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.

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.

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.

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.

Corrigendum: Identification of volatile organic compounds in extremophilic bacteria and their effective use in biocontrol of postharvest fungal phytopathogens.

[This corrects the article DOI: 10.3389/fmicb.2021.773092.].

Changes in phenolic compounds in garlic (Allium sativum L.) owing to the cultivar and location of growth.

The contents of total phenolic compounds, flavonoids, and phenolic acids were determined in selected garlic cultivars grown at four locations. The total phenolic content varied from 3.4 mg gallic acid equivalents (GAE)/g of dry matter (dm) to 10.8 mg GAE/g of dm with a mean value of 6.5 mg GAE/g of dm. The myricetin, quercetin, kaempferol, and apigenin flavonoids were not detected in any of the samples. Caffeic acid and ferulic acid were the major phenolic acids found with mean values of 2.9 mg/kg of dm and 2.6 mg/kg of dm, respectively. The mean contents of vanillic, p-hydroxybenzoic, and p-coumaric acids were comparable (0.4-0.8 mg/kg of dm), and the level of sinapic acid was negligible (< 0.1 mg/kg of dm). There was a significant effect of location but an insignificant effect of genotype on contents of caffeic, vanillic, p-hydroxybenzoic, and p-coumaric acids. However, genotype but not location affected the contents of total phenolics and ferulic acid. On average, the white garlic cultivars and Chinese garlic cultivars contained higher contents of total phenolics and ferulic acid than the purple garlic cultivars. However, the differences in the total phenolic content between the purple and white garlic cultivars were not significant.

Packing black ripe olives in acid conditions.

The type of container (airtight and pouches with different O2 permeability) and packing conditions (cover brine, air or N2 atmosphere) has been studied to preserve black ripe olives in acid medium for a year. Unlike the traditional sterilized product, these acidified olives only needed pasteurization to assure its microbial safety, the absence of acrylamide being an additional advantage. Surprisingly, an increase in the oxygen diffusion through the films (i) faded the black color of the olives, (ii) softened the fruit that lost around 33% of its initial firmness in only 6 months, and (iii) produced the lipid́s oxidation forming volatile compounds that transmitted an abnormal flavor which tasters identified as rancid. Therefore, ripe olives in acid medium must be packed in airtight containers such as glass jars, cans o metallic pouches with cover brine or N2 atmosphere. The addition of calcium is recommended to avoid olive softening.

Multi-Statistical Approach for the Study of Volatile Compounds of Industrial Spoiled Manzanilla Spanish-Style Table Olive Fermentations.

Table olives can suffer different types of spoilage during fermentation. In this work, a multi-statistical approach (standard and compositional data analysis) was used for the study of the volatile organic compounds (VOCs) associated with altered (butyric, sulfidic, and putrid) and non-altered (normal) Manzanilla Spanish-style table olive fermentations. Samples were collected from two industrial fermentation yards in Seville (Spain) in the 2019/2020 season. The VOC profiles of altered (n = 4) and non-altered (n = 6) samples were obtained by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Ninety-one VOCs were identified and grouped into alcohols (30), esters (21), carbonyl compounds (12), acids (10), terpenes (6), phenols (6), sulfur compounds (2), and others (4). The association of the VOCs with spoilage samples depended on the standard or compositional statistical methodology used. However, butyric spoilage was strongly linked by several techniques to methyl butanoate, ethyl butanoate, and butanoic acid; sulfidic spoilage with 2-propyl-1-pentanol, dimethyl sulfide, methanol, 2-methylbutanal, 2-methyl-2-butenal, ethanol, 2-methyl-3-buten-2-ol, and isopentanol, while putrid was mainly related to D-limonene and 2-pentanol. Our data contribute to a better characterisation of non-zapatera spoiled table olive fermentations and show the convenience of using diverse statistical techniques for a most robust selection of spoilage VOC markers.

Industrial Processing of Algerian Table Olive Cultivars Elaborated as Spanish Style.

Olives from the Sigoise, Verdale, and Sevillana cultivars were elaborated as Spanish-style table olives by four Algerian factories, and the quality and food safety of the industrial table olives have been studied by the analysis of physicochemical and microbiological parameters. Differences were observed between the treatments carried out by the different factories throughout the manufacturing process, especially during the washing stage, but no significant differences were found between the analyzed samples for the concentration of sugars and polyphenols. The final pH values reached at the end of fermentation ranged between 5.04 and 4.27, and the titratable acidity was above 0.4% for all samples. Lactic and acetic acids were produced in mean concentrations of 0.68% and 0.21% as a result of lactic acid bacteria (LAB) and yeast metabolism, respectively. However, the presence of butyric, isobutyric, and propionic acids was also detected, and was related to the growth of undesirable spoilage microorganisms, responsible for secondary fermentations. The high-throughput sequencing of bacterial DNA suggested the dominance of LAB species belonging to genera Lactiplantibacillus, Leuconostoc, Pediococcus, Oenococcus, or Enterococcus. The Enterobacteriaceae family was detected during the first days of brining and in only one sample after 120 days of fermentation. Other spoilage microorganisms were found, such as Lentilactobacillus buchneri or the Pectinatus and Acetobacter genera, capable of consuming lactic acid and these played an essential role in the onset of spoilage. The Clostridium and Enterobacter genera, producers of butyric and propionic acids, were responsible for the malodorous fermentation present in the industrial samples that were analyzed. The study concluded that the safety of the table olives analyzed was compromised by the presence of undesirable microorganisms and microbial stability was not guaranteed. The elaboration process can be improved by reducing the washing steps and the time should be reduced to avoid the loss of fermentable matter, with the goal of reaching a pH < 4.0 after the fermentation and preventing the possibility of the growth of spoilage microorganisms and foodborne pathogens.

Identification of Volatile Organic Compounds in Extremophilic Bacteria and Their Effective Use in Biocontrol of Postharvest Fungal Phytopathogens.

Phytopathogenic fungal growth in postharvest fruits and vegetables is responsible for 20-25% of production losses. Volatile organic compounds (VOCs) have been gaining importance in the food industry as a safe and ecofriendly alternative to pesticides for combating these phytopathogenic fungi. In this study, we analysed the ability of some VOCs produced by strains of the genera Bacillus, Peribacillus, Pseudomonas, Psychrobacillus and Staphylococcus to inhibit the growth of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Monilinia fructicola, Monilinia laxa and Sclerotinia sclerotiorum, in vitro and in vivo. We analysed bacterial VOCs by using GC/MS and 87 volatile compounds were identified, in particular acetoin, acetic acid, 2,3-butanediol, isopentanol, dimethyl disulphide and isopentyl isobutanoate. In vitro growth inhibition assays and in vivo experiments using cherry fruits showed that the best producers of VOCs, Bacillus atrophaeus L193, Bacillus velezensis XT1 and Psychrobacillus vulpis Z8, exhibited the highest antifungal activity against B. cinerea, M. fructicola and M. laxa, which highlights the potential of these strains to control postharvest diseases. Transmission electron microscopy micrographs of bacterial VOC-treated fungi clearly showed antifungal activity which led to an intense degeneration of cellular components of mycelium and cell death.

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.

Panel and Panelist Performance in the Sensory Evaluation of Black Ripe Olives from Spanish Manzanilla and Hojiblanca Cultivars.

There is vast experience in the application of sensory analysis to green Spanish-style olives, but ripe black olives (≈1 × 106 kg for 2016/2017) have received scarce attention and panelists have less experience on the evaluation of this presentation. Therefore, the study of their performance during the assessment of this presentation is critical. Using previously developed lexicon, ripe olives from Manzanilla and Hojiblanca cultivars from different origins were sensory analysed according to the Quantitative Descriptive Analysis (QDA). The panel (eight men and six women) was trained, and the QDA tests were performed following similar recommendations than for green olives. The data were examined while using SensoMineR v.1.07, programmed in R, which provides a diversity of easy to interpret graphical outputs. The repeatability and reproducibility of panel and panelists were good for product characterisation. However, the panel performance investigation was essential in detecting details of panel work (detection of panelists with low discriminant power, those that have interpreted the scale in a different way than the whole panel, the identification of panelists who required training in several/specific descriptors, or those with low discriminant power). Besides, the study identified the descriptors of hard evaluation (skin green, vinegar, bitterness, or natural fruity/floral).

Influence of processing conditions on acrylamide content in black ripe olives.

The presence of acrylamide was investigated in different presentations of commercial black ripe olives, a well-known sterilized alkali-treated product. The analysis was performed by gas chromatography-mass spectrometry (GC-MS) after bromination of acrylamide, using (13C3)acrylamide as internal standard. In-house validation data for commercial ripe olives showed good precision and accuracy of the method, with repeatability below 3% and recoveries between 94 and 105%. Acrylamide was detected in all samples, but its concentration varied significantly from 176 to 1578 microg/kg of pulp. The effects of different processing conditions (two preservation methods and three darkening methods), cultivar (Hojiblanca or Manzanilla), and presentation form (pitted or sliced olives) on acrylamide content were evaluated in experiments performed in an olive-processing plant. All canned samples were sterilized at 121 degrees C for 30 min. Statistical analysis of the data indicated that the effects of darkening method and olive cultivar were the most pronounced. Acrylamide contents did not significantly differ after 6 months of storage. The small amounts of free amino acids and reducing sugars found in olives before sterilization did not significantly correlate with the acrylamide formed.

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.

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.

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.

Fatty acid profile of table olives and its multivariate characterization using unsupervised (PCA) and supervised (DA) chemometrics.

The fatty acid composition of 67 commercial presentations of table olives was determined. The most abundant fatty acids, in decreasing order of presence, were C18:1, C16:0, C18:2 n-6, and C18:0. The ranges, expressed as grams of fatty acids per 100 g of edible portion, for the different nutritional fractions were as follows: saturated fatty acids, 2.07-5.99; monounsaturated fatty acids, 5.67-19.42; polyunsaturated fatty acids, 0.52-3.87; and trans-fatty acids, 0.08-0.44. Principal component analysis of the matrix of the fatty acid composition led to the deduction of new factors. The first accounted for 55.10% of the total variance and was mainly related to C16:10, C18:0, C20:0, C22:0, C24:0, C18:1, C18:1t, and C20:1. The second factor accounted for 10.33% of variance and was related to C16:1 and C18:2 n-6. They did not permit differentiation among elaboration types or cultivars. However, discriminant analysis was successfully applied for this objective. The fatty acids that most contributed to discriminate among elaboration styles were C17:1, C18:1, C16:0, C17:0, and C18:0 (function 1) and C17:0, C17:1, C20:0, C16:0, C18:1, and C24:0 (function 2). In the case of cultivars, they were C20:0, C18:1, C17:1, C18:2 n-6, C18:1t, and C18:2t (function 1); C18:2 n-6, C18:1, C16:0, C20:0, C18:0, and C18:2t (function 2); and C17:0, C18:3 n-3, and C17:1 (function 3). Results from this study have shown differences among the fatty acid composition and fat content of the diverse commercial presentations of table olives, which can be applied in predictive and classification discriminant analysis.