Yeast Ecology of Fermented Table Olives: A Tool for Biotechnological Applications Yeast Ecology of Fermented Table Olives: A Tool for Biotechnological Applications

The table olive is considered to be a traditional fermented vegetable in the Mediter­ ranean 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 essen ­ tial technological applications in this fermented vegetable. On the other hand, spoil­ age 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 prop­ erties. 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


Introduction
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 [1]. 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 [2]. Other countries such as USA, Peru, Argentina and Australia, are also rising as competitive producers [3].
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 bio synthesis, because they can be used firstly as carbon sources and eventually as energetic cur rency 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 metabo lism 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 [13], 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 activ ity of yeasts together with heterofermentative bacteria [12].
On the contrary, methanol, detected in brine [14], 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 condi tions [15].

Enzymatic activities
Yeasts ensure their survival in tissues, and are connected with the digestion of host's pro teins and other organic molecules due to a large array of physiological traits and enzy matic 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 isola tion and selection of useful microorganisms [20], it is the secretion of extracellular prote ases 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 pro duced by molds and bacteria [27], 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 endotryptase. However, all kind of yeasts do not comprise the same range of enzymes. Hence, different yeasts are func tioning differently toward the degradation of sugars. It is generally accepted that one yeast can ferment one sugar [30]. Since today, it is known that three of the aldo-hexose sugars, called dextrose, dmannose, and dgalactose, are exclusively fermentable by yeasts. It has been reported that yeasts which ferment dextrose, can also use mannose and laevulose [30]. 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.

Functional properties
Despite the fact that research has been focused almost exclusively on LAB, as being extremely useful in table olives microbiota [31], recent studies have been negotiating the significant con tribution 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 [32].
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][33][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 pro cess [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, pro biotics are microorganisms which could have beneficial impact on the human organism [38]. 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 nonfood 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 [39]. Thus, finding other yeast strains with probiotic character istics 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, [35] 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. [8] 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 CaCO 2 cell line and present an immunostimulatory activity [40][41][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 [43]. 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 [40]. Secondly, phytate has a strong che lating 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 dephosphory lation 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 [41].
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 Bcomplex vita mins [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 freeradicalscavenging activ ity. For instance, W. anomalus produced the highest activity in a laboratory medium [46]. The production of bioactive antioxidants may retard the oxidative degeneration of fatty sub stances 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 activi ties. The former has frequently been detected, while the isolation of strains with lipoytic activities has been reported to a lesser extent [7]. 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.

Yeast ecology of table olives
According to Ref. [4] it has been reported, that yeasts are responsible for the fermentation pro cess 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 ole aginosus, Pichia anomala, C. boidinii and T. delbrueckii [47][48][49][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 [3].
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 meth ods comparing the obtained results with diverse taxonomic keys [10,61]. However, molecu lar 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 [62], and (ii) the direct sequencing of the D1/D2 domains of the 26S rRNA gene amplified with primers NL1 and NL4 [63] or the 5.8S-ITS region amplified with primers ITS1 and ITS4 [62].
The information following below is a short description of microbial ecology studies at dif ferent 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).

Spain
According to Ref. [12], S. cerevisiae, Issatchenkia occidentalis and Geotrichum candidum were iden tified 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. anoma lus, 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 [54]. According to Ref. [7], 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 abun dant 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 [57] used a culture-independent approach based on the PCR-DGGE analysis for the identifica tion 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 [34].
To summarize, the hierarchically most prominent yeast on Spanish

Greece
In relevant scientific work of Greek scientists it has been discovered that Metschnikowia pulcher rima, D. hansenii and Aureobasidium pullulans were the dominant yeast species at the beginning of the fermentation process of Greek Conservolea black olives [55]. These researchers found a new yeast species associated with this type of fermentation, named Candida olivae. Species het erogeneity 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 [60]. Summarizing P. mem branifaciens is the dominant yeast on Conservolea black olives followed by P. anomala.

Italy
In Ref. [56], 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 fer mentation period is clear. In a similar work [58] also in Sicily with green table olives of cultivar Nocellara dell' Etna, researchers found S. cerevisiae, P. anomala, C. diddensiae, and I. orientalis dur ing the process. Finally, when researchers [6] studied the technological and spoiling character istics 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 biodiver sity in green olives is seems that P. guilliermondii and P. kluyveri are likely to play a crucial role in their fermentation.

France
In Ref. [51] it has been identified that P. anomala, C. boidinii and Debaryomyces etchelsii were dominant species in French black olives of Nyons area (South France).

Portugal
According to Ref. [8], 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 [59], 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 biodiver sity 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 [8].
Concluding this brief overview of yeast ecological studies in fermented

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][65][66][67][68][69]. In table olive fermentations, yeasts are an important group of microorganisms that act as both desirable and spoilage microor ganisms 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 char acter and it has been found, that W. anomalus protects olives from unsaturated fatty acid oxi dation 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 fermenta tion [72]. It has been reported that some oliverelated yeast strains, such as W. anomalus, can produce enzymes that could cause softening of the olives as an unfavorable property [33].
According to Ref. [73], 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 [74]. Moreover, L. pentosus's performance was rapidly improved, with the use of S. cerevisiae as a starter in green table olive solutions [73]. In the same study, it has been proved that the production of lactic acid was increased, as well. Yeasts seem to be active microorganisms dur ing 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 [75].
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 envi ronmental variables on yeast growth [5]. A problem usually occurring in such applications is to find the appropriate methodology to manage such a large amount of data, which is neces sary 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 [6] has been used. Principal Component Analysis (PCA) clearly differentiated and assisted the selection and discrimi nation of several W. anomalus and C. boidinii isolates with high global desirable activity levels [76].
However, in some cases, dominant yeasts could create products with milder taste and less selflife preservation [4,15]. This problem was reported in [65] 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 CO 2 resulting in penetrating olives and damaging the fruits [64]. 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. [33], 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 [77].
Moreover, yeasts present in packed olives can produce an excess gas (CO 2 ) leading to swollen containers, clouding of the brines, or produce off-flavors and off-odors [4]. 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 [78]. Fortunately, yeasts from

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 hav ing 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 preva lence 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 introduc tion 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 qual ity 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 sub ject with most recent the work enhancing the knowledge on fungal communities in directly brined Alorena de Malaga green olives fermentations using metabarcoding analysis [83]. 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, visualiz ing and exploring the food bacterial communities based on network analysis has announced preliminary results [84]. FoodMicrobionet, the platform prepared by the inclusion of 17 bacte rial 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 webbased 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 through put screening technique for aroma formation like the one used in [85] may provide exciting results with a plethora of new interrelationships. Similarly, a recent work with Sicilian table olives (cv. Nocellara Etnea) [86] 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.