This study uses an “omics” approach to evaluate the bacterial biodiversity changes during fermentation process of natural green cracked Aloreña de Málaga table olives, from raw material to fermented ...fruit. For this purpose, two industries separated by almost 20km in Guadalhorce Valley (Málaga, Spain) were analysed for obtaining both brines and fruit samples at different moments of fermentation (0, 7, 30 and 120days). Physicochemical and microbial counts during fermentation showed the typical evolution of this type of processes, apparently dominated by yeasts. However, high-throughput barcoded pyrosequencing analysis of V2–V3 hypervariable region of the bacterial 16S rRNA gene showed at 97% identity the presence of 131 bacterial genera included in 357 operational taxonomic units, not detected by the conventional approach. The bacterial biodiversity was clearly higher in the olives at the moment of reception in the industry and during the first days of fermentation, while decreased considerably as elapse the fermentation process. The presence of Enterobacteriaceae and Lactobacillaceae species was scarce during the four months of study. On the contrary, the most important genus at the end of fermentation was Celerinatantimonas in both brine (95.3% of frequency) and fruit (89.4%) samples, while the presence of well-known spoilage microorganisms (Pseudomonas and Propionibacterium) and halophilic bacteria (Modestobacter, Rhodovibrio, Salinibacter) was also common during the course of fermentation. Among the most important bacterial pathogens related to food, only Staphylococcus genus was found at low frequencies (<0.02% of total sequences). Results show the need of this type of studies to enhance our knowledge of the microbiology of table olive fermentations. It is also necessary to determine the role played by these species not previously detected in table olives on the quality and safety of this fermented vegetable.
•Pyrosequencing analysis revealed 131 bacterial genera in fermented olive and brines.•The biodiversity was higher at the onset of fermentation compared to the end.•Celerinatantimonas was the most important genera found at the end of fermentation.•Pseudomonas and Propionibacterium were also common during course of fermentation.•Food may be considered safe by the practical absence of foodborne pathogens.
Yeasts are unicellular eukaryotic microorganisms isolated from many foods, and are commonly found in table olive processing where they can play a double role. On one hand, these microorganisms can ...produce spoilage of fruits due to the production of bad odours and flavours, the accumulation of CO2 leading to swollen containers, the clouding of brines, the softening of fruits and the degradation of lactic acid, which is especially harmful during table olive storage and packaging. But on the other hand, fortunately, yeasts also possess desirable biochemical activities (lipase, esterase, β-glucosidase, catalase, production of killer factors, etc.) with important technological applications in this fermented vegetable. Recently, the probiotic potential of olive yeasts has begun to be evaluated because many species are able to resist the passage through the gastrointestinal tract and show beneficial effects on the host. In this way, yeasts may improve consumers' health by decreasing cholesterol levels, inhibiting pathogens, degrading non assimilated compounds, producing antioxidants and vitamins, adhering to intestinal cells or by maintaining epithelial barrier integrity. Many yeast species, usually also found in table olive processing, such as Wicherhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens and Kluyveromyces lactis, have been reported to exhibit some of these properties. Thus, the selection of the most appropriate strains to be used as starters, alone or in combination with lactic acid bacteria, is a promising research line to develop in a near future which might improve the added value of the commercialized product.
► Yeasts are part of the usual microbiota found in table olive processing. ► These microorganisms may improve the technological process but also the human health. ► However, certain species under certain conditions can produce the spoilage of the product. ► Diverse statistical tools have been used for the selection of the most relevant yeasts strains.
This work studies the effects of different sodium (in the range of 4–10%), potassium (0–4%) and calcium (0–6%) chloride salt mixtures on the fermentation profile of Gordal olives processed according ...to the Spanish style. For this purpose, response surface methodology based on a simplex centroid mixture design with constrain (sum of salt percentages = 10%) was used. All treatments reached appropriate titratable acidity levels, but this parameter could not be related to the initial chloride salt concentration. The presence of CaCl2 led to lower initial and after-fermentation pHs, delayed sugar diffusion into the brine, its maximum concentration and titratable acidity formation. CaCl2 also delayed Enterobacteriaceae and yeast sprang, decreasing their overall growth. This chloride salt also showed a tendency to reduce overall lactic acid bacteria growth. KCl had a similar behaviour to NaCl but, in general, increased overall microbial growth. Thus, a partial substitution of NaCl in Spanish-style green olives with KCl and CaCl2 does not substantially modify the fermentation profile but does produce some changes, which, when properly managed, could help to improve product processing.
► The use of statistical modelling techniques allowed the optimisation of the fermentative process as a function of diverse chloride salts combinations. ► Results show that an appropriate lactic acid process can also be obtained with lower sodium content. ► CaCl2 affected the microbiological and physic-chemical profile of fermentation, reducing overall microbial growth. ► The partial substitution of NaCl by KCl or CaCl2 did not represent a microbial safety risk for the fermented olives.
Statistical modelling techniques were used in the present study to assess the individual effects of temperature and NaCl concentration on the growth of 10 lactic acid bacteria and 6 yeast strains ...mostly isolated from different forms of table olive processing and belonging to the species Lactobacillus pentosus, Lactobacillus plantarum, Saccharomyces cerevisiae, Wickerhamomyces anomalus and Candida boidinii. The mathematical models obtained in synthetic laboratory media show that yeasts, except for C. boidinii, were more resistant to a high salt concentration than lactic acid bacteria, with an MIC value ranging from 163.5 (S. cerevisiae) to 166.9 g/L (W. anomalus); while for L. pentosus and L. plantarum this parameter ranged from 110.6 to 117.6 g/L, respectively. With regards to temperature, lactic acid bacteria showed a slight trend towards supporting higher temperature values than yeasts, with the exception of S. cerevisiae. The maximum temperatures for growth of L. pentosus and L. plantarum were 41.9 and 43.0 °C, respectively; while for W. anomalus and C. boidinii they were 38.2 and 36.5 °C. The optimum temperatures for growth were also higher for L. pentosus and L. plantarum (35.5 and 32.9 °C), compared to W. anomalus and C. boidinii (29.3 and 26.9 °C, respectively). Additional experiments carried out in natural olive brines confirmed previous results, showing that high NaCl concentrations clearly favoured yeast growth and that at high temperatures LAB slightly overcame yeasts. Results obtained in this paper could be useful for industry for a better control of both table olive fermentation and packaging.
► A total of 16 table olive related microorganisms were modelled in the present study in laboratory media. ► Overall, yeasts were more resistant to high salt concentrations than LAB. ► On the contrary, LAB showed a slight trend to support higher temperatures than yeasts. ► Experiments carried out in natural olive brines confirmed these results.
This work examines the formation of poly-microbial communities adhered to the surface of Manzanilla olive fruits processed according to the Spanish style. The experimental design consisted of four ...pilot fermenters inoculated with four Lactobacillus pentosus strains, plus another fermenter which was not inoculated and fermented spontaneously. Lactic acid bacteria and yeasts were analysed in depth on olive epidermis throughout fermentation by plate count, molecular techniques and scanning electron microscopy. Data show that in all cases high population levels (above 8 log10 CFU per olive) were reached for both groups of microorganisms at the second week of fermentation and that these counts never fell below 6 log10 CFU per olive during the 3 months that fermenters were monitored. In situ observation of olive epidermis slices revealed a strong aggregation and adhesion between bacteria and yeasts by the formation of a matrix which embedded the microorganisms. Geotrichum candidum, Pichia galeiformis and Candida sorbosa were the main yeast species isolated from these biofilms at the end of fermentation (confirmed by RFLP analysis of the 5.8S-ITS region), while molecular characterization of lactobacilli isolates by means of RAPD-PCR with primer OPL5 showed in many cases a high similarity in their banding profiles with the inoculated strains. Results obtained in this survey show the importance of studying the olive epidermis throughout fermentation, because ultimately, olives are ingested by consumers.
► Lactic acid bacteria and yeasts were analysed in depth on fermented olive epidermis. ► A strong aggregation and adhesion between bacteria and yeasts on olive epidermis was noticed. ► Microorganisms were embedded by an apparent exopolysaccharide matrix forming a complex biofilm. ► In the majority of cases, the inoculated Lactobacillus pentosus strains were imposed over the native LAB population.
This work studies the effects of the partial substitution of NaCl with potassium and calcium chloride salts on the fermentation profile of Spanish-style green Manzanilla olives. For this purpose, ...response surface methodology based in an enlarged simplex centroid mixture design with constrain (∑salts = 100 g/L) was used. Regarding to physicochemical characteristics, pH decreased when CaCl2 increased, titratable acidity was lower in presence of KCl while combined acidity increased as the contents of KCl and CaCl2 were close to the barycentre of the experiment (∼33.33% each salt). Regarding to microbiological profile, Enterobacteriaceae growth was slight stimulated in presence of high CaCl2 contents, yeast patterns were not linked to the initial brine compositions, while the maximum lactic acid bacteria population decreased slightly as KCl and CaCl2 increased in the proportion 1:1, although a moderate (equilibrated) content of both may be stimulating. Results obtained in this work show that Spanish-style green Manzanilla cv. can be fermented in diverse mixtures of chloride salts, albeit the initial CaCl2 should be limited to 20–30 g/L to prevent excessive Enterobacteriaceae growth; combining it with a similar proportion of KCl may also improve LAB predominance.
•The reduction of NaCl concentration in table olives would be favourable for consumers.•The partial substitution of NaCl with KCl and CaCl2 on table olive fermentation was studied.•Olive fermentation can be achieved in presence of mixtures of diverse chloride.•CaCl2 should not exceed 20–30 g/L to prevent excessive Enterobacteriaceae growth.•A similar proportion of KCl and CaCl2 may improve LAB imposition.
This work evaluates the use of two multifunctional starters of Lactobacillus pentosus species (TOMC LAB2 and TOMC LAB4) during elaboration of Manzanilla olive fruits processed according to the ...Spanish-style. Data show that the use of inocula at the onset of fermentation led to a proper acidification and sugar consumption of brines compared to the spontaneous process, obtaining in a shorter period of time the maximum population for lactic acid bacteria. Both inoculated L. pentosus strains were recovered at high frequencies at the end of fermentation on the olive surface, which was corroborated by RAPD-PCR analysis. In situ observation of olive epidermis slices by scanning electron microscopy revealed a strong aggregation and adhesion between microorganisms, which reached population levels of approximately 6 and 7 log10 cfu/cm2 for yeasts and lactic acid bacteria, respectively. Enterobacteriaceae on the olive surface were also found at the onset of fermentation (∼9 log10 cfu/cm2), but they declined during the process and were below the detection limit at the end of fermentation. Results obtained in this study show the advantage of using multifunctional starters with the ability to adhere to the olive epidermis because, ultimately, the fruits are the food ingested by consumers.
•Two Lactobacillus pentosus strains were assayed in this work as multifunctional starter in olive fermentations.•Microbial and physicochemical changes were properly modeled during fermentation.•Starters led to a proper acidification and consumption of sugars in brines.•The inoculated L. pentosus strains were recovered at the end of fermentation.•Observation of olive epidermis slices by SEM revealed the presence of biofilms.
This study assesses the technological characteristics and the dominance in brines of four selected potential probiotic Lactobacillus pentosus strains during green table olive Spanish-style ...fermentations. When compared with the spontaneous process, the use of these microorganisms as starter led to higher lactic acid bacteria than yeast populations, a decrease in Enterobacteriaceae levels and a faster acidification of the brines. Microbial changes were properly modelled with equations including both growth and death phases. Diverse genetic profiles among the lactic acid bacteria populations were found at the end of fermentation. Hence, inoculation using the current green olive processing technology guaranteed the development of the typical Spanish-style with, as shown by discriminant function analysis, some distinctive characteristic processes, according to strains, but did not assure per se the imposition of the selected strain in the cover brines.
•In this work, four L. pentosus strains were assayed as starter cultures in olive fermentations.•Microbial changes were properly modelled for both growth and death phases.•Starters led to high LAB and low Enterobacteriaceae levels and fast acidification.•Inoculation did not assure per se the imposition of the particular strain in the brines.•However, distinctive characteristic processes were obtained by the use of starters.
The present study focused on investigating a peculiar spoilage of traditional Aloreña de Málaga table olives characterized by the formation of whitish and soft regions on the olive surface. To ...determine its causes, the main microbiological and physicochemical changes in 50 commercial packages were monitored until the alteration in appearance (63 days). Colour and firmness of fruits deteriorated progressively in the packaged olives during storage. Sugar in brines (7.5 g/L) remained stable during the first month and then decreased gradually (5.0 g/L) while, in parallel, the lactic acid bacteria population and titratable acidity increased. After two months of storage, evidence of spoilage was noticed, coinciding with the maximal lactic acid bacteria populations in fruit (>6.5 log10 CFU/g). The spoilage affected ∼25% of the fruits within packages. The microbial species detected in the product after packaging were Enterobacter gergoviae and Lactobacillus plantarum among bacteria, and Candida tropicalis, Candida parapsilosis, and Lodderomyces elongisporus among the yeasts while Lactobacillus pentosus was the dominant species in the spoilt packages. A specific biotype of L. pentosus was only detected in the damaged fruits. Further studies will be made to confirm the association between the spoilage and the presence of this L. pentosus biotype.
•This study reports a new spoilage in olive packages and its characterization.•The genotypes of the microbial populations were determined by molecular techniques.•Enterobacteriaceae, yeasts and LAB were initially present, but only LAB counts increased.•Packaging conditions favour Lactobacillus pentosus growth against the initial Lactobacillus plantarum.•A specific biotype of Lactobacillus pentosus was predominant in the spoilt product.
For the first time, functional data analysis (FDA) was used to assess the effects of different treatments on Protection Denomination of Origin Aloreña de Málaga table olive fermentations, focusing on ...the evolution of yeast population. The analysis of fermentation by a conventional approach led to scarce information. However, the transformation of microbial (and also physicochemical) data into smooth curves allowed the application of a new battery of statistical tools for the analysis of fermentations (functional pointwise estimation of the averages and standard deviations, maximum, minimum, first and second derivatives, functional regression, and functional F and t-tests). FDA showed that all the treatments assayed led to similar trends in yeast population while changes in pH and titratable acidity profiles led to several significant differences. Therefore, FDA represents a promising and valuable tool for studying table olive fermentations and for food microbiology in general.
•Functional data analysis (FDA) was used for the first time in food fermentations.•FDA led to the estimation of microbiological and physicochemical functional curves.•FDA also estimated the area under such curves and their means, minima, and maxima.•Yeast growth was not affected by treatments, according to functional ANOVA.•Changes in pH and acidity were affected by treatments, according to functional ANOVA.
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