International Journal of Food Microbiology 128 (2008) 189196

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Review

Role of yeasts in table olive production

F.N. Arroyo-Lpez a,, A. Querol b, J. Bautista-Gallego c, A. Garrido-Fernndez c
a b c

Institut Cavanilles de Biodiversitat i Biologa Evolutiva, Universitat de Valncia, Edici d'Instituts, Parc Cientc de Paterna, P.O. Box 22085, E-46071 Valncia, Spain Departamento de Biotecnologa de Alimentos, Instituto de Agroqumica y Tecnologa de los Alimentos (CSIC), PO Box 73. E-46100 Burjassot, Valencia, Spain Departamento de Biotecnologa de Alimentos, Instituto de la Grasa (CSIC), Avda. Padre Garca Tejero, 4. 41012, Sevilla, Spain

a r t i c l e

i n f o

a b s t r a c t
Table olives are a traditional fermented vegetable of the Mediterranean countries, but their production and consumption are now spread all around the world. Yeasts can play a double role in this food. They are present throughout the fermentative process and it is generally accepted that they can produce compounds with important organoleptic attributes determining the quality and avour of the nal product. However, yeasts can also be spoilage microorganisms in olive fermentation/storage and packing causing gas pockets, swollen containers, cloudy brines and off-avours and off-odours. Candida boidinii, Debaryomyces hansenii, Pichia anomala, P. membranifaciens, Rhodotorula glutinis and Saccharomyces cerevisiae are species isolated with a high frequency from olive processes. Scarce information is still available about the ecology, biochemistry and molecular biology of these important microorganisms in table olives. A wider knowledge about these aspects could facilitate the possible application of yeasts as a starter culture, due to their ability to produce aromatic compounds, antioxidants, enzymes, and improve the growth of lactic acid bacteria. 2008 Elsevier B.V. All rights reserved.

Article history: Received 11 June 2008 Received in revised form 7 August 2008 Accepted 29 August 2008 Keywords: Table olives Yeasts Spoilage Fermentation Packing

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yeasts isolated from table olives. . . . . . . . . . . . . . . . . . . . . . . . . Role of yeasts in table olive fermentations . . . . . . . . . . . . . . . . . . . . 3.1. Biochemical characteristics of yeasts isolated from table olive fermentations 3.2. Yeast and lactic acid bacteria interactions in table olive fermentations . . . . 4. Role of yeasts in table olive packing . . . . . . . . . . . . . . . . . . . . . . . 4.1. Growth of yeasts in table olive packing . . . . . . . . . . . . . . . . . . 4.2. Spoilage by yeasts in table olive packing . . . . . . . . . . . . . . . . . 5. Future and current research topics with yeasts from table olives . . . . . . . . . 5.1. Predictive models with yeasts from table olives . . . . . . . . . . . . . . 5.2. Yeasts as biocontrol agents in table olives. . . . . . . . . . . . . . . . . 5.3. Study of the biochemical characteristics of yeasts from table olives . . . . . 5.4. Genetics of yeasts associated with table olives . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 190 191 191 192 193 193 193 193 193 193 194 194 194 194

1. Introduction Yeasts are unicellular eukaryotic microorganisms classied in the kingdom Fungi, with about 1500 species described (Kurtzman and Fell, 2006). They are characterised by a wide dispersion in natural

Corresponding author. Tel.: +34 963 543668; fax: +34 963 543670. E-mail address: fnarroyo@cica.es (F.N. Arroyo-Lpez). 0168-1605/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.08.018

habitats but are most frequently isolated from sugar-rich substrates. However, several species have been able to adapt to different environments or ecological niches provided by human activity. In some cases, these yeasts have been unconsciously selected by humans for thousands of years for their splendid properties in the elaboration of different foods, a process known as domestication (Barrio et al., 2006). In this way, yeasts are well known for their enormous importance in food and beverage production. The fermentation of sugars by yeast is an old and well known technology where carbohydrates are

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transformed into different compounds such as water, ethanol, carbon dioxide, etc. Several species (fundamentally of the genus Saccharomyces) are involved in the fermentation of wine, beer, bread, caper, cucumber and other vegetables. However, they are also signicant as spoilage microorganisms, especially in food and beverages with a low pH, high salt concentrations and low temperatures (Stratford, 2006). This is the case for table olive production, where a habitual low pH and high NaCl concentration is occurred in the nal product (Garrido Fernndez et al., 1997). The olive fruit is a drupe. It has a bitter component (oleuropein), a low sugar concentration (2.66.0%) and high oil content (1230%), although these values can change with maturity and olive variety (Garrido Fernndez et al., 1997). Such characteristics prevent olives from being consumed directly from the tree and it has promoted a series of processes to make them eatable that differ considerably from region to region. The International Olive Oil Council (IOOC, 2008) estimates that table olives' world production reached around 1,823,000 tons in the 2006/2007 crop year. The most important industrial preparations are: a) the green Spanish style, with about 60% of the production, b) ripe olives by alkaline oxidation (the so-called Californian style) and c) naturally black olives (also known as Greek style) (Fernndez Dez et al., 1985; Garrido Fernndez et al., 1997; Panagou et al., 2008). Recently, other products such as green seasoned table olives are gaining the favour of consumers due to a progressive awareness for traditional and natural foodstuffs (Arroyo Lpez et al., 2005). The IOOC (2004) was sensible to their progressive importance by including them under the heading Specialties in the current Trade Standard Applying to Table Olives. In brief, the procedure for preparing green Spanish-style olives consists of treating the fruits with a dilute NaOH solution (23%) to degrade polyphenols and to increase the permeability of the cell wall, followed by water washes to remove the excess of alkali, and brining (initial concentration of 912% NaCl), where olives undergo lactic acid fermentation. When the substrates are exhausted, the fruits are graded, sorted and mechanized (pitted, stuffed, etc.). The commercial presentations of green olives are numerous and include the use of many stufng materials. Olives for producing ripe olives (by alkaline oxidation) are previously preserved in an aqueous solution (brine, acidic water, etc.) and darkened throughout the year. Darkening consists of several treatments of dilute NaOH solutions and water washes between them. During the oxidation process, air is passed throughout the suspension of the olives in the liquid. Once the olives obtain the proper color ring around the outer surface, this is xed by immersion in a lactate or gluconate iron solution. These olives are usually packed in light brine. Their commercial presentations are limited to plain (whole), pitted, sliced, and, sometimes, olive paste. Untreated olives (green, turning colour or naturally black) are directly brined after picking. In brine, olives undergo a fermentation, which characteristics depend on the physico-chemical conditions, cultivar, temperature, and salt content. The fruits are maintained in this solution until they lose their natural bitterness at least partially. As the market demands, olives are sorted, graded and packed. In some commercial presentations, they can be broken or cut along their higher longitudinal diameter and/or seasoned with natural products or their avours. There are many other traditional/industrial ways of processing table olives according to fermentation conditions (temperature, levels of salt, and type of acid) and raw material (green, turning colour or black olives). A complete description of the different types of olive processing can be found in Fernndez Dez et al. (1985), Garrido Fernndez et al. (1997), Snchez Gmez et al. (2006) and Garrido Fernndez et al. (2006). However, it must be emphasised that scarce information is available about the effects of yeasts on the organoleptic properties of the fruits and their relations with other microorganisms present during olive fermentations.

The aim of this paper is to review the role of yeasts during table olive processing and packing, given their double importance as fermentative and spoilage microorganisms in this food. We also want to assess the possible utilization of yeasts in other applications during the fermentation/storage of olives due to their ability to produce aromatic compounds, enzymes, vitamins, antioxidants and improve the growth of lactic acid bacteria, expounding at the same time possible future research topics with these signicant microorganisms. 2. Yeasts isolated from table olives Several studies have focused on the detection of yeasts adhered to the surface of olive fruits. Florenzano et al. (1973) reported the presence of yeasts among the microbiota found on the surface of fresh mature olives. Pelagatti (1978) made a detailed study of the microorganisms adhered to 12 fresh green Italian cultivars, isolating 56 pure cultures of yeasts. Deiana et al. (1992) observed that the species found depended on the maturation degree of the olive. However, yeast counts on surface of fresh olive fruits are generally low (b1 log10 CFU g 1), as was reported by Arroyo Lpez (2007) with ManzanillaAlorea olives during three consecutive seasons. In the past, the characterisation of yeasts associated with table olives has mainly been made by biochemical and morphological methods, using the taxonomic keys of Barnett et al. (1990) and Kurtzman and Fell (1998). The process was complex, laborious and time consuming, although the methodology permitted the isolation and characterization of a great variety of genera and species around the world. Mrak et al. (1956) described the presence of Candida krusei, C. parapsilosis, C. rugosa, Pichia membranifaciens and Rhodotorula glutinis during the fermentation and storage of green olives in the USA. Gonzlez Cancho (1965, 1966a, b) found yeasts of the genera Candida (C. tropicalis, C. parapsilopsis, C. rugosa), Pichia (P. anomala, P, membranifaciens) as well as Saccharomyces cerevisiae from green olive Spanish style fermentations. Pelagatti (1978) and Marquina et al. (1992) isolated several species of the genera Candida, Debaryomyces, Kluyveromyces, Pichia, Rhodotorula and Saccharomyces from directly brined green and turning colour olives. Balatsouras (1967) reported the presence of yeasts of the genera Trichosporon, Candida, Pichia, Kloeckera, Torulopsis and Debaryomyces during the fermentation of directly brined natural black olives from Greek cultivars, and Borcakli et al. (1993) isolated species of Debaryomyces from Turkish cultivars. Gonzlez Cancho et al. (1975) identied S. cerevisiae and P. anomala as the main species in Spanish natural black olive fermentations. When these olives were fermented in the presence of air the species present were C. saitoana, D. hansenii, P. membranifaciens and Williopsis saturnus var. mrakii (Durn Quintana et al., 1986). Kotzekidou (1997) identied Torulaspora delbrueckii, D. hansenii and Cryptococcus laurentii as the predominant species in Greek-style black olives, while Marquina et al. (1997) in studies carried out with olive brines from seven locations in Morocco isolated C. boidinii, P. membranifaciens and T. delbrueckii. Hernndez et al. (2007) found P. anomala, Kluyveromyces marxianus, D. hansenii and S. cerevisiae to be the main species present during the processing of directly green table olives from Portugal. Recently, molecular methods were used for the identication of yeast associated with table olives. These techniques confer a higher accuracy degree in the nal identication than classical biochemical methods. In this way, Arroyo Lpez et al. (2006) identied the species S. cerevisiae, Issatchenkia occidentalis and Geotrichum candidum from green seasoned table olives, and Candida boidinii and Hanseniaspora guilliermondii from processed black olives by means of the restriction pattern analysis of the 5.8S rRNA gene and the intergenetic spacers ITS together with the sequencing of the D1/D2 region of the 26S rRNA gene. Coton et al. (2006) identied P. anomala, C. boidinii and Debaryomyces etchelsii as the predominant species in black olive natural fermentations from France using the same methodology. Finally, Hurtado et al. (2008) found the species C. boidinii, C. diddensiae, C.

F.N. Arroyo-Lpez et al. / International Journal of Food Microbiology 128 (2008) 189196 Table 1 The most signicant and commonly reported table olives yeast species according to the type of preparation and country where were isolated Spanish style green olives Specie C. krusei Country Spain USA Spain USA Spain USA Spain Spain Spain USA USA Spain References (d),(e),(g) (o) (f),(h) (o) (f),(h) (o) (f),(g) (d),(e),(f),(g) (d),(e),(f),(h) (o) (o) (d),(e),(f) Directly brined green olives Specie C. boidinii Country Spain Portugal Morocco Spain Portugal Spain Spain Spain Portugal Portugal Spain Spain Portugal Morocco Spain Portugal Spain Portugal Morocco References (k) (m) (n) (a),(k) (j),(m) (a) (a) (k) (j) (j),(m) (k) (k) (m) (n) (k) (j) (a), (e) (j), (m) (n) Black olives Specie C. boidinii Country Spain France Spain Greece France Spain Greece Spain France Spain Spain Spain Spain

191

References (a) (b) (c) (l) (b) (c) (l), (p) (a) (b) (i), (e) (c) (a) (i)

C. parapsilosis C. rugosa C. tropicalis P. anomala P. membranifaciens R. glutinis S. cerevisiae

C. diddensiae D. hansenii G. candidum I. occidentalis K. lactis K. marxianus P. anomala P. kluyveri P. membranifaciens

C. saitoana Cryptococcus laurentis D. etchelsii D. hansenii H. guilliermondii P. anomala P. membranifaciens R. glutinis S. cerevisiae

R. glutinis R. minuta S. cerevisiae T. delbrueckii

T. delbrueckii W. saturnus var. mrakii

Greece Spain

(l) (c)

References in Table 1: (a) Arroyo Lpez et al. (2006); (b) Coton et al. (2006); (c) Durn Quintana et al. (1986); (d) Fernndez Dez et al. (1985); (e) Garrido Fernndez et al. (1997); (f) Gonzlez Cancho (1965); (g) Gonzlez Cancho (1966a); (h) Gonzlez Cancho (1966b); (i) Gonzlez Cancho et al. (1975); (j) Hernndez et al. (2007); (k) Hurtado et al. (2008); (l) Kotzekidou (1997); (m) Marquina et al. (1992); (n) Marquina et al. (1997); (o) Mrak et al. (1956); (p) Panagou et al. (2002).

membranifaciens, Kluyveromyces lactis, P. membranifaciens, P. kluyveri, and R. glutinis during processing of Arbequina table olives in Spain. Molecular techniques are rapid, easy and more precise, eliminating part of the subjectivity that usually accompanies the output of the biochemical test. In general, S. cerevisiae, P. membranifaciens, P. anomala and R. glutinis are yeasts isolated practically from all olives preparations, while C. boidinii is isolated normally from both directly brined green and black olives. D. hansenii is a species related to high salt concentration preparations. Table 1 summarizes the most commonly reported table olive yeast species according to processing styles and countries. 3. Role of yeasts in table olive fermentations 3.1. Biochemical characteristics of yeasts isolated from table olive fermentations The presence of yeasts during the fermentation of green Spanish style olives was reported in the earliest studies of this product (Gonzlez Cancho, 1965). This fermentation is carried out by lactic acid bacteria (LAB) but yeasts are present throughout the process and reach populations which can range from 4 to 6 log10 CFU ml1 (Garrido Fernndez et al., 1997). The principal aim of the fermentation of green olives is to achieve the preservation of the fruits by means of the production of lactic acid, especially by strains of Lactobacillus pentosus and L. plantarum (Garrido Fernndez et al., 1997), while maintaining the desirable organoleptic attributes. A stable and safe product must have a low pH (b4.5), otherwise the growth of Gram-negative bacteria and the germination of spores of Clostridium botulinum is possible (Nout, 1994). When LAB outgrow yeasts, high free acidity levels and low pH are reached (pH b 4.5). However, in some cases, yeasts can become dominant and cause a product with a milder taste and less self preservation (Garrido Fernndez et al., 1997; Panagou et al., 2008). This problem was reported by Tassou et al. (2002) during the processing of naturally black olives fermented at different NaCl levels and temperatures. Moreover, an excessive growth of fermentative yeast (N 7 log10 CFU ml 1) could also produce a vigorous production of

CO2 that may penetrate olives and damage the fruits (Fernndez Dez et al., 1985). Durn Quintana et al. (1979) related this alteration with the presence of various yeast strains of S. cerevisiae and P. anomala in black olive fermentations. The use of a high level of NaCl during fermentation (N8% in the equilibrium) could favour the growth of yeasts against LAB (Garrido Fernndez et al., 1997; Tassou et al., 2002). Another unfavourable property of some yeasts is their polysaccharolytic activity. This is the production of enzymes that cause the polysaccharides of the olive fruit cell wall to degrade. Hernndez et al. (2007) found some yeast strains of Rhodotorula minuta and D. hansenii in green table olive fermentations with this capacity. Finally, strains of R. glutinis, R. minuta and R. rubra can grow and form pellicles in olive brines and produce polygalacturonases which cause a softening of olives kept in storage (Vaughn et al., 1969). In other fermented vegetables, yeasts can also play a negative role. For example, in cucumber fermentation (a process very similar to table olive production) yeasts coexist with LAB during the primary fermentation and they are the prevalent population all through the secondary fermentation (Fleming et al., 1995). Among the fermentative species reported are P. anomala, and Saccharomyces rosei. In open tanks, surface growth of oxidative yeasts (C. krusei, D. hansenii and Rhodotorula species) may occur during post-fermentation. To offset the potential growth of yeasts and softening of cucumbers, potassium sorbate is usually added to the brine (Fleming et al., 1995). However, in their benecial aspect, yeasts can produce compounds such as ethanol, glycerol, higher alcohols, esters, and other volatile compounds with an important role in avour generation and texture maintenance during fermentation/storage (Garrido et al., 1995; Garrido Fernndez et al., 1997). For instance, the production of glycerol by S. rosei in cucumber fermentations has been reported by Daeschel et al. (1988). In wines, among the 1,000 volatile compounds identied, more than 400 are produced by yeasts (Querol and Fleet, 2006). Esters produced by yeasts can contribute to both aroma and avour in the case of alcoholic and non-alcoholic fermentations. Several factors can contribute to aroma production by yeasts. These include changes in fermentation conditions such as temperature, pH, aeration, and the nature and concentration of the substrate used

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Table 2 Main characteristics of the positive and negative roles of yeasts isolated from table olive fermentation and packing Fermentation Positive role Yeast species Negative role Yeast species S. cerevisiae (c) P. anomala (c) D. hansenii (f) R. minuta (f) R. glutinis (k) R. minuta (k) R. rubra (k) Not related clearly with yeast species Production of desirable volatile Not related clearly with yeast Gas pocked spoilage/excessive compounds and metabolites species production of CO2 Antioxidant activity P. anomala (e) Polysaccharolytic activity Improvement of the lactic acid bacteria growth Killer activity D. hansenii (j) S. cerevisiae (i) D. hansenii (g) K. marxianus (g) P. membranifaciens (h) Polygalactunorase activity Packing Negative role Production of CO2 Increase of the cell number (clouding of the brines) Production of off-avours and off odours Softening of fruits Product with a milder taste a less self preservation Yeast species P. anomala (l) S. cerevisiae (a) (l) I. occidentalis (a) S. cerevisiae (a) Not related clearly with yeast species P. anomala (l) S. cerevisiae (l)

Resistance to high I. occidentalis (b) preservative concentrations Biodegradation of polyphenols C. tropicalis (d) References in Table 2: (a) Arroyo Lpez et al. (2006); (b) Arroyo Lpez et al. (2008); (c) Durn Quintana et al. (1979); (d) Ettayebi et al. (2003); (e) Gazi et al. (2001); (f) Hernndez et al. (2007); (g) Hernndez et al. (2008); (h) Santos et al. (2000); (i) Segovia Bravo et al. (2007); (j) Tsapatsaris and Kotzekidou (2004); (k) Vaughn et al. (1969); (l) Vaughn et al. (1972).

(Suomalainen and Lehtonen, 1979). In table olives, Snchez et al. (2000) reported the formation of acetic acid, succinic acid, formic acid and ethanol during green olive fermentations. Montao et al. (2003) also found the production of high amounts of ethanol, methanol and acetic acids in industrially fermented green olives, with acetaldehyde in small amounts. However, the formation of these compounds in table olives has not yet been linked with yeast growth, and far from it, with the yeast species. Hernndez et al. (2007) studied the esterase and lipase activity of several yeasts isolated from green table olives. Most of the yeast strains investigated showed esterase activity, but fewer cases had lipase activity. So the yeast population may therefore contribute to increasing the free fatty acid content in olives during fermentation. This case has been reported in olive oil by Ciafardini et al. (2006) who found only two strains of Williopsis californica and S. cerevisiae with lipase activity. Yeasts can also synthesize a number of bioactive compounds which can serve as antioxidants. Several Candida and Saccharomyces species produce compounds such us carotenoids, citric acid, glutathione and tocopherols with interesting antioxidant properties (Abbas, 2006). The formation of these substances can be induced in yeasts grown under stressing conditions or in response to fermentation medium ingredients such us phenolics or additives that are known to be toxic to cells grown aerobically (Cruz et al., 1999). The screening of yeasts for freeradical-scavenging activity is an active area of research (Gazi et al., 2001). These authors reported that P. anomala, widely isolated from table olive fermentations (Garrido Fernndez et al., 1997), produced the highest activity in a laboratory medium. Therefore, yeasts from table olives could produce bioactive antioxidants, retarding oxidative degeneration of fatty substances and improving human health. The role of yeasts is especially important in directly brined green and natural black olives (Fernndez Dez et al., 1985). In these preparations, olives are not treated with NaOH, a previous step for debittering green olive fruits devoted to the Spanish style, so LAB are partially inhibited due to the presence of phenolic compounds. In some cases, yeasts can be the predominant microorganisms during fermentation if the levels of NaCl used are high (8% NaCl in the equilibrium). Arroyo Lpez et al. (2007a) found populations of yeasts around 6.5 log10 CFU ml 1 in directly brined green cracked olives of the ManzanillaAlorea variety with 11% initial NaCl. In Greek black dry-salted olives (cv. Thassos), yeasts became the dominant microbial group, with a population which ranged from 4.7 to 6.0 log10 CFU g 1 in olives with 7.5% NaCl (Panagou, 2006). Tassou et al. (2002) reported that only yeasts were able to grow in naturally black olives (Conservolea cv.) when the NaCl concentration was 8%. During the processing of black table olives of Hojiblanca cv. by means of both anaerobic and aerobic processes with 4% NaCl and 0.3% acetic acid,

yeasts reached their maximum populations 10 days after brining, with 7 log10 CFU ml 1 for the aerobic process and 4 log10 CFU ml 1 for the anaerobic process (Arroyo Lpez et al., 2006). zay and Borcakli (1996) also found a population of yeast of 6 log10 CFU ml 1 as predominant microorganisms in naturally black olives of the Gemlik cv. The yeast population during the fermentation of naturally black olives can be reduced by the use of sorbic and benzoic acids without affecting the LAB population, although a marked concentration of both preservatives in olive esh was observed (Turantas et al., 1999). There are few studies on the dynamics of the diverse species of yeast populations in table olives but it has been demonstrated that the initial proportion of some of them may condition the relative presence or extinction of others and the type and yield of the nal product (Jamec and Raspor, 2005). In this context, Hurtado et al. (2008) studied the yeast population dynamic during the processing of Arbequina table olives and reported that, at the beginning of the fermentation, C. diddensiae was the most abundant species in brine, although the presence of R. glutinis was also relevant. After the disappearance of these species, yeast population was mainly composed of P. kluyveri and C. boidinii. Later, when the LAB counts overcame the yeast population, P. membranifaciens and Kluyveromyces lactis appeared and remained until the end of the process. 3.2. Yeast and lactic acid bacteria interactions in table olive fermentations Recent studies have shown that the growth of LAB could be improved by the use of yeast. L. plantarum improved its growth when D. hansenii was inoculated 48 h before in olive juice (Tsapatsaris and Kotzekidou, 2004). Co-culture with S. cerevisiae improved L. pentosus performance in green table olive solutions (Segovia Bravo et al., 2007). In this case, the production of lactic acid was also slightly improved. Yeasts appear to be active microorganisms synthesising substances such as vitamins, amino acids and purines, or breakdown complex carbohydrates, which is essential for the growth of Lactobacillus species that need a more complex medium for optimal growth (Viljoen, 2006). Among the vitamins and other enzyme cofactors that are accumulated and/or synthesized by yeast are thiamine (vitamin B1), nicotinic acid, pyridoxine (vitamin B6) and pantothenic acid (Abbas, 2006). Moreover, LAB excrete lactic acid which leads to a lowering of the pH, which either inhibits the growth on undesired pathogens (enterobacteria and clostridia) or promotes yeast growth (Viljoen, 2006). In cucumber fermentations, another favourable aspect of the mixed culture of LAB and yeasts (S. cerevisiae and S. rosei) was the complete exhaustion of sugars at the end of the process (Daeschel et al., 1988). However, some studies have also reported the inhibition of the growth of yeasts (S. cerevisiae, S. exiguus, and T. delbrueckii) in

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fermented salads by the use of lactic starters which led to pH b 4.2 and lactic acid percentages of 0.280.43% (Bonestroo et al., 1993). The production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for the reduction in yeast growth and nal ethanol yield when LAB were present (Narendranath et al.,1997). In sourdough microbiota, the coexistence of LAB and yeasts is also common as in table olives. Coculture of L. brevis or L. plantarum with S. cerevisiae or S. exiguus showed different behaviour. The lack of competition for maltose when S. exiguus was present in coculture with both LAB enhanced the bacterial cell yield and lactic in acetic acid production; S. cerevisiae competed greatly with each of the LAB for glucose and only with L. plantarum for fructose, causing a decrease in the bacterial cell number and acid production (Gobbeti et al., 1994). The importance of antagonistic and synergistic interactions between Lactobacillus species and yeasts in sourdough microbiota has also been reported by De Vuyst and Neysens (2005). Therefore, the interrelationship between LAB and yeasts in table olives may also play also an essential role in product preservation and must be studied in more detail. Table 2 summarizes the main characteristics of the positive and negative roles of yeasts in table olive fermentations. 4. Role of yeasts in table olive packing 4.1. Growth of yeasts in table olive packing On the contrary, the role of yeasts as spoilage microorganims in table olive packing seems very clear. Packed olives can suffer spoilage due to yeasts in spite of the habitual low pH obtained in the nal product (Garrido Fernndez et al., 1997). Yeasts can grow if a residual sugar concentration is present in the packed olives, where they can reach population levels of 6 log10 CFU ml1 (Arroyo Lpez et al., 2005). Yeasts in table olives can also use the normal lactic and acetic acids produced during fermentation or added for nal storage as a substrate in aerobic conditions (Ruiz Cruz and Gonzlez Cancho, 1969). However, they are not able to use these acids under anaerobic conditions. This means that in the usual anaerobic conditions in which these products are packed, products could be stable even in the presence of a population of yeasts. Their stabilisation during shelf life may be affected by diverse environmental conditions. Panagou et al. (2002) reported that a CO2 atmosphere was the most effective procedure to keep the number of yeasts low during storage of dry-salted black olives. All the yeast strains isolated by these authors were identied as C. famata (the imperfect form of D. hansenii). A review of the characterisation and ecology of spoilage yeasts for the improvement of their diagnosis and control has recently been published (Loureiro, 2000). Betts et al. (1999) studied the effect of pH, NaCl, and temperature on the growth of ve genera, including Debaryomyces, Pichia, Candida and Saccharomyces closely related to table olives. They suggest a synergy between pH and NaCl at low temperatures that could be used for assessing the spoilage potential of new and existing product formulation. Their results are in agreement with those reported by Durn Quintana et al. (2003) with respect to P. anomala, P. membranifaciens, P. minuta, S. cerevisiae, C. diddensiae, and D. hansenii isolated from table olives. The most vigorous species in a laboratory medium were P. anomala, C. diddensiae, and D. hansenii, which were able to grow at 7 C and 8% NaCl; however, selected combinations of pH and NaCl prevented the growth of all of them at 7 C. Frequently, acetic acid is also used in packed olives, particularly in seasoned olives. The effect of this acid on the growth rate of S. cerevisiae was studied, among others, by Arneborg et al. (1995). However, its effect was strongly inuenced by the buffering capacity of the medium. Finally, Panagou (2004) also reported that untreated green olives packed in acidied brines under aerobic conditions or modied atmospheres showed a slight increase of about 1.0 to 1.5 log10 CFU g 1 in yeast counts.

4.2. Spoilage by yeasts in table olive packing Yeasts present in packed olives can produce an excess gas (CO2) leading to swollen containers, clouding of the brines, or produce offavours and off-odours (Fernndez Dez et al., 1985; Garrido Fernndez et al., 1997). In any case, the package is returned to the factory with the corresponding economic loss. Vaughn et al. (1972) reported that yeasts identied 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 and benzoic acids and their salts on yeasts growth to stabilise table olive packing were reported by Rodriguez de la Borbolla y Alcal et al. (1961), Marsilio and Cichelli (1992) and Arroyo Lpez et al. (2008). Table 2 summarizes the negative role of yeast species in table olive packing. 5. Future and current research topics with yeasts from table olives 5.1. Predictive models with yeasts from table olives Modelling the response of spoilage yeast as a function of different environmental variables (temperature, pH, NaCl) or preservatives (sorbic and benzoic acids, metabisulphite, etc) is very useful to nd packing conditions which can inhibit yeast growth. In this context, predictive microbiology is a powerful tool. Arroyo Lpez et al. (2008) studied the effects of sorbic and benzoic acids on a native yeast cocktail from table olives composed of S. cerevisiae, P. anomala, I. occidentalis, and C. diddensiae. Arroyo Lpez et al. (2007b,c) used a logistic regression to estimate the growthno growth interface of S. cerevisiae and I. occidentalis as a function of NaCl, acid type (citric, lactic, acetic, HCl) and potassium sorbate concentrations. These new studies that link spoilage yeast with predictive microbiology can help to optimise the concentrations of preservatives in packing, and, at the same time, uncover possible interactions between them. Following this methodology, I. occidentalis has been reported as a very resistant yeast to weak acids in brine olives (Arroyo Lpez et al., 2008). Studies carried out by Laencina et al. (1985) showed a preliminary effect of essential oils from lemon, fennel, rosemary, lavender, and diverse kinds of thyme on some yeasts isolated from table olives (P. membranifaciens, P. fermentans, P. anomala, C. guillermondii, and C. tropicalis, among others). Given the current preference of consumers for natural and seasoned products, a deeper study of their effects on nal product stabilisation, using the recent predictive microbiology modelling would be of interest for extending the commercial presentation on offer. Modelling during storage/fermentation can be another challenge. In the case of desirable yeasts, the predictive models can be used to improve yeast growth, or to optimize the production of certain metabolites. Tsapatsaris and Kotzekidou (2004) used a central composite design and response surface methodology to study the effects of environmental variables on the fermentation of olive juice by L. plantarum and D. hansenii. However, information on yeast growth or inhibition modelling during olive fermentations is still scarce and requires further research. 5.2. Yeasts as biocontrol agents in table olives Since the rst description of the killer factor of S. cerevisiae, many halotolerant killer yeasts, which show killer activity in the presence of NaCl were isolated from many fermented foods like miso, soy sauce and salted vegetables. Among them are: D. hansenii, P. anomala and C. farinosa. Furthermore, 76% of the salted vegetables contained killer yeasts mainly D. hansenii strains (Suzuki et al., 1989). Subsequently, several works have focused on the use of yeasts as biocontrol agents in table olives (Marquina et al., 1997; Santos et al., 2000; Hernndez et al., 2008). Killer yeasts can produce toxic proteins or glycoproteins (socalled killer toxins) that can cause death in other sensitive (killersensitive) yeast strains. Yeasts with killer activity may improve table

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olive packing by reducing the requirement for salt and preservatives. This property has been reported for various authors from yeast isolated from table olives (Llorente et al.,1997; Marquina et al.,1997; Santos et al., 2000; Hernndez et al., 2008). Hernndez et al. (2008) found that Debaryomyces was the genus with the highest percentages of killer strains tested, and they suggest that starter cultures of these isolates could be used as a biocontrol method against spoilage yeasts. In other studies performed with yeasts isolated from the spontaneous fermentations of olive brines, strains of the genera Pichia, Kluyveromyces, Candida, and Torulaspora were also killer against killer-sensitive strains of S. cerevisiae (Marquina et al., 1992, 1997). Santos et al. (2000) described that a P. membranifaciens strain isolated from fermenting olives produce a killer toxin that binds primarily to the (1 6)--D-glucan of the cell wall of a sensitive yeast (C. boidinii IGC 3430) and this toxin shows its maximum killer activity in the presence of NaCl. Absorption of most of the killer toxin to the (16)--D-glucan was complete within 2 min. However, a possible drawback to the utilization of killer yeasts as biocontrol agents is that the killer activity is related with the pH and NaCl concentration of the brine (Hernndez et al., 2008). 5.3. Study of the biochemical characteristics of yeasts from table olives The inuence of yeasts on the avour and texture of olives is an important factor that must be studied in more detail. As mentioned above, Hernndez et al. (2007) reported the presence of yeasts with esterase and polysaccharolytic activities in green olive fermentations. Their results show that the activity of these yeasts may play an important role in the nal organoleptic characteristics of the fermentations. Strains of Rhodotorula species produce polygalacturonases which cause a softening as reported by Vaughn et al. (1972). Therefore, the use of a starter culture consisting of a well-adapted yeast strain with low polyssacharolytic activity and desirable biochemical properties could be of interest (Hernndez et al., 2007). However, scarce information is still available about the compounds and enzymes produced by the yeast population during fermentation and their concentrations in packed olives, and this is a vital preliminary step. Olives are fruits with high fat concentrations. The presence of lipolytic yeast in table olives could modify the nutritional composition of the fruits and their organoleptic characteristics. Many questions must still be answered concerning this point, and probably a study of enzyme activities involving yeasts isolated from olive fermentations can help to x the role of the non-Saccharomyces versus Saccharomyces species during this process. The identication and comparison of yeasts between spoilage versus non-spoilage processes will also help to know the contribution of each species in the nal quality and aroma of table olives. Biodegradation of polyphenols by yeast is another interesting aspect. Olives have oleuropein which is responsible for their bitter taste. During the processing of green Spanish style, fruits are treated with NaOH to degrade oleuropein and to sweeten the olives. Large quantities of olive wastewater are produced containing high levels of phenolic compounds, which is a serious environmental problem in almost all Mediterranean countries. Therefore, there is a potential demand for yeasts with a capacity to reduce the amount of polyphenols in olives, olive brines and their wastewater. Some strains of C. tropicalis and Yarrowia lipolytica have shown capacity to reduce the chemical oxygen demand, monophenols and polyphenols in olive mill wastewater (Ettayebi et al., 2003; Lanciotti et al., 2005). C. tropicalis has also been isolated from processing of black olives by Durn Quintana et al. (1986). 5.4. Genetics of yeasts associated with table olives Another aspect that must be researched in detail is the genetics of yeasts associated with table olives. A yeast strain can inuence the nal result of a fermentation as has been shown in wines (Querol and Fleet, 2006), and for this reason different methods have been developed for the strain characterisation (Querol et al., 1992;

Fernndez Espinar et al., 2006). A preliminary observation carried out on 10 isolates of S. cerevisiae from green seasoned olives, showed the presence of four different proles when total DNA was digested with the enzyme HinfI (Arroyo Lpez et al., 2006). Results showed the presence of a heterogeneous population of S. cerevisiae in table olives, but no tests were carried out to check if some type of ecological succession existed and their possible inuence on the organoleptic properties of the product. Intensive research has focused on other foods (wines and beer) to elucidate the molecular mechanisms (genome renewal, gene duplication, polyploidization, etc.) involved in yeast adaptation to the industrial process (Barrio et al., 2006). Yeast strains isolated from industrial fermentations are generally diploids, prototrophic (no nutritional requirements), homothallic with high heterosigosity and low sporulation efciency. However, no information about the genetic characteristics of Saccharomyces isolates from olives is available. A detailed study on this matter could be convenient for the development of an eventual starter culture which could improve fermentation. For instance, rare-mating between different yeasts with desirable properties is a safe method to obtain hybrids with characteristics of both parents, and this methodology is already being used in wines (Barrio et al., 2006). However, a previous screening to detect candidate yeast and their molecular characterisation is still necessary. A DNA sequence analysis of selected loci (multilocus sequence typing) has been used to assess the genealogical relationships between wild and industrial S. cerevisiae strains in population-based studies (Fay and Benavides, 2005). Overall DNA divergence between strains was estimated at about 0.42% (bp per 100 bp) for industrial strains (isolated from fermentative processes) and at 0.14% for wild strains (isolated from grapes), suggesting lower diversity in the wild strains. Population genomics, which aims to measure the diversity within a given species by sequencing partial or whole genomes for several isolates, is an emerging eld, and as far as we know no sequences from Saccharomyces isolates from olives are included in these studies. This information could contribute to clarifying the origin of these isolates and to knowing whether they have the same or different wine origin. In conclusion, harnessing and exploiting the activities of yeasts in table olives require fundamental knowledge on their ecology, physiology, biochemistry and molecular biology. This knowledge will provide the base for improving the strategies performed on this food. Undoubtedly, the full potential of the commercial value of yeasts in table olive fermentations has not been fully determined and many challenges are awaiting research and exploitation. Acknowledgements This work was supported by the Spanish Government (projects AGL2003-00779 and AGL2006-03540-ALI, partially nanced by European regional development funds, ERDF). Authors wish to recognize to Dra. M.C. Durn-Quintana for her full dedication to the study of table olive yeasts. F.N. Arroyo-Lpez and J. Bautista-Gallego also want to thank a Juan de la Cierva postdoctoral research contract and JAE fellowship from MEC and CSIC, respectively. References
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