Effect of Sequential Inoculum of Beta-Glucosidase Positive and Probiotic Strains on Brine Fermentation to Obtain Low Salt Sicilian Table Olives.

In the present study, the β-glucosidase positive strain Lactobacillus plantarum F3. 3 was used as starter during the fermentation of Sicilian table olives (Nocellara Etnea cultivar) at two different salt concentrations (5 and 8%), in order to accelerate the debittering process. The latter was monitored through the increase of hydroxytyrosol compound. In addition, the potential probiotic Lactobacillus paracasei N24 strain was added after 60 days of fermentation. Un-inoculated brine samples at 5 and 8% of salt were used as control. The fermentation was monitored till 120 days through physico-chemical and microbiological analyses. In addition, volatile organic compounds and sensorial analyses were performed during the process and at the end of the fermentation, respectively. Lactic acid bacteria and yeasts were, in depth, studied by molecular methods and the occurrence of the potential probiotic N24 strain in the final products was determined. Results highlighted that inoculated brines exhibited a higher acidification and debittering rate than control ones. In addition, inoculated brines at 5% of salt exhibited higher polyphenols (hydoxytyrosol, tyrosol, and verbascoside) content compared to samples at 8% of NaCl, suggesting a stronger oleuropeinolytic activity of the starter at low salt concentration. Lactobacilli and yeasts dominated during the fermentation process, with the highest occurrence of L. plantarum and Wickerhamomyces anomalus, respectively. Moreover, the potential probiotic L. paracasei N24 strain was able to survive in the final product. Hence, the sequential inoculum of beta-glucosidase positive and potential probiotic strains could be proposed as a suitable technology to produce low salt Sicilian table olives.

Introduction

Among fermented vegetables, table olives are widespread in the Mediterranean area with increasing consumption in both European and non-European countries (International Olive Council, 2016). Olives are intrinsically health thanks to the high content of fiber, vitamins, and polyphenols which play a very important role, exhibiting pharmacological properties and antioxidants effects. In particular, hydroxytyrosol scavenges free radicals, inhibits human low-density lipoprotein (LDL) oxidation, inhibits platelet aggregation, and discloses anticancer activity by means of pro-apoptotic mechanisms (Raederstorff, 2009; Allouche et al., 2011; Buckland and Gonzalez, 2015).

In Sicily, table olives fermentation is mainly performed under traditional methods exploiting the fermentative action of the autochthonous microbiota. Olives are directly brined without previous debittering treatment; therefore, the indigenous microorganisms and the effect of the physico-chemical conditions of brine (pH, salt, presence/absence O2, etc.) are mainly responsible of the hydrolysis of the oleuropein and of other β-glucosides. The oleuropein, a β-glucoside compound lending the strong “bitterness” aroma to the olive fruit, is hydrolyzed by β-glucosidases enzymes with the release of glucose and aglycones which are degraded, by an esterase, in the no-bitter phenols hydroxytyrosol, and elenolic acid (Bianchi, 2003). The spontaneous debittering is time consuming and not predictable, and it is strongly influenced by physico-chemical parameters, by the presence of fermentable substrates, and by the autochthonous microbiota. Starter cultures with oleuropein degrading activity were extensively applied in order to reduce the debittering time and to control the fermentation process (Panagou et al., 2003, 2008; Marsilio et al., 2005; Servili et al., 2006; Bevilacqua et al., 2013; Bonatsou et al., 2015), and, among them, the use of β-glucosidase positive strains could be promising (Ghabbour et al., 2011; Tataridou and Kotzekidou, 2015).

Recently, salt intake consumption hypertension and cardiovasular diseases [U.S. Dept. of Agriculture and U.S. Dept. of Health and Human Services, 2010; World Health Organization (WHO), 2012].

Recently, high attention was paid to salt intake since its overmuch intake is considered a risk factor for the onset of hypertension and cardiovascular diseases [U.S. Dept. of Agriculture and U.S. Dept. of Health and Human Services, 2010; World Health Organization (WHO), 2012]. Indeed, the set-up of table olives with low NaCl content is an issue of great interest for the sector (Bautista-Gallego et al., 2013a). However, a complete removal of salt may lead to an increased risk in the survival/ growth of spoilage or food pathogen microorganisms and may also alter food flavor, causing important economic losses. Hence, several studies evaluated the use of KCl, CaCl2, and ZnCl2 as NaCl replacers. It is well-demonstrated that table olives dealt with NaCl reduction and partial substitution with other salts have a more equilibrated mineral composition, enhancing the consumers' acceptance (Bautista-Gallego et al., 2010, 2011a, 2013b). Nevertheless, the effect of NaCl replaces on sensorial aspects is still controversial and strongly influenced by the concentration of the salt mixture used (Zinno et al., 2017). Recently, Pino et al. (2018a) demonstrated that the reduction of NaCl content to 5%, without any NaCl replacers, did not negatively affect the Nocellara Etnea table olives fermentation, obtaining a microbiologically safe product with appreciate sensorial traits.

Another challenge for vegetable product industry is to satisfy the increasing consumer demand for healthier products. Numerous studies demonstrated that table olives are promising carrier for probiotic strains being able to support their survival, probably thanks to the release of prebiotic substances from fruits. Additionally, their microstructure, in terms of roughness of olive surface, promotes the formation of biofilm that seem to protect probiotic bacteria from stressful conditions (such as acidic environment), favoring their survival through the human gastrointestinal tract (De Bellis et al., 2010; Arroyo-López et al., 2012a; Blana et al., 2014; Randazzo et al., 2017; Rodríguez-Gómez et al., 2017). According to that, the present study was aimed to evaluate the effect of a sequential inoculum of a β-glucosidase positive strain and probiotic bacteria on brine fermentation in order to set up a low salt Sicilian table olives.

Materials and Methods

Pilot Scale Olives Processing

Traditionally Sicilian–style table olives from Nocellara Etnea cultivar, provided from a local company, located in Paternò (Sicily), were processed without any lye treatment. Olives were pre-treated as previously reported (Pino et al., 2018a) and directly immersed in sterilized brine, containing 5 or 8% (w/v) of NaCl. The β-glucosidase positive strain Lactobacillus plantarum F3.3, previously isolated from fermented table olives was used as starter. The strain was previously characterized for the presence of bglH gene according to Marasco et al. (1998) and its β-glucosidase activity was evaluated by enzymatic assay, according to Sestelo et al. (2004). To set-up probiotic table olives, the potential probiotic Lactobacillus paracasei N24, belonging to the Di3A microbial collection was added. This strain was selected according to its technological and probiotic features (Pitino et al., 2010; Randazzo et al., 2010) and for its good ability to survive in table olives (Randazzo et al., 2017; Pino et al., 2018a). Both microorganisms were applied as lyophilized strains.

The experimental fermentation design comprised 8 treatments: 4 fermentations at 5% of NaCl with (F5A; F5B) and without (F5C; F5D) the addition of L. plantarum F3.3 strain, and 4 fermentations at 8% of NaCl with (F8A; F8B) and without (F8C; F8D) the addition of L. plantarum F3.3. Brine samples F5A; F5B; F8A; F8B were inoculated with the L. plantarum starter culture, to a final cell density of 7 log cfu/ml, directly after brining. The potential probiotic L. paracasei N24 strain was inoculated in F5B; F5D; F8B; F8D samples after 60 days of brining (at final cell density of 9 log cfu/ml). All fermentations were done at room temperature (18 ± 2°C), and followed up to 120 days. Marine salt was periodically added to maintain the initial concentration and fresh brine was supplied to keep olives totally dipped. Each fermentation was carried out in triplicate.

Physico-Chemical Analysis

The pH of the brines was detected by using a MettlerDL25 pHmeter (MettlerDL25, Mettler-Toledo International Inc.). Titratable acidity was determined by titring brine samples with 0.1 N NaOH and was expressed as lactic acid (g/100 ml).

The olive brines were filtered through PTFE filters (Millipore, 0.45 μm) and injected in the chromatographic system to analyze the phenol fraction. The HPLC instrument consisted of a chromatography Waters Alliance 2695 HPLC equipped with a Waters 996 photodiode array detector (PDA) set at 280 nm. The column used was a Luna C18 (250 mm × 4.6 mm i.d., 5 μm, 100 Å, Phenomenex, Torrence, CA) which was maintained at 30°C in an oven. The flow rate was 1 mL/min. Separation was obtained by elution gradient using an initial composition of 95% of A solution (2% acetic acid in water) and 5% of B solution (methanol). The concentration of B solution was increased to 30% in 15 min and to 70% in 25 min and then, after 2 min in isocratic condition, the mobile phase was set at the same initial concentration in 8 min. Phenolic compounds were identified by injecting the pure standards of oleuropein, verbascoside, tyrosol, and hydroxytyrosol and by comparing their retention time and UV-Vis spectra. All the analyses were performed in triplicate.

Microbiological Analyses

Brine samples at 1, 30, 60, 90, and 120 days of fermentation were subjected to microbiological analysis as previously described (Pino et al., 2018a). Mannitol Salt Agar (MSA), incubated at 32°C for 48 h, was used to isolate both coagulase positive and negative staphylococci. All media were provided from Oxoid (Italy) with the exception of YM provided from Difco (Italy). Microbiological analyses were performed in triplicate and results were expressed as log cfu/ml ± standard deviation.

LAB Isolation and Identification

For each brine sample (F5A, F5B, F5C, F5D, F8A, F8B, F8C, and F8D) and each sampling time (1, 30, 60, 90, and 120 days), 20% of the total number of colonies, recovered on MRS agar plate, were randomly selected, purified, checked for catalase activity and Gram reaction, and microscopically examined before storing in liquid culture using 20% (v/v) glycerol at −80°C. Six-hundred (600) LAB isolates were purified and subjected to total genomic DNA (gDNA) extraction following the method described by Pino et al. (2018b). gDNA concentration and quality were evaluated using the Fluorometer Qubit (Invitrogen, Carlsbad, 278 CA, USA). Multiplex RecA and Tuf gene species-specific PCR were performed as previously described (Torriani et al., 2001; Ventura et al., 2003), respectively. Strains not identified at species level with species-specific PCR were subjected to 16S rRNA gene PCR-RFLP analysis according to Pino et al. (2018b). For each PCR-RFLP cluster, the 16S rRNA gene PCR amplicon of one representative strain was purified using the Qiaquick PCR purification kit (Qiagen Hilden, Germany) and was subjected to sequencing and Blast analysis.

Isolation and Genotypic Identification of Yeasts

From each brine samples at each sampling time, as previously reported, 200 colonies were randomly isolated from YM medium, purified, and microscopically examined prior to storing in liquid culture using 20% (v/v) glycerol at −80°C. For the yeasts characterization, DNA was extracted according to Ruiz-Barba et al. (2005) and subjected to repetitive element palindromic (rep)-PCR analysis by using GTG5 primer. The PCR reaction was carried out in a final volume of 25 μl, containing: 5 μl of DNA, 5 μl 5X PCR Buffer, 1 μl of primer GTG5 (5-GTGGTGGTGGTGGTG-3), 13.9 μl of filtered water on 0.1 μl of Taq polymerase (Invitrogen, Italy). The amplification program was as follows; an initial denaturation (95°C, 5 min) followed by 30 cycles of denaturation (95°C, 30 s), annealing (40°C, 1 min), and extension (65°C, 8 min) with a single final extension (65°C, 16 min). PCR products were electrophoresed in a 2 % agarose gel in 1X TAE buffer, stained with ethidium bromide (30 min) and visualized under ultraviolet light. The resulting fingerprints were digitally captured and analyzed with the Bionumerics 6.6 software package (Applied Maths, Kortrijk, Belgium). Dendrogram for clustering comparison was built with UPGMA (Unweighted Pair Group Method) method and Pearson correlation.

To validate the clustering analysis and for identification of strains, the 26S rDNA gene of all isolates was further sequenced. The gDNA amplification was performed according to Porrua et al. (2018). PCR products were resolved by electrophoresis on agarose gel (1% w/v) stained with ethidium bromide. DNA ladder plus (Invitrogen, USA) was used to evaluate the molecular weight of amplified DNA. PCR products were purified using Isolate DNA kit (Bioline, USA) according to the manufacturer's instructions and quantified by agarose gel electrophoresis (1% w/v) in 0.5X TBE buffer (89 mM Tris-borate, 2 mM EDTA pH 8). An amount of 10 μl of purified product with forward primer NL1 was used for sequencing by Stab Vida (Lisbon, Portugal). Nucleotide sequences were aligned with the software Molecular Evolution Genetic Analysis (MEGA).

Rep-PCR for Detecting the Presence of Probiotic L. paracasei N24 Strain

Rep-PCR genomic fingerprinting was performed on 79 L. paracasei strains, isolated from F5B, F5D, F8B, and F8D brine samples at 120 days of fermentation, using the (GTG)5-primer, as described by Versalovic et al. (1994). PCR was carried out in a 20 μl reaction mixture containing 1x Thermo Green buffer (Thermo Scientific, Waltman, MA, USA), 3.0 mM MgCl2, 200 μM of each dNTP (Fermentas), 1 U of Taq polymerase (Thermo Scientific, Waltman, MA, USA), 2 μM (GTG)5 primer and 50 ng gDNA. Amplifications were performed in a MyCycler thermal cycler (BioRad, Hercules, CA). The PCR cycling parameters and gel running conditions were set according to Solieri et al. (2012). The only modification was the change of annealing temperature from 40 to 45°C. The GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific, Waltman, MA, USA) was used as a molecular size marker. BioDoc Gel Analyzer device (Biometra GmbH, Germany) was used to capture DNA fingerprint images which were then processed through the BioNumerics software v3.0 (Applied Maths, Sint-Martens-Latem, Belgium). Repeatability of rep-PCR was assessed using the inoculated strain N24 as internal control. Pearson's correlation similarity coefficient was chosen to calculate bands patterns similarity matrix with optimization and curve smoothening values at 1%. Unweighted pair group method with arithmetic mean (UPGMA) analysis was exploited to build the (GTG)5-based dendrogram.

Analysis of Volatile Organic Compounds (VOCs)

VOCs analysis was performed on brine samples at 1, 60, and 120 days of fermentation following method and conditions previously described (Randazzo et al., 2017; Pino et al., 2018a) using a gas chromatography-mass-spectrometry (GC-MS). All analyses were performed in triplicate and the results were expressed as means in μg/l of brine.

Sensory Evaluation of Table Olives

Table olives at 120 days of fermentation were subjected to sensory evaluation by trained panelists (6 females and 4 males, aged from 22 to 40 years). Sensory panel was conducted according to the International Olive Council method (International Olive Council, 2011). Descriptors related to negative sensations, gustatory, and kinaesthetic perceptions were evaluated as previously described (International Olive Council, 2011, 2016; Pino et al., 2018a). In addition, the overall acceptability descriptor, such an indication of the overall quality, was also scored. Sensory data were acquired by a direct computerized registration system (FIZZ Biosystemes. Couternon, France).

Statistical Analysis

Microbiological and chemical (i.e., single phenol compounds, pH and acidity) data were analyzed by ANOVA (One-way Analysis of Variance) using Tukey's post-hoc test, in order to assess the overall differences among treatments. The reference level of significance was 0.01 for chemical assay and 0.05 for VOCs and microbiolgical assays. All statistical analyses were performed using MATLAB software (MathWorks, version 8.5.0), while sensory data were analyzed using the software package Statgraphics® Centurion XVI (Statpoint Technologies, INC.) setting samples as treatments. Data correlations between brine samples differently treated and VOCs were computed using XLStat software (version 2016.1).

Results

Physico-Chemical Data

In Table 1, the results of pH and titratable acidity detected in brine samples analyzed thought the fermentation are shown. At the beginning of fermentation, pH values ranged from 5.8 to 6.3, and then they decreased after 30 days. Differences among samples become more appreciable after 60 days of fermentation with the lowest values showed by F5B and F8C samples. At the end of fermentation (120 days) the pH fitted the hygienic limit of 4.3 in all samples (Table 1).

Table 1

Results of pH and titratable acidity values in olive brines expressed as means and standard deviations at different time of fermentations.

Days of fermentation
	1	30	60	90	120
pH
F1	5.9 ± 0.02ab	4.6 ± 0.01bc	4.4 ± 0.01bc	4.4 ± 0.03c	4.2 ± 0.03b
F2	6.3 ± 0.02c	4.5 ± 0.01ab	4.3 ± 0.01ab	4.2 ± 0.02a	4.2 ± 0.01b
F3	6.1 ± 0.01abc	4.4 ± 0.03a	4.4 ± 0.02bc	4.3 ± 0.01b	4.3 ± 0.03b
F4	5.9 ± 0.02ab	4.4 ± 0.02a	4.4 ± 0.03bc	4.2 ± 0.01a	4.0 ± 0.02a
F5	5.9 ± 0.01ab	4.6 ± 0.01bc	4.5 ± 0.07c	4.4 ± 0.01c	4.3 ± 0.03b
F6	6.1 ± 0.02abc	4.7 ± 0.07c	4.5 ± 0.03c	4.4 ± 0.03c	4.2 ± 0.02b
F7	6.2 ± 0.03bc	4.5 ± 0.04ab	4.2 ± 0.01a	4.5 ± 0.03d	4.3 ± 0.04b
F8	5.8 ± 0.03a	4.6 ± 0.04bc	4.4 ± 0.01bc	4.3 ± 0.02b	4.3 ± 0.08b
ACIDITY (g LACTIC ACID 100/ml)
F1	0.0225 ± 0.003a	0.356 ± 0.001bc	0.401 ± 0.001ab	0.436 ± 0.015bc	0.413 ± 0.016abc
F2	0.1010 ± 0.015b	0.367 ± 0.017c	0.430 ± 0.007b	0.526 ± 0.016d	0.528 ± 0.013d
F3	0.0135 ± 0.002a	0.307 ± 0.007a	0.368 ± 0.020a	0.379 ± 0.032ab	0.436 ± 0.015bc
F4	0.0082 ± 0.002a	0.329 ± 0.008ab	0.363 ± 0.003a	0.458 ± 0.016c	0.458 ± 0.017c
F5	0.0133 ± 0.003a	0.374 ± 0.008c	0.396 ± 0.007ab	0.419 ± 0.008bc	0.385 ± 0.007a
F6	0.0067 ± 0.001a	0.385 ± 0.007c	0.408 ± 0.08ab	0.419 ± 0.008bc	0.396 ± 0.007ab
F7	0.0082 ± 0.002a	0.318 ± 0.008a	0.372 ± 0.005a	0.352 ± 0.008a	0.396 ± 0.008ab
F8	0.0077 ± 0.001a	0.318 ± 0.008a	0.363 ± 0.008a	0.396 ± 0.007abc	0.420 ± 0.007abc

Data in the same column with different letters are significantly different at P < 0.01.

The titratable acidity values exhibited an increasing trend in all samples with the exception of F8A and F8B brines, which slightly decreased in acidity at 120 days. The highest acidity values were detected in brines at 5% of NaCl (F5A, F5B, F5C, and F5D). In addition, a significant increase in acidity was recorded after the addition of N24 strain (90 days) mainly in F5B and F5D samples (Table 1).

Results of hydroxytyrosol, tyrosol, oleuropein, and verbascoside quantification are shown in Table 2. Overall, all polyphenols analyzed showed an increasing trend during the fermentation mainly in 5% NaCl brine samples inoculated with starter culture. The highest values of hydroxytyrosol, tyrosol, and verbascoside were mainly recorded in brines inoculated with L. plantarum F3.3 starter culture already at 30 days. Similar behavior was observed for oleuropein.

Table 2

Results of the analyzed phenols in olive brines expressed as means (mg/l) and standard deviations at different time of fermentations.

Days of fermentation
	1	30	60	90	120
HYDROXYTYROSOL
F5A	4.77 ± 0.01c	104.13 ± 0.78e	128.14 ± 0.36c	199.81 ± 0.69c	267.34 ± 5.56f
F5B	5.24 ± 0.11d	95.68 ± 0.34d	101.38 ± 3.86a	166.81 ± 0.79b	180.12 ± 0.63d
F5C	3.14 ± 0.04b	86.86 ± 0.16c	112.14 ± 0.23b	137.87 ± 1.38a	174.32 ± 1.37cd
F5D	3.25 ± 0.06b	84.93 ± 0.17bc	113.47 ± 0.41b	151.95 ± 0.49ab	160.50 ± 3.87ab
F8A	4.97 ± 0.01c	94.16 ± 2.28d	121.63 ± 1.75c	191.93 ± 0.11c	192.66 ± 0.30e
F8B	4.98 ± 0.02c	89.57 ± 1.67cd	112.21 ± 1.99b	153.63 ± 0.47ab	172.98 ± 0.15cd
F8C	3.05 ± 0.08b	56.89 ± 3.17a	112.21 ± 0.49ab	142.40 ± 0.47a	167.71 ± 2.05bc
F8D	2.54 ± 0.07a	80.65 ± 0.88b	102.57 ± 2.00a	147.98 ± 0.69ab	152.78 ± 0.90a
TYROSOL
F5A	0.0 ± 0.0	8.89 ± 0.01d	10.29 ± 0.06b	14.77 ± 0.80e	19.56 ± 0.16d
F5B	0.0 ± 0.0	8.32 ± 0.37d	10.28 ± 1.12b	14.17 ± 0.09de	13.52 ± 0.01bc
F5C	0.0 ± 0.0	7.36 ± 0.02c	9.37 ± 0.05ab	11.29 ± 0.16a	13.62 ± 0.14c
F5D	0.0 ± 0.0	7.45 ± 0.01c	9.66 ± 0.02ab	12.58 ± 0.04bc	12.66 ± 0.30abc
F8A	0.0 ± 0.0	6.79 ± 0.27bc	9.35 ± 0.06ab	13.56 ± 0.24cd	13.51 ± 0.35bc
F8B	0.0 ± 0.0	6.62 ± 0.21b	8.03 ± 0.36a	10.98 ± 0.03a	12.08 ± 0.06a
F8C	0.0 ± 0.0	4.80 ± 0.06a	8.58 ± 0.08a	11.29 ± 0.04a	12.57 ± 0.45ab
F8D	0.0 ± 0.0	6.78 ± 0.04bc	8.32 ± 0.05a	11.80 ± 0.04ab	11.66 ± 0.24a
OLEUROPEIN
F5A	0.0 ± 0.0	16.07 ± 2.85b	21.97 ± 0.39e	35.60 ± 1.61c	50.62 ± 0.59d
F5B	0.0 ± 0.0	15.41 ± 2.12b	11.51 ± 0.71bc	20.47 ± 0.93ab	21.41 ± 0.18ab
F5C	0.0 ± 0.0	9.08 ± 0.22a	13.51 ± 0.36d	19.86 ± 2.14ab	23.43 ± 0.62abc
F5D	0.0 ± 0.0	8.30 ± 0.11a	12.01 ± 0.33bc	15.09 ± 0.45a	18.81 ± 1.30a
F8A	0.0 ± 0.0	9.06 ± 0.51a	12.26 ± 0.05bcd	26.99 ± 3.80b	28.48 ± 1.22bc
F8B	0.0 ± 0.0	8.32 ± 0.18a	9.99 ± 0.15a	22.38 ± 2.03ab	24.64 ± 0.50abc
F8C	0.0 ± 0.0	7.55 ± 0.10a	12.79 ± 0.03cd	19.22 ± 0.56a	31.94 ± 3.60c
F8D	0.0 ± 0.0	9.13 ± 0.24a	11.19 ± 0.01b	18.84 ± 1.29a	24.02 ± 1.27abc
VERBASCOSIDE
F5A	0.0 ± 0.0	31.69 ± 2.39cd	32.36 ± 1.95ab	50.73 ± 0.17bc	65.39 ± 0.41d
F5B	0.0 ± 0.0	31.61 ± 1.10cd	28.91 ± 3.11a	54.53 ± 0.63cd	58.57 ± 1.78c
F5C	0.0 ± 0.0	30.03 ± 0.21cd	31.72 ± 0.06ab	33.32 ± 0.42a	44.04 ± 0.16a
F5D	0.0 ± 0.0	29.41 ± 0.25bc	37.59 ± 0.71bc	55.40 ± 0.93de	52.57 ± 0.68b
F8A	0.0 ± 0.0	32.32 ± 0.91cd	36.45 ± 3.38bc	59.66 ± 0.90e	57.66 ± 1.39c
F8B	0.0 ± 0.0	33.91 ± 1.27d	40.85 ± 1.22c	59.75 ± 1.56e	56.92 ± 1.75bc
F8C	0.0 ± 0.0	18.37 ± 0.40a	32.29 ± 0.23ab	35.25 ± 2.31a	42.53 ± 1.64a
F8D	0.0 ± 0.0	25.26 ± 0.41b	31.93 ± 0.18ab	47.92 ± 0.81b	45.75 ± 0.74a

a–f: for each phenols, data in the same column with different superscript letters are significantly different P < 0.01.

Microbial Count

Table 3 shown microbial counts of brine samples at both 5 and 8% of NaCl, which is expressed as log cfu/ml. Viable mesophilic bacteria showed different trend among samples. In detail, brines at 5% of NaCl (F5A–F5D), from an initial average value of 7.11 log unit, exhibited a steady trend during the fermentation with slight decrease of cell density after 60 days. Similar behavior was observed for brine samples at 8% of NaCl (F8A–F8D) which showed a mean initial value of 6.73 log unit and a final mean value of 5.56 log unit (Table 3). Regarding LAB population, all brine samples inoculated with starter culture (F5A, F5B, F8A, F8B) presented, at the beginning of fermentation, higher cell density than spontaneous ones (F5C, F5D, F8C, F8D). From 60 to 120 days, LAB reached the highest values in samples inoculated with the potential probiotic N24 strain (F5B, F5D, F8B, F8D). Yeasts were present at an initial level of about 3 log cfu/ml in all experimental brines with the exception of F5A and F5B samples which exhibited initial value of 4.04 and 4.37 log cfu/ml, respectively (Table 3). The yeasts cell densities significantly increased thought the fermentation process, achieving, at 120 days, an average value of 7.37 log unit and 6.64 log unit in brine samples at 5 and 8% of NaCl, respectively. Regarding the staphylococci count, only coagulase negative staphylococci, forming red colonies in the medium, were enumerated and their level, at the beginning of the fermentation, was quite similar among all samples. After a slight increase till 60 days, a decrease to final average values of 3.0 and 3.8 log cfu/ml was achieved in 5 and 8% brine samples, respectively. Similar behavior was observed for enterobacteria counts, which significantly decreased through the fermentation process. At the end of fermentation (120 days) this microbial group was detected, at value below 2 log, in brine samples at 8% of NaCl and below the detection limit in samples at 5% of NaCl, with the exception of the F5D sample. E. coli was never detected in any brine samples analyzed (data not shown).

Table 3

Microbial counts expressed as log10 CFU/ml of 3 replicates ± standard deviation of the main microbial groups detected in experimental brine samples during the fermentation process.

Microbial groups	Days of fermentation
	1	30	60	90	120
MESOPHILIC BACTERIA
F5A	7.34 ± 0.03f	7.14 ± 0.03d	7.44 ± 0.03f	6.79 ± 0.05e	5.86 ± 0.04c
F5B	7.28 ± 0.02bd	6.99 ± 0.10d	7.95 ± 0.03g	7.43 ± 0.02f	6.43 ± 0.01e
F5C	6.90 ± 0.07f	6.57 ± 0.10c	6.35 ± 0.03d	5.82 ± 0.03c	5.63 ± 0.02d
F5D	6.93 ± 0.02be	6.44 ± 0.07c	6.12 ± 0.06c	6.15 ± 0.08d	6.94 ± 0.06f
F8A	6.72 ± 0.07acd	5.84 ± 0.08ab	5.76 ± 0.06b	5.35 ± 0.03ab	4.95 ± 0.03a
F8B	6.87 ± 0.03bc	5.72 ± 0.03a	6.86 ± 0.02e	6.94 ± 0.03e	6.47 ± 0.09e
F8C	6.58 ± 0.10a	5.95 ± 0.02b	5.38 ± 0.07a	5.26 ± 0.05a	5.21 ± 0.05b
F8D	6.74 ± 0.03acde	5.64 ± 0.11a	5.51 ± 0.06a	5.51 ± 0.08b	5.61 ± 0.06d
LACTOBACILLI
F5A	7.11 ± 0.07eg	7.73 ± 0.11h	7.44 ± 0.06c	6.92 ± 0.05d	6.26 ± 0.13a
F5B	7.20 ± 0.06fg	7.74 ± 0.11h	8.83 ± 0.07ef	8.49 ± 0.09h	8.29 ± 0.04c
F5C	6.09 ± 0.06a	6.97 ± 0.06cfg	6.78 ± 0.06b	6.21 ± 0.42ac	6.02 ± 0.11a
F5D	6.22 ± 0.06ab	6.73 ± 0.08deg	8.64 ± 0.10d	7.86 ± 0.06ef	7.16 ± 0.09b
F8A	7.68 ± 0.18h	6.69 ± 0.05bcd	7.03 ± 0.08ab	6.38 ± 0.09bc	6.18 ± 0.06a
F8B	7.02 ± 0.08def	6.74 ± 0.01bef	8.14 ± 0.09fg	7.98 ± 0.09fg	7.10 ± 0.10b
F8C	6.59 ± 0.15bc	6.25 ± 0.10a	6.56 ± 0.06a	6.00 ± 0.07ab	5.87 ± 0.03a
F8D	6.68 ± 0.07cd	6.12 ± 0.05a	8.58 ± 0.12eg	7.63 ± 0.38eg	6.94 ± 0.10b
YEASTS
F5A	4.04 ± 0.04de	4.53 ± 0.05ce	5.03 ± 0.04b	7.22 ± 0.10d	8.39 ± 0.12c
F5B	4.37 ± 0.12e	4.56 ± 0.10c	6.80 ± 0.04d	7.14 ± 0.12d	7.07 ± 0.13b
F5C	3.77 ± 0.10cd	4.11 ± 0.14be	6.12 ± 0.12c	6.42 ± 0.09c	7.11 ± 0.14b
F5D	3.47 ± 0.10bc	6.06 ± 0.08f	5.79 ± 0.04c	5.55 ± 0.05a	6.93 ± 0.09b
F8A	3.04 ± 0.04a	3.16 ± 0.12a	4.37 ± 0.03a	5.64 ± 0.03a	7.06 ± 0.12b
F8B	3.05 ± 0.03ab	4.03 ± 0.03b	6.13 ± 0.14c	6.51 ± 0.03bc	6.04 ± 0.04a
F8C	3.35 ± 0.14abc	4.64 ± 0.04c	6.06 ± 0.06c	6.65 ± 0.03bc	7.22 ± 0.10b
F8D	3.24 ± 0.12ab	3.42 ± 0.13a	4.66 ± 0.13ab	5.33 ± 0.03a	6.24 ± 0.05a
STAPHYLOCOCCI
F5A	4.08 ± 0.07a	4.40 ± 0.13c	4.62 ± 0.03a	4.25 ± 0.11c	3.74 ± 0.20bc
F5B	4.03 ± 0.09a	4.70 ± 0.13d	4.80 ± 0.10ab	3.42 ± 0.21ab	3.04 ± 0.06a
F5C	4.15 ± 0.20a	4.30 ± 0.24a	4.41 ± 0.15a	3.68 ± 0.52ab	3.21 ± 0.08a
F5D	4.06 ± 0.14a	4.80 ± 0.05e	4.79 ± 0.20ab	3.27 ± 0.27a	3.07 ± 0.11a
F8A	4.85 ± 0.08b	5.28 ± 0.22f	4.83 ± 0.16ab	4.81 ± 0.10d	4.28 ± 0.14d
F8B	4.21 ± 0.10a	4.85 ± 0.07b	5.03 ± 0.15b	4.77 ± 0.17d	3.98 ± 0.03cd
F8C	4.80 ± 0.23b	5.38 ± 0.10b	5.01 ± 0.09b	3.76 ± 0.14b	3.51 ± 0.29ab
F8D	4.31 ± 0.10a	4.71 ± 0.24b	5.08 ± 0.12b	4.47 ± 0.20cd	3.48 ± 0.19ab
ENTEROBACTERIA
F5A	2.66 ± 0.31a	1.19 ± 0.15a	<1	<1	<1
F5B	2.29 ± 0.09a	1.49 ± 0.31a	<1	<1	<1
F5C	2.28 ± 0.17a	1.36 ± 0.41a	<1	<1	<1
F5D	4.01 ± 0.11cd	3.38 ± 0.29c	2.18 ± 0.09a	1.63 ± 0.15a	1.55 ± 0.19a
F8A	3.31 ± 0.21b	3.06 ± 0.10bc	2.16 ± 0.15a	1.38 ± 0.27a	1.21 ± 0.15a
F8B	3.88 ± 0.16bc	3.32 ± 0.19bc	2.57 ± 0.23a	1.14 ± 0.10a	1.63 ± 0.14a
F8C	3.41 ± 0.15bd	2.72 ± 0.39b	2.00 ± 0.18a	1.31 ± 0.17a	1.27 ± 0.17a
F8D	3.43 ± 0.11bd	2.75 ± 0.11b	2.04 ± 0.06a	1.28 ± 0.20a	1.24 ± 0.14a

a–h: for each medium, data in the same column with different superscript letters are significantly different (P < 0.05).

Molecular Identification of LAB

Six hundred isolates from MRS agar plates were considered LAB based on their positive Gram reaction, non-motility, absence of catalase activity and spore formation, as well as rod or coccal shape. RecA and Tuf gene species-specific PCRs revealed the presence of strains belonging to L. plantarum, Lactobacillus pentosus, L. paracasei, and Lactobacillus casei species. The distribution of lactobacilli at different sampling times is reported in Figure 1A and their occurrence in the different brine samples is showed in Figure 1B. Isolates not identified at species level were indicated as “others.” Evaluating the distribution of LAB strains through the fermentation, results indicated that L. plantarum and L. pentosus represented the dominant species at the beginning of the process (till 30th days) and a high occurrence of L. paracasei species was detected up to 60 days. Whereas, the highest occurrence of L. casei strains was achieved at 120 days (Figure 1A). Zooming on the fermentation at different salt content (5 and 8%) differently inoculated, results showed the dominance of L. plantarum strains in samples inoculated with starter, the highest occurrence of L. casei and L. pentosus in spontaneous samples (F5C, F8C, F5D, F8D) and of L. paracasei in samples inoculated with the probiotic strain N24 (Figure 1B).

Figure 1

Isolation frequency, expressed in percentage, of LAB species throughout the fermentation (A) and in the experimental brine samples (B).

Isolates not identified initially with recA and Tuf gene primer pairs were subjected to PCR-RFLP analysis of the 16SrDNA and clustered into four different groups (data not shown). One representative isolate for each cluster was identified by 16S rRNA gene sequencing and were deposited in the GenBank database. The species attribution and the accession numbers of the sequenced strains were as follows (isolates code in parentheses): Leuconostoc mesenteroides MK085109 (F5C.1), Lactococcus lactis MK085110 (F5B.38), Lactobacillus brevis MK085111 (F5D.44), and Enterococcus faecium MK085112 (F5A.21).

Molecular Identification of Yeasts

Two-hundred yeast isolates were randomly obtained during the fermentation process. The dendogram generated by rep-PCR with primer GTG5 showed that the isolates formed 17 groups clearly differentiated. The most numerous groups belonged to the Wickerhamomyces anomalus and Candida boidinii species, although representatives of Candida diddensiae, Pichia kluyveri and Meyerozyma guillermondii were also identified (Figure 2A).

Figure 2

Dendrogram generated after bioinformatics analysis with Bionumerics 6.6 software package of the rep-PCR profiles obtained with GTG5 (A). Isolation frequency (%) of yeasts during the fermentation time of Nocellara Etnea table olives (B). Distribution of yeast species in different experimental brine samples (C).

The evolution of the different yeast species throughout the fermentative process is presented in Figure 2B. At this regard, W. anomalus and C. boidinii formed a stable dual species consortium through the fermentation, since they were both detected more frequently than others species, with a mean frequency of 49 and 37%, respectively. Indeed, these species were dominant in all brine samples differently treated (Figure 2C). The rest of the species were isolated at very low mean frequencies; in particular, M. guillermondii (1.97%) was detected only in samples at 8% of NaCl till 30th days of fermentation, whereas, P. kluyveri (2.98%) in samples F5A, F5D, and F8D till 60 days and C. diddensiae (5.87%) was detected starting from the 30th day of fermentation only in brines at 8% of NaCl (Figure 2C).

Detection of L. paracasei N24 Strain at 120 Days of Fermentation

The presence of inoculated strain N24 was assessed at 120 days of fermentation by rep-PCR with primer GTG5 on a pool of 79 L. paracasei isolates from samples F5B, F5D, F8B, and F8D. Preliminarily, the rep-PCR repeatability was evaluated using gDNA from strain N24 as internal control in four different gels, obtaining a similarity of 74.3% (data not shown). Accordingly, this value was retained as similarity threshold to establish the identity of isolates compared to the rep-PCR profile of promising probiotic strain N24. The dendrogram generated using the GTG5-based patterns of L. paracasei isolates revealed the presence of four major clusters (from A to D) and four singleton L. paracasei isolates below 74.3% similarity (Figure 3; Supplementary Figure 1). The cluster analysis showed that the major cluster A grouped both the inoculated strain N24 and 48 out of 79 isolates, indicating that these isolates were assimilated to the N24 strain profile. The majority of them were isolated from samples F5B and F8B, over the indigenous L. paracasei isolates. In detail, out of 48, 18 strains were isolated from F5B, 12 from F8B, 10 from F5D, and 8 from F8D. The remaining isolates belonged to clusters B, C, and D.

Figure 3

Dendrogram generated after cluster analysis of the digitized GTG5-PCR fingerprints of the Lactobacillus paracasei strains isolated from F5B, F5D, F8B, and F8D brine samples at 120 days of fermentation.

Volatile Organic Compounds (VOCs)

Results of VOCs of different brine samples at 60 and 120 days of fermentation are reported in Table 4. Overall, 46 compounds as acids, alcohols, esters, aldehydes, and phenols were identified, exhibiting a growing trend through the fermentation, with the exception of samples F5A, F5D, F8A, and F8D. The highest value was registered in samples F8C, with a value of 2,739.17 μg/l. Alcohols were the most abundant compounds, followed by esters and acids, whereas aldehydes and phenols were detected at lower concentrations. Focusing on each compound, among alcohols, ethanol dominated the fermentation process showing an increase only in spontaneous samples (F5C and F8C), followed by isoamylalcohol and phenylethylalcohol, which registered a variable trend through the fermentation (Table 4). Ethyl-acetate and methyl 2-methylbutanoate were the main detected esters. Among acids, the acetic acid was the most abundant compound, with the highest value in F5C and F8C samples. The most abundant aldehydes and phenols were nonanal, benzaldehyde and creasol, respectively (Table 4).

Table 4

Volatile organic compounds (VOCs) expressed as μg/l of experimental brine samples at 60 and 120 days of fermentation.

Compounds	rt	T0	F5A 60	F5A 120	F5B 60	F5B 120	F5C 60	F5C 120	F5D 60	F5D 120	F8A 60	F8A 120	F8B 60	F8B 120	F8C 60	F8C 120	F8D 60	F8D 120
Acids		0.00	48.46	92.24	70.48	150.75	74.29	124.08	49.57	40.69	57.77	35.47	81.06	111.28	58.14	135.11	35.60	50.30
Acetic acid	24.78	0.00a	26.43b	58.74e	46.26d	77.75f	72.30f	96.15g	44.02d	39.67c	29.76b	35.47c	50.02de	62.01e	58.14e	106.25h	35.60c	34.45c
Propionic acid	29.74	0.00a	4.15d	3.62d	5.22e	32.50f	1.01b	2.31c	0.00a	1.02b	5.84e	0.00a	2.06c	0.65b	0.00ha	3.14d	0.00a	0.96b
Isobutyric acid	31.43	0.00a	0.91b	0.25b	1.28bc	3.10c	0.98b	1.98c	0.00a	0.00a	1.02b	0.00a	6.56d	2.01c	0.00a	2.26c	0.00a	0.00a
Butanoic acid	35.01	0.00a	0.32b	0.62b	0.90b	4.37c	0.00a	0.15b	5.55c	0.00a	0.62b	0.00a	1.05b	10.60d	0.00a	0.96b	0.00a	0.00a
Hexanoic acid	36.19	0.00a	8.96b	27.89f	7.61b	31.99f	0.00a	13.67cd	0.00a	0.00a	10.54c	0.00a	18.21e	34.12f	0.00a	12.63c	0.00a	14.89a
2-Ethyleptanoic acid	57.20	0.00a	7.69d	1.12b	9.21e	1.04b	0.00a	9.82e	0.00a	0.00a	9.99e	0.00a	3.16c	1.89b	0.00a	9.87e	0.00a	0.00
Alcohols		63.03	519.08	287.57	494.50	437.32	914.38	1662.74	276.30	152.56	539.05	312.90	962.57	995.99	960.57	1842.36	229.37	191.88
Ethanol	3.33	6.77a	290.25c	151.13b	315.24c	162.54b	582.12d	1325.14e	164.48b	83.96b	305.17c	164.48b	639.44d	669.21d	615.98d	1485.32e	152.36b	125.87b
Isoamylalcohol	11.58	0.00a	79.84d	2.61a	48.91c	69.71d	78.83d	118.13e	22.14b	20.16b	83.78d	3.01a	75.12d	78.65d	78.01d	129.58e	1.03a	23.04b
1-Hexanol	19.23	0.00a	9.52bc	8.44b	12.47c	15.86d	13.15c	20.14e	13.52c	11.33c	10.03c	7.55b	18.39e	15.19d	13.84c	21.06e	3.69a	3.52a
cis Hexen 1 ol	20.84	0.00a	21.01d	22.63d	29.82e	28.56e	59.62g	83.22i	14.25c	7.86b	22.97d	31.24e	71.98h	52.12f	61.24g	89.61i	5.12b	1.14a
3-Octenol	25.13	0.00a	0.00a	0.96a	0.86a	1.99b	2.05b	2.63bc	0.00a	0.00a	0.00a	0.62a	3.92d	1.84b	1.16a	2.98c	0.00a	0.00a
1-Eptanol	25.79	2.98b	3.10b	3.54b	5.01c	5.03c	12.13e	5.14c	1.07a	0.93a	2.44b	2.58b	8.52d	3.01b	9.84d	6.23c	0.00a	0.00a
1-Octanol	31.12	19.58f	68.11i	34.99h	2.84b	11.84d	23.14g	3.21c	0.86a	0.51a	65.28i	37.37h	9.68c	8.72c	23.69g	3.02b	14.32e	0.00a
1-Nonanol	35.91	4.11c	0.00a	1.05b	0.00a	0.00a	23.96d	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	22.10d	0.00a	0.00a	0.00a
Benzyl alcohol	47.77	0.00a	7.42c	14.26d	9.63c	13.65d	13.68d	1.01a	4.42b	3.63b	8.12c	9.08c	29.63g	22.13f	16.54e	0.00a	0.00a	0.00
Phenylethyl alcohol	50.96	0.00a	39.83c	47.96cd	69.72d	128.14ef	102.56e	103.89e	55.56d	24.18b	41.26c	56.97d	105.89e	145.12f	114.21e	104.56e	52.85d	38.31a
1-U < 1ecanol	53.54	27.49c	0.00a	0.00a	0.00a	0.00a	3.14b	0.23a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	3.96b	0.00a	0.00a	0.00a
Esters		4.09	181.46	200.51	283.60	421.49	334.35	448.16	284.96	363.63	274.22	358.87	287.75	433.17	378.62	579.82	306.71	308.86
Ethyl acetate	2.75	0.00a	140.11b	145.91b	129.71b	149.52b	147.22b	248.78d	136.82b	298.72e	205.62c	244.42d	132.71b	178.12c	205.61c	325.16e	222.53	195.93
Ethyl propanoate	3.56	0.00a	5.71b	1.49a	0.00a	7.02b	0.00a	0.00a	0.00a	0.00a	7.69b	7.71b	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a
Ethyl butanoate	4.07	0.00a	0.00a	2.50a	1.88a	9.02c	3.96b	1.02a	0.00a	0.00a	7.53c	0.00a	2.48a	10.86c	8.88c	0.00a	0.00a	0.00a
Methyl 2-methylbutanoate	5.44	0.00a	0.00a	8.37b	15.12c	91.13g	12.33c	61.20e	11.72c	0.00a	0.00a	22.94d	6.42b	83.25g	25.84d	70.58f	10.83bc	14.18c
Methyl 3-methylbutanoate	5.85	0.00a	0.00a	0.72a	12.09e	61.05g	1.04a	8.52d	0.00a	1.14a	0.00a	16.24f	6.23c	16.14f	2.51b	10.58e	0.00a	15.42f
Isoamylacetate	7.47	0.00a	8.52c	0.44a	8.13c	10.28c	105.31l	39.18f	96.21i	25.47e	16.18d	38.55f	5.62b	18.12d	3.46b	44.01g	54.99h	57.92h
Methyl hexanoate	9.90	0.00a	0.00a	2.43a	0.00a	0.00a	0.00a	0.21a	0.00a	0.47a	14.83b	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a
Ethyl hexanoate	12.29	0.00a	0.00a	0.46a	7.58c	4.02b	3.65b	3.68b	0.00a	0.00a	0.00a	0.00a	11.13e	9.06d	9.01d	5.12b	0.00a	0.00a
Ethyl lactate	18.49	0.00a	0.00a	6.14b	29.34e	39.42f	10.97c	31.74e	0.00a	5.11b	0.00a	0.00a	32.67e	52.36h	23.12d	45.49g	0.00a	0.00a
Methyl 2-hydroxy-3-methylbutanoate	23.10	0.00a	0.00a	0.00a	0.00a	3.96b	0.21a	7.76c	0.00a	0.00a	0.00a	11.09d	0.00a	4.05b	0.00a	8.23c	0.00a	0.00a
Ethyl octanoate	23.44	0.00a	0.00a	2.95c	1.87b	0.26a	1.48b	1.02b	0.00a	4.60c	0.00a	0.00a	3.02c	0.75b	9.09d	1.81b	0.00a	0.00a
Ethyl-3-hydroxybutyrate	28.92	0.00a	18.04d	9.20c	12.47c	6.99b	0.00a	0.80a	0.00a	8.17b	0.00a	0.00a	3.82b	6.31b	0.00a	1.63b	0.00a	10.85c
Ethyl 2-hydroxy-4-methylpentanoate	30.00	0.00a	1.50a	10.49e	6.15c	7.54d	3.96b	7.14d	9.38e	4.67c	3.60b	5.06c	11.36f	12.12f	6.77cd	14.75g	3.54b	5.60c
Methyl decanoate	32.95	2.95b	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.00a	0.23a	0.00a	0.00a
Ethyl decanoate	35.77	0.00a	0.00a	0.00a	1.24b	0.33a	1.97b	2.23b	0.00a	0.00a	0.00a	0.00a	1.68b	1.46b	1.86b	4.96c	0.00a	0.00a
Ethyl benzoate	36.74	0.00a	0.00a	0.95b	13.82d	3.68c	1.45b	1.88b	0.00a	0.00a	0.00a	0.00a	14.58d	5.02c	2.41b	5.12c	0.00a	0.00a
Acetic acid, 2 phenylethyl	43.01	0.00a	0.00a	1.68b	7.28d	2.13b	0.68a	1.63b	0.00a	0.00a	0.00a	0.00a	1.73b	2.36b	1.72b	4.16c	0.00a	0.00a
Methyl hydrocinnamate	45.91	1.14b	0.00a	3.65c	0.00a	7.61e	5.23d	2.22b	0.00a	0.00a	0.00a	0.00a	10.03f	12.13f	10.36f	5.16d	0.00a	0.00a
Ethyl dodecanoate	46.20	0.00a	0.00a	0.00a	8.36c	0.48a	0.71a	9.04c	6.03b	0.00a	8.55c	0.00a	12.12d	1.58a	1.12a	21.85	0.00	0.00
Ethyl hydrocinnamate	48.97	0.00a	7.58c	3.12b	28.56l	17.05g	34.18m	20.11h	24.80i	15.28f	10.22cd	12.86e	32.15m	19.48h	66.86n	10.98d	14.82f	8.96c
Aldheydes		149.90	87.40	62.52	36.83	72.14	44.29	50.71	32.71	1.40	120.28	117.41	53.09	34.44	42.84	71.32	45.34	16.55
Octanal	14.81	14.12e	10.58d	6.70c	6.52c	5.98bc	6.48c	5.12b	0.00a	0.61a	9.21d	10.64d	13.58e	5.86b	4.47b	9.36d	0.00a	0.00a
Nonanal	20.78	65.32l	40.78h	4.25b	11.93d	31.85g	14.56d	18.17e	13.25d	0.00a	32.76g	52.07i	19.63e	9.86c	14.02d	25.12f	24.46f	1.76b
3-Octanal	21.64	0.00a	0.00a	1.90b	1.58b	1.96b	2.36b	2.36b	1.74b	0.79a	0.00a	26.47e	2.56b	1.02a	2.36b	2.15b	12.78d	5.27c
Decanal	26.85	68.82h	30.75f	41.89g	6.78b	18.44d	9.57c	9.99a	7.46b	0.00a	24.09e	0.00a	5.69b	5.68b	11.00c	18.96d	0.00a	7.47b
Benzaldehyde	28.08	1.64b	5.29c	7.78d	10.02e	13.91f	11.32e	15.07f	10.26e	0.00a	54.22h	28.23g	11.63e	12.02e	10.99e	15.73f	8.10d	2.05b
Phenols		0.00	102.59	49.90	21.94	144.69	116.02	146.22	26.95	72.45	137.38	147.21	7.80	43.19	112.46	110.56	5.61	2.24
Guaiacol	47.25	0.00a	0.00a	2.99b	0.00a	29.08d	57.16e	61.12f	12.85c	3.58b	0.00a	0.00a	0.00a	11.02c	56.23e	53.01e	0.00a	0.00a
Creosol	52.57	0.00a	102.59h	43.69e	10.50b	98.65g	17.89bc	44.32e	12.54a	52.36	95.11	106.62	3.16	23.14	19.67	28.15	0.00	0.00
Phenol	55.36	0.00a	0.00a	0.63a	1.33b	3.56c	5.69d	9.75e	1.56b	2.58c	0.00a	0.00a	0.23a	1.02b	4.50d	10.42e	0.00a	0.00a
4-Ethyl phenol	63.20	0.00a	0.00a	2.59b	10.11d	13.40e	35.28g	31.03h	0.00a	13.93e	42.27h	40.59h	4.41c	8.01d	32.06g	18.98f	5.61c	2.24b
Total		217.02	938.99	692.74	907.35	1226.39	1483.33	2431.91	670.49	630.72	1128.70	971.86	1392.27	1618.07	1552.63	2739.17	622.63	569.83

a–h: for each compound different superscript letters in the same raw are significantly different (P < 0.05). Bold value indicates VOCs class and the total of them.

Figure 4 shows correlation between VOCs and brine samples differently treated. Overall, it is possible to point out that the salt concentration did not influence the VOCs formation through fermentation in brine samples, which were mainly grouped based on the treatment (starter and/or probiotic addition and spontaneous). In detail, samples inoculated with starter (F5A and F8A) were clustered together, showing a negative correlation with alcohol and ester compounds; spontaneous brine samples inoculated with the probiotic strain N24 (F5D and F8D) were negatively correlated to phenols, aldehydes and alcohols. Different correlations were detected for samples inoculated with both starter and probiotic strains (F5B and F8B). In particular, sample F8B at 60 and 120 days of fermentation were grouped together, exhibiting a positive correlation with alcohols and acids and a negative correlation with phenols and aldehydes (Figure 4). Evaluating sample F5B, it is possible to assert that VOCs formation was strongly influenced by the fermentation time. In fact, the sample F5B at 120 days revealed a distinct VOCs profile, displaying a positive correlation with esters, acids, and phenols. Similarly, spontaneous fermentation samples (F5C and F8C) were grouped based on fermentation time, showing a divergent VOCs profile through the fermentation.

Figure 4

Heat map of correlation between VOCs and F5 (A-D) and F8 (A-D) brine samples at 60 and 120 days of fermentation. The colors of the scale bar denote the nature of the correlation, with 1 indicating a perfectly positive correlation (green) and −1 indicating a perfectly negative correlation (red) between VOCs and brine samples.

Sensory Data

Table 5 shown results of sensory analysis. Overall, none negative sensation was perceived, as deduced by the low scores attributed by panelist to these descriptors. No statistically significant differences were achieved among samples for hardness, fibrousness, and crunchiness. Among gustatory descriptors, higher scores for acidity were attributed to un-inoculated brine samples at both 5 and 8% of NaCl (F5C and F8C), while higher bitterness score was observed in samples without the addition of the β-glucosidase L. plantarum strain (F5C, F5D, F8C, and F8D). Finally, F5B and F8B samples received higher scores for the overall acceptability descriptor.

Table 5

Sensory data obtained for the evaluation of fruits in the different treatments assayed.

	Descriptors
	Abnormal fermentation							Gustatory sensations			Kinaesthetic sensations			Overall acceptability
Samples	Musty	Rancid	Cooking effect	Soapy	Metallic	Earthy	Winey-vinegary	Acidity	Saltiness	Bitterness	Hardness	Fibrousness	Crunchiness
F5A	1.1 ± 0.18a	0.6 ± 0.29a	0.6 ± 0.22a	0.2 ± 0.14a	0.6 ± 0.26a	1.6 ± 0.12a	1.3 ± 0.24a	2.7 ± 0.18a	5.4 ± 0.11a	2.3 ± 0.09a	6.4 ± 0.24b	2.1 ± 0.06a	7.2 ± 0.26a	7.5 ± 0.04a
F5B	1.1 ± 0.37a	0.5 ± 0.43a	0.6 ± 0.16a	0.3 ± 0.11a	0.8 ± 0.21a	1.5 ± 0.29a	1.2 ± 0.28a	2.5 ± 0.26a	5.3 ± 0.33a	2.0 ± 0.14a	6.0 ± 0.21a	2.8 ± 0.45a	7.5 ± 0.19a	9.8 ± 0.41b
F5C	1.3 ± 0.21a	0.6 ± 0.34a	0.8 ± 0.24a	0.4 ± 0.21a	0.6 ± 0.09a	1.6 ± 0.26a	1.0 ± 0.31a	7.9 ± 0.12b	5.4 ± 0.25a	6.7 ± 0.24b	5.9 ± 0.33a	2.2 ± 0.28a	7.6 ± 0.21a	6.9 ± 0.11a
F5D	1.1 ± 0.38a	0.5 ± 0.26a	0.7 ± 0.26a	0.3 ± 0.04a	0.7 ± 0.19a	1.6 ± 0.61a	0.8 ± 0.21a	2.9 ± 0.21a	5.8 ± 0.34a	6.9 ± 0.29b	6.3 ± 0.25a	2.5 ± 0.36a	7.7 ± 0.28a	6.7 ± 0.32a
F8A	1.1 ± 0.26a	0.4 ± 0.22a	0.5 ± 0.18a	0.3 ± 0.04a	0.5 ± 0.24a	1.4 ± 0.58a	1.2 ± 0.39a	2.7 ± 0.61a	5.4 ± 0.10a	2.5 ± 0.09a	6.2 ± 0.18a	2.7 ± 0.29a	7.6 ± 0.35a	7.6 ± 0.09a
F8B	1.3 ± 0.24a	0.6 ± 0.28a	0.8 ± 0.23a	0.2 ± 0.35a	0.5 ± 0.22a	1.4 ± 0.29a	0.9 ± 0.15a	2.6 ± 0.86a	5.6 ± 0.06a	2.6 ± 0.32a	5.9 ± 0.24a	2.1 ± 0.43a	7.5 ± 0.32a	9.3 ± 0.45b
F8C	1.4 ± 0.27a	0.4 ± 0.21a	0.5 ± 0.26a	0.4 ± 0.25a	0.7 ± 0.13a	1.5 ± 0.54a	1.3 ± 0.44a	8.8 ± 0.37c	5.7 ± 0.21a	7.3 ± 0.43b	6.2 ± 0.05a	2.7 ± 0.51a	7.6 ± 0.33a	6.4 ± 0.42a
F8D	1.4 ± 0.32a	0.3 ± 0.35a	0.7 ± 0.28a	0.4 ± 0.23a	0.8 ± 0.31a	1.4 ± 0.61a	1.3 ± 0.43a	2.9 ± 0.53a	5.7 ± 0.34a	7.2 ± 0.32b	6.4 ± 0.09a	2.5 ± 0.44a	7.8 ± 0.31a	6.8 ± 0.38a

a–c: for each descriptor, in the same column, with different superscript letters are significantly different (P < 0.05).

Discussion

A current challenge in the processing technology of table olives is the selection of starter cultures able to fasten and safely drive the fermentation process. In contrast to industrial starter cultures, autochthonous strains, that naturally dominate spontaneous fermentation, tend to have high metabolic capacities, which can beneficially affect the quality of the final product. In addition, it was already established that the microbial dynamics through fermentation is influenced by the technology applied (e.g., salt reduction). One of the most widely employed strategies to reduce sodium content in table olives is the use of NaCl substitutes, which can be added alone or in combination with other salts (Bautista-Gallego et al., 2013a). Few studies have evaluated the possibility to setup low NaCl table olives without any salt replacement. Based on our previously reported data (Randazzo et al., 2017; Pino et al., 2018a), in the present study a wild β-glucosidase positive strain was used both as debittering and as driven agent during olive fermentations at lowered salt content (5%). It is well-known that β-glucosidase enzyme is important for oleuropein hydrolysis and, among lactobacilli, L. plantarum species has been successfully used as starter for its strong ability to break the glycosidic bond of oleuropein (Ciafardini et al., 1994; Tataridou and Kotzekidou, 2015) and for its high versatility. Overall, our data revealed that all brine samples reached a pH value ≤ 4.3 and exhibited a good acidification rate, indicating the success of the fermentation and ensuring the microbiological safety of the final product, in accordance to other researches (Corsetti et al., 2012; Martorana et al., 2017). In particular, samples inoculated with the β-glucosidase positive strain exhibited a more pronounced reduction of the fermentation time, with a higher content of hydroxytyrosol, tyrosol, and verbascoside compounds from 30 days of fermentation than un-inoculated ones, according to other studies (Romero et al., 2004; Ben Othman et al., 2009; Pistarino et al., 2013; Kaltsa et al., 2015). In addition, the autochthonous strain exhibited a better adaptation/growth rate in brine samples at 5% of salt. It is well-established that autochthonous strains are generally more adapted to harsh conditions of raw material than allochthonous ones, and, therefore, to dominate the microbiota, driving the fermentation and counteracting spoilage microorganisms (Di Cagno et al., 2008; Bevilacqua et al., 2015). Microbiological data indicated a significant reduction of Enterobacteriaceae starting from 30 days of fermentation, with an improvement of the safety of the final product, as previously reported (Pino et al., 2018a). It is interesting to point out that results revealed a high occurrence of yeasts as part of table olive natural microbiota, coexisting with LAB during the whole fermentation process (Arroyo-López et al., 2008, 2012a,c), which could be related to the geographic area, and cultivars (Bleve et al., 2014). Our data registered an unusual yeast count at the end of the process, higher than Spanish-style and Sicilian-style table olives, which generally reached value of 4-5- log CFU/ml. This could be linked to the processing technology applied in the present study. Yeasts favor bacteria growth, enhancing lactic acid production to inhibit spoilage microorganisms and affect flavor and texture of the final products (Arroyo-López et al., 2008, 2012b; Bevilacqua et al., 2013). As reported in a recent review, Candida boidinii, Debaryomyces hansenii, and Pichia membranifaciens were revealed as the most geographically diffused species (Campus et al., 2018). Evaluating yeast behavior, although it is noteworthy that yeast development is related to high salt level and phenolic compounds or low pH. In the present study, W. anomalus, and C. boidinii were the species mainly detected in brines processed at 5% of salt, whereas C. diddensae and M. guilliermondii were mainly revealed in brines at 8% NaCl. Several studies reported strong β-glucosidase activity for W. anomalus species (Bautista-Gallego et al., 2011b; Arroyo-López et al., 2012c; Romero-Gil et al., 2013; Bonatsou et al., 2015), and strong lipase and esterase activities for C. boidinii species, which positive impacts to fruity and olive flavor (Hernández et al., 2007; Bautista-Gallego et al., 2011b; Arroyo-López et al., 2012c; Pereira et al., 2015). In contrast to previously published data (Hurtado et al., 2008; Bautista-Gallego et al., 2011b), C. diddensae, which is generally associated to the early stage of fermentation, was detected at the highest frequency at 120 days. Focusing on LAB population, L. plantarum, L. pentosus, and L. paracasei were the main species found in all brine samples, confirming both their key role in table olive fermentation and biofilm formation with yeasts. In addition, a high survivability of the potential probiotic L. paracasei N24 strain was depicted in the final products, mainly in samples at 5% of NaCl. This evidence confirms its suitability to growth in harsh environment, such as brines, and that table olives are able to support probiotic survival (Lavermicocca et al., 2005; Rodríguez-Gómez et al., 2017; Pino et al., 2018a). In fact, nutrients and prebiotics released into the brines favor the biofilm formation, protecting bacteria from acidic environment and enhancing their passage through human gastrointestinal (GI) tract (De Bellis et al., 2010; Ranadheera et al., 2010; Arroyo-López et al., 2012a; Blana et al., 2014; Rodríguez-Gómez et al., 2014a,b, 2017; Grounta et al., 2015). It is interesting to point out that the addition of the potential probiotic strain at 60 days of fermentation in brine samples processed at low salt content and with starter, significantly modified the VOCs pattern. In particular, compounds responsible for floral and fruity notes, such as phenylethyl alcohol and methyl 2-methylbutanoate, highly increased, while ethanol and isoamyl-alcohol significantly deceased compared to un-inoculated samples. The high content of alcohols in un-inoculated brine samples could be related to yeast metabolic activities (Bleve et al., 2014; Randazzo et al., 2017). This evidence was in accordance to sensory data since panelists attributed the higher score to the bitterness and acid descriptors in un-inoculated samples. Finally, data obtained from correlation between VOCs and brine samples differently treated revealed that the VOCs formation was mainly influenced by the starter and/or probiotic addition instead of salt content.

Conclusion

The effects of a sequential inoculum of β-glucosidase positive and potential probiotic strains on the fermentation of Sicilian table olives were investigated. Remarkably, results demonstrate that the technology applied, based on the sequential inoculum and the brines fermentation at low salt content, without any salt replacement, did not increase the risk of microbial spoilage, nor the overgrowth of foodborne pathogens. Indeed, the composition and the dynamics of brine microbiota, mainly constituted by LAB and yeasts consortium, significantly affected the composition of the VOCs and the sensorial traits of the final products, which were confirmed by a panel of trained assessors. Hence, the results of the present study are promising, suggesting the possibility to formulate table olives with reduced salt content.

Author Contributions

AP, FR, AT, AV, and LS performed the experiments and analyzed the data. AP, JB-G, and CR wrote the manuscript. CR, JB-G, and CC designed the study. CR, JB-G, and FA-L contributed to data interpretation. All authors revised the manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.