J. Bautista-Gallego

Role of yeasts in table olive production

Table olives are a traditional fermented vegetable of the Mediterranean countries, but their production and consumption are now spread all around the world. Yeasts can play a double role in this food. They are present throughout the fermentative process and it is generally accepted that they can produce compounds with important organoleptic attributes determining the quality and flavour of the final product. However, yeasts can also be spoilage microorganisms in olive fermentation/storage and packing causing gas pockets, swollen containers, cloudy brines and off-flavours and off-odours. Candida boidinii, Debaryomyces hansenii, Pichia anomala, P. membranifaciens, Rhodotorula glutinis and Saccharomyces cerevisiae are species isolated with a high frequency from olive processes. Scarce information is still available about the ecology, biochemistry and molecular biology of these important microorganisms in table olives. A wider knowledge about these aspects could facilitate the possible application of yeasts as a starter culture, due to their ability to produce aromatic compounds, antioxidants, enzymes, and improve the growth of lactic acid bacteria.

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.

Fermentation profiles of Manzanilla-Aloreña cracked green table olives in different chloride salt mixtures.

NaCl plays an important role in table olive processing affecting the flavour and microbiological stability of the final product. However, consumers demand foods low in sodium, which makes necessary to decrease levels of this mineral in fruits. In this work, the effects of diverse mixtures of NaCl, CaCl(2) and KCl on the fermentation profiles of cracked directly brined Manzanilla-Aloreña olives, were studied by means of response surface methodology based in a simplex lattice mixture design with constrains. All salt combinations led to lactic acid processes. The growth of Enterobacteriaceae populations was always limited and partially inhibited by the presence of CaCl(2). Only time to reach half maximum populations and decline rates of yeasts, which were higher as concentrations of NaCl or KCl increased, were affected, and correspondingly modelled, as a function of salt mixtures. However, lactic acid bacteria growth parameters could not be related to initial environmental conditions. They had a longer lag phase, slower growth and higher population levels than yeasts. Overall, the presence of CaCl(2) led to a slower Enterobacteriaceae and lactic acid bacteria growth than the traditional NaCl brine but to higher yeast activity. The presence of CaCl(2) in the fermentation brines also led to higher water activity, lower pH and combined acidity as well as a faster acidification while NaCl and KCl had fairly similar behaviours. Apparently, NaCl may be substituted in diverse proportions with KCl or CaCl(2) without substantially disturbing water activity or the usual fermentation profiles while producing olives with lower salt content.

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.

Biofilm formation on abiotic and biotic surfaces during Spanish style green table olive fermentation

In this work, the establishment of polymicrobial communities on the surfaces which come into contact with the brine during Spanish style Gordal cv. green olive fermentation when subjected to spontaneous or controlled processes (inoculated with Lactobacillus pentosus LPCO10 or 128/2) was studied. Scanning electron microscopy showed that L. pentosus and yeast populations were able to form mixed biofilms throughout the fermentation process on both abiotic (glass slide) and biotic (olive skin) surfaces. The biofilm architectures in both supports were completely different: on the glass slides only aggregates of L. pentosus and yeasts without any polymeric matrix surrounding them were found while on the skin of the fruits, true mature biofilms were observed. During fermentation, the lactic acid bacteria (LAB) population on the olives remained similar while that of yeasts increased progressively to reach similar levels at the end of the process (8-9 log CFU/cm(2)). Molecular analysis showed that different populations of L. pentosus and yeasts were the only microbial members of the biofilm formed during fermentation, regardless of inoculation. Hence, the green olive surface provides an appropriate environmental condition for the suitable development and formation of complex biofilms during controlled or natural table olive processing.

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.

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.

Modelling the inhibition of sorbic and benzoic acids on a native yeast cocktail from table olives

The single and combined effects, in a synthetic medium at selected pH values, of sorbic and benzoic acids on a yeast cocktail (Saccharomyces cerevisiae, Pichia anomala, Issatchenkia occidentalis, and Candida diddensiae, isolated from table olives) have been studied. Applying the checkerboard method the minimum inhibitory concentration (MIC) obtained for the respective individual preservatives (expressed as undissociated acid) were: sorbic acid, 5.94, 3.85 and 3.19 mM at pH of 4.5, 4.0, and 3.5, respectively; and benzoic acid, not detected (at total 20.5 mM), 10.40 and 6.83 mM, respectively, for the same pH levels. The estimated fractional inhibitory concentrations (FIC) indexes showed additive effects between inhibitors. Fractional area (fa), modelled by the (extended) Lambert and Lambert [2003. A model for the efficacy of combined inhibitors. J. Appl. Microbiol. 95, 734-743] equation (ELPM), also showed additives of both preservatives but different shapes in the dose-response curves; the individual MIC (as undissociated acid) deduced from this method were: 5.60, 3.31, and 3.26 mM for sorbic acid at pH of 4.5, 4.0, and 3.5, respectively; and 29.65 (extrapolated), 10.00, and 6.25 mM for benzoic acid at the same pH levels. Mixtures above the curves connecting the limits (MIC) at each pH were also inhibitory. There was agreement between MIC values from FIC and ELPM, although the last one provided further information on the inhibition behaviour. I. occidentalis was the most resistant yeast of the cocktail to sorbic and benzoic acids.

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.