F. Rodríguez-Gómez

Lipolytic activity of the yeast species associated with the fermentation/storage phase of ripe olive processing

Ripe olives account for ca. 30% of the worlds table olive production. Fruits intended for this type of product are preserved in an aqueous solution (acidic water or brine) for several months, where they may undergo a spontaneous fermentation. Enterobacteriaceae and lactic acid bacteria were not detected in the present survey during storage. Thus, the work focused on studying the yeast microflora associated with the ripe olive storage of Manzanilla and Hojiblanca cultivars in acidified brines. A total of 90 yeast isolates were identified by means of molecular methods using RFLP analysis of the 5.8S-ITS rDNA region and sequencing of the D1/D2 domains of the 26S rDNA gene. The two most important species identified in both cultivars were Saccharomyces cerevisiae and Pichia galeiformis, which were present throughout the storage period, while Candida boidinii was detected during the later stages of the process. The species Pichia membranifaciens was detected only in the early stages of the Hojiblanca cultivar. The lipase assays performed with both extracellular and whole cell fractions of the yeast isolates showed that neither of the S. cerevisiae and P. galeiformis species had lipase activity, while the P. membranifaciens isolates showed a weak activity. On the contrary, all C. boidinii isolates gave a strong lipase activity. Change in olive fat acidity was markedly higher in the presence of the yeast population than in sterile storage, indicating that lipases produced by these microorganisms modify the characteristics of the fat in the fruit.

Exploring the yeast biodiversity of green table olive industrial fermentations for technological applications

In recent years, there has been an increasing interest in identifying and characterizing the yeast populations associated with diverse types of table olive elaborations because of the many desirable technological properties of these microorganisms. In this work, a total of 199 yeast isolates were directly obtained from industrial green table olive fermentations and genetically identified by means of a RFLP analysis of the 5.8S-ITS region and sequencing of the D1/D2 domains of the 26S rDNA gene. Candida diddensiae, Saccharomyces cerevisiae and Pichia membranifaciens were the most abundant yeast species isolated from directly brined Aloreña olives, while for Gordal and Manzanilla cultivars they were Candida tropicalis, Pichia galeiformis and Wickerhamomyces anomalus. In the case of Gordal and Manzanilla green olives processed according to the Spanish style, the predominant yeasts were Debaryomyces etchellsii, C. tropicalis, P. galeiformis and Kluyveromyces lactis. Biochemical activities of technological interest were then qualitatively determined for isolates belonging to all yeast species. This preliminary screening identified two isolates of W. anomalus with interesting properties, such as a strong β-glucosidase and esterase activity, and a moderate catalase and lipolytic activity, which were also confirmed by quantitative assays. The results obtained in this survey show the potential use that some yeast species could have as starters, alone or in combination with lactic acid bacteria, during olive processing.

Formation of lactic acid bacteria-yeasts communities on the olive surface during Spanish-style Manzanilla fermentations.

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 log(10) CFU per olive) were reached for both groups of microorganisms at the second week of fermentation and that these counts never fell below 6 log(10) 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 OPL(5) 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.

Multivariate analysis to discriminate yeast strains with technological applications in table olive processing

This survey uses a multivariate classification analysis to discriminate yeast strains with interesting biochemical activities for the processing of table olives among a collection of 32 isolates belonging to 16 different yeast species. Lipase, esterase and β-glucosidase activities (desirable characteristics) were quantitatively evaluated in both extracellular and cellular fractions for all isolates in different types of culture media. The study of the quantitative data by cluster and principal component analyses led to the identification of several Wickerhamomyces anomalus,Candida boidinii and Candida diddensiae isolates with promising characteristics (the best global activity levels), clearly differentiated from the rest of the yeasts. The results obtained in this work open up new alternatives to this methodology for the study, classification and selection of the most suitable yeasts to be used as starters, alone or in combination with lactic acid bacteria, during table olive processing.

Selection of yeasts with multifunctional features for application as starters in natural black table olive processing

Yeasts are unicellular eukaryotic microorganisms with a great importance in the elaboration on many foods and beverages. In the last years, researches have focused their attention to determine the favourable effects that these microorganisms could provide to table olive processing. In this context, the present study assesses, at laboratory scale, the potential technological (resistance to salt, lipase, esterase and β-glucosidase activities) and probiotic (phytase activity, survival to gastric and pancreatic digestions) features of 12 yeast strains originally isolated from Greek natural black table olive fermentations. The multivariate classification analysis carried out with all information obtained (a total of 336 quantitative input data), revealed that the most promising strains (clearly discriminated from the rest of isolates) were Pichia guilliermondii Y16 (which showed overall the highest resistance to salt and simulated digestions) and Wickerhamomyces anomalus Y18 (with the overall highest technological enzymatic activities), while the rest of strains were grouped together in two clearly differentiated clusters. Thus, this work opens the possibility for the evaluation of these two selected yeasts as multifunctional starters, alone or in combination with lactic acid bacteria, in real table olive fermentations.

Potential benefits of the application of yeast starters in table olive processing

Yeasts play an important role in the food and beverage industry, especially in products such as bread, wine, and beer, among many others. However, their use as a starter in table olive processing has not yet been studied in detail. The candidate yeast strains should be able to dominate fermentation, together with lactic acid bacteria, but should also provide a number of beneficial advantages. Technologically, yeasts should resist low pH and high salt concentrations, produce desirable aromas, improve lactic acid bacteria growth, and inhibit spoilage microorganisms. Nowadays, they are being considered as probiotic agents because many species are able to resist the passage through the gastrointestinal tract and show favorable effects on the host. In this way, yeasts may improve the health of consumers by means of the degradation of non-assimilated compounds (such as phytate complexes), a decrease in cholesterol levels, the production of vitamins and antioxidants, the inhibition of pathogens, an adhesion to intestinal cell line Caco-2, and the maintenance of epithelial barrier integrity. Many yeast species, usually found in table olive processing (Wickerhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens, and Kluyveromyces lactis, among others), have exhibited some of these properties. Thus, the selection of the most appropriate strains to be used as starters in this fermented vegetable, alone or in combination with lactic acid bacteria, is a promising research line to develop in the near future.

Evaluating the individual effects of temperature and salt on table olive related microorganisms

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.

Influence of Ripe Table Olive Processing on Oil Characteristics and Composition As Determined by Chemometrics

The changes in ripe olive fat produced by processing were studied according to cultivars using the general linear model, principal component analysis (PCA), predictive discriminant analysis (DA), and hierarchical clustering. Acidity, peroxide value, K(270), and DeltaK increased during storage. Acidity also increased after sterilization, whereas K(270) decreased after darkening; K(232) showed a progressive decrease during processing. Fatty acids (except C17:0, C18:0, and C24:0), triacylglycerols (except PLLn, OOLn+PoOL, PLL+PoPoO, SOO, and POS+SLS), polar compounds, diacylglycerol, and monoacylglycerols also suffered statistically significant changes during processing. A PCA discriminated between cultivars and, within the same cultivar, among the raw materials from the rest of the treatments. Using fatty acid and triacylglycerol compositions, predictive DA discriminated between cultivars (100% correct), but high discriminant capacity among processing steps (95% correct assignation and 87% in cross-validation) was achieved only with fatty acids. A hierarchical clustering analysis successfully grouped cultivars and processing steps according to overall olive oil composition and quality.

Growth/no growth interfaces of table olive related yeasts for natamycin, citric acid and sodium chloride

The present work uses a logistic/probabilistic model to obtain the growth/no growth interfaces of Saccharomyces cerevisiae, Wickerhamomyces anomalus and Candida boidinii (three yeast species commonly isolated from table olives) as a function of the diverse combinations of natamycin (0-30 mg/L), citric acid (0.00-0.45%) and sodium chloride (3-6%). Mathematical models obtained individually for each yeast species showed that progressive concentrations of citric acid decreased the effect of natamycin, which was only observed below 0.15% citric acid. Sodium chloride concentrations around 5% slightly increased S. cerevisiae and C. boidinii resistance to natamycin, although concentrations above 6% of NaCl always favoured inhibition by this antimycotic. An overall growth/no growth interface, built considering data from the three yeast species, revealed that inhibition in the absence of citric acid and at 4.5% NaCl can be reached using natamycin concentrations between 12 and 30 mg/L for growth probabilities between 0.10 and 0.01, respectively. Results obtained in this survey show that is not advisable to use jointly natamycin and citric acid in table olive packaging because of the observed antagonistic effects between both preservatives, but table olives processed without citric acid could allow the application of the antifungal.

Fortification of table olive packing with the potential probiotic bacteria Lactobacillus pentosus TOMC-LAB2

Dairy products are currently the main carriers of probiotic microorganisms to the human body. However, the development of new matrices for probiotic delivery is convenient for intolerant to milk (or its derivatives) and those requiring low-cholesterol diet consumers. The present work focused on the fortification of previously fermented green Spanish style olives with the autochthonous putative probiotic bacteria Lactobacillus pentosus TOMC-LAB2. The fortification was carried out by inoculating the bacteria into the packing brines using Manzanilla fruits from three different processes: (i) spontaneously fermented (F1), (ii) fermented using L. pentosus TOMC-LAB2 as starter (F2), and (iii) spontaneously fermented and then thermally treated (F3). Data showed that all inoculated treatments had higher population levels (5.49, 4.41, and 6.77 log10 cfu/cm2) than their respective controls (1.66, 4.33, and 0.0 log10 cfu/cm2, for F1, F2, and F3 treatments, respectively). The presence of L. pentosus TOMC-LAB2 on olive surface was confirmed by rep-PCR, with a recovery frequency at the end of the shelf life (200 days) of 52.6, 57.9, and 100.0% for F1, F2, and F3 treatments, respectively. Thus, results obtained in this work show the ability of this microorganism to survive under packing conditions for long period of times as well as to colonize the olive surface which is the food finally ingested by consumers. This opens the possibility for the development of a new and simply probiotic fortified olive product.