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.

Mechanisms involved in the antiproliferative and proapoptotic effects of unsaponifiable fraction of extra virgin olive oil on HT-29 cancer cells.

Extra virgin olive oil (EVOO) has demonstrated great oncostatic and antiinflammatory properties. Nowadays, it is clear that unsaponifiable fraction (UF) as well as other minor EVOO components have a key role in these beneficial effects. The present study was designed to evaluate UF effect in HT-29 human colon adenocarcinoma cells. Cell growth and viability assays were determined by sulphorhodamine B test at different time points (24, 48, and 72 h). The proapoptotic effect was evaluated by flow cytometric studies and different protein expression were determined by immunoblotting. UF µg/mL concentrations' range significantly reduced the growth of HT-29 cell line. Moreover UF induced intrinsic apoptotic pathway in HT-29 human colon adenocarcinoma cells through PPARγ and NFκB signaling pathways coming up to COX-2 downregulation and modulating p53 suppressor protein levels. The results suggest that UF of EVOO may exert an important role in the anticancer effect of EVOO and provide a natural resource for the prevention or treatment of human colon cancer.

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.].

Effect of Spanish-Style Table Olive Processing on Fatty Acid Profile: A Compositional Data Analysis (CoDA) Approach.

This manuscript considers that the composition of Manzanilla and Hojiblanca fats are compositional data (CoDa). Thus, the work applies CoDa analysis (CoDA) to investigate the effect of processing and packaging on the fatty acid profiles of these cultivars. To this aim, the values of the fat components in percentages were successively subjected to exploratory CoDA tools and, later, transformed into ilr (isometric log-ratio) coordinates in the Euclidean space, where they were subjected to the standard multivariate techniques. The results from the first approach (bar plots of geometric means, tetrahedral plots, compositional biplots, and balance dendrograms) showed that the effect of processing was limited while most of the variability among the fatty acid (FA) profiles was due to cultivars. The application of the standard multivariate methods (i.e., Canonical variates, Linear Discriminant Analysis (LDA), ANOVA/MANOVA with bootstrapping and n = 1000, and nested General Linear Model (GLM)) to the ilr coordinates transformed data, following Ward's clustering or descending order of variances criteria, showed similar effects to the exploratory analysis but also showed that Hojiblanca was more sensitive to fat modifications than Manzanilla. On the contrary, associating GLM changes in ilr with fatty acids was not straightforward because of the complex deduction of some coordinates. Therefore, according to the CoDA, table olive fatty acid profiles are scarcely affected by Spanish-style processing compared with the differences between cultivars. This work has demonstrated that CoDA could be successfully applied to study the fatty acid profiles of olive fat and olive oils and may represent a model for the statistical analysis of other fats, with the advantage of applying appropriate statistical techniques and preventing misinterpretations.

Quality parameters and fatty acid and triacylglycerol profiles of directly brined table olive fat throughout processing and shelf life.

The transformations that may suffer directly brined table olive fat during processing were studied using cracked Aloreña de Málaga olive as a model. The classical studies showed that storage increased acidity and K270, but not peroxide value, K232 and ΔK. FA profiles, nutritional fat subclasses, and TAGs suffered several significant changes along processing, although some could be spurious. Compositional data (CoDa) analysis identified C18:2n-6 and C18:1c, and their corresponding TAGs as the most affected compounds, conclusion that was particularly evident after weighted CoDa log-ratio analysis (LRA). Thus, CoDa analysis is a promising alternative statistical tool to study table olive FA, TAG profiles and fats in general. Most of the quality parameters' values and FA and TAG concentrations were compatible with Virgin Olive Oil (VOO), showing that the fat quality and nutritional characteristics of natural green olives in brine could be comparable to that of VOO.

Chemometric characterisation of the fats released during the conditioning processes of table olives.

This work deals with the characterisation of five fat types released during the conditioning process of table olives, pitting green olives (PGF), pitting ripe olives (PRF), pitting/stuffing green olives with vegetable origin products (PSGVF) and pitting/stuffing green olives with animal origin materials (PSGAF) along with the fat collected at the end of the factory sewer (WF). The parameters analysed included: acidity, peroxide value, spectrophotometric measurements (K232, K270 and ΔK), fatty acid composition of the raw material and the neutral fat, triacylglycerol composition of the neutral fat as well as the proportion and composition of the polar fraction. A chemometric analysis of specific groups of compounds was able to establish differences among the types of fats; the overall analysis showed two major groups: PGF, PRF and PSGVF were only moderately degraded, and PSGAF and WF were highly degraded. The information provided can help industries and authorities to handle these residues properly.

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.

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.

Impact of Processing and Extraction on the Minor Components of Green Spanish-Style Gordal Table Olive Fat, as Assessed by Innovative Approaches.

This work aims to study the effect of the green Spanish-style table olive processing and extraction method of fat on its minor components. For this purpose, it uses standard multivariate analysis (developed for Euclidean space), Compositional Data (CoDa) analysis (for data in the simplex) and Multiple Factor analysis (MFA). Overall, processing had a scarce effect on most of the minor components except ethyl and methyl esters and diacylglycerols, which markedly increased during fermentation; however, these compounds in table olive do not have the negative connotations that those in olive oil do since they are normal metabolites from the yeast microflora habitually present during the process. The work also showed that the quantification of minor components in table olive fat was an extraction-dependent method since Soxhlet increased the concentrations of fatty alcohols, triterpene dialcohols, sterols, waxes and polar compounds. Regarding statistical methods, CoDa analysis strategies were successfully applied to produce more appropriate clustering and Principal Component Analysis (PCA) segregation than standard tools. Moreover, MFA allowed for study of the components individually and by groups; the relationships among groups led to the most appropriate clustering and PCA segregation of samples and revealed the effect of the chemical groups' evolution on the similarity/dissimilarity between samples. Therefore, MFA was the statistical analysis that led to the most information on the effect of processing and extraction methods. Its combination with appropriate CoDa logratios could be an exciting challenge.

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).

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.

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.

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).

Assessment of the Minor-Component Transformations in Fat during the Green Spanish-Style Table Olive Processing.

There is an increasing interest of consumers for natural and healthy products. This work assesses the transformations that green Spanish-style processing of Manzanilla and Hojiblanca table olives produces on the minor components of their fat. Discriminant analysis showed that most of the variability was not due to processing (24.4%) but to differences between cultivars (59.2%). Therefore, the final products have a similar quality to the original olive fat; that is, the quality of the fat was scarcely affected. The only systematic trends observed were the decrease in hexacosanol, tetracosanol, and octacosanol (fatty alcohols) and C46 (wax), after lye treatment, and the high levels of alkyl esters in the packaged product. Thus, minor-component levels in green table olives are, in general, within the limits established for extra virgin olive oil since the alkyl esters should be considered habitual products of fermentation and not as an alteration as in olive oil.

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.

Effect of green Spanish-style processing (Manzanilla and Hojiblanca) on the quality parameters and fatty acid and triacylglycerol compositions of olive fat.

This work studies the effect of processing Manzanilla and Hojiblanca olives as green Spanish-style on the quality parameters and fatty acid and triacylglycerol compositions of their oils. Lye treatment reduced the values of most quality parameters while fermentation/packaging increased acidity, K232 and K270. Processing did not cause any systematic effect on fatty acids (FA), triacylglycerols or nutritional fat subclasses but significant differences between cultivars were observed. Principal component analysis (PCA) confirmed that most of the variation among oil characteristics was due to cultivars and only a limited proportion (∼22% and ∼14% variance for FA and triacylglycerols, respectively) to processing. Furthermore, the levels of the quality parameters and fatty acids with restrictions in the legislation were below the limits established in the Commission Implementing Regulation (EU) 1348/2013 for extra virgin olive oil (EVOO), except for C18:3n-3 in Hojiblanca. Therefore, the fat of processed olives was compatible with EVOO.

Chemometric classification of the fat residues from the conditioning operations of table olive processing, based on their minor components.

The work characterizes the unsaponifiable matter of fats released in pitting green (GP) and ripe (RP) olives, in pitting/stuffing green olives with vegetable (GPSV) and animal (GPSA) products and in the fat settled at the end of the factory sewer system (W). The unsaponifiable matter ranged from 1.94% (RP) to 5.91% (GPSA); total sterols from 1319 mg/kg fat (GPSV) to 2002 mg/kg fat (RP), with ß-sitosterol as the most abundant. Fatty alcohols ranged from 242 mg/kg (GP) to 556 mg/kg (W), with C22 as the most abundant. Triterpene diols were found only in GPSV (erythrodiol + uvaol, 0.80%). Wax was not present in the fats from GP and RP but increased with the general fat degradation in the order GPSV (128 mg/kg), GPSA (171 mg/kg) and W (263 mg/kg). Chemometric analysis was able to detect differences among the diverse fats; sterols + fatty alcohols cluster analysis was useful for fat grouping.