Table olives are fermented vegetables very popular in the world and especially in the Mediterranean countries. Five main styles (Spanish or Sevillian, Castelvetrano, Siciliano, Californian, and Greek) are diffused to produce commercial products, beside several traditional styles. Although the main preparation methods of table olives are known for a long time, they are not yet optimized systems, and each of them is characterized by advantages and disadvantages. The use of NaOH for green olive debittering is responsible for the elimination of many aroma compounds and nutritionally important molecules. High volumes of heavily contaminated wastewaters are produced during olive processing. Spontaneous fermentation processes used to ferment black or green olives are difficult either to monitor or control. Microbial starters, selected for specific bio/technological and safety traits, can be useful to (i) improve the table olives organoleptic characteristics, (ii) control the fermentation process and significantly reduce the time to obtain a final product, (iii) monitor the correct evolution of the process, (iv) ensure the maintenance and/or improvement of nutritional and healthy features of the product, (v) protect table olives from undesired spoilage and pathogenic microorganisms, (vi) produce table olives as a carrier of microorganisms with probiotics characters, and (vii) enhance product stability and shelf life.
- table olives
- organoleptic traits
- nutritional characteristics
Table olives are one of the most important and popular fermented vegetables in Western world and in particular in Southern European countries. Table olives world production was estimated to be 2,742,500 tons in 2015–2016 season (Figure 1).
The 29% of this production (796,000 tons) is located in the European Union (EU). Spain has a leading position in table olive production with 514,000 tons, followed by Greece (210,000 tons), Italy (50,000 tons), and Portugal (17,500 tons) . Among the countries of the Mediterranean Basin, Egypt, Turkey, Algeria, and Morocco are the main producers and consumers (Figure 2).
According to International Olive Council Standard, the term “table olive” means the product prepared from the sound fruits of varieties of the cultivated olive trees that are chosen for their production of olives whose volume, shape, flesh-to-stone ratio, fine flesh, taste, firmness, and ease of detachment from the stone make them particularly suitable for processing; treated to remove its bitterness and preserved by natural fermentation, or by heat treatment with or without the addition of preservatives; packed with or without covering liquid.
Table olives are classified according to the degree of ripeness of the drupes (green olives, olives turning color, and black olives), trade preparations (treated olives, natural olives, dehydrated and/or shriveled olives, olives darkened by oxidation, and specialties), and styles (whole, pitted, stuffed, salad, and other). They are produced by processing raw olives with the objective of eliminating their natural bitterness, which is mainly due to oleuropein and other phenolics . The main commercial types of table olives are processed according to five styles: Spanish (or Sevillian), Castelvetrano, Siciliano, Californian, and Greek , although several other traditional styles also exist for the preparation of treated and natural table olives .
The two main commercial table olives preparations, lye-treated olives (Spanish and Castelvetrano styles) and brine-soaked olives (Greek style) are industrially produced by spontaneous fermentation, but, currently, these processes are difficult to be monitored and controlled . The spontaneous process cannot ensure either the correct evolution of the process or the good quality and safety standards of the final product. Controls of the presence of biogenic amines and toxins in table olives commercial preparations need to be increased .
In green olives productions, the NaOH is used as chemical debittering system. This treatment is economic, characterized by a simple implementation, and an easy standardization. However, simultaneously to the debittering effect, it causes the elimination of many aroma compounds together with nutritional and health important molecules. The process produces also high volumes of heavily contaminated wastewaters. Besides, the use of NaOH for debittering organic table olives is prohibited in many countries .
The employment of starter cultures of
It has also been demonstrated that yeasts, producing desirable metabolites and volatile compounds, are able to improve the organoleptic properties. Yeasts can also enhance the growth of lactic acid bacteria (LAB) and degrade phenolic compounds. A role of yeasts as starters has been recently proposed for production of table olive [11–15].
There is an increasing interest in lowering NaCl concentration (now 8–10%) and in shortening the fermentation time (8–12 months) in order to obtain a healthier product suitable to reach the market very soon.
Recently, Bleve et al. described a novel method based on the sequential use of autochthonous yeast and bacterial strains to shorten the time of fermentation, to standardize the process, and to improve organoleptic and nutritional properties of olives .
The future challenges will be to investigate some strains for their probiotic characteristics, in order to produce functional olives. Indeed, several studies demonstrated that the use of LAB as starter for table olive production can produce beneficial effects on human health [17, 18]. Also yeast strains have been evaluated for their probiotic properties [13, 15]. The nutritional and health-related compounds associated to fermented table olives (or derivatives) could be assessed by
Table olives are considered by many food scientists as the “food of the future” owing to the healthy bioactive compounds they contain. In fact, table olives, together with olive oil, represent an important food of the Mediterranean diet and are perceived to have positive nutritional and therapeutic effects. Monounsaturated fatty acids, as found in olives are known to be healthier than polyunsaturated and saturated fats. In addition, epidemiological studies indicate that olive biophenols have a role in lowering incidence of several chronic and heart diseases [19, 20].
2. Production methods
Table olives, directly harvested from trees, need to be processed in order to reduce or eliminate their bitter taste and to obtain a product ready to be consumed. Different commercial preparations of table olives are produced using procedures inherited and opportunely modified from traditional methods. As previously extensively described by Boskou et al. , the main methods including fermentation steps to obtain the final product are water-cured olives produced by soaking olives in water over a week or more and then placing them in brine where a fermentation process can occur; Greek-style or “natural” olives and Sicilian-style green olives spontaneously fermented in brines; lye-treated olives (Spanish or Sevillian style, Castelvetrano method) produced by a first treatment with alkali (NaOH) and, after washing olives with water to remove NaOH, by a second step in brine to obtain a partial or complete fermentation of the drupes.
Other methods for black olives not involving fermentation are known as Californian and Spanish styles. The drupes are debittered by lye and soaking in brine. They are also aerated insufflating air to oxidize the pigments and immersed in ferrous gluconate or ferrous lactate solution in order to stabilize a uniform black color. Table olives can also be produced by traditional methods diffused in Mediterranean Basin using lime (CaO) and olive wood ash, or they can be dried and debittered without chemicals by using salt or heat treatment. Some cultivars of olives resulted naturally debittered also by parasite fungi directly on the tree without necessity of further treatment. They can also undergo a natural sweetening during ripening on the tree, although genetic and biochemical mechanisms involved in this last phenomenon are until now unknown.
3. Biotechnological approaches to produce table olives
The fermentation process, generally performed by indigenous microorganisms, is one of the best and oldest procedures of treating food products to transform and preserve them. However, as already demonstrated in several food products (wine, beer, bread, yogurt, cheese, sake, chocolate, etc.), spontaneous fermentations are uncontrolled and not predictable. These spontaneous processes are inefficient since they do not ensure the expected quality and safety characteristics of the final product, the sensorial and structure features, the limitation, or absence of growth of harmful or undesired spoilage organisms .
In order to obtain a more controlled process and to improve the quality and safety levels of table olives, the selection and use of starter cultures is diffusing. In fact, several studies demonstrated the usefulness and the benefits of starters in table olives production [5, 9, 15, 23].
3.1. Starter selection
The microbiota associated to olives can be different among the different cultivars. Microorganisms detected in table olives and brines belong to members of bacteria (lactic acid bacteria, Enterobacteriaceae,
The presence of hazardous pathogens such as
The most studied group of bacteria is lactic acid bacteria (homo and hetero fermentative), since they are responsible for the sugar conversion to organic acids and in particular to lactic acid. The different table olives production methods can influence microbial population present in raw olives and their evolution during fermentation (Figure 3).
Yeasts found in olives belong to the genera
Also the mould genera
The introduction of LAB and yeasts starter cultures in table olives production can also be motivated by the difficulty to monitor and control spontaneous fermentation in the industrially production of black as well as several cultivars of green olives [33–35]. Starter cultures are preparations of microorganisms, live, or resting, generally present in high cell number, which can be added to enhance, accelerate, and improve a fermentation process by their metabolic activities.
LAB have been considered very important since they are able to debitter olives, low brine pH, limit the spoilage and the presence of pathogens, and develop a correct flavor and texture in the final product. Several studies proposed the use of
The use of yeasts as starters cultures has been recently proposed for production of table olive [11, 13, 14, 35, 39], since they can improve the organoleptic properties [11, 40], enhance the growth of LAB [12, 41], and biodegrade phenolic compounds .
Moreover, the possibility to use simultaneous or sequential inocula of yeasts and LAB in green and black olives has been proposed [12, 15, 37, 41, 43]. The presence of yeasts together with LAB can produce a significant improvement of the sensorial quality of olives. They can also favor LAB growth rate, help in Enterobacteriaceae reduction, sensitively shorten the time needed to obtain the final product.
Moulds can be responsible of undesirable effects on table olives quality. They can alter olive taste and appearance and can be responsible of mycotoxins production . These microorganisms need to be deeply studied in order to evaluate their possible positive role in table olive processing.
LAB and yeast strains to be used as starter cultures can be selected among microorganisms associated to a spontaneous fermentation. In a first step, they can be selected on the basis of their characteristics and abilities: (i) to lower pH (by homo or hetero-fermentative metabolism for LAB, by fermentative metabolism for yeasts); (ii) to survive and to grow in the presence of different constraints (poor nutrient substrate like olives in brine, low pH, high salt level, presence of phenols, wide range of temperatures), (iii) to produce lactic acid and other organic acids; (iv) to metabolize phenols and, in particular, to degrade oleuropein, which is the main compound responsible for the bitter taste in olives; (v) to develop desired flavors (volatile compounds); (vi) to produce no biogenic amines, which represent an emerging concern in table olives, wine and other fermented products [44–46]; (vii) to possess esterase and lipase activities that have a role in improving the aromatic profile of fermented olives (by increasing their free fatty acid content); (viii) to have no proteolytic and pectolytic activities, which could have a negative impact on olive quality since they are related to olive softening; (ix) to have functional (probiotics and health-promoting) properties. Several laboratory tests have been developed to select yeasts and LAB for all of these features. In a laboratory-scale, the most promising isolates can be tested for their ability to dominate the indigenous microbiota by predominant growth or by production of antagonistic substances during table olives fermentation.
The selected LAB and yeast isolates can be then tested in a pilot-scale fermentation (200 kg) in order to mimic the industrial conditions of fermentation. The best performing isolates can be proposed for industrial-scale fermentation in tanks of 3–8 tons.
3.2. Influence of starter cultures on table olives chemical and aromatic profile
The distribution and structure of the chemical constituents of olive fruit is complex and depend on variety, cultivation practices, geographical origin, and the level of maturation. Olive fruit’s average composition is water (50%), protein (1.6%), fat (22%), carbohydrate (19.1%), cellulose (5.8%), inorganic substances (1.5%) and phenolic compounds (1–3%).
Both in green and black olive fermentations, lactic, citric, tartaric, and acetic acids were found to be the major metabolic products in drupes and in the brines [9, 47, 48], responsible for a decrease in pH value (about 4.0), satisfactory for naturally black olive fermentation [29, 30].
Although in the literature there are many data about aroma compounds in olive oil, very little is known about the quali-quantitative composition of volatile compounds in table olives. Among table olives, more attention has been placed on the characterization of the volatile fraction of the fermented black olives. Little is known about volatile fraction of green olives, probably because their volatile profiles are less rich, due to the NaOH treatment; the latter affects many precursors of the volatile compounds.
The formation of flavor compounds in table olives is a dynamic process mainly occurring during fermentation carried out by LAB and yeasts, along with a variety of contaminating microorganisms, which produce a variety of volatile compounds . Volatile and semivolatile organic compounds are responsible for the olive complex flavor that in turn can influence the consumer’s preference. The “green odor” of unripe olives was associated to the presence of C5 and C6 volatile compounds (alcohols and aldehydes) originating from the activity of lypoxygenase metabolic pathway . Hexanol and 2-hexenal are the major contributors to the characteristic green odor of olives and of many fruit and vegetable fermented foods. In spontaneous fermentation of black olives, the main product is ethanol that derives from the metabolic activity of different yeasts and hetero-fermentative LAB and is very important for the organoleptic properties of the final product .
C6 alcohols such as 1-hexanol and
Fatty acids, formed enzymatically during fermentations constitute an important group of aroma compounds that can contribute to the aroma complexity of table olives [29, 30]. Terpenes production is closely linked to cultivars, geographical area, climatic conditions and proliferation of specific pests and microorganisms characteristic of a given production area . The presence of styrene can increase during fermentation . This compound could be linked to an environmental contaminants and/or produced by L-phenylalanine deamination and decarboxylation of
The use of selected starter cultures has been proposed for Spanish- and Greek-type, green and black, olives to improve fermentation performance. Starters can accelerate and control the process, reduce undesired off-flavors and enhance quality of the final product by the development of typical and peculiar sensorial and taste characteristics.
In the evolution of volatile compounds during spontaneous fermentation of different black olive cultivars (Leccino, Cellina di Nardò, Conservolea and Kalamàta), Tufariello et al.  identified three main temporary steps characterized by the presence of chemical descriptors: aldehydes at the first stage (30 days), higher alcohols and styrene in the middle (90 days), and ethyl esters and fatty acids at the end of fermentation (third fermentation stage, 180 days). These descriptors could help in monitoring the fermentation process of other black olive cultivars as well as of naturally fermented green olives.
In starter-driven fermentations carried out by sequential inoculum of yeast LAB strain, three main stages have been described. The first stage (30 days) is characterized by high aldehydes content, compounds responsible of herbaceous flavors in fruits and vegetables. The second stage (60 days) is characterized by the presence of higher alcohols, styrene  and terpenes, compounds correlated with the metabolic activities of inoculated yeast starter strains. The third final fermentation stage (90 days), mainly characterized by the presence of acetate esters (isoamyl acetate, ethyl acetate), esters (ethyl hexanoate and ethyl octanoate), and acids, probably due to the different pathways undertaken by LAB enzymes.
The use of starter microorganisms significantly reduced the time of fermentation process from 180 to 90 days. The first stage of fermentation shifted from 90 (spontaneous fermentation) to 60 days (starter-driven fermentation) and the second step shifted from 180 (spontaneous fermentation) to 90 days (starter-driven fermentation). The use of sequential inoculation strategy of selected yeast and LAB starters produced a volatile profile richer in compounds that can be associated to attributes such as fruity, winey-sweet and herbaceous. A significant reduction of volatile phenols and hydrocarbons was observed.
For Moresca and Kalamàta table olives inoculated with selected starter cultures of
Grounta et al.  demonstrated that the use of
In green olives, most studies have been carried out for the selection of starter cultures able to control fermentation in brines after lye treatment. This process generally needs 3–7 months to be completed. It is mainly driven by LAB belonging to lactobacilli,
In order to improve the fermentation of directly brined green olives, the application of the “pied de cuve” technology has been proposed . Partially fermented brines deriving from a previous spontaneous fermentation were used to produce green olives with improved aroma and taste complexity. In comparison with brines deriving from previous fermentations performed with the starter
Volatile compounds confer peculiar sensorial characteristics and contribute to the aroma “fingerprint” of single table olives variety. Then, it could be important to promote the use of new descriptors other than those linked to taste (crispness, sourness, bitterness, and astringency), appearance (brightness, intense green color, etc.) . New descriptors should be able to describe floral, fruity, green, winey as well other similar notes (Figure 5).
To link chemical data to sensory data, it is necessary to evaluate the perception thresholds of the volatiles, defined as the lowest concentration capable of producing a sensation. The contribution of each volatile compound to odor profile can be quantified by its odor activity value (OAV). OAV is the ratio of the compound concentration to its odor threshold. In the table olives sector, these thresholds are not available, so it is not still possible to establish the role of each volatile compound as odorant in the multiplicity of olives aroma components.
3.3. Influence of microbial fermentation on table olives nutritional profile
In the past decades, olive oil and table olives have been attracting interest, mostly due to their beneficial effects on health. Table olives contain several nutritional components that largely depend on the olive variety, the cultivation conditions, the maturation stage of the olive fruit, and the processing method. The consumption of table olives thus allows the dietary introduction of bioactive components, such as triterpenic acids, α-tocopherol, biophenols, and fatty acids. These compounds are known to be responsible for a variety of health benefits. More specifically, olive fruits are remarkably rich in maslinic and oleanolic acids . These triterpenic acids are located in the epicarp of the olive fruit and they constitute the main substances of the surface waxes [61, 62]. Some studies indicate that maslinic and oleanolic acids possess health beneficial activities such as anti-inflammatory, antioxidant [63–65], antimicrobial , antiviral , cardioprotective , antihypertensive , antihyperlipidemic [70, 71], antidiabetic [72, 73], and even antitumor [73–77]. It is worthwhile noting that the content of these bioactive compounds in table olives is significantly higher than in olive oil .
NaOH treated green and black olives contain low levels of these compounds in comparison with naturally fermented olives . Indeed, the NaOH treatment leads to the solubilization of maslinic and oleanolic acids into the alkaline and washing solutions. The resulting final product contained significantly reduced levels of these compounds.
In Greek-style preparations, the fermentation of black table olives driven by selected starter cultures can preserve the triterpenic acid content. The amount of these molecules was around 1000–2000 mg/kg olive flesh, much higher than the values observed in extra virgin olive oils [Bleve G., unpublished]. These observations confirm that table olives can be considered a dietary natural source of triterpenic acids.
The health benefits of olive oil and table olives are also attributed to their high content in monounsaturated fatty acid (MUFA). Commonly recognized as a high-fat food (about 80–85% of the calories in olives come from fat), olives provide a high content of oleic acid. Linoleic acid and α-linolenic acid are present in small amounts. Owing to the content of MUFA, the consumption of table olives can prevent and reduce the risk of cardiovascular diseases, regulate cholesterol levels, stimulate transcription of LDL-cholesterol receptor mRNA, and reduce breast cancer risks [79–81]. In Spanish-, Californian-, and Greek-style processes, triglylglycerols composition remains unaffected, although fatty acid composition of both green and black olives, shows differences depending on the ripeness degree. The concentration of oleic acid, the most abundant fatty acid in green and black olives, showed differences depending on the stage of maturity the producing methods . When considering the PUFA/SFA ratio, green and directly brined table olives showed a value >0.4. This is a value recommended by the nutritional guidelines . In particular, a significantly high PUFA/SFA ratio was found in directly brined olives [84, 85]. The use of selected starter cultures for black olives fermentation ensured a PUFA/SFA ratio >0.4 in the final product.
The olive fruit is also highly valuable for the presence of α-tocopherol (TC), β-carotene (BC) , and biophenols. TC acts as the major radical scavenging antioxidant and efficiently interrupts the propagation of lipid oxidation chain . Several works described a protective action of TC on human health against different pathologies. It contributes to reduce the effects of inflammations and it defends the body against the negative effects of free radicals . When cultivars of black table olives were fermented using selected starter cultures, Vitamin E and carotenes levels were found more constant.
Among biophenols, tyrosol, hydroxytyrosol, luteolin, and oleuropein are the main species found in olives [88–93]. The latter compound is mainly responsible for the bitter taste of unprocessed olives. Other phenolic compounds are verbascoside, 3,4-dihydroxyphenylglycol , anthocyanins, flavonoids, and phenolic acids . Mechanisms postulated for chemical and microbial oleuropein degradation and the effects on valuable phenols caused by different table olives processing methods are described in details by Boskou et al. .
In Greek-style fermented olives (driven by natural microbiota or by starter cultures), a higher content of total phenols was detected than that observed in lye treated olives [15, 29, 30, 37, 95, 96]. During the process, a complete hydrolysis of oleuropein and its aglycone takes place in olive flesh by yeasts and LAB β-glycosidase and esterase activities. In fact, high levels of hydroxytyrosol and tyrosol, together with verbascoside, caffeic acid, vanillic acid, and hydrocaffeic acid were detected in the final products. Table olives are a very good source of hydroxytyrosol which is known to possess a high antioxidant and free radical scavenging activity [95, 97]. Table olives together with virgin olive oil are the only edible source of hydroxytyrosol; in olive oil, however, the bound forms of this compound prevail.
Changes in the profiles of bioactive compounds caused by metabolic activities of microbial starters can produce variations in the bioaccessibility and/or bioavailability of these metabolites. The role of microorganisms and the effects of their activities on these bioactive compounds need to be further elucidated. Table olives can be used to ensure a “positive” or “optimal” dietary intake of these compounds. They represent a source of phytochemicals useful for the prevention of several diseases and the promotion of human health (Figure 6).
3.4. Use of starter cultures as probiotics
Another important character for the selection of potential starter cultures is referred to probiotic traits that beneficially influence intestinal microflora and health . LAB, mainly the strains belonging to genera
Probiotic characteristics are strongly required for microorganisms to be used for food production. These attributes can be conferred to a food by microbial preparations different or by the same microorganisms used as starter cultures. In the first case, the interaction between probiotics strains and traditional starter cultures must be considered, since some probiotics strains may have effects on organoleptic properties of the food product or can influence negatively the starter culture bacteria . In the latter case, strains to be proposed as starters need to be selected also for probiotics traits.
Recent studies have focused on the use of table olives as a carrier of LAB probiotic strains as well as on the evaluation of the fermentation performances of these probiotics [39, 101, 104, 105].
As proposed by different authors [14, 23, 109], for these microorganisms, probiotics traits are: (i) the resistance and/or survival to gastric pH conditions and to bile salts, (ii) the capacity to adhere to intestinal mucosa, and (iii) the antimicrobial activities against intestinal and food-borne pathogens. In addition, several other health promoting factors can be considered in order to promote yeast starters as probiotics. They are the production of B vitamins and the reduction of the intestinal proinflammatory response as an antagonistic effect of yeasts or probiotic bacteria toward pathogen; the ability to reduce cholesterol serum levels ; the ability to biodegrade phytate complexes, responsible for sequestering nutritional divalent minerals; the capability to synthesize natural folates, essential cofactors in the biosynthesis of nucleotides and crucial for cellular replication and growth ; the ability to produce a number of bioactive compounds. Anyway, in order to obtain probiotics and healthy olives, it is necessary that yeasts adhere to olive skin and survive during storage/packaging. The copresence of yeasts and LAB in the biofilm associated to epidermis of natural black olives (Greek-style fermented Conservolea) and Spanish-style olives (Gordal and Manzanilla) indicate that the coinoculation of yeasts and LAB as multifunctional starter is a good strategy for carrying probiotics by table olives [35, 58].
3.5. Influence of starters in bioremediation of table olive processing wastewaters (TOPW)
During table olives processing, clean water is used and a large quantity of wastewater is produced depending on cultivar, maturity, and type of treatment from 0.5 l/kg to 6 l/kg. In 2013/2014, 2.7 million tons of table olives were produced in the world. The production of 1.2–14 million tons of wastewaters can be estimated. The volumes of wastewaters depends on the different table olives processing methods: for Spanish style 2–3.5 l/kg of olives; for California green ripe olives 1.5–3.5 l/kg of olives, for California black ripe olives 2–6.5 l/kg of olives, for naturally black olives (Greek style) 1 l/kg of olives . The availability of the water as a resource and the environmental impact deriving from its use are very important matters for many table olives producer countries. The main problems associated to TOPW are their high chemical oxygen demand (COD) up to 35 g/l, biological oxygen demand (BOD) ranging from 0.6 to 38.3 g/l, different pH values (alkaline, up to 9–13, for waters deriving by lye-treatment and acidic, 3.6–4.4, for fermentation brines), the presence of several water-soluble phenols and polyphenols, the high salt levels (56–77 g/l) in Greek-style olives.
The different composition of TOPW (produced by different table olives processing methods) requires the development of diverse approaches for their management and treatment. The contemporary presence of high organic matter content (reducing sugars, organic acids), suitable to be used by microorganisms, and of compounds that affect microbial growth and metabolism (phenols), renders biological approaches for their remediation very difficult.
There are different methods based on the use of aerobic and anaerobic processes of TOPW [113, 114]. There are also strategies that combine chemical and biological processes, using a pretreatment with
The use of starter microorganisms in table olive processing can represent a useful system to mitigate the presence of chemical pollutants in TOPW. They are phenols (such as oleuropein and derivates, anthocyanins), NaOH in the lye and sodium content in brine. Selected microorganisms, by their metabolic activities can reduce the presence of phenols. They can also efficiently dominate the spontaneous microflora and facilitate reduction of NaCl concentration in brines. Concerning NaOH in lye, a future challenge can be to develop new systems able to reduce or eliminate the use of NaOH in lye. These strategies can allow to perform a more natural transformation process by the combination of technological and biotechnological approaches.