Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Aloreña green table olives

International Journal of Food Microbiology 144 (2011) 487–496

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Aloreña green table olives Hikmate Abriouel, Nabil Benomar, Rosario Lucas, Antonio Gálvez ⁎ Área de Microbiología, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaen, 23071-Jaen, Spain

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Article history: Received 30 June 2010 Received in revised form 4 November 2010 Accepted 4 November 2010 Keywords: Table olives Spontaneous fermentation Bacteria Yeasts and molds Archaea Culture-independent analysis PCR-DGGE

a b s t r a c t Aloreña table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The present study focused on Aloreña table olives manufactured by small and medium enterprises (SMEs) from Valle del Guadalhorce (Southern Spain) under three different conditions (cold storage, and ambient temperature fermentations in small vats and in large fermentation tanks). The microbial load of brines during fermentation was studied by plate counting, and the microbial diversity was determined by a culture-independent approach based on PCR-DGGE analysis. The viable microbial populations (total mesophilic counts, yeasts and molds, and lactic acid bacteria — LAB) changed in cell numbers during the course of fermentation. Great differences were also observed between cold, vat and tank fermentations and also from one SME to another. Yeasts seemed to be the predominant populations in cold-fermented olives, while LAB counts increased towards the end of vat and tank fermentations at ambient temperature. According to PCR-DGGE analysis, microbial populations in coldfermented olives were composed mostly by Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. together with halophilic archaea (mainly by haloarchaeon/Halosarcina pallida and uncultured archaeon/uncultured haloarchaeon/Halorubrum orientalis) and yeasts (Saccharomyces cerevisiae and Candida cf. apicola). Vat-fermented olives stored at ambient temperature included a more diverse bacterial population: Gordonia sp./Pseudomonas sp., Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. and Thalassomonas agarivorans together with halophilic archaea and yeasts (mainly S. cerevisiae and C. cf. apicola, but also Pichia sp., and Pichia manshurica/Pichia galeiformis). Some LAB were detected towards the end of vat fermentations, including Lactobacillus pentosus/Lactobacillus plantarum and Lactobacillus vaccinostercus/Lactobacillus suebicus. Only the tank fermentation showed a clear predominance of LAB populations (Lactobacillus sp., Lactobacillus paracollinoides, and Pediococcus sp.) together with some halophilic archaea and a more selected yeast population (P. manshurica/P. galeiformis). The present study illustrates the complexity of the microbial populations in naturally-fermented Aloreña table olives. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Mediterranean culture has been focused for many centuries on the extraction of olive oil and the elaboration of table olives, thus olive farming has been the most robust agricultural practice in Mediterranean countries such as Spain, Italy and Greece. Many different industries manufacture table olives according to the same protocol (known as “Spanish style”) by the transformation of the bitter and unpalatable olive fruit into a stabilised fermented product. Such transformation is achieved by a debittering step before brining by treatment with NaOH. The fruits are then put in brines (6–8% w/v NaCl) where the olives ferment. The fermentation process could be for 3 to 7 months, after which olives are packaged in new acidified brine ⁎ Corresponding author. Área de Microbiología, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus Las Lagunillas s/n. 23071-Jaén, Spain. Tel.: + 34 953 212160; fax: + 34 953 212943. E-mail address: [email protected] (A. Gálvez). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.11.006

and sold. In general, green table olive fermentation is a natural process in which the spontaneous fermentation results from the competitive activities of the indigenous microbiota (lactic acid bacteria and yeasts) together with a variety of contaminating microorganisms from different sources. This spontaneous fermentation starts as soon as olives are put in brines (Garrido-Fernández et al., 1997). Many investigations have been carried out on table olives over the past 100 years, most of them dealing with green table olives (García García et al., 1992; Durán Quintana et al., 1999; Panagou et al., 2003; RuizBarba et al., 1994; Spyropoulou et al., 2001). However, many aspects of traditional preparations are unexplored and need to be improved with the aim to achieve a better socio-economic development and to open new international market for unknown local products with a high nutritional and safety values. Aloreña table olives are a typical product of “Guadalhorce” region in Malaga (Spain). This variety of olives has peculiar characteristics which are linked with the area (climate, pedology and geographical location) and make them different from other green table olives.

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Aloreña table olives or “naturally-fermented olives” are characterized by their green–yellow colour, an excellent quality, a good flesh-tostone ratio, and taste. Furthermore, the manufacturing process of this type of olives does not include any treatment with NaOH (Garrido-Fernández et al., 1997) as debittering step because this variety of olives is characterized by a low proportion of oleuropein (the main bitter glucoside of olives). Removal of the remaining oleuropein is done only by repeated washing treatments with water. Aloreña table olives are also frequently seasoned with fennel, thyme, garlic and pepper, making them rich in aroma. In spite of the importance of Aloreña table olives, their microbiology is not yet investigated and the microorganisms implicated in this fermentation are unknown until the present. The manufacturing processes of Aloreña table olives are carried out by small and medium enterprises (SMEs) from Guadalhorce valley (Malaga, Spain), and currently have several unsolved problems derived from the lack of suitable scientific and technological knowledge about the microbiota present. After harvest, olives are stored in brines either in the cold or at ambient temperature in the presence of 4–10% NaCl. Cold-stored olives undergo very slow fermentation, resulting in a final high sugar content which derives in abundant gas formation during distribution and the consequent softening and browning of the fruits. This natural, traditional fermentation process has not been studied yet at microbiological level. The aim of the study presented here was to assess and compare the diversity of bacteria, archaea and yeasts and molds in different olive fermentations by a culture-independent molecular approach based on PCR-DGGE. The further identification of the predominant species by DNA sequencing will allow a more precise estimation of the distribution of different organisms in Aloreña table olives.

2.2. Microbiological analyses of culturable microbiota

2. Material and methods

2.3.2. PCR amplification of the microbial community For the Bacteria domain, Denaturing Gradient Gel Electrophoresis (DGGE) samples were prepared by two successive PCR amplifications (nested PCR) by using the primer pairs described by Ogier et al. (2002). First, a 700-bp fragment of the 16S rRNA gene including the V3 region was amplified by using WO1 and WO12 primers (Ogier et al., 2002). Second, the 700-bp fragment was used to amplify the V3 region with GC-clamp HDA1 and HDA2 (Ogier et al., 2002). For the Archaea domain, DNA fragments encoding 16S rRNAs amplified by using the primers (Arch344F with the GC clamp and Arch915R) and PCR conditions described by Casamayor et al. (2000). For the Bacteria and Archaea domains, PCR amplification was performed with 50 μl mixtures containing 2 μl of template DNA, 1× Taq buffer (Invitrogen), each deoxynucleoside triphosphate at a concentration of 250 μM, 25 pmol of each primer, 2.5 U of native Taq DNA polymerase (Invitrogen). For yeast and molds, the D1/D2 domain of the 26S rDNA was amplified by a two-step, nested PCR using NL1 and NL4 primers in the first PCR, and the GC-clamp NL1 and LS2 primers in the second PCR as described by Cocolin et al. (2000). PCR products of different amplifications procedures were analyzed by electrophoresis in 2% (wt/vol) agarose gels.

2.1. Olive brine samples The samples analyzed in this study were obtained from olive fermentations carried out by four different SMEs of Guadalhorce valley in Malaga (Spain). Olives were harvested by hand and put immediately in brines. Three different manufacturing processes were employed by the local industries: A) Green olives were cracked, placed in 150-liter vats filled with brine (9–11%) brine and stored at 10 °C for several months. After cold fermentation, olives were washed with tap water and packaged in 5% brine with added 1% citric acid, and sold (SMEs 1, 2 and 3). These cold-fermented table olives are distributed locally and are highly prone to gas production in package due to the high amounts of residual sugars. B) Vat-fermented olives (SMEs 1 to 4). Green olives were placed in 150-liter vats filled with tap water, added with salt 8–10% (wt/ vol) and allowed to ferment at ambient temperature for three to six months. C) Tank-fermented olives. Green olives were placed in 6000-liter glass fibre tanks added with salt (6% wt/vol) and 0.8% (vol/vol) of acetic acid to promote the beginning of fermentation. Olives were allowed to ferment for 4 to 7 months. While cold fermentation (A) and vat fermentation (B) processes were carried out by all enterprises, only SME1 made tank-fermented olives. Sampling of the brines was performed in depth from two vats under aseptic conditions (one sample per vat per time point) during 6 months of fermentation in brine. Two vats from each SME were sampled at each time and combined to yield a single point sample. Exceptions were the large fermentation tanks, which were sampled and analyzed in duplicate. The pH of the brine samples was monitored through the fermentation process.

Olive brine samples serially diluted in sterile saline solution and plated in triplicate on the following media: TSA (Scharlab, Barcelona, Spain) for estimation of total aerobic mesophilic bacteria; YMA (Scharlab) supplemented with chloramphenicol (150 mg/l; Sigma) for yeasts and molds, and MRS agar (Scharlab) supplemented with 0.4 g/l sodium azide (Sigma, Madrid) for lactic acid bacteria (LAB). Counts were obtained after 48 h and 5 days of incubation at 30 °C. Results were calculated as the means of three determinations. 2.3. Culture independent study of microbial populations during fermentation 2.3.1. DNA isolation Total genomic DNA was isolated from fermented olive brine samples corresponding to different enterprises and fermentation steps. Samples (5 ml each) were centrifuged for 10 min at 13,000 rpm in a microcentrifuge. The cell pellets were kept overnight at − 20 °C until use. Total DNA was extracted with guanidium thiocyanate as described previously by Abriouel et al. (2006). The resulting DNA solution was stored at − 20 °C. For DNA preparation from pure cultures, total DNA was extracted from overnight cultures by the method of De los Reyes-Gavilan et al. (1992). In both cases, the resulting DNA solution was treated with RNase (50 μg/ml) and incubated for 15 min at 37 °C. The quality of the DNA recovered was routinely checked on agarose gels. DNA concentration and quality were assessed by measuring optical density at 260 and 280 nm with a SmartSpec™ spectrophotometer (BioRad).

2.3.3. DGGE analysis The GC-clamp PCR products (40 μl) obtained from 16S rRNAs region (Bacteria and Archaea domains) and D1/D2 domain (yeasts and molds) amplification as described above were subjected to DGGE analysis by using the Dcode universal mutation detection system (Bio-Rad, Richmond, Ca) on 8% polyacrylamide gels as described elsewhere (Abriouel et al., 2006). DNA sequencing and analysis of sequence data were carried out as described elsewhere (Abriouel et al., 2006). DGGE fragments were excised and the DNA was eluted in 20 μl of sterile water 2 h at 4 °C. One microliter of the eluted DNA of each DGGE band was reamplified using the same primers without GC-clamp (HDA1 and HDA2 for Bacteria, Arch340F and Arch915R

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for Archaea, NL1 and LS2 for yeasts and molds). DNA sequencing was carried out on a CEQ 2000 XL DNA Analysis System (Beckman Coulter, CA). A search for homology of the DNA sequence was done using the BLAST algorithm available at the National Center for Biotechnology Information (NCBI, USA). Identity values with closest relatives were 100% in all cases. New DNA sequences were deposited in the database of the European Molecular Biology Library (EMBL) under the following accession numbers FR692003 to FR692017 (for Bacteria), FR692018 to FR69020 (for Archaea) and FR692023 (for yeasts and molds). 3. Results 3.1. Monitoring of microbial count changes during fermentation Microbial counts during fermentation of olive samples changed greatly depending on the fermentation time, temperature and SMEs (Fig. 1). For the cold-fermented olives, total viable mesophilic counts and yeasts and molds reached their maximum (3–5 log10 units) after 1– 2 months fermentation at 10 °C in for the three SMEs (Fig. 1A to C). Viable counts decreased in most samples by the end of fermentation, especially for the yeasts and molds. LAB did not seem to proliferate in the cold-stored olives, and were detected at very low numbers after 2 months fermentation in only two SMEs (SME 1 and SME 2). Samples from olives fermented in vats at ambient temperature exhibited great variation in viable cell counts between the four SMEs sampled (Fig. 1E to H). Yeasts and molds reached highest viable counts within 1–2 months of storage and decreased afterwards. It could also be noticed that total mesophilic counts and yeasts and molds tended to be high for the whole storage period in SMEs 2 and 4 (with some exceptions at month 3) while SMEs 1 and 3 showed much lower and variable counts. LAB were detected, in general, towards the end of the fermentation period (months 2 to 6). The lowest counts of LAB were reported for SMEs 1 and 3, while SME 4 reached the maximum (up to 5.6 log10 units). In the tank fermentation, total viable mesophilic counts remained very high from the beginning (6.2 to 7 log10 units) and decreased at month 6 (Fig. 1D). The population of yeasts and molds was also high at the beginning of fermentation, but rapidly decreased within the first month. This was followed by a gradual increase in counts (peaking at month 3 with 6.86 log10 units) and a final decrease. LAB were detected from month 1 till the end of fermentation, and in this period they followed a similar trend as yeasts and molds, peaking at month 3 with 5.28 log10 units. Regarding the pH profile of the different olive samples, greatest changes were monitored in tank fermented olives (SME1) with the large pH drop from 5.2 to 3.9 after six months fermentation in parallel to the development of LAB (Fig. 1D). For the vat and cold-fermented samples, final pH values were slightly higher in the intervals of pH 4.2 to 4.6 (Fig. 1). 3.2. Monitoring of changes in microbial diversity during fermentation of Aloreña table olives PCR-DGGE analysis of olive brines from the different enterprises revealed substantial differences in microbial composition that depended mostly on the fermentation method (Table 1, Fig. 2). Small differences were also detected between the different SMEs for the same fermentation method. In most samples, DNA corresponding to a variety of bacteria, together with archaea and yeasts as well as some molds was detected. 3.2.1. Cold-fermented olives Three SMEs (SME1, SME2, and SME3) were sampled for coldfermented olives. The DGGE profiles for the domain Bacteria from

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cold-fermented olives revealed that Gordonia sp./Pseudomonas sp. and Sphingomonas sp. were the predominant bacteria from the beginning to the end of the cold-storage period for most of the samples, especially from SMEs 1 and 2 (Table 1). Other DNA bands corresponding to bacteria such as Sphingomonas sp./Sphingobium sp./Sphingopyxis sp., Pantoea agglomerans, Enterobacter sp., and uncultured cyanobacteria or halophilic bacteria were detected sporadically. Listeria monocytogenes/Listeria innocua was also detected in one of the samples from SME3. Surprisingly, lactic acid bacteria were not detected in the cold fermentation olive brines, except for the uncultured Weissella sp. detected in one of the samples from SME3. Several bands corresponding to archaea (uncultured archaeon/ uncultured haloarchaeon/Halorubrum sp., as well as uncultured archaeon/uncultured Haloarchaeon/Halorubrum orientalis) were detected in many of the samples from the three SMEs (Table 2). Archaea were detected more frequently at the end of the cold-storage period, being detected in samples from two SMEs after three months of storage and in samples from the three SMEs after six months of storage. The yeast species Saccharomyces cerevisiae and Candida cf. apicola (C. apicola) were found in samples from the three SMEs at the beginning of the cold-storage period, and were also detected as the predominant yeast species in samples from SME2 till the end of sampling period (Table 3). However, they were detected much more randomly in the other two SMEs. Pichia sp. was also detected in samples from SMEs 1 and 2, and Cryptococcus macerans was detected at the end of fermentation in SME3. 3.2.2. Vat-fermented samples Samples from olives fermented in vats showed greatest differences in bacterial composition between the four SMEs studied (Table 1). Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./ Sphingopyxis sp. were detected most frequently in samples from SME1 for most of the fermentation period. Thalassomonas agarivorans was also detected very frequently, for samples from SMEs 1 and 4. DNA bands corresponding to the lactic acid bacteria (LAB) group were detected in samples from SME 2 (Lactococcus lactis/Lactobacillus coryniformis) and SME 3 (Lactobacillus pentosus/Lactobacillus plantarum) at months 3 and 6 of fermentation. In SME4, LAB were detected after month 2 of fermentation (Lactobacillus pentosus/Lactobacillus plantarum and Lactobacillus vaccinostercus/Lactobacillus suebicus). The profiles of DNA bands corresponding to archaea of vat fermented samples did not differ greatly from the cold fermentations. The uncultured archaeon/uncultured haloarchaeon/Halorubrum sp., as well as uncultured archaeon/uncultured Haloarchaeon/Halorubrum orientalis were detected in almost all samples within the first three months of sampling, but disappeared in samples taken at month 3 in all cases (Table 2). One sample (SME2) showed additional bands that corresponded to an uncultured haloarchaeon/Haloarchaeon. Bands corresponding to archaea were detected again at the end of sampling (month 6) only in SMEs 2 and 4. The yeasts species S. cerevisiae and C. apicola were present from the beginning in vat samples from SMEs 1, 2, and 4 (Table 3) and were also detected in most of the samples taken from the same SMEs from the beginning to the end of sampling period. Pichia manshurica/Pichia galeiformis were detected in most samples from SME 2 (instead of S. cerevisiae), while Pichia sp. was found in most samples from SME 4. Samples from SME 3 showed a completely different profile, with only a single species (Zygosaccharomyces mrakii) being detected at 0 and 2 months of fermentation. 3.2.3. Tank-fermented olives This type of fermentation was carried out only by SME 1. At the beginning, the only DNA bands for bacteria detected corresponded to Vibrio sp., but after one month the bacterial community changed

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Fig. 1. Changes in microbial counts and pH of Aloreña table olive brines from different SMEs during fermentation under cold storage (A, B, C), in tank (D) or in vats at room temperature (E, F, G, H). Total aerobic mesophilic bacteria, yeasts and molds and lactic acid bacteria were determined.

completely to the predominance of lactic acid bacteria (LAB) such as Lactobacillus sp. and Pediococcus sp., though one sample also revealed a T. agarivorans band (Table 1). The bacterial diversity profile

remained stable from months 2 to month 6 of fermentation, with predominating bands that corresponded to Lactobacillus sp., Lactobacillus paracollinoides, and Pediococcus sp.

Table 1 Bacteria detected along the different fermentations of Aloreña table olives by culture-independent methods. Sample

Cold fermentation SME1

Time of fermentation (months) 1 (November)

2 (December)

3 (January)

6 (April)

Gordonia sp./Pseudomonas sp. Sphingomonas sp. Uncultured bacterium

Gordonia sp./Pseudomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Gordonia sp./Pseudomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Gordonia sp./Pseudomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Gordonia sp./Pseudomonas sp. Uncultured bacterium Enterobacter agglomerans Pantoea sp. Pantoea agglomerans Uncultured Sphingomonas Sp. Listeria monocytogenes/Listeria innocua Uncultured bacterium Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Gordonia sp./Pseudomonas sp. Sphingomonas sp.

Gordonia sp./Pseudomonas sp. Uncultured bacterium Uncultured Enterobacter sp. Uncultured bacterium

Uncultured bacterium Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. Uncultured cyanobacterium Uncultured halophilic bacterium

Uncultured bacterium Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Uncultured bacterium Gordonia sp./Pseudomonas sp. Uncultured Sphingomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Gordonia sp./Pseudomonas sp. Thalassomonas agarivorans Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Lactococcus lactis/ Lactobacillus coryniformis

Lactococcus lactis/ Lactobacillus coryniformis Erythrobacter sp. Anoxybacillus sp. Uncultured bacterium Lactobacillus pentosus/ Lactobacillus plantarum Lactobacillus vaccinostercus/ Lactobacillus suebicus Lactobacillus plantarum Uncultured bacterium Shewanella sp.

SME2

Gordonia sp./Pseudomonas sp. Uncultured bacterium Uncultured Sphingomonas sp.

Gordonia sp./Pseudomonas sp. Sphingomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. Gordonia sp./Pseudomonas sp. Uncultured bacterium Uncultured Sphingomonas sp.

SME3

Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Uncultured Weissella sp. Gordonia sp./Pseudomonas sp.

Gordonia sp./Pseudomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Gordonia sp./Pseudomonas sp. Uncultured Sphingomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. Thalassomonas agarivorans

SME2

Gordonia sp./Pseudomonas sp. Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.

Thalassomonas agarivorans

SME3

Gordonia sp./Pseudomonas sp. Sphingomonas sp. Uncultured bacterium Pseudomonas sp. ncultured Thalassomonas Thalassomonas agarivorans Uncultured bacterium

–

Vat fermentation SME1

SME4

Tank fermentation SME1 Vibrio sp.

Enterococcus sp. Pseudomonas sp./Pseudomonas fluorescens/Pseudomonas orientalis Thalassomonas agarivorans Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. Thalassomonas agarivorans

Sphingomonas sp. Thalassomonas agarivorans Uncultured bacterium

Gordonia sp./Pseudomonas sp. Sphingomonas sp. Lactobacillus plantarum Thalassomonas agarivorans Uncultured bacterium

Lactobacillus pentosus/ Lactobacillus plantarum Lactobacillus vaccinostercus/ Lactobacillus suebicus Uncultured bacterium Shewanella sp.

Lactobacillus sp./Pediococcus sp. Pediococcus sp. Thalassomonas agarivorans

Lactobacillus sp./Pediococcus sp. Lactobacillus paracollinoides Pediococcus sp.

Lactobacillus sp. Lactobacillus paracollinoides Pediococcus sp.

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0 (October)

Lactobacillus sp. Lactobacillus paracollinoides Pediococcus sp.

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Fig. 2. Illustrative DGGE profiles of Bacteria (A), Archaea (B) and yeasts and molds (C) from Aloreña table olives. The letters on lanes indicate fermentation conditions: a, cold fermentation; b, vat fermentation; c, tank fermentation. Band numbers in A: 1 and 2, Gordonia sp./Pseudomonas sp.; 3, Sphingomonas sp.; 4, uncultured bacterium; 5, Enterobacter agglomerans; 6, Pantoea sp.; 7, Pantoea agglomerans; 8, Enterococcus sp.; 9, Pseudomonas sp./Pseudomonas fluorescens/Pseudomonas orientalis; 10, Thalassomonas agarivorans; 11, Sphingomonas sp./Sphingobium sp./Sphingopyxis sp.; 12 to 14, Lactobacillus pentosus/Lactobacillus plantarum; 15, Lactobacillus sp.; 16, Lactobacillus paracollinoides; 17, Pediococcus sp. Band numbers in B: 1, Halobacteriaceae archaeon/Halosarcina pallida; 2, uncultured archaeon; 3 and 4, uncultured archaeon/uncultured haloarchaeon/Halorubrum orientalis; 5 to 7, uncultured haloarchaeon/haloarchaeon. Band numbers in C: 1 and 4, Candida cf. apicola; 2, Saccharomyces cerevisiae; 3, Pichia sp.; 5 to 8, Zygosaccharomyces mrakii; 9, 11 and 12, Aureobasidium pullulans/Kabatiella microsticta/Discosphaerina fagi; 10, Exophiala alcalophila.

The presence of archaea was more limited in the tank-fermented olives. Positive bands were identical to the vat and cold fermentations, but disappeared after month 3 in the two fermenters sampled (Table 2). The profiles of yeasts and molds for tank-fermented olives differed greatly from cold or vat-fermented samples. At the beginning, the predominant species in the tanks corresponded to filamentous fungi such as Aureobasidium pullulans/Kabatiella microsticta/Discosphaerina fagi and Exophiala alcalophila (Table 3). However this profile became much more homogeneous for the remaining fermentation period, in which P. manshurica/P. galeiformis was the main yeast species detected. 4. Discussion Naturally-fermented Aloreña table olive brines contain different groups of microorganisms at the beginning of fermentation: bacteria, archaea and yeasts and molds as revealed by DGGE analysis. Previous studies have shown that the natural fermentation of green table olives involves lactic acid bacteria and yeasts, while some Gram-negative species may also be found, especially at the beginning. Relatively low viable counts of lactic acid bacteria (2–3 log10 CFU/ml), Enterobacteriaceae (2–3 log10 CFU/ml), yeasts (2–3 log10 CFU/ml) and Pseudomonas sp. (1–3 log10 CFU/ml) have been reported previously in Conservolea, Manzanilla, Gemlik and Edincik green and black table olives from Spain and Turkey (Borcakli et al., 1993; Durán Quintana et al., 1999; Nychas et al., 2002; Özay and Borcakli, 1996; Ruiz-Barba et al., 1994). However, Aloreña table olive counts obtained in this study were different from those reported above and were highly dependent on the SME and the fermentation conditions. In this case,

the fermentation seemed to be carried out mainly by yeasts present during all the fermentation process for all samples, except for the olives fermented in tank (SME1) which were previously acidified with acetic acid. This acidification may have favoured the fermentation by LAB although this fermentation was also carried out by yeasts in cooperation with LAB. The LAB found in Aloraña table olives by culture-independent analysis, especially those belonging to the genus Lactobacillus, both homofermentative (L. plantarum and L. pentosus) and heterofermentative (L. paracollinoides) differed from the usual bacterial profile detected in other green table olive fermentations in which L. plantarum is the predominant microorganism (Garrido-Fernández et al., 1997). In our study L. plantarum was only detected in some olive brines fermented at room temperature, in which it prevailed till the end of fermentation or was replaced by L. vaccinostercus/L. suebicus. L. paracollinoides (considered to be a spoilage bacterium in brewery) together with pediococci were also detected in the tank fermentations, indicating its greater complexity as far as LAB are concerned. By contrast, the low numbers or complete absence of LAB in olives stored at cold temperature could be explained by their lower growth capacity at this temperature and the use of available sugars by competing microorganisms. Bacterial species such as Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./Sphingopyxis were detected by PCR-DGGE in all samples fermented at room and cold temperature except in the tank fermentations. Although PCR-DGGE has emerged as one of the more promising culture-independent molecular methods for profiling the microbial ecology of food and beverage ecosystems (Ampe et al., 1999; Cocolin et al., 2002; Ercolini et al., 2001; Giraffa and Neviani, 2001), it has a limited capacity to discriminate between species with highly similar 16S rDNA sequences as shown in the present study. Other targets (such as rpoB gene) have been proposed for PCR-DGGE community analysis in order to avoid some of the limitations of 16S rDNA target (Dahllöf et al., 2000). Recently, barcoded pyrosequencing has emerged as an alternative approach for culture-independent analysis with a higher sensitivity of detection but with a resolution power only at genus level (Liu et al., 2008; Humblot and Guyot, 2009; Roh et al., 2009). At present, it seems that more studies are required to elucidate the advantages/disadvantages/ complementarity of PCR-DGGE and barcoded pyrosequencing for culture-independent analysis of microbial communities in food systems. The presence of Pseudomonas sp. in olive brines was confirmed by real-time PCR with genus-specific primers (data not shown). Pseudomonas spp. has been detected previously on the surface of unfermented black olives (Ercolini et al., 2006) and also in brines from early fermentation of naturally black olives in which they were suppressed as the LAB and yeast populations increased (Nychas et al., 2002). The development of proteolytic microorganisms like Pseudomonas spp. in table olives, followed by decarboxylation and deamination of the resulting amino acids by heterofermentative lactobacilli could cause an unusual type of spoilage characterized by a decrease in the acidity of brines and swelling (Harmon et al., 1987), and could also lead to biogenic amine formation. However, the presence of Pseudomonas and Sphingomonas sp./Sphingobium sp./Sphingopyxis through the whole fermentation process of olives has never been reported before. These aerobic bacteria have been detected in several other microaerophilic or anaerobic environments such as anaerobic digestion of cassava effluents (Carbone et al., 2002) or submerged biofilters for olive mill wastewater treatment (Pozo et al., 2007) to cite some examples. Both Sphingomonas spp. and Sphingobium sp. can reduce nitrates (Liang and Lloyd-Jones, 2010; Patureau et al., 2000). Anaerobic growth of Pseudomonas requires the presence of the alternative electron acceptors NO3, NO2 or arginine for substrate-level phosphorylation (Schreiber et al., 2007; Vander Wauven et al., 1984). P. aeruginosa also performs pyruvate fermentation, which is essential for long-term anaerobic survival (Eschbach et al.,

Table 2 Archaea detected along the different fermentations of Aloreña table olives by culture-independent methods. Samples

Time of fermentation (months) 0 (October)

2 (December)

3 (January)

6 (April)

–

–

BC SME2

Haloarchaeon/Halosarcina pallida Uncultured archaeon

–

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/ Halorubrum orientalis Uncultured archaeon/Uncultured Haloarchaeon/ Halorubrum orientalis

SME3

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

–

Haloarchaeon/Halosarcina pallida Uncultured archaeon Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis Uncultured haloarchaeon/Haloarchaeon Haloarchaeon/Halosarcina pallida Uncultured archaeon

–

Uncultured archaeon/Uncultured Haloarchaeon/ Halorubrum orientalis

–

Uncultured archaeon/Uncultured Haloarchaeon/ Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

–

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/ Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

–

–

Vat fermentation SME2 Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

SME3

Haloarchaeon/Halosarcina pallida Uncultured archaeon

SME4

Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

Tank fermentation SME1 Haloarchaeon/Halosarcina pallida Uncultured archaeon Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis Haloarchaeon/Halosarcina pallida Uncultured archaeon Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

Haloarchaeon/Halosarcina pallida Uncultured archaeon Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis –

Haloarchaeon/Halosarcina pallida Uncultured archaeon/Uncultured Haloarchaeon/ Halorubrum orientalis

H. Abriouel et al. / International Journal of Food Microbiology 144 (2011) 487–496

1 (November)

Cold fermentation SME1 Uncultured archaeon/Uncultured Haloarchaeon/Halorubrum orientalis

493

SME3 SME4

SME2

Pichia manshurica/Pichia galeiformis Pichia manshurica/Pichia galeiformis Pichia manshurica/Pichia galeiformis Pichia manshurica/Pichia galeiformis Tank fermentation SME1 Aureobasidium pullulans/ Kabatiella microsticta/Discosphaerina fagi Exophiala alcalophila

– –

Pichia manshurica/Pichia galeiformis Candida cf. apicola Zygosaccharomyces mrakii Saccharomyces cerevisiae Pichia sp.

–

Saccharomyces cerevisiae Candida cf. apicola Pichia manshurica/Pichia galeiformis Candida cf. apicola – Saccharomyces cerevisiae Candida cf. apicola Pichia sp. – –

Saccharomyces cerevisiae Candida cf. apicola Pichia manshurica/Pichia galeiformis Candida cf. apicola – Saccharomyces cerevisiae Candida cf. apicola Pichia sp. Saccharomyces cerevisiae Candida cf. apicola Pichia manshurica/Pichia galeiformis Candida cf. apicola Zygosaccharomyces mrakii Saccharomyces cerevisiae Vat fermentation SME1

– Saccharomyces cerevisiae Candida cf. apicola SME3

SME2

Saccharomyces cerevisiae Candida cf. apicola Pichia sp. Saccharomyces cerevisiae Candida cf. apicola

–

Saccharomyces cerevisiae Candida cf. apicola

–

Saccharomyces cerevisiae Candida cf. apicola Pichia sp Saccharomyces cerevisiae Candida cf. apicola

3 (January) 2 (December) 1 (November)

Saccharomyces cerevisiae Candida cf. apicola Pichia sp. Saccharomyces cerevisiae Candida cf. apicola Cold fermentation SME1 Saccharomyces cerevisiae Candida cf. apicola

0 (October)

Time of fermentation (months) Sample

Table 3 Yeasts and molds detected along the different fermentations of Aloreña table olives by culture-independent methods.

Saccharomyces cerevisiae Candida cf. apicola Pichia sp. Saccharomyces cerevisiae Candida cf. apicola Cryptococcus macerans –

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6 (April)

494

2004). The mechanisms accounting for the presence of these bacterial genera in table olive fermentations should be further investigated. The presence of T. agarivorans (non-fermentative Gram-negative marine agarolytic bacterium from coastal water) was probably due to the use of marine salt used in brines. This bacterium only was detected in brines stored at room temperature, since optimal growth temperature is about 25 °C in the presence of salt (Jean et al., 2006). Potentially pathogenic bacteria such as Vibrio sp. and L. monocytogenes/L. innocua were also detected in two olive samples. Vibrio sp. was only detected at the beginning of fermentation. However, the acidic pH and anaerobic conditions of table olive fermentation do not favour the survival/proliferation of this accidental contaminant acclimated to grow under aerobic conditions and neutral to slightly alkaline pH. In contrast, a previous study reported the presence of Listeria spp. in green table olives prepared by different methods (Caggia et al., 2004), demonstrating that the product can support survival of Listeria, despite its low pH, low aw, and high salt concentration. Being a psychrotrophic bacterium, Listeria could find some advantage for survival/proliferation in cold-fermented olives like in the present study. Although there are no previous reports on listeriosis outbreaks associated to consumption of table olives, the incidence of this bacterium in Aloreña table olives (which are consumed without pasteurisation) should be further investigated. An unusual finding of the present study was the detection of archaea in brines for the different types of fermented olives, at least at some points during the fermentation. The presence of slightly extreme conditions like high salt concentration (10%) and low pH could allow the prevalence of halophilic archaea. DGGE analysis of archaea indicates the dominance of Halorubrum sp./uncultured haloarchaeon. This is the first culture-independent study of archaea in table olives. The disparate copies of rRNA genes found in single genomes among the archaea may lead to an overestimate of diversity (Boucher et al., 2004; Mylvaganam and Dennis, 1992). However, only one archaeon type was detected in all samples and through the fermentation process. The absence of diversity changes in archaea along the sampling period suggests that Halorubrum sp. (which exhibits little capacity for fermentation) did not contribute to olive fermentation and their presence is only an artefact of salt product used in brine preparation, as observed for T. agarivorans. Salt products that contain high number of halophilic archaea can decrease the quality of foods such as cheese, olives, tomato paste, meat and fish, due to the production of gelatinase, lipase and proteases (Birbir et al., 2004; Elevi et al., 2004). The diversity of yeast and mold species associated with brines during fermentation of Aloreña table olives was determined by sequencing of the D1/D2 of the large-subunit 26S ribosomal DNA widely accepted as a standard procedure for yeast identification (Kurtzman and Robnett, 1998; Scorzetti et al., 2002). The results obtained by DGGE analysis showed that S. cerevisiae, C. apicola and Pichia sp. (P. manshurica/P. galeiformis, and Pichia sp.) were generally the main yeast species present through the fermentation processes regardless of the storage temperature and SME carrying out the fermentation procedure. The same composition was reported with culture-dependent methods by Durán Quintana et al. (2005), who found Pichia anomala, Pichia membranaefaciens, Pichia minuta, S. cerevisiae, Candida diddensii, Candida famata, and Debaryomyces hansenii as the main yeast species from various types of table olives stored at low temperature (7 °C). In the present study, other yeasts such as Z. mrakii and Cryptococcus macerans were detected in vat and cold-stored olives respectively. Also, some filamentous fungi were detected at the beginning of tank fermentation (like A. pullulans/K. microsticta/D. fagi and E. alcalophila), most probably from epiphytic microflora or environment. However, the tank fermentation showed a much more homogeneous yeast population, and only Pichia sp. prevailed until the end of fermentation. This selection was most probably due to the added acetic acid and by the higher decrease of pH

H. Abriouel et al. / International Journal of Food Microbiology 144 (2011) 487–496

(reaching a final value of 3.9). These results agree with those reported by Durán Quintana et al. (2005). S. cerevisiae was also inhibited by lactic acid produced as end product of lactic acid fermentation (Narendranath et al., 2001a,b; Thomas et al., 2002) as occurred in tank fermented samples. The occurrence and the significance of these yeasts in green olive brines during fermentation have been reported previously by González Cancho (1965). Yeasts present in vegetable fermentation play a beneficial role, which is important in terms of lactic acid bacteria nutritional requirements towards vitamins produced by yeasts as in the case of L. plantarum (Ruiz-Barba and Jimenez-Diaz, 1994). In Aloreña table olives, yeasts were associated with LAB in tank and vat fermentations at room temperature. In most cases, however, yeasts were associated with Gram-negative bacteria in most of the vat-fermented samples. The predominance of Gramnegative bacteria and yeasts in the cold fermentation (together with unspent carbohydrates) leads to explosive gas formation once the olives are exposed to room temperature, suffering an undesirable secondary fermentation at retail which leads to the running over from the brine in the packed olives. Furthermore, some non-fermentative Gram-negative bacteria (as in our case) may cause spoilage by alkalinisation of brines (data not shown). Brine alkalinisation not only opens the door for proliferation of spoilage (and maybe even pathogenic bacteria) but it also favours darkening of the brines and the fruit (Garrido-Fernández et al., 1997), decreasing the olive acceptability by consumers. In conclusion, DGGE analysis carried out on Aloreña table olives during fermentation revealed higher differences in microbial diversity between vats, SMEs and storage conditions. Spontaneous fermentation of table olives relies upon microflora naturally present on raw material or in the containers in which the olives were stored (Tassou, 1993). This practice could lead to variation in the quality and flavour of the product and to the spoilage of olives (Lanciotti et al., 1999). Thus, this natural fermentation remains an empirical process which should be standardized by the development of starter cultures and new biotechnological processes for improving its organoleptic quality, its safety aspects and thus avoiding important economic losses related to several problems. In this way, those traditional processes should be replaced by a controlled fermentation by using the autochthonous homofermentative lactic acid bacteria isolated in further studies from the same olive brines during fermentation. Acknowledgements This work was supported by research project Bioándalus 08/18/ L4.3 (Instituto Andaluz de Biotecnología), and Junta de Andalucía research actions (AGR230, PAI-05-0107). Dr Abriouel was financed by a contract from Ramón y Cajal Research Programme. We also acknowledge financial support from the University of Jaén (Plan de Apoyo a la Investigación). We also thank Grupo de Desarrollo Rural Valle del Guadalhorce and associated SMEs for their collaboration.

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