zink, R. (2003). Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification. Applied and Environmental Microbiology, 69, 6908e6922. Zanoni, P., Farrow, J. A. E., Phillipps, B. A., & Collins, M. D. (1987). Lactobacillus pentosus (Fred, Peterson, and Anderson) sp nov., nom. rev. International Journal of Systematic Bacteriology, 37, 339e341.
Contents lists available at SciVerse ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Research note
Molecular characterization of lactic acid bacteria isolated from industrially fermented Greek table olives Agapi I. Doulgeraki a, Paraskevi Pramateftaki b, Anthoula A. Argyri b, George-John E. Nychas a, Chrysoula C. Tassou b, Efstathios Z. Panagou a, * a b
Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece Hellenic Agricultural Organisation ‘Demeter’, Institute of Technology of Agricultural Products, Sofokli Venizelou 1, Lycovrissi 14123, Attica, Greece
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2012 Received in revised form 28 June 2012 Accepted 5 July 2012
A total of 145 lactic acid bacteria (LAB) isolates have been recovered from fermented table olives and brine and characterized at strain level with molecular tools. Pulsed-Field Gel Electrophoresis (PFGE) of ApaI macrorestriction fragments was applied for strain differentiation. Species differentiation was based either on Denaturing Gradient Gel Electrophoresis (PCR-DGGE) (black olives) or on restriction analysis of the amplified 16S rRNA gene (PCR-ARDRA) (brine and green olives). Species identification was based on sequence analysis of 16S rRNA gene. When the data were insufficient to resolve the species level of the isolates, specific multiplex PCR assays targeting the recA or tuf genes were employed. From 145 LAB isolates, 71 different strains were recovered from fermented olive and brine samples; 17 strains were assigned to Leuconostoc mesenteroides, 51 were grouped in Lactobacillus plantarum group (including 13 L. plantarum, 37 Lactobacillus pentosus, 1 Lactobacillus paraplantarum), 2 Lactobacillus paracasei subsp. paracasei and 1 Leuconostoc pseudomesenteroides. L. plantarum was recovered mainly from green olive fermentation, whereas in black olives the main species identified were L. pentosus and Ln. mesenteroides). These observations reveal that olives are a highly diverse ecosystem regarding the presence of LAB, which may affect the quality of the final fermented product. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Table olives Fermentation Denaturing Gradient Gel Electrophoresis Pulsed-Field Gel Electrophoresis Multiplex PCR
1. Introduction The cultivation of table olives is widespread throughout the Mediterranean basin and holds a major role not only through its contribution to rural economy but also from the cultural and environmental point of view. The estimated world production of table olives for the period 2011/12 amounted to 2.6 million tonnes approximately and has shown an increasing trend over the last years (IOC, 2012). Today, the market offers a wide variety of table olive elaborations that are able to satisfy all consumer tastes, even the most sophisticated ones. However, there are three basic commercial preparations of table olives that have attracted the interest of the scientific community, namely Spanish style green olives, naturally black olives (Greek style) and black ripe olives (Californian style) for which processing conditions are well established in the literature (Sánchez Gómez, García García, & Rejano Navarro, 2006). A basic step in table olive processing, especially for Spanish style green olives and natural black olives, is
* Corresponding author. Tel.: þ30 210 5294693. E-mail address: [email protected] (E.Z. Panagou). 0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lwt.2012.07.003
fermentation. This process is carried out primarily by the spontaneous microbiota of lactic acid bacteria encountered on olive drupes and in the brine environment aiming at the biotransformation of fermentable substrates (mainly reducing sugars) in olives to organic acids (primarily lactic acid and acetic acid) that are released into the fermentation brine. The net effect is an accumulation of acids and a lowering of brine pH which in combination with the salt content of brine ensures the microbiological safety of the product and at the same time provides the desirable sensory attributes of the final product (Garrido Fernández, Fernández Díez, & Adams, 1997). Moreover, LAB produce small amounts of ethanol and other volatile compounds that make a significant contribution to the final flavor of table olives (for a review on lactic acid bacteria and table olive fermentation see Hurtado, Reguant, Bordons, & Rozès, 2012). It is generally accepted that the table olive industry is highly traditional and even in our days table olive processing remains craft and empirical despite its economic importance. However, new identification and typing techniques for LAB have been continuously developed and implemented to identify the ecological diversity of this microbial group in table olive processing using both culture dependent and independent techniques (Abriouel, Benomar, Lucas, & Gálvez, 2011; Ercolini, Villani, Aponte,
354
A.I. Doulgeraki et al. / LWT - Food Science and Technology 50 (2013) 353e356
& Mauriello, 2006; Hurtado et al., 2011; Hurtado, Reguant, Esteve Zarzoso, Bordons, & Rozès, 2008; Panagou, Schillinger, Franz, & Nychas, 2008; Randazzo, Restuccia, Daniele Romano, & Caggia, 2004). The aim of the present research note is to report on the characterization of LAB isolates from industrially fermented Spanishstyle green olives and natural black olives in Greece using molecular identification techniques. Until now such information came only from small-scale experimental fermentations and there was a lack of LAB characterization from industrial installations. The results obtained in this work will be employed in further research focusing on the investigation of the technological properties of the isolated strains (e.g. probiotic potential, bacteriocin production) for the selection of potential starter cultures with improved characteristics in table olive fermentation. 2. Materials and methods 2.1. Isolation of LAB Brine and olive samples from Spanish-style green olives of cv. Conservolea and Halkidiki, as well as natural black olives from cv. Conservolea and Kalamata were collected from six processing plants in different regions of Greece (Peloponnese, Sterea Ellada). Olive samples were taken directly from fermentation vessels at the end of fermentation process and transported to the laboratory with minimum delay for further analysis. Olive samples (25 g) and brine (1 ml) were aseptically transferred to 225 ml or 9 ml of sterile quarter strength Ringer’s solution, respectively. Decimal dilutions were prepared in the same Ringer’s solution and duplicate 1 ml samples of the appropriate dilutions were mixed on de ManeRogosaeSharp medium (MRS; Merck 1.10660, Darmstadt, Germany), overlaid with 10 ml of the same medium and incubated at 25 C for 48e72 h, to quantify the population of LAB. All plates were examined visually for typical colony types and morphological characteristics that were associated with the growth medium. Moreover, the selectivity of the MRS medium was routinely checked by Gram staining and microscopic examination of smears prepared from randomly selected colonies. LAB were isolated from the highest dilution of MRS growth medium. From each of the aforementioned samplings 10% of the colonies (i.e., 10e15 colonies) were randomly selected and purified. Pure cultures were stored at 80 C in MRS broth medium supplemented with 20% (v/v) glycerol (Serva, Heidelberg, Germany). Before experimental use each isolate was subcultured twice, while the purity of the culture was always checked. A total of 145 pure cultures were finally picked from both olives and brine and subjected to molecular analysis. 2.2. Pulsed Field Gel Electrophoresis (PFGE) Pulsed Field Gel Electrophoresis was performed in order to determine the LAB heterogeneity at strain level. In brief, genomic DNA extraction was performed from all isolates as reported elsewhere (Doulgeraki, Paramithiotis, Kagkli, & Nychas, 2010). The restriction enzyme ApaI (10U) (New England Biolabs, Ipswich, MA, USA) was applied according to the recommendations of the manufacturer. Restriction fragments were separated in 1% PFGE grade agarose gel in 0.5 mM TriseBorate buffer on CHEF-DRII (Bio-Rad, Hercules, CA, USA) equipment with the following running parameters: 6 V cm1, 1 s initial switching time, 10 s final switching time, and 16 h of total run at 14 C. Gels were then stained with ethidium bromide (0.5 mg mL1) in water for 1 h and distained for 2 h before being photographed using a GelDoc system. Conversion, normalization and further analysis were performed using the Pearson
coefficient and UPGMA cluster analysis with Bionumerics software, version 6.1 (Applied Maths, Sint-Martens-Latem, Belgium). 2.3. Identification and characterization of strains Following PFGE differentiation, the different isolates were subjected to PCR-DGGE e(33 isolates from black olives) (Cocolin et al., 2004) and PCR-ARDRA (38 isolates from brine and green olives) (Rodas, Ferrer, & Pardo, 2003), respectively for species differentiation. The identification of a representative number of the different patterns detected in the later methods was achieved by sequence analysis of V1eV6 region of 16S rRNA gene (Doulgeraki et al., 2010). In the case where the acquired data were insufficient to resolve the species level of the bacteria isolates, for the differentiation of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum or Lactobacillus casei and Lactobacillus paracasei, specific multiplex PCR assays targeting the recA gene (Torriani, Felis, & Dellaglio, 2001) or tuf gene (Ventura, Canchaya, Meylan, Klaenhammer, & Zink, 2003) were employed. In all cases, DNA was extracted according to Doulgeraki, Paramithiotis, and Nychas (2011). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences are JX129193 to JX129206. 3. Results and discussion The microbiological analysis of olive samples resulted in average LAB populations of 6.03 (1.03) and 5.73 (0.35) log cfu g1 for black and green olives, respectively, whereas in the brines the respective average values were 6.15 (0.57) and 6.48 (0.83) log cfu ml1. The application of PFGE-ApaI macrorestriction analysis to a total of 145 isolates resulted in 71 different fingerprints (Fig. 1). In total 33 (black olives) and 38 (brine and green olives) different fingerprints were subjected to PCR-DGGE and PCR-ARDRA which resulted in 3 and 2 different patterns, respectively. The results from sequence analysis of a representative number of isolates revealed that the three different patterns detected in PCR-DGGE were assigned to Leuconostoc mesenteroides (17 fingerprints), Leuconostoc pseudomesenteroides (1 fingerprint) and L. plantarum group (15 fingerprints). Additionally, the two patterns detected in PCRARDRA were assigned to L. plantarum group (36 fingerprints) and L. casei group (2 fingerprints). For the differentiation of isolates assigned to L. plantarum group and L. casei group, multiplex PCR assays targeting the recA and tuf genes, respectively were employed, resulting in 13 L. plantarum, 37 L. pentosus, 1 L. paraplantarum and 2 L. paracasei subsp. paracasei. Table 1 summarizes the prevalence of the different genera detected in the different samples. The aforementioned species have been previously associated with the microbiota of spontaneous fermentation of table olives. More accurately, L. plantarum and L. pentosus have been reported as the predominant species in the fermentation of table olives (Abriouel et al., 2011; Asehraou, Peres, Faid, & Brito, 2002; Chamkha, Sayadia, Brub, & Godon, 2008; De Bellis, Valerio, Sisto, Lonigro, & Lavermicocca, 2010; Hurtado et al., 2008; Mourad & Nour-Eddine, 2006; Ruiz-Barba & JiménezDíaz, 1995). It has to be noted that L. pentosus was the most common species detected with a great diversity at strain level. Similarly to these results, L. pentosus has been reported as the most frequently isolated species and found to show a high strain diversity throughout fermentation of green olives (De Bellis et al., 2010), black cv. Conservolea olives (Doulgeraki, Hondrodimou, Iliopoulos, & Panagou, 2012) and brine of green olives (Hurtado et al., 2008). It needs to be noted that L. plantarum was considered to be the dominant LAB species in table olive fermentation (Garrido Fernández et al., 1997). However, when L. pentosus was first described as a new species (Zanoni, Farrow, Phillipps, & Collins,
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355
Fig. 1. Cluster analysis of PFGE ApaI digestion fragments of the lactic acid bacteria isolates recovered from different samples of olives (green and black) and brine calculated by the unweighted average pair grouping method. The distance between the pattern of each strain is indicated by the mean correlation coefficient (r%).
1987), several L. plantarum isolated from the table olive environment have been re-identified as L. pentosus. Additionally, Ln. mesenteroides was detected on green (De Bellis et al., 2010) and black olive fermentation (Ercolini et al., 2006). In
the same studies Ln. pseudomesenteroides was also recovered, while it was the most frequent species detected in the latter study. Furthermore, L. paracasei (De Bellis et al., 2010; Mourad & NourEddine, 2006; Oliveira et al., 2004; Van den Berg et al., 1993) and
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Table 1 Lactic acid bacteria isolated from different olive and brine samples. Species
Source Green olives
Leuconostoc mesenteroides L. pseudomesenteroides Lactobacillus plantarum L. pentosus L. paraplantarum L. paracasei subsp. paracasei
22 1
Total
23
Black olives 20 1 1 21 1 44
Brine
Total
5
20 1 46 72 1 5
78
145
23 50
L. paraplantarum have been previously recovered from olive fermentation (Doulgeraki et al., 2012; Hurtado, Reguant, Bordons, & Rozès, 2009; Hurtado, Reguant, Bordons, & Rozès, 2010; Hurtado et al., 2008). L. casei was found to be the most common species recovered from naturally fermented green Sicilian olives (Randazzo et al., 2004). From Table 1 it can also be inferred that there is greater diversity of LAB in black olives compared to green olives as black olive processing does not include a lye treatment step as in the case of Spanish-style green olives. This observation is in good agreement with a recently published report on the role of lactic acid bacteria in table olive fermentation (Hurtado et al., 2012). The cluster analysis of PFGE ApaI digestion fragments of the LAB isolates showed two major clusters (Fig. 1). The upper cluster contains L. mesenteroides that was recovered almost entirely from black olives. However, no specific information could be provided from the clustering in the second branch which included isolates recovered from brine, green and black olives. The macrorestriction analysis of DNA used for the differentiation of microbiota at strain level was found to be able to distinguish even the L. plantarum from L. pentosus and L. paraplantarum. Similar results have been reported previously (Doulgeraki et al., 2012). PCR-DGGE and PCR-ARDRA were unable to differentiate these species as reported previously for PCR-DGGE (Abriouel et al., 2011; Ercolini et al., 2006), as well as RFLP (Hurtado et al., 2008; Randazzo et al., 2004) and REP-PCR (De Bellis et al., 2010). In conclusion, the above observations reveal that olives are a high diverse ecosystem concerning the biodiversity of LAB, which may affect the quality and/or safety of the final fermented product. To extend the results obtained in this work, future research is necessary to define the technological properties of these strains, such as probiotic potential and bacteriocin production for the selection of certain strains as potential starter cultures in order to enhance the fermentation process and produce consistent and high quality final products. Acknowledgments The research leading to these results has received funding from the EU (FP7/2007e2013), under grant agreement n 243471PROBIOLIVES (www.probiolives.eu). The information in this document reflects only the authors’ views and the Community is not liable for any use that may be made of the information contained therein. References Abriouel, H., Benomar, N., Lucas, R., & Gálvez, A. (2011). Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Alorena green table olives. International Journal of Food Microbiology, 144, 487e496. Asehraou, A., Peres, C., Faid, M., & Brito, D. (2002). Reducing the bloater spoilage incidence in fermented green olives during storage. Grasas y Aceites, 53, 330e334.
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