Microbial and Chemical Characterization of Natural-Style Green Table Olives from the Gordal, Hojiblanca and Manzanilla Cultivars

Citation: Ruiz-Barba, J.L.; Sánchez, A.H.; López-López, A.; CortésDelgado, A.; Montaño, A. Microbial and Chemical Characterization of Natural-Style Green Table Olives from the Gordal, Hojiblanca and Manzanilla Cultivars. Foods 2023,12, 2386. https://doi.org/10.3390/ foods12122386 Academic Editor: Barbara Lanza Received: 15 May 2023 Revised: 13 June 2023 Accepted: 14 June 2023 Published: 15 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article Microbial and Chemical Characterization of Natural-Style Green Table Olives from the Gordal, Hojiblanca and Manzanilla Cultivars JoséLuis Ruiz-Barba, Antonio Higinio Sánchez , Antonio López-López , Amparo Cortés-Delgado and Alfredo Montaño * Food Biotechnology Department, Instituto de la Grasa (CSIC), Carretera de Utrera, Km. 1, 41013 Seville, Spain; [email protected] (J.L.R.-B.); [email protected] (A.H.S.); [email protected] (A.L.-L.); [email protected] (A.C.-D.) *Correspondence: [email protected]; Tel.: +34-954-611-550; Fax: +34-954-616-790 Abstract: Microbial and biochemical changes in the brine during the spontaneous fermentation of Gordal, Hojiblanca and Manzanilla olive cultivars processed according to the natural style were monitored. The microbial composition was assessed through a metagenomic study. Sugars, ethanol, glycerol, organic acids and phenolic compounds were quantified by standard methods. In addition, the volatile profiles, contents of phenolic compounds in the olives and quality parameters of the final products were compared. Fermentation in Gordal brines was conducted by lactic acid bacteria (mainly Lactobacillus and Pediococcus) and yeasts (mainly Candida boidinii,Candida tropicalis and Wickerhamomyces anomalus). In Hojiblanca and Manzanilla brines, halophilic Gram-negative bacteria (e.g., Halomonas,Allidiomarina and Marinobacter) along with yeasts (mainly, Saccharomyces) were responsible for the fermentation. Higher acidity and lower pH values were reached in Gordal brines compared to Hojiblanca and Manzanilla. After 30 days of fermentation, no sugars were detected in Gordal brine, but residual amounts were found in the brines from Hojiblanca (<0.2 g/L glucose) and Manzanilla (2.9 g/L glucose and 0.2 g/L fructose). Lactic acid was the main acid product in Gordal fermentation, whereas citric acid was the predominant organic acid in the Hojiblanca and Manzanilla brines. Manzanilla brine samples showed a greater concentration of phenolic compounds than Hojiblanca and Gordal brines. After a 6-month fermentation, Gordal olives were superior compared to the Hojiblanca and Manzanilla varieties regarding product safety (lower final pH and absence of Enterobacteriaceae), content of volatile compounds (richer aroma), content of bitter phenolics (lower content of oleuropein, which resulted in less perceived bitterness) and color parameters (more yellow and lighter color, indicating a higher visual appraisal). The results of the present study will contribute to a better understanding of each fermentation process and could help to promote natural-style elaborations using the above-mentioned olive cultivars. Keywords: green table olives; Manzanilla cultivar; Gordal cultivar; Hojiblanca cultivar; microbiota; spontaneous fermentation; metagenomics 1. Introduction Table olives are one of the most popular fermented foods in the Mediterranean area, mainly Spain, Italy and Greece. According to the Association of Exporters and Industrialists of Table Olives (ASEMESA), the average production of table olives in Spain in the last five seasons was 561,100 tons, which represents 19.7% of the world production of this product [ 1 ]. The most important olive cultivars dedicated to green table olive production in Spain are Hojiblanca (46% of the total Spanish production), Manzanilla (36%) and Gordal (7%) [ 2 ]. The Hojiblanca (Olea europaea arolensis) olive is located in the provinces of Cordoba and Malaga and, to a lesser extent, in those of Seville and Granada. It obtained its name from the whitish color on the underside of its leaves and has double aptitude, presenting good characteristics for the production of olive oil and table olives. The fruit has a regular Foods 2023,12, 2386. https://doi.org/10.3390/foods12122386 https://www.mdpi.com/journal/foods Foods 2023,12, 2386 2 of 18 shape and its size varies greatly, ranging from 230 to 700 olives/kg. The Manzanilla ( Olea europaea pomiformis ) olive, also known as Manzanillo, is cultivated widely throughout Spain, mainly in the province of Seville. This variety of olive is characterized by its high productivity and harvest, which is conducted by hand, to avoid any damage to the fruit. The Gordal (Olea europaea regalis) olive, internationally known as Sevillano, is mainly cultivated in Andalusia, particularly in the province of Seville. It is a vigorous cultivar appreciated mainly for the size of its fruits that reach an average weight of 12.5 g [ 3 ]. The main characteristics of these varieties are: Hojiblanca: size, highly variable ( 230–700 olives/kg ); flesh-to-stone ratio, 5:1 to 6.5:1; shape, regular; oil content, 23–29% of fresh weight (f.w.); Manzanilla: size, medium (200–280 olives/kg); flesh-to-stone ratio, 6:1; shape, apple-like; oil content, around 15% f.w.; and Gordal: size, large (100–120 olives/kg); flesh-to-stone ratio, 7.5:1; shape, ellipsoidal: oil content, <10% f.w. [4]. The above-mentioned olive cultivars, when marketed as green table olives, are mainly processed according to the Spanish-style method, which includes an alkaline treatment and washing with water prior brining. Surprisingly, the industrial production of naturalstyle olives is relatively scarce, in spite of this type of table olives being very appreciated by consumers and being popular in different areas using autochthonous olive cultivars (e.g., Gordal in the province of Seville, Arbequine in the region of Catalonia and Aloreña in the province of Málaga). In addition, the natural-style table olive is the only one recognized as an organic product, since it only uses olives, water and common salt as raw materials; it is nutritionally superior to the Spanish style due to its higher content of poliphenols, sterols, total fatty acids fractions and fiber [5]. In general, the natural fermentation process is conducted by the indigenous microbiota, mainly yeast, without the addition of starters [ 6 ]. Physicochemical conditions and olive cultivar strongly affect the growth of lactic acid bacteria (LAB) in the natural-style table olives [ 7 ]. Recently, we made a comprehensive study of the microbial and biochemical changes during the spontaneous fermentation of the natural-style green olives from the Manzanilla cultivar [ 8 ]. This cultivar is known to present relatively high concentrations of polyphenols (mainly represented by oleuropein, which is responsible for the bitterness taste), which appears to be the main reason for the usual absence of LAB growth throughout the fermentation in this type of elaboration [ 9 ]. Furthermore, this fact makes the fermentation time required for an acceptable debittering particularly long (>8 months), since the debittering process relies on diffusion phenomena and on the presence of yeast species with β -glucosidase and esterase activities, but it lacks the contribution of LAB species with such enzymatic activities [ 10 ]. In this sense, the Gordal and Hojiblanca cultivars could be more adequate for the natural-style elaboration, as these cultivars present a lower content of oleuropein than Manzanilla [ 11 ]. However, to the best of our knowledge, no detailed study on the natural fermentation of these two varieties has been published to date. In addition to polyphenols, the profile of volatile compounds, generally related with the aroma in foodstuffs, is important to evaluate the quality of the final product. Hence, different research groups included the analysis of volatile compounds in their studies on table olives from different olive cultivars in recent years [ 9 , 12 – 19 ]. The objectives of the present work are to study the microbial community dynamics and biochemical changes in the brines of the natural-style fermentation using the cultivars Gordal, Hojiblanca and Manzanilla, and to compare the main characteristics of the final products, including the contents of volatile compounds, polyphenols and quality parameters. The results of the present study will contribute to a better understanding of each fermentation process and could help to promote the natural-style elaboration using the above-mentioned olive cultivars. This can have a positive impact both on the consumer (consumption of organic food with a high content of beneficial nutrients) and on the industry (lower production costs and a smaller volume of wastewater than the Spanish-style elaboration). Foods 2023,12, 2386 3 of 18 2. Materials and Methods 2.1. Olive Processing The olives (Gordal, Hojiblanca and Manzanilla cultivars) were harvested in Arahal (Seville province, Spain) at their mature-green stage (Gordal, on 14 September; Manzanilla, on 28 September; and Hojiblanca, on 14 October 2021–2022 season) and transported to our laboratories to be processed. Olives from each cultivar were subjected to quality control to remove damaged fruits and then placed into cylindrical plastic vessels (3.3 kg of fruits plus 2.1 L of liquid each). Then, the olives were directly immersed in a brine containing 10% (w/v) of NaCl. For each cultivar, fermentations were conducted in duplicate at ambient temperature, which was in the range of 8–22 ◦C. 2.2. Sampling Brine samples from each vessel were taken during fermentation to control the main chemical and microbiological characteristics. Microbial DNA extraction from the fermenting brines were conducted at days 60, 120 and 180. Non-volatile compounds, including sugars, organic acids and phenolic compounds, in brine were analyzed at days 30, 60, 120 and 180. In addition, at the final sampling, brines were analyzed for volatile compounds and olive samples were analyzed for phenolic compounds, color parameters and firmness. 2.3. Chemical and Microbiological Analyses in Brine 2.3.1. Physicochemical Analyses The titratable acidity, pH, combined acidity and salt content of the olive brines were determined as described in [ 20 ]. A Metrohm 670 titroprocessor (Herisau, Switzerland) was used for the measurements of pH, titratable acidity and combined acidity. The titratable acidity was determined by titrating up to pH 8.3 with 0.2 N NaOH and expressed as g/100 mL of lactic acid. The combined acidity was determined with 2 N HCl until the pH value reached 2.6 and was expressed as the equivalent of NaOH per liter. Sodium chloride was determined by titration with silver nitrate. 2.3.2. Analysis of Sugars, Organic Acids, Ethanol and Glycerol Major sugars in brines (glucose, fructose, mannitol and sucrose) were determined by HPLC following a previous method [ 21 ] with modifications. A brine sample (0.25 mL) and 0.75 mL of internal standard (xylitol, 2 g/L) were applied to an SPE cartridge (Sep-Pak Vac, tC18, 500 mg, Waters) that had been previously conditioned with methanol (2 mL) and water (10 mL). The SPE eluate was collected in a test tube, and then 1 mL water was applied to the cartridge and the eluate was collected in the same test tube. The solution was then desalted by adding 1 g of a strongly acidic resin (Amberlite IR-120, Sigma-Aldrich, St. Louis, MO, USA) and 1 g of a weakly basic resin (Amberlite IRA-96, Sigma-Aldrich, St. Louis, MO, USA). Samples were shaken occasionally during a 60 min desalting period. Finally, an aliquot of the solution was filtered through a 0.45 µ m nylon filter, and 50 µ L of the filtrate was injected into the chromatograph. The separation was performed on a Rezex RCM Monosaccharide column (300 × 7.8 mm i.d., Phenomenex, Torrance, CA, USA) at 80 ◦ C, using deionized water as the mobile phase at a flow rate of 0.6 mL/min and a refractive index detector. Lactic acid, acetic acid, citric acid, succinic acid, ethanol and glycerol were quantified by HPLC following a previous method [ 22 ] with modifications. The separation was conducted on a Rezex ROA-Organic Acid H+ (300 × 7.8 mm i.d.) column (Phenomenex, Torrance, CA, USA) held at 50 ◦ C, using 0.005 M H 2 SO 4 as the mobile phase at a flow rate of 0.6 mL/min and a refractive index detector. Brine samples (20 µ L) were directly injected into the chromatograph after filtration through a 0.45 µ m nylon filter. Concentrations were calculated by the comparison of the peak areas with those of the external standards for each compound. All samples were analyzed in duplicate. Foods 2023,12, 2386 4 of 18 2.3.3. Analysis of Phenolic Compounds Phenolic compounds (oleuropein, hydroxytyrosol, tyrosol and verbascoside) in brine were analyzed by HPLC. A mixture of 0.2 mL of brine, 0.1 mL of internal standard (100 mg/L gallic acid) and 0.2 mL of water was filtered through a 0.45 µ m pore size nylon filter and an aliquot (20 µ L) was injected into the chromatograph. The HPLC system consisted of a Waters 2695 separation module (Waters Assoc., Milford, MA, USA) connected to a Waters 996 photodiode array detector and controlled with Empower 2 software (Waters). The chromatographic conditions were as follows: column, Luna Phenyl-Hexyl (5 µ m, 250 × 4.6 mm, Phenomenex, Torrance, CA, USA); column temperature, 35 ◦ C; flow rate, 1 mL/min; mobile phase, (A) water adjusted to pH 2.0 with phosphoric acid and (B) methanol; gradient solvent program, 0–10 min, 90% A to 70% A; 10–30 min, 70% A; 30–40 min, 70% A to 60% A; 40–45 min, 60% A; 45–50 min, 60% A to 50% A; 50–66 min, 50% A to 10% A; and 66–67 min, 10% A to 90% A. The detection of phenolic compounds was conducted at 280 nm. All phenolic compounds were identified by comparing their retention times and UV-visible spectra to those of authentic standards. For quantification by the external standard method, the calibration curves of each phenolic compound in methanol were used. 2.3.4. Analysis of Volatile Compounds The volatile compounds were determined by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME/GC-MS). Briefly, 2 mL of brine was placed into a 15 mL glass vial with 20 µ L of internal standard (6-chloro-2hexanone, 20 mg/L) and volatiles were extracted, identified and quantified according to the method described in [ 9 ] with several modifications (1 cm–80 µ m Divinylbenzene/Carbon Wide Range/Polydimethylsiloxane (DVB/CWR/PDMS) fiber (Agilent Technologies, Santa Clara, CA, USA), SPME autosampler (PAL3, Agilent), desorption time of 5 min). The volatile compounds were semi-quantified by the comparison of the peak areas to those of the internal standard (6-chloro-2-hexanone). Each sample was analyzed in duplicate. 2.3.5. Microbial Counts during Fermentations The populations of the main groups of microorganisms were determined by plating the brines and their decimal dilutions (in 0.9% NaCl) on the appropriate solid media with a spiral plater (Easy Spiral Dilute, Interscience, Saint Nom la Bretèche, France). The culture media used were de Man–Rogosa–Sharpe agar (MRS, Oxoid, Basingstoke, UK) supplemented with 0.02% (w/v) sodium azide (Sigma-Aldrich, St. Louis, MO, USA) and 0.05% (w/v) L-cysteine (AppliChem GmbH, Darmstadt, Germany) for LAB; glucoseyeast extract agar containing oxytetracycline (0.01% w/v; AppliChem; OGYE) for yeast; VRBG agar (Laboratorios Conda S.A., Torrejón de Ardoz, Spain) for Enterobacteriaceae; and plate count agar (PCA, Labkem, Premiàde Dalt, Spain) for mesophilic aerobic bacteria (MAB). MRS plates were incubated under anaerobic conditions using a DG250 Anaerobic Workstation (Don Whitley Scientific Ltd., Shipley, West Yorkshire, UK), with a gas mixture consisting of 10% H 2 -10% CO 2 -80% N 2 at 30 ◦ C for 72 h. OGYE plates were incubated aerobically at 25 ◦ C for 72 h. VRBG plates were incubated aerobically at 37 ◦ C for 24 h. PCA plates were incubated aerobically at 22 ◦ C for 72 h. The resulting numbers of colonyforming units were counted with a Scan 500 (Interscience, Saint-Nom-la-Bretèche, France) colony counter. The detection limit was established in 10 CFU/mL. 2.3.6. Metagenomic Analysis At each sampling point, 200 mL of the fermenting olive brines were collected and centrifuged to obtain a microbial pellet. The pellets were washed with 6% (w/v) NaCl and centrifuged again to obtain a final pellet, which was stored at − 30 ◦ C until use. Total DNA from the preserved, defrosted pellets was extracted and purified using the DNeasy PowerFood Microbial Kit (Qiagen, Germantown, MD, USA). The metagenomic analyses Foods 2023,12, 2386 5 of 18 were conducted as described in a previous work [ 8 ]. The distribution of reads in the quality check protocol and DADA2 routine is shown in Table S1. 2.4. Analyses in Olive Fruit 2.4.1. Analyses of Phenolic Compounds The polyphenols in pulp were extracted by liquid–liquid extraction following the procedure of McDonald et al. [ 23 ] with some modifications. A homogenized olive pulp (10 g) was placed into a beaker and 50 mL of methanol–water (60:40, v/v) was added. After 2 min of intense homogenization with Ultra-Turrax (IKA Labortechnik, Staufen, Germany), the paste was filtered and washed several times with methanol–water (60:40, v/v) until 100 mL of the extract was collected. An aliquot of the extract (20 mL) was extracted with hexane (2 × 20 mL) to remove oil, and the hydroalcoholic extract was then filtered through a nylon filter (0.45 µ m). An aliquot (0.2 mL) of the filtrate was used for analysis by HPLC, for the analysis in brine mentioned above. In addition, this filtrate was analyzed for total phenols using a Folin–Ciocalteu reagent as reported in [ 24 ], and the results are expressed as gallic acid equivalents (mg/kg pulp). 2.4.2. Quality Parameters The color of the olives was measured using a Color-View model 9000 spectrophotometer (BYK-Gardner, Inc., Silver Spring, MD, USA) with a measurement area of 11 mm diameter, 45 ◦ circumferential illumination and an observation angle of 0 ◦ . All measurements were conducted on the CIE 1976 L*a*b* scale using illuminating conditions CIE type C, 10 ◦ observer. For each sample, the results corresponded to the mean of 20 measurements, each made on one olive. Chroma was calculated as [(a*) 2 + (b*) 2 ] 1/2 . Hue angle was calculated as tan−1(b*/a*). The color index iwas obtained by the following equation: i= (4R635 + R590 −2R560)/3 , where R635, R590 and R560 are the values of reflectance at 635, 590 and 560 nm, respectively. The firmness of olives was determined using a Kramer shear compression cell coupled with TA.TXplus Texture Analyser (Stable Micro System, Surrey, UK). The cross-head speed was 200 mm/min. The firmness was the mean of 10 replicate measurements, each of which was performed on three pitted olives, and expressed as Newton/100 g pitted olives. The sensory analysis of the olive bitterness was evaluated by 14 members of the sensorial panel of the Instituto de la Grasa staff. The perceived bitter taste was quantified on a non-structured line scale from 1 (absence of bitterness) to 11 (strong presence of the bitter taste). The following caffeine solutions were used as reference: 40 mg/mL and 100 mg/mL, corresponding to 4 5 and 7 5 scores on the unstructured scale. Each sample ( 3–5 olives ) was served to panelists in a cupping glass, which were coded with three randomly chosen digits, in separate booths in conditions of normal daylight and room temperature. Between the tests, the panelists were provided with tap water to cleanse the palate. Samples were analyzed in triplicate. The results corresponded to mean scores (panel average). 2.5. Statistical Analyses A one-way analysis of variance (ANOVA, Duncan’s test) was applied to the collected data for microbial counts, volatile compounds, phenolic compounds, color parameters and firmness in order to determine differences among cultivars or sampling times. Significant differences were determined at the p< 0.05 level. The ANOVA was performed using SPSS software v. 23.0 (IBM Corp., Armonk, NY, USA). 3. Results and Discussion 3.1. Changes in Microbiological Counts and Physicochemical Characteristics during Fermentation The microbiological counts are shown in Table 1.Enterobacteriaceae were not detected in any sample, while LAB growth was only detected in the fermenting brines of the Gordal cultivar. In contrast, yeasts were present in all samples, being the dominant microbial group Foods 2023,12, 2386 6 of 18 in the brines of the Hojiblanca and Manzanilla cultivars. In contrast, LAB were dominant in the Gordal brines, so that after 60 days, their populations reached 6.2 ± 0.4 log CFU/mL and hardly changed for the rest of the fermentation. The absence of LAB growth in the Manzanilla brines was somewhat expected, as it is supported by previous studies that point to the higher content in antimicrobial compounds derived from phenolics as the main cause [ 9 , 25 – 27 ]. In the case of the Hojiblanca variety, seasonal variations in the content of essential nutrients and/or the presence of high concentrations of natural inhibitory compounds (mainly phenolics) in the fruits used for the experiment could contribute to the absence of LAB growth in this cultivar. It has been reported that the phenolic content of the olive fruits increases from the Gordal variety (low) to Hojiblanca (medium) and Manzanilla (high) [ 11 ]. The yeast population showed similar counts in the three fermentations after 60 days (around 5.7 log CFU/mL) with a slight decrease (1 log in Gordal and lower than 1 log in Manzanilla and Hojiblanca) afterwards. The counts in PCA for MAB were virtually the same as those of the dominant group for each olive variety: LAB in Gordal or yeast in the Hojiblanca and Manzanilla brines, indicating that these dominant microorganisms were the main responsible microorganisms for the actual counts observed in this non-selective culture medium, which allows for the growth of both bacteria and yeast on it. Table 1. Microbial counts (log CFU/mL) in the brines during the fermentation of the Gordal, Hojiblanca and Manzanilla cultivars processed in the natural style a. Gordal Hojiblanca Manzanilla 60 days Yeast 5.6 ±0.5 a,B 5.6 ±0.3 a,B 5.7 ±0.2 a,B LAB 6.2 ±0.4 A nd nd MAB 6.1 ±0.3 b,A 5.4 ±0.0 a,B 5.5 ±0.0 a,A 120 days Yeast 4.6 ±0.1 a,A 5.4 ±0.1 b,B 5.5 ±0.2 b,AB LAB 6.3 ±0.1 A nd nd MAB 6.4 ±0.2 b,AB 5.3 ±0.0 a,B 5.5 ±0.3 a,A 180 days Yeast 4.7 ±0.1 a,A 5.0 ±0.0 b,A 5.3 ±0.1 c,A LAB 6.7 ±0.1 B nd nd MAB 6.7 ±0.1 c,B 4.9 ±0.1 a,A 5.2 ±0.1 b,A a Values are means ± SD of duplicate fermentations, each analyzed in duplicate (n= 4). Means in the same row labelled with different lower-case letters are significantly different (p< 0.05). For a given microorganism group, means in the same column labelled with different capital letters are significantly different (p< 0.05). nd = not detected (<10 CFU/mL). The changes in the physicochemical characteristics of brines are shown in Table 2. Higher acidity and lower pH values were reached in the Gordal brines compared to Hojiblanca and Manzanilla (0.91% acidity–pH 3.46 in Gordal versus 0.34–4.66% and 0.42–4.61% in Hojiblanca and Manzanilla, respectively, after 180 days of fermentation). Gordal brines showed these acidity and pH values due to the substantial growth of LAB from a very early stage in the fermentation process, in contrast to their absence in the brines of the two other varieties. The combined acidity, which refers to the organic acid salts present in brine, was significantly lower (e.g., 2-fold lower at day 30) in Gordal than in Hojiblanca or Manzanilla. As a consequence, the Gordal brine presented a lower buffer capacity, which explains that, with the same titratable acidity, the pH obtained is lower in this olive variety (see data at day 60). The concentration of NaCl in the brines reached final values of 5.6–6.0% NaCl in the three olive cultivars. As the required minimum salt concentration for this product is 6.0% [ 28 ], the addition of salt might be required in some cases to guarantee product safety. Foods 2023,12, 2386 7 of 18 Table 2. Physico-chemical characteristics of the brines during the fermentation of Gordal, Hojiblanca and Manzanilla cultivars processed in the natural style a. Gordal Hojiblanca Manzanilla 30 days pH 4.76 ±0.03 a 4.74 ±0.05 a 4.75 ±0.04 a Titratable acidity (% lactic acid) 0.13 ±0.00 a 0.21 ±0.00 b 0.23 ±0.01 c Combined acidity (N) 0.023 ±0.000 a 0.047 ±0.000 b 0.047 ±0.004 b Salt content (% NaCl) 6.52 ±0.09 b 5.74 ±0.23 a 6.37 ±0.01 b 60 days pH 3.77 ±0.03 a 4.74 ±0.01 b 4.70 ±0.04 b Titratable acidity (% lactic acid) 0.36 ±0.01 a 0.36 ±0.01 a 0.31 ±0.04 a Combined acidity (N) 0.030 ±0.001 a 0.058 ±0.000 b 0.062 ±0.006 b Salt content (% NaCl) 5.83 ±0.02 a -b6.14 ±0.08 b 120 days pH 3.59 ±0.02 a 4.68 ±0.00 b 4.61 ±0.04 b Titratable acidity (% lactic acid) 0.72 ±0.02 c 0.37 ±0.00 a 0.47 ±0.01 b Combined acidity (N) 0.042 ±0.001 a 0.068 ±0.000 b 0.071 ±0.008 b Salt content (% NaCl) - 5.66 ±0.06 a - 180 days pH 3.46 ±0.00 a 4.66 ±0.01 b 4.61 ±0.04 b Titratable acidity (% lactic acid) 0.91 ±0.01 c 0.34 ±0.01 a 0.42 ±0.02 b Combined acidity (N) 0.048 ±0.000 a 0.072 ±0.000 b 0.073 ±0.008 b Salt content (% NaCl) 5.56 ±0.01 a 5.58 ±0.06 a 5.98 ±0.18 b a Values are means ± standard deviation of two biological replicates, each analyzed in duplicate. Means in the same row labelled with different letters are significantly different (p< 0.05). b= not analyzed. It can be also noted that the final pH values in Hojiblanca and Manzanilla olives were slightly over the pH required by the International Olive Council (IOC) normative [ 28 ] (i.e., pH < 4.3), although the final titratable acidity values were above the required value (0.3% as lactic acid). Therefore, in order to improve the product safety by reaching pH values lower than 4.3, the addition of lactic acid into the initial brine, as previously conducted by Aponte et al. [ 25 ], or into the fermenting brine at the beginning of the fermentation would be advisable in natural-style green olives from the Hojiblanca and Manzanilla cultivars. However, such acidification could affect the yeast biota [ 29 ]. Further research is needed to confirm this point. 3.2. Changes in Fermentation Substrates and Major End-Products The concentrations of single carbohydrates in raw olives (g/100 g wet pulp) were as follows: 0.2% sucrose, 4.5% glucose, 0.9% fructose and 2.2% mannitol in Gordal; 0.3% sucrose, 2.2% glucose, 0.2% fructose and 1.3% mannitol in Hojiblanca; and 0.4% sucrose, 4.0% glucose, 0.9% fructose and 0.7% mannitol in Manzanilla. These carbohydrates diffused into the brines and were subsequently metabolized during fermentations through the microbial activity. Sucrose was not detected in brine in any case. While mannitol was not consumed in any case, which is in accordance with our own previous studies [ 8 , 9 ], glucose and fructose were metabolized differently by the microbiota growing in the brines of each olive variety. As shown in Figure 1a, the highest consumption rate of these sugars was observed in the Gordal variety, so that no glucose or fructose was detected after 30 days of fermentation. At this sampling time, small amounts of glucose (<0.2 g/L) were measured in the Hojiblanca brine, whereas 2.9 g/L glucose and 0.2 g/L fructose were still found in the Manzanilla brine. The presence of LAB at high numbers early in the fermentation of the Gordal variety is most probably the cause for such a quick sugar consumption. Instead, the low levels of sugar observed for the Hojiblanca variety after 30 days of fermentation could be explained by the low initial content of sugars in the fruits in addition to yeast metabolism. Foods 2023,12, 2386 8 of 18 Foods 2023, 12, x FOR PEER REVIEW 8 of 19 days of fermentation. At this sampling time, small amounts of glucose (<0.2 g/L) were measured in the Hojiblanca brine, whereas 2.9 g/L glucose and 0.2 g/L fructose were still found in the Manzanilla brine. The presence of LAB at high numbers early in the fermentation of the Gordal variety is most probably the cause for such a quick sugar consumption. Instead, the low levels of sugar observed for the Hojiblanca variety after 30 days of fermentation could be explained by the low initial content of sugars in the fruits in addition to yeast metabolism. The major organic acids produced in the different fermentations are shown in Figure 1b. Lactic acid was only detected in the Gordal fermentations, being the major end-product, followed by acetic and citric acids, reaching final concentrations of 7.9, 0.8 and 0.3 g/L, respectively. This result clearly reflects the lactic acid fermentation conducted by the LAB of the genera Lactobacillus and Pediococcus detected through the metagenomic study (Figure 2). In contrast, citric acid was the predominant organic acid produced in the Hojiblanca and Manzanilla brines, reaching final concentrations of 1.8 and 1.6 g/L, respectively, whereas lactic acid was detected in trace amounts (<0.02 g/L). In the absence of LAB, explaining the lack of lactic acid production, yeast genera, such as Candida, Pichia and Saccharomyces, detected in high proportions in these brines (Figure 2), are known to produce citric acid [30]. As no acetic acid bacteria were identified in the metagenomic study, acetic acid must be the physiological by-product of the alcoholic fermentation conducted by yeast, such as Saccharomyces [31], which was present in these brines (Figure 2). Finally, succinic acid was found at low concentrations (<0.2 g/L) in all the three olive fermentations. Other major end-products, such as ethanol, reached final concentrations of 4.7, 3.9 and 4.1 g/L in the Gordal, Hojiblanca and Manzanilla brines, respectively (Figure 1c). Glycerol was also detected in these brines at the final concentrations of 0.3, 0.7 and 1.3 g/L, respectively. The formation of ethanol, glycerol, succinic acid, citric acid and acetic acid in the Hojiblanca and Manzanilla olives can be attributed to sugar metabolism by yeasts [32– 34]. However, in the Gordal olives, LAB could also contribute to the formation of acetic acid [35]. Figure 1. Changes in the composition of ( a ) carbohydrates, ( b ) organic acids and ( c ) ethanol and glycerol during the fermentations of Gordal, Hojiblanca and Manzanilla olive cultivars processed in the natural style. Points are means of duplicate fermentations. Where error bars are not visible, determinations were within the range of the symbols on the graph. The major organic acids produced in the different fermentations are shown in Figure 1b. Lactic acid was only detected in the Gordal fermentations, being the major end-product, followed by acetic and citric acids, reaching final concentrations of 7.9, 0.8 and 0.3 g/L, respectively. This result clearly reflects the lactic acid fermentation conducted by the LAB of the genera Lactobacillus and Pediococcus detected through the metagenomic study (Figure 2). In contrast, citric acid was the predominant organic acid produced in the Hojiblanca and Manzanilla brines, reaching final concentrations of 1.8 and 1.6 g/L, respectively, whereas lactic acid was detected in trace amounts (<0.02 g/L). In the absence of LAB, explaining the lack of lactic acid production, yeast genera, such as Candida,Pichia and Saccharomyces, detected in high proportions in these brines (Figure 2), are known to produce citric acid [ 30 ]. As no acetic acid bacteria were identified in the metagenomic study, acetic acid must be the physiological by-product of the alcoholic fermentation conducted by yeast, such as Saccharomyces [ 31 ], which was present in these brines (Figure 2). Finally, succinic acid was found at low concentrations (<0.2 g/L) in all the three olive fermentations. Other major end-products, such as ethanol, reached final concentrations of 4.7, 3.9 and 4.1 g/L in the Gordal, Hojiblanca and Manzanilla brines, respectively (Figure 1c). Glycerol was also detected in these brines at the final concentrations of 0.3, 0.7 and 1.3 g/L, respectively. The formation of ethanol, glycerol, succinic acid, citric acid and acetic acid in the Hojiblanca and Manzanilla olives can be attributed to sugar metabolism by yeasts [32–34] . However, in the Gordal olives, LAB could also contribute to the formation of acetic acid [ 35 ]. Foods 2023,12, 2386 9 of 18 Foods 2023, 12, x FOR PEER REVIEW 9 of 19 Figure 1. Changes in the composition of (a) carbohydrates, (b) organic acids and (c) ethanol and glycerol during the fermentations of Gordal, Hojiblanca and Manzanilla olive cultivars processed in the natural style. Points are means of duplicate fermentations. Where error bars are not visible, determinations were within the range of the symbols on the graph. Figure 2. Evolution of (a) bacteria and (b) fungi in brines from the Gordal, Hojiblanca and Manzanilla cultivars processed in the natural style. Only microbial OTUs with abundances >1% in at least 2 samples were considered. The abundance of OTUs in the 2 biological replicates was averaged. (a) Bacteria (b) Fungi 0 10 20 30 40 50 60 70 80 90 100 60 120 180 Relative abundance (%) Gordal Others Pediococcus Pediococcus ethanolidurans Lactobacillus Time (days) 0 10 20 30 40 50 60 70 80 90 100 60 120 180 Relative abundance (%) Manzanilla Others Pseudomonas Salegentibacter Methylophaga sp. Marinicauda algicola Flavobacteriales Idiomarina Salinicola Chromohalobacter Alcanivorax venustensis Marinobacter Marinimicrobium agarilyticum Aliidiomarina Halomonas Time (days) 0 10 20 30 40 50 60 70 80 90 100 60 120 180 Relative abundance (%) Hojiblanca Others Pseudomonas Salegentibacter Methylophaga sp. Marinicauda algicola Flavobacteriales Kosakonia Klebsiella Idiomarina Enterobacteriaceae Chromohalobacter Alcanivorax venustensis Marinobacter Marinimicrobium agarilyticum Aliidiomarina Halomonas Time (days) 0 10 20 30 40 50 60 70 80 90 100 60 120 180 Relative abundance (%) Gordal Others Pichia membranifaciens Candida diddensiae Wickerhamomyces anomalus Candida tropicalis Candida boidinii Time (days) 0 10 20 30 40 50 60 70 80 90 100 60 120 180 Relative abundance (%) Manzanilla Others Pichia mandshurica Nakazawaea Candida diddensiae Saccharomyces Time (days) 0 10 20 30 40 50 60 70 80 90 100 60 120 180 Relative abundance (%) Hojiblanca Others Penicillium Torulaspora delbrueckii Pichia mandshurica Candida boidinii Saccharomyces Candida diddensiae Nakazawaea Time (days) Figure 2. Evolution of ( a ) bacteria and ( b ) fungi in brines from the Gordal, Hojiblanca and Manzanilla cultivars processed in the natural style. Only microbial OTUs with abundances >1% in at least 2 samples were considered. The abundance of OTUs in the 2 biological replicates was averaged. 3.3. Changes in Phenolic Compounds in Brines during Fermentation The concentrations of the main phenolic compounds (hydroxytyrosol, tyrosol, oleuropein and verbascoside) in brines exhibited notable differences between cultivars during fermentation (Table 3). The Manzanilla brine samples showed a greater concentration of phenolic compounds than the Hojiblanca and Gordal brines. In particular, in Manzanilla, the main phenolic compound throughout fermentation was oleuropein, the bitter-tasting secoroid glucoside, reaching a maximum value of 2262 ± 31 mg/L after 120 days and then slightly decreased by the end of the fermentation. However, oleuropein reached about 100 mg/L in Hojiblanca, while it was undetectable in the Gordal brines. These differences in oleuropein concentrations in brine are consistent with the higher bitterness of Manzanilla compared to other Spanish cultivars, such as Hojiblanca, Gordal and Aloreña [ 36 ]. Hydroxytyrosol predominated throughout the fermentation process in the Hojiblanca and Gordal cultivars, which is in agreement with previous studies using other olive cultivars, Foods 2023,12, 2386 16 of 18 Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods12122386/s1. Table S1: Distribution of reads in the quality check protocol and DADA2 routine of samples of the fermenting brines of olives subjected to the metataxonomy analysis of 16S rRNA and ITS amplicons; Table S2: Number of reads, OTUs, Good’s coverage and different alpha diversity estimators of the metataxonomy analysis of 16S rRNA (Bacteria and Archaea) and ITS (Fungi) amplicons of the fermenting brines from the Gordal, Hojiblanca and Manzanilla cultivars processed in the natural style; Figure S1: Volatile profiles in the fermenting brines of olives from the Gordal, Hojiblanca and Manzanilla cultivars processed in the natural style at the end of fermentation (180 days). Values are means of 2 biological replicates. Error bars represent standard deviations (n= 4). Author Contributions: Conceptualization, A.M. and J.L.R.-B.; Formal analysis, A.H.S., A.L.-L., J.L.R.-B. and A.C.-D.; Methodology, A.M. and J.L.R.-B.; Investigation, A.M., J.L.R.-B., A.H.S. and A.L.-L.; Resources, A.H.S. and A.L.-L.; Data curation, A.M., J.L.R.-B., A.H.S. and A.C.-D.; Supervision, A.M.; Writing—original draft, A.M.; Writing—review and editing, A.M. and J.L.R.-B.; Funding acquisition, A.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Junta de Andalucia (project P20-00071) and the Spanish Government (grant number: PID2020-116314RB-I00). These projects included European Regional Development Funds (ERDF). Data Availability Statement: The data used to support the findings of this study can be made available by the corresponding author upon request. Conflicts of Interest: The authors declare no conflict of interest. References 1. ASEMESA (Association of Exporters and Industrialists of Table Olives). 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