Scientific reports of microbial ecology studies reporting the isolation of yeasts from different cultivars of table olives and different fermentation processes and different Mediterranean countries.
The table olive is considered to be a traditional fermented vegetable in the Mediterranean countries and its production and consumption is recently spreading all around the world. The presence of yeasts is very important during olive fermentation due to their double role. On one hand, yeasts maintain desirable biochemical activities (lipase, esterase, β-glucosidase, catalase, production of killer factors, etc.) with essential technological applications in this fermented vegetable. On the other hand, spoilage activity may be shown. However, recent studies have reported that yeasts coming from table olives would be a new source of potential probiotics. Indeed, many yeast species found in table olive processing, have been reported to demonstrate such properties. Thus, starter cultures technology will play significant role, not only in olive fermentation by controlling the safety and the quality of the final product, but also in consumer’s health.
- table olives
- starter cultures
Table olives are one of the most important and well known fermented vegetables, with an estimated worldwide production currently reaching 2.5 million tons per year . Not only the production, but also the consumption of this food is closely related to the culture and diet of some Mediterranean countries (Greece, Spain, Italy, France, Portugal, Egypt, and Turkey), which are the main producers . Other countries such as USA, Peru, Argentina and Australia, are also rising as competitive producers .
Yeasts play a vital role during table olives fermentation process principally on the safety, the quality and the flavor of the final product . On one hand, yeasts are responsible for properly driving the fermentation, due to their constant presence throughout the fermentative process. It is generally accepted, that they can produce compounds with important organoleptic attributes determining the quality and flavor of the final product . More specifically, they are able to produce desirable biochemical activities such as lipase, esterase, β-glucosidase, catalase, production of killer factors, which have several technological applications in the fermented table olives. On the other hand, yeasts may also cause spoilage of olives, during fermentation, packing and storage, due to the production of bad odors and flavors, the accumulation of CO2 leading to swollen containers and gas pocket effects, the clouding of brines, the softening of fruits and the degradation of lactic acid. The technological implications and functional properties of yeasts have been extensively reviewed [5–9]. It has been reported that they could exert a fundamental role for the fermentation of green-treated olives and black-naturally fermented ones . Moreover, many yeast species, which were found in table olive processing, such as Wickerhamomyces anomalus, Saccharomyces cerevisiae, Pichia membranifaciens and Kluyveromyces lactis, have been reported to exhibit some interesting properties, such as enhancement of the organoleptic characteristics of the fruits, degradation of polyphenols, enhancement of the growth of lactic acid bacteria (LAB) and antioxidant action [5, 9]. Therefore, the selection of the most appropriate yeast strains to be used as starters, in combination with LAB or not, is a promising future target which may improve the added value of the final product .
2. Biology and physiology of yeasts from fermented table olives
Yeasts are single-celled microbes, larger than bacteria, belonging to the kingdom of Fungi. Nowadays 1500 species are identified which corresponds approximately to 1% of all described fungal species . Both their survival and growth are ensured by consuming starches and sugars. Plenty of yeasts are commercially available and can provide an inexpensive source for biochemical and biotechnological applications (classic and molecular) .
Generally, in the heterotrophic organisms, such as yeasts, the energy and carbon metabolism have an interconnection between them, i.e., anabolism is connected with catabolism. ATP is provided by the oxidation of organic molecules which also play an important role in the biosynthesis, because they can be used firstly as carbon sources and eventually as energetic currency for all kinds of cellular work. Although, for instance, table olive yeasts species contain a wide set of carbon sources (polyols, alcohols, organic acids and amino acids) that can help in their growth, they prefer to metabolize sugars. There are a lot of data related to the metabolism of different carbon sources, with the most widely studied sugars being hexoses (glucose, fructose, galactose or mannose) and disaccharides (maltose or sucrose), as well as compounds with two carbons (ethanol or acetate) [5, 12].
The metabolic pathway of the central carbon metabolism is mainly identical between different yeast species, suggesting that these microorganisms might constitute a metabolic homogenous group. Nevertheless, the mechanisms for nutrient uptake, the number of different isoenzymes and the regulation of fermentation and respiration differ substantially , making yeasts a highly heterogeneous and complex metabolic group.
Moreover, the presence of volatile compounds, such as ethanol, glycerol, higher alcohols, and, to a lesser extent, acetaldehyde in brines can be directly attributed to the metabolic activity of yeasts together with heterofermentative bacteria .
On the contrary, methanol, detected in brine , is not related with the yeast metabolism, because its activity is due to pectinolytic enzymes. The presence of these enzymes in the drupes is favored by an improper handling of raw material and by poor fermentation conditions .
2.1. Enzymatic activities
Yeasts ensure their survival in tissues, and are connected with the digestion of host’s proteins and other organic molecules due to a large array of physiological traits and enzymatic activities [16, 17]. It is characteristic that the kind of medium used for their isolation and growth is an important adaptive factor which determines the technological activities of the strains selected [18, 19]. Although many surveys have underlined the potential of unconventional and extreme environments as a source of natural biodiversity for the isolation and selection of useful microorganisms , it is the secretion of extracellular proteases which has been studied extensively in yeasts isolated from these environments [21, 22]. Esterases from yeasts are also gaining industrial interest with applications in laundry detergents and in dairy industries [23, 24], while little attention has been paid to lipases from yeasts [7, 25, 26]. Although peptic enzymes for industrial uses have been so far produced by molds and bacteria , the pectinolytic activity of yeasts has, however, been studied with ambiguous results [28, 29].
Yeasts can also produce plenty of other interesting enzymes like invertase, zymase, hexose phosphatase, maltase, reductase, carboxylase, melibiase, and endo-tryptase. However, all kind of yeasts do not comprise the same range of enzymes. Hence, different yeasts are functioning differently toward the degradation of sugars. It is generally accepted that one yeast can ferment one sugar . Since today, it is known that three of the aldo-hexose sugars, called dextrose, d-mannose, and d-galactose, are exclusively fermentable by yeasts. It has been reported that yeasts which ferment dextrose, can also use mannose and laevulose . Due to the enzyme invertase, yeasts can firstly reverse and secondly ferment cane-sugar. However, they are unable to ferment milk-sugar, because of the lack of the enzyme lactase.
2.2. Functional properties
Despite the fact that research has been focused almost exclusively on LAB, as being extremely useful in table olives microbiota , recent studies have been negotiating the significant contribution of yeasts to table olives fermentation [3, 5, 9], during the whole process and adding value to the final product. The selection of yeasts to be used as starter cultures is an unclear and complicated procedure, which includes three main steps. The first one is the selection of promising species, which should have some interesting properties. The second step is the validation of those microorganisms on laboratory scale and finally the third one, and the most important, is the demonstration at large scale .
Starter cultures must be selected for their functional traits, and for their ability to dominate at the fermentation process, as well as leading to a stable final product. Thus, the screening of those yeast strains is very important. Esterase and lipase enzymatic activities are desirable in yeasts because of their ability to improve the organoleptic characteristics of olives through the configuration of volatile compounds that can be produced by the catabolism of free fatty acids [9, 32–34].
Yeasts with β-glucosidase activity are also good candidates, because they can be used for the hydrolysis of oleuropein, removing the natural bitterness present in brined olives and thus, avoiding the placement of olives into large amounts of water [3, 35]. However, limited data are available in the literature about the contribution of these species on table olive fermentation process [7, 17, 34]. Furthermore, very few strains which produce nontoxic compounds, like biogenic amines (BA) (spermine, spermidine, agmatine) was proposed as a new criterion for the selection of yeasts as starter cultures for many fermented products [36, 37]. Many yeast species which have the ability to produce BA can be found in olives. As a coincidence, this trait should be investigated as another critical point for the selection of proper starter cultures in the near future.
It is noteworthy to mention the probiotic profile of some table olives yeasts. Generally, probiotics are microorganisms which could have beneficial impact on the human organism . This positive impact would exist only if those microorganisms are safe for the host, metabolically active within the gastrointestinal tract and are being consumed, either as food components or as non-food preparations. For instance, Saccharomyces boulardii, a member of S. cerevisiae species, is the only yeast with clinical role and proven probiotic efficiency in double blind clinical studies . Thus, finding other yeast strains with probiotic characteristics and especially those isolated from table olives is of great interest.
The probiotic potential of yeasts isolated from fermented table olives has been documented to some extent [8, 35]. In particular, Psani & Kotzekidou,  found Torulaspora delbrueckii and Debaryomyces hansenii strains to be quite tolerant in high bile salt concentrations and low pH values. Moreover, the fact that the inoculation of these strains were able to inhibit food borne pathogens such as Listeria monocytogenes, Bacillus cereus and Salmonella typhimurium is remarkable. Furthermore, data from Ref.  showed that P. membranifaciens and Candida oleophila strains have similar properties but with a different spectrum of inhibition zones (for Escherichia coli, Salmonella enteritidis and Staphylococcus aureus). Another significant number of yeast species, such as K. lactis, D. hansenii, T. delbrueckii and S. cerevisiae, have shown tolerance to traverse the gastrointestinal tract, inhibit the enteropathogens, adhere the intestinal CaCO2 cell line and present an immunostimulatory activity [40–42]. In particular, S. cerevisiae has shown its ability to prevent the survival of E. coli O157:H7 under simulated gastrointestinal conditions by the production of ethanol . Although research of olive yeast strains with the properties mentioned above is a promising task, it is important for olive yeasts to adhere on to the olive skin and survive during storage and/or packaging, in order to be ingested by consumers at elevated numbers.
Except from their probiotic properties, yeasts could positively affect human health in other ways, as well. For instance, diverse strains of K. lactis, S. cerevisiae, and Issatchenkia orientalis show great ability to reduce cholesterol serum levels . Secondly, phytate has a strong chelating ability to form insoluble complexes with divalent minerals of nutritional importance such as zinc, calcium, magnesium and iron. Due to a lack of some required enzymes, humans cannot degrade phytate complexes in the gastrointestinal tract. It is known that dephosphorylation of phytate is catalyzed by phytases, which are widespread in yeast species such as I. orientalis, W. anomalus, S. cerevisiae, T. delbrueckii, and K. lactis [41, 44]. Thus, yeasts could be included in humans’ diet in order to help them to do so.
Folates (vitamin B9) are considered essential co-factors in the biosynthesis of nucleotides and play an important role in cellular replication and growth. It is common that mammals cannot synthesize folates and a potential solution is for yeasts to help them to do so because they contain a folate biosynthesis pathway and can produce natural folates. Some of those species which have a high folate biosynthesis pathway are the S. cerevisiae and Candida glabrata .
Other species of yeasts, such as the diverse yeast strains isolated from table olives, belonging to the P. membranifaciens and P. farinosa species have the ability to produce B-complex vitamins [8, 45]. This means that yeasts can synthesize a number of bioactive compounds which can serve as natural antioxidants.
Moreover, researchers are interested in screening of yeasts for free-radical-scavenging activity. For instance, W. anomalus produced the highest activity in a laboratory medium . The production of bioactive antioxidants may retard the oxidative degeneration of fatty substances and improve human health. In any case, probiotic yeasts could be able to adhere onto the olive epidermis, and thus, be ingested by consumers.
Once researchers are very interested in the organoleptic features of olives, yeasts have some very essential and strain-specific metabolic properties, such as esterase and lipolytic activities. The former has frequently been detected, while the isolation of strains with lipoytic activities has been reported to a lesser extent . Enterase positive yeasts are covetable because the fact that they are able to meliorate the flavor of olives from the formation of esters coming from free fatty acids. Strong lipase activity, as well as, weak activity have been detected both in vitro in some yeast species. The former has been reported in Candida boidinii, D. hansenii and T. delbrueckii while the latter only in P. membranifaciens [34, 35]. Those authors have emphasized the change of the free fatty acids composition of olives in the presence of yeast populations in contrast with sterile conditions, indicating that lipases produced by these microorganisms modify the characteristics of fruit lipids and therefore its organoleptic characteristics.
3. Yeast ecology of table olives
According to Ref.  it has been reported, that yeasts are responsible for the fermentation process of natural black olives. However, to green olives, the fermentation is driven by LAB. Black olive fermentations have been studied in different countries, mainly in the Mediterranean (Italy, Spain, Portugal, Greece, and Morocco) and showed some yeast species biodiversity. Despite this fact, a few species were dominant, such as P. membranifaciens, Saccharomyces oleaginosus, Pichia anomala, C. boidinii and T. delbrueckii [47–50]. However, since LAB are partially inhibited in directly brined green and natural black olives due to the presence of phenolic compounds, yeasts became highly important for the fermentation process .
Nowadays, several studies have focused on yeasts microbiota situated on the surface of olive fruits. Some of those studies are summarized in Table 1. In the past, the characterization of yeasts associated with table olives was mainly made by morphological and biochemical methods comparing the obtained results with diverse taxonomic keys [10, 61]. However, molecular methods have started to be used recently, for the identification of yeasts coming from table olives fermentation. One of the most applicable methods is the Denaturating Gradient Gel Electrophoresis (DGGE-PCR), which is more precise than any classical method and is based on (i) restriction fragment length polymorphism (RFLP) analysis obtained after cutting the amplified 5.8S rRNA gene and the associated intergenic spacers ITS with endonucleases , and (ii) the direct sequencing of the D1/D2 domains of the 26S rRNA gene amplified with primers NL1 and NL4  or the 5.8S-ITS region amplified with primers ITS1 and ITS4 .
|Saccharomyces cerevisiae, Issatchenkia occidentalis, Geotrichum candidum (during process)||Spanish green seasoned table olives||Sequencing of 26S rRNA, D1/D2 region|||
|Candida boidinii, Hanseniaspora guilliermondii (final product)||Ripe black olives||Sequencing of 26S rRNA, D1/D2 region|||
|P. anomala, C. boidinii, Debaryomyces etchelsii (during process)||French black olives||RFLP, Sequencing of D1/D2 region|||
|C. boidinii, Candida sorsoba, Candida diddensiae, P. membranifaciens, Kluyveromyces lactis, P. anomala, P. kluyveri, Rhodotorula glutinis (during process)||Spain, Arbequina table olives||RFLP||[52, 53]|
|Pichia caribbica, Lachancea fermentati, Nakazawaea holstii (during process)||Spain, Arbequina and Cornicabra||Sequencing of 5.8 rRNA, ITS1-ITS2 region|||
|Metschnikowia pulcherrima, Debaryomyces hansenii, Aureobasidium pullulans (during process)||Greece, Conservolea black olives||Sequencing of 5.8 rRNA, ITS1-ITS2 region|||
|P. membranifaciens, P. anomala (final product)||Greek black olives||Sequencing of 5.8 rRNA, ITS1-ITS2 region|||
|Candida parapsilosis, P. guilliermondii, P. kluyveri (during process)||Sicilian green table olives.||RFLP, Sequencing of 26 s rRNA D1/D2 region|||
|S. cerevisiae, Pichia galeiformis, C. boidinii (final product), and P. membranifaciens (during possess)||Hojiblanca||RFLP, Sequencing of 5.8 s rRNA, ITS1-ITS2 region|||
|C. diddensiae, S. cerevisiae and P. membranifaciens (during process)||Spain/ Alorena||RFLP, Sequencing of 26 s rRNA D1/D2 region|||
|Candida tropicalis, P. galeiformis and P. anomala (during process)||Spain/Gordal and Manzanilla||RFLP, Sequencing of 26 s rRNA D1/D2 region|||
|S. cerevisiae and Candida apicola (during process)||Spain/ Alorena de Malaga||PCR-DGGE of 26SrRNA|||
|S. cerevisiae, P. anomala, C. diddensiae, Issatchenkia orientalis (during process)||Sicilian green table olives||PCR-DGGE of 26 s rRNA|||
|P. membranifaciens, Pichia fermentans, S. cerevisiae, Candida oleophila. (during process)||Portuguese brined olive||RFLP, Sequencing of 26 s rRNA D1/D2 region|||
|Citeromyces matritensis, Zygotorulaspora mrakii, S. cerevisiae (during process)||Portugal, Manzanilla||Sequencing of 26 s rRNA D1/D2 region|||
|P. membranifaciens (during process)||Greece, Conservolea||Sequencing of 5.8 s rRNA, ITS1-ITS2 region|||
The information following below is a short description of microbial ecology studies at different cultivars and processing steps within the major table olives production countries of Mediterranean in an attempt to draw conclusions on their yeast colonization (Figure 1).
According to Ref. , S. cerevisiae, Issatchenkia occidentalis and Geotrichum candidum were identified from Spanish naturally green seasoned table olives (cv. Alorena). In the same study the researchers identified C. boidinii and Hanseniaspora guilliermondii from the preservation stage of ripe black olives. During the fermentation of Arbequina naturally green table olives in Spain [52, 53], C. boidinii, C. sorsoba, Candida diddensiae, K. lactis, P. membranifaciens, W. anomalus, P. kluyveri, and Rhodotorula glutinis were found. In a study looking at the Spanish yeast biodiversity of oleic ecosystems, the yeast biodiversity in the fresh table olive, crushed olives and olive pomace from Arbequina and Cornicabra varieties, has been found to contain Pichia caribbica, Lachancea fermentati and Nakazawaea holstii, as the most important isolated species . According to Ref. , researchers carried out the molecular identification by means of a RFLP analysis and sequencing of a total of 199 yeast isolates obtained from Spanish industrial green table olive fermentation. C. diddensiae, S. cerevisiae and P. membranifaciens were the most abundant yeast species isolated from directly brined Alorena olives, but for Gordal and Manzanilla cultivars, Candida tropicalis, P. galeiformis and W. anomalus were found. Recently, other scientists  used a culture-independent approach based on the PCR-DGGE analysis for the identification of yeasts associated with Alorena de Malaga olive fermentation, and found that in cold fermented olives the most essential yeasts were S. cerevisiae and Candida apicola. Finally, it has been shown that the most important species throughout the storage period of table olives were S. cerevisiae and Pichia galeiformis, although C. boidinii was present at the last stages of the process and P. membranifaciens was detected at an earlier stage of the Hojiblanca cultivar storage .
To summarize, the hierarchically most prominent yeast on Spanish table olives are P. membranifaciens, C. boidinii, C. diddensiae, S. cerevisiae and P. anomala.
In relevant scientific work of Greek scientists it has been discovered that Metschnikowia pulcherrima, D. hansenii and Aureobasidium pullulans were the dominant yeast species at the beginning of the fermentation process of Greek Conservolea black olives . These researchers found a new yeast species associated with this type of fermentation, named Candida olivae. Species heterogeneity changed during fermentation and both P. membranifaciens and P. anomala became the only dominant yeasts at the end of the fermentation. A similar work focusing on microbial heterogeneity during aerobic and modified atmosphere packaging storage of Conservolea natural black olives found that P. membranifaciens was dominated in all pouches regardless of gas composition with a frequency more than 80% during storage . Summarizing P. membranifaciens is the dominant yeast on Conservolea black olives followed by P. anomala.
In Ref. , it has been revealed that in Sicilian environment in four Italian olive cultivars (Bradofina, Castriciana, Nocellara del Belice and Passalunara) and one Spanish (Manzanilla) the presence of P. kluyveri followed by Candida parapsilosis and P. guilliermondii during the entire fermentation period is clear. In a similar work  also in Sicily with green table olives of cultivar Nocellara dell’ Etna, researchers found S. cerevisiae, P. anomala, C. diddensiae, and I. orientalis during the process. Finally, when researchers  studied the technological and spoiling characteristics of yeast microflora isolated from cultivar Bella Di Cerignola table olives, most prominent yeasts isolated were Candida famata and C. guilliermondii. Summarizing the Italian yeast biodiversity in green olives is seems that P. guilliermondii and P. kluyveri are likely to play a crucial role in their fermentation.
In Ref.  it has been identified that P. anomala, C. boidinii and Debaryomyces etchelsii were dominant species in French black olives of Nyons area (South France).
According to Ref. , researchers studied the yeast population associated with Portuguese brined green olives of cultivars Galega and Cordovil, fermentation was mainly driven by P. membranifaciens, P. fermentans, S. cerevisiae and C. oleophila. In a similar work , it has been found that during the initial phases of cracked green Manzanilla olive fermentation a great diversity of yeasts was observed; however, as the process was evolving, the biodiversity decreased with the fermentative yeasts Citeromyces matritensis, Zygotorulaspora mrakii and S. cerevisiae becoming the dominant species. These species though are reported as high risk spoilage microorganisms contrary to P. membranifaciens reported by .
Concluding this brief overview of yeast ecological studies in fermented table olives around the Mediterranean it becomes apparent that among Candida species, C. boidinii, C. diddensiae, C. famata (formerly D. hansenii), C. guilliermondii (formerly H. guilliermondii), and C. oleophila are the most prominent. Among Pichia species, P. membranifaciens, Pichia anomala (W. anomalus) and P. fermentans are the most prominent. From other species we tend to isolate more A. pullulans, Debaryomyces etchellsii, G. candidum, I. occidentalis, K. lactis, Rhodotorula spp., S. cerevisiae and Z. mrakii. These yeasts are plentiful for detailed studies on their role during table olive fermentations and could offer great opportunities for biotechnological tools as starter cultures.
4. Biotechnological applications of table olives yeast strains
Yeast microbiota in olives is very heterogeneous and can be altered depending on the olive cultivar, region, type of fermentation process, salt concentration, pH, nutrients, oxygen and interactions with other microorganisms [5, 57, 64–69]. In table olive fermentations, yeasts are an important group of microorganisms that act as both desirable and spoilage microorganisms and it is important to evaluate their biodiversity in table olive fermentations [3, 4]. As a positive effect, some yeast species isolated from table olives, such as Debaryomyces, Pichia and Candida are known to include a considerable number of strains with a killer character and it has been found, that W. anomalus protects olives from unsaturated fatty acid oxidation and peroxide formation [3, 70, 71], relating also to the antioxidant activity of yeasts [7, 8]. Moreover, it has been reported that W. anomalus and S. cerevisiae isolated from diverse table olive fermentations, have phytase enzymes that are required for the degradation of phytate complexes [3, 41, 44]. On the other hand, W. anomalus is important yeast for olive fermentation, but it may also have a role in the deterioration of olives at the end of fermentation . It has been reported that some olive-related yeast strains, such as W. anomalus, can produce enzymes that could cause softening of the olives as an unfavorable property .
According to Ref. , the combination of yeasts with LAB, has resulted to an improvement in growth of the LAB. As a result, the production of lactic acid was improved, thanks to the greater availability of the necessary nutrients by the yeasts activity and lysed cells. Indeed, recent studies have shown that the growth of LAB could be stimulated by the use of yeasts, as for example L. plantarum, improved its growth when D. hansenii was inoculated in olive brine . Moreover, L. pentosus’s performance was rapidly improved, with the use of S. cerevisiae as a starter in green table olive solutions . In the same study, it has been proved that the production of lactic acid was increased, as well. Yeasts seem to be active microorganisms during the fermentation of table olives, synthesizing substances such as vitamins, amino acids and purines, or breakdown complex carbohydrates, which are essential for the growth of Lactobacillus species .
Therefore, selection of the most appropriate yeasts for starters should be firstly based on strains possessing the best enzymatic activities as mentioned earlier and secondly to their ability to predominate during fermentation. Moreover, it is needed to have a high resistance to salt and low pH values. Predictive microbiology is seemed to be a valuable tool for the discrimination and selection of the most promising strains, determining the influence of environmental variables on yeast growth . A problem usually occurring in such applications is to find the appropriate methodology to manage such a large amount of data, which is necessary when researchers have to analyze several biochemical activities or growth data from a considerable number of strains.
Multivariate analysis techniques offer a viable approach in solving this setback. For instance, multivariate analysis approach to study growth and qualitative activity data of a number of yeasts isolated from Bella di Cerignola table olives  has been used. Principal Component Analysis (PCA) clearly differentiated and assisted the selection and discrimination of several W. anomalus and C. boidinii isolates with high global desirable activity levels .
However, in some cases, dominant yeasts could create products with milder taste and less self-life preservation [4, 15]. This problem was reported in  during fermentation of natural black olives at different NaCl levels and temperatures. Moreover, an excessive growth of fermented yeast (7 log10 CFU mL−1) could produce a vigorous production of CO2 resulting in penetrating olives and damaging the fruits . The use of high levels of NaCl during fermentation (8% in the equilibrium) could privilege the growth of yeasts against LAB [4, 65]. In accordance with Ref. , it has been found that some strains of Rhodotorula minuta and D. hansenii in green table olive fermentations are having this ability. Finally, strains of R. glutinis, R. minuta and R. rubra could grow, form pellicles in olive brines and produce polygalacturonases causing a softening of olives kept in storage .
Moreover, yeasts present in packed olives can produce an excess gas (CO2) leading to swollen containers, clouding of the brines, or produce off-flavors and off-odors . Furthermore, it has been reported that yeasts identified currently as S. cerevisiae and P. anomala spoiled the olives through a combination of gas-pocket formation and softening . Fortunately, yeasts from table olives are almost entirely nonpathogenic. The inhibitory effect of sorbic acid, benzoic acid and their salts on yeasts growth to stabilize table olive packing, were reported previously [5, 79, 80].
5. Future perspectives
At this moment, the scientific community has gathered significant amount of knowledge about the ecology of fermented table olives, physiology, biochemistry and genetics of yeasts isolated from table olives fermentation and more data are soon going to be added with the use of high throughput sequencing techniques. Despite the large amount of beneficial properties that these microorganisms could offer to the final product, recent findings showed that yeasts are not only in the cover brines, but also on the fruit epidermis, which indicates a promising source of probiotic strains [43, 81]. The production of an innovative and functional food having the advantages of probiotic yeasts, can contribute to a final product with added value, even higher than it already has. From the research so far, it is clear that yeasts from table olives show an interesting technological and probiotic profile. However, further research is needed as far as their physiology, ecology, biochemistry is concerned. Also the study of yeasts are needed in molecular level throughout the fermentation, in order to be explained their prevalence or not in the final product, especially when it has to do with probiotic microorganisms. Undoubtedly, the full potential of table olive related yeasts have not been fully determined and many challenges are awaiting research, dissemination and industrial exploitation.
The microbiota of olives varies somewhat from region to region, from cultivar to cultivar and from type to type of processing [5, 12, 33, 34, 76]. For this reason and due to the importance of yeasts to the final product, it is a challenge to investigate and combine the diversity of yeasts in table olives around regions as a geographical indication. This could lead to the introduction of new table olives as PDO or PGI products, especially for EU countries. Finally, starter cultures can play an essential role in olive fermentation by controlling the safety and the quality of the final product. For extensive information on selection of yeasts as starter cultures for table olives, Refs. [32, 82] are thorough reviews with step by step procedures to follow.
Next Generation Sequencing (NGS) approaches have recently started to appear in the subject with most recent the work enhancing the knowledge on fungal communities in directly brined Alorena de Malaga green olives fermentations using metabarcoding analysis . Along these lines more publications are expected in the forthcoming years.
However, NGS technologies are providing massive amount of data, many times difficult to interpret at logical, applied level. Recently, a very important effort on databasing, visualizing and exploring the food bacterial communities based on network analysis has announced preliminary results . FoodMicrobionet, the platform prepared by the inclusion of 17 bacterial studies on dairy products, dairy starter cultures, raw and fermented meat, doughs and sourdoughs and fermented vegetables is attempting to analyze nodes and network properties while building an interactive web-based visualization. By this the researcher can explore the relationships between Operational Taxonomic Units (OTUs) and samples in order to identify core and sample specific bacterial communities. A similar approach will be very much in use for yeast/fungal studies of various matrices. The above if combined with a high throughput screening technique for aroma formation like the one used in  may provide exciting results with a plethora of new interrelationships. Similarly, a recent work with Sicilian table olives (cv. Nocellara Etnea)  investigates the bacterial community and its dynamics during the fermentation of the olives and its effect on metabolome formation.
In the following years we expect more detailed studies in the field of table olives microbial ecology and biotechnology, as well as gaining valuable knowledge for the related microbial functions that can be applied far beyond olive fermentations.