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

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.

Microbial community and volatilome changes in brines along the spontaneous fermentation of Spanish-style and natural-style green table olives (Manzanilla cultivar).

Microbial community and volatilome of brines were monitored during the spontaneous fermentations of Spanish-style and Natural-style green table olives from Manzanilla cultivar. Fermentation of olives in the Spanish style was carried out by lactic acid bacteria (LAB) and yeasts, whereas halophilic Gram-negative bacteria and archaea, along with yeasts, drove the fermentation in the Natural style. Clear differences between the two olive fermentations regarding physicochemical and biochemical features were found. Lactobacillus, Pichia, and Saccharomyces were the dominant microbial communities in the Spanish style, whereas Allidiomarina, Halomonas, Saccharomyces, Pichia, and Nakazawaea predominated in the Natural style. Numerous qualitative and quantitative differences in individual volatiles between both fermentations were found. The final products mainly differed in total amounts of volatile acids and carbonyl compounds. In addition, in each olive style, strong positive correlations were found between the dominant microbial communities and various volatile compounds, some of them previously reported as aroma-active compounds in table olives. The findings from this study provide a better understanding of each fermentation process and may help the development of controlled fermentations using starter cultures of bacteria and/or yeasts for the production of high-quality green table olives from Manzanilla cultivar.

Production of volatile compounds by wild-type yeasts in a natural olive-derived culture medium.

The production of volatile compounds in naturally fermented green table olives from Manzanilla cultivar was investigated. A total of 62 volatile compounds were detected after 24 weeks of fermentation. To clarify the contribution of yeasts to the formation of these compounds, such microorganisms were isolated from the corresponding fermenting brines. Five major yeast strains were identified: Nakazawaea molendinolei NC168.1, Zygotorulaspora mrakii NC168.2, Pichia manshurica NC168.3, Candida adriatica NC168.4, and Candida boidinii NC168.5. When these yeasts were grown as pure cultures in an olive-derived culture medium, for 7 days at 25 °C, the number of volatiles produced ranged from 22 (P. manshurica NC168.3) to 60 (C. adriatica NC168.4). Contribution of each yeast strain to the qualitative volatile profile of fermenting brines ranged from 19% (P. manshurica NC168.3) to 48% (Z. mrakii NC168.2 and C. adriatica NC168.4). It was concluded that C. adriatica NC168.4 presented the best aromatic profile, being a solid candidate to be part of a novel starter culture to enhance the organoleptic properties of naturally fermented green table olives.

Aroma profile and volatile composition of black ripe olives (Manzanilla and Hojiblanca cultivars).

The aroma profile and volatile composition of 8 samples of black ripe olives from Manzanilla and Hojiblanca cultivars were analyzed with the aim to characterize this type of table olive. The aroma of samples was described by a sensory panel using quantitative descriptive analysis (QDA), whereas the volatiles were analyzed by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). Eleven odor descriptors (briny, sautéed mushroom, earthy/soil-like, oak barrel, nutty, artificial fruity/floral, natural fruity/floral, vinegary, alcohol, fishy/ocean-like, and cheesy) were evaluated, of which only one descriptor (briny) showed a significant difference between cultivars. A total of 74 volatile compounds were identified in the headspace of samples, of which 12 were identified as significant volatiles contributing to the discrimination between Manzanilla and Hojiblanca black ripe olives. Partial least squares (PLS) regression was able to predict one odor descriptor (nutty) with sufficient accuracy and allowed identifying the volatiles that highly contributed to three odor descriptors of black ripe olives (nutty, natural fruity/floral, and cheesy).

Changes in volatile composition during the processing and storage of black ripe olives.

The present study revealed the effects of each step of black ripe olive processing (preservation, darkening, packing + sterilization) and storage on the volatile composition of two olive cultivars (Manzanilla and Hojiblanca). The preservation step enriched the volatile profile of the olives, mainly in ethyl acetate, methyl acetate, and ethanol. The darkening step produced the total or partial elimination of 55-65% of the volatiles identified before this step. Around 70% of the volatiles in the final products corresponded to compounds that were formed or increased significantly as a result of the sterilization treatment at 121 °C. Although differences in the volatile compositions and contents between Manzanilla and Hojiblanca were found, the dominant volatiles in both cultivars were benzaldehyde, dimethyl sulfide and ethyl acetate. Storage for 8 months had little influence on their volatile profiles, although the stability of individual volatiles in Manzanilla was better than that in the Hojiblanca cultivar.

Effect of Spanish-style processing steps and inoculation with Lactobacillus pentosus starter culture on the volatile composition of cv. Manzanilla green olives.

The effects of the main steps of Spanish-style processing (alkaline treatment and fermentation) on the volatile composition of cv. Manzanilla green olives were studied. Both spontaneous and controlled fermentations were considered. In the latter case, a Lactobacillus pentosus strain from green olive fermentation brine was used as starter culture. The volatile profile was determined by headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). Most of the volatile compounds detected in fresh olives decreased or were undetected after alkaline treatment, while several compounds (mostly acetic acid, 2-methylbutanoic acid, and ethanol) were formed as a result of this treatment. Over 50 new volatile components, mostly esters and phenols, appeared as a result of fermentation. The most outstanding finding was a considerable increase in 4-ethyl phenol (almost 100-fold increase) in inoculated olives compared to the uninoculated product. However, a sensory panel did not find significant differences in odor perception.

Microbiota and Metabolite Profiling of Spoiled Spanish-Style Green Table Olives.

The aim of the present study was to assess the malodorous spoilages of Spanish-style green table olives through microbial and metabolite composition using current measuring techniques (e.g., high-throughput DNA sequencing, headspace solid-phase microextraction combined with gas chromatography-mass spectrometry). Under different alkaline and washing conditions, the spoilage fermentations were reproduced with Gordal and Manzanilla olive cultivars using a low salt concentration (71 g L-1 NaCl) in the initial brine. The degradation of lactic acid and significant increases in volatile fatty acids and phenols were found in all the spoiled samples in comparison with the unspoiled control samples. According to high-throughput DNA sequencing, Cardiobacteriaceae and Ruminococcus were the dominant bacteria in the spoiled samples. PLS regression and Pearson's correlation coefficient analyses revealed positive and negative correlations among microbial communities, metabolites, and sensory spoilage descriptors. Notably, the "zapatera" descriptor was significantly associated with Propionibacterium, which was positively correlated with acetic acid, propionic acid, succinic acid, and methyl propanoate; while the "butyric" descriptor exhibited a significant positive relationship with the genus Ruminococcus, which gave an almost significant correlation with propionic and butyric acids.

Effect of post-fermentation and packing stages on the volatile composition of Spanish-style green table olives.

The volatile profile of Spanish-style green table olives after fermentation and the changes in volatile compounds that occurred as a result of the post-fermentation and subsequent packing stage were explored by solid phase micro-extraction (SPME) and gas chromatography coupled to mass spectrometry (GC-MS). Three olive cultivars (Manzanilla, Gordal, and Hojiblanca) were processed and olive samples were taken at three different times throughout the elaboration: after fermentation, after post-fermentation, and after packing. A total of 132 volatile compounds were identified, including 10 phenols, 25 alcohols, 11 acids, 39 esters, 8 hydrocarbons, 14 carbonyl compounds, 17 terpenes, and 6 other compounds. A varying number of compounds from each chemical family underwent significant changes because of the post-fermentation and packing stages. Among them, some typical reaction products of lipid oxidation (e.g. (E)-2-decenal and (E,E)-2,4-decadienal) increased with the post-fermentation in Manzanilla cultivar, and also as a result of packing in all three cultivars.

Stability of color in Spanish-style green table olives pasteurized and stored in plastic containers.

BACKGROUND: There is an increasing interest in the use of pasteurizable plastic packaging by the olive industry. In order to investigate the change from traditional glass or varnished can containers to plastic packaging, the proper plastic material that is compatible with fermented olives while maintaining color quality during pasteurization treatment and storage must be selected. This work is focused on color stability in two distinct pasteurizable plastic containers with different oxygen permeability. RESULTS: In PET + MDPE/EVOH (polyethylene terephthalate + medium-density polyethylene/ethylene vinyl alcohol) pouches, pasteurization provoked severe browning which drastically decreased their color shelf life (<6 weeks). However, this browning did not occur in the unpasteurized product without preservatives owing to the presence of microorganisms. In AlOx-coated PET + MDPE (aluminum oxide coating on polyethylene terephthalate + medium-density polyethylene) pouches, color changes were small or negligible throughout storage, especially if ascorbic acid was added to the packing solution (shelf life > 6.5 months). CONCLUSION: The plastic material had a significant effect on the retention of color of the pasteurized product. The use of AlOx-coated PET + MDPE pouches could be an alternative to traditional packaging for the pasteurization and storage of Spanish-style green olives from a color quality standpoint. © 2017 Society of Chemical Industry.

Assays to control the development of the green staining alteration in Spanish-style green olives of the Gordal variety.

BACKGROUND: Olives of the Gordal variety processed according to the Spanish style sometimes develop an alteration in color known as green staining (GS), due to the formation of harmless copper-chlorophyll complexes, which makes the product less valuable. The aim of this study was to investigate methods to minimize the impact that this alteration has for the table olive industry. RESULTS: Calcium chloride, sorbic, benzoic and ascorbic acids and SO2 did not inhibit the development of the alteration in olives packed under their own fermentation brine or new fresh brine. It was also discovered that the incubation of olive samples at 45 °C for 20 days accelerates the formation of GS and can be a very useful tool to predict the incidence of the alteration in advance. By applying this test to numerous industrial tanks for four consecutive seasons, it was found that GS was mainly present in olives harvested at the beginning of the season. CONCLUSION: The formation of GS in olives of the Gordal variety is time- and temperature-dependent, and none of the additives tested avoided or retarded the development of the alteration. However, an accelerated test to predict the development of GS formation has been proposed that could contribute to minimize the effects of the alteration. © 2015 Society of Chemical Industry.

Stability of monosodium glutamate in green table olives and pickled cucumbers as a function of packing conditions and storage time.

The effects of different packing conditions and storage times on the stability of monosodium glutamate (MSG) added to two different fermented vegetables (Spanish-type green table olives and pickled cucumbers) were studied. Factors such as packaging material (glass bottle versus plastic pouch), heat treatment (pasteurisation versus non-pasteurisation), and the presence or not of a preservative compound (potassium sorbate) were considered. The MSG content of pickled cucumbers was stable for up to 1 year of storage in all packing conditions studied. The MSG content also remained stable in pasteurised green table olives. On the contrary, MSG was extensively degraded (>75% degradation) after 54 weeks of storage in unpasteurised green olives with a higher degradation rate in glass bottles compared with plastic pouches. In the presence of potassium sorbate, MSG was also considerably degraded in olives packed in plastic pouches (>50% degradation), but hardly degraded in glass bottles. The results indicate that MSG degradation in olives is due to the action of both lactic acid bacteria and yeasts, with the formation of γ-aminobutyric acid as the major end-product.

Comparative study of the use of sarcosine, proline and glycine as acrylamide inhibitors in ripe olive processing.

The main purpose of this study was to evaluate the inhibitory effect on acrylamide (AA) formation and the impact on sensory characteristics in ripe olives of three selected amino acids (sarcosine, proline and glycine), which previously showed high AA inhibition rates in an olive model system. Each amino acid was separately added to packing solutions to give 100 or 200 mM at equilibrium, prior to a sterilisation treatment at 121°C. The results showed that sarcosine at 100 mM may be a good candidate for reducing the AA content in ripe olives with a limited effect on sensory characteristics. Studies with a model solution of AA and sarcosine heated at 121°C for 30 min suggested that the main mechanism for the inhibitory effect of sarcosine on AA formation was the Michael reaction.

Degradation of ascorbic acid and potassium sorbate by different Lactobacillus species isolated from packed green olives.

The aim of this research was to ascertain the lactic acid bacteria responsible for the degradation of ascorbic acid and/or potassium sorbate, isolated from packed green olives where these additives had diminished. A total of 14 isolates were recovered from samples of different green olive containers. According to partial sequencing of the 16S rRNA coding gene, Lactobacillus parafarraginis, Lactobacillus rapi, Lactobacillus pentosus, Lactobacillus paracollinoides, and Pediococcus ethanolidurans were identified. With the exception of L. pentosus and L. paracollinoides, the other species had not been mentioned in table olives before this study. Only three of the 14 isolates metabolized ascorbic acid in MRS broth, and the products from ascorbic acid in modified MRS broth without carbon sources were acetic and lactic acids. Except for the two L. rapi and the two P. ethanolidurans strains, the remaining 10 isolates depleted potassium sorbate added into MRS broth to some extent. The product generated by three of these strains was confirmed to be trans-4-hexenoic acid. The degradation of ascorbate or sorbate by lactic acid bacteria should be taken into account when these additives are used in food products where this group of bacteria may be present.

Effect of processing and storage time on the contents of organosulfur compounds in pickled blanched garlic.

The influence of processing, with and without fermentation, on the contents of organosulfur compounds, namely, γ-glutamyl peptides, S-alk(en)yl-L-cysteine sulfoxides (ACSOs), and S-allyl-L-cysteine (SAC), in pickled blanched garlic was evaluated. For each processing type, the effect of the preservation method and storage time was also analyzed. Blanching in hot water (90 °C for 5 min) hardly affected the individual organosulfur compound content. The fermentation and packing steps negatively affected the levels of all compounds except for SAC. The content of this compound increased during storage at room temperature whereas γ-glutamyl peptides and ACSOs were degraded to various extents. The pasteurization treatment itself had no significant effect on the concentrations of organosulfur compounds. Use of the corresponding fermentation brine in the case of the fermented product in conjunction with refrigerated storage was found to be the best method to preserve the levels of organosulfur compounds in pickled garlic stored for up to one year.

Fermented vegetables containing benzoic and ascorbic acids as additives: benzene formation during storage and impact of additives on quality parameters.

Chemical and sensorial changes related to the use of benzoates and ascorbic acid as additives in packed fermented vegetables were investigated. For this, three selected vegetables (green olives, cucumbers, and caperberries) stored under different conditions (glass or plastic containers, ambient or refrigerated storage) were used. In all cases, benzoic acid remained unchanged (glass bottle) or decreased slightly (plastic pouch) at prolonged storage. Ascorbic acid was partially or totally degraded during storage, the degradation rate depending on the storage conditions and the vegetable matrix. Benzene levels higher than 10 µg/L were found in cucumbers and caperberries containing both additives, but only when packed in plastic pouches and after prolonged storage at room temperature. In these conditions, an appreciable browning of brine, related to AA degradation, was also found. The use of benzoate alone had a significant influence on vegetable color, but flavor was not significantly affected at the benzoate levels tested. On the basis of the present study, benzoates should be removed from fermented vegetable formulations containing ascorbic acid to eliminate possible benzene formation during long-term storage.

D-amino acid formation in sterilized alkali-treated olives.

The occurrence of d-amino acids in commercial ripe olives, a well-known sterilized alkali-treated product, was investigated by high-performance liquid chromatography (HPLC) with precolumn automatic derivatization. Absolute amounts of D-amino acids were in total 18.6-38.2 mg/100 g edible portion. The major D-amino acids were D-aspartic acid, D-glutamic acid, D-serine, and D-leucine. Furthermore, to evaluate the effects of sterilization time and olive pH on amino acid racemization, a simulated processing of green ripe olives was carried out. Serine (both free and bound form) was the most-racemized amino acid after heat treatment. Sterilization (15-35 min at 121 degrees C) increased the racemization values of both free and protein-bound amino acids, although in case of protein-bound phenylalanine the increase was not statistically significant. With an increase of pH from 8 to 10 units, the racemization values of all amino acids increased significantly, except for free forms of aspartic and glutamic acids. In general, the effects of the sterilization time and olive pH on total concentration (L + D enantiomers) of each amino acid were also significant.

Vitamin content and amino acid composition of pickled garlic processed with and without fermentation.

The effect of processing, with and without fermentation, upon the nutritional composition of pickled garlic was evaluated. On a dry basis, the fermented product had a higher content of riboflavin, alpha-tocopherol, and most individual amino acids but a lower thiamin level than the unfermented product. Ascorbic acid was totally lost during processing. The chemical scores for the unfermented and fermented product were 88 and 108%, respectively, with the limiting amino acid being leucine. Water blanching (90 degrees C for 4 min) affected only the ascorbic acid content, whereas fermentation significantly affected the contents of thiamin, ascorbic acid, and alpha-tocopherol, as well as glutamic acid and arginine. For each processing type, the effect of the preservation method and storage time on vitamins and amino acid composition was also analyzed. In the case of the fermented product, usage of the corresponding fermentation brine plus refrigerated storage was also assayed as the packing/preservation method and was found to give the best result from a nutritional standpoint.