**2.1. Role of yeasts**

322 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

toxicity effects toward several microorganisms (Sayadi et al., 2000).

hygienic limit to avoid microbiological risks for consumers.

because it is an expensive procedure.

other soluble compounds. Olive fermentation is considered to be over when the sugars are totally consumed by microorganisms. The months necessary for this process might change depending on several factors, such as the variety and olive size, the salt concentration and temperature (Cardoso et al., 2010). Nowadays there are neither physico-chemical nor microbiological controls to objectively determine the end of fermentation and producers decide, according to personal criteria, when olives are ready to eat (Hurtado et al., 2008).

Other important components of olives are polyphenols. The most important classes of phenolic compounds in table olives are phenolic acids, phenolic alcohols, flavonoids, and secoiridoids (Sousa et al., 2006). The olive phenols are nutritionally interesting due to their antioxidant activities, moreover, these compounds are determinant in the shelf-life of olive oil and sensory qualities of both oil and table olives. Some of these, such as oleuropein and its hydrolysis derivatives, have antimicrobial activities against a wide variety of microorganisms, including lactic acid bacteria (LAB). The inhibiting effect of many polyphenols on LAB growth has been widely studied (Fleming et al., 1973; Ruiz-Barba et al., 1993). Moreover, the increase of oleuropein content in the growth medium reduces the activity of bacteria to hydrolyse this glycoside (Romeo & Poiana, 2007). Recently several studies on antimicrobial activity of olive products have been carried out, namely with olive leaves, olive fruits and their pure compounds, such as oleuropein, hydroxytyrosol and aliphatic aldehydes (Sousa et al., 2006) and it has been found that ferulic acid exhibits

Compared to the relatively few microbial species employed in other fermented foods, microorganisms evolved in vegetable fermentations are many and different. In olive fruits the epiphytic microbial population consists of yeast, fungi, and both Gram positive and Gram negative bacteria but throughout the fermentation process, *Enterobacteriaceae*, LAB

It has been generally established that LAB are responsible for the fermentation of treated olives. While LAB and yeasts compete for the fermentation of untreated olives, and in some

LAB usually isolated from fermentation brines of treated olives include both heterofermentative and homofermentative species (Hutkins, 2006), and *Lactobacillus plantarum* is considered essential to produce the lactic acid needed for preservation and typical flavour. *L. plantarum* generally coexists with a yeast population until the end of the fermentation process and during storage, although other microorganisms may be involved depending on the applied parameters of the process. Organic acids, such as lactic acid, and sodium chloride are primary preservatives for table olives. Olives show a water activity greater than 0.85 and a final pH close to 4.6 or below, which represent the most important

The control of temperature during the fermentation steps often led to beneficial effects, especially in those region where the fermentation temperature follows environmental fluctuations. Unfortunately, in most companies the temperature control is not applicable

and yeasts are the most relevant microorganisms (Garrido-Fernández et al., 1997).

cases yeasts can be exclusively responsible for fermentation on untreated olives.

The positive role of yeasts in table olive fermentation has recently been reconsidered. Yeasts are especially relevant in directly brined green and black natural olive fermentations, where fruits are not treated with NaOH solutions. In these conditions, in the first fermentation step the LAB growth is slow because of the presence of phenolic compounds in brine. Growth of oxidative yeasts and molds may occur in brine surfaces if the tanks are open. To prevent this growth, the air layer between the liquid and the top of tank must be reduced as much as possible.

The main roles of yeasts in the processing of fermented olives, are associated with the production of alcohols, ethyl acetate, acetaldehyde and organic acids, compounds that are relevant for the development of taste and aroma and for the preservation of the typical characteristics of this fermented food (Alves et al., 2012).

Some yeast species seem to improve the growth of LAB. Yeasts are able to synthesize substances such as vitamins, amino acids and purines, or breakdown complex carbohydrates, which are essential for the growth of *Lactobacillus* species that request a nutritionally rich environment for optimal growth (Viljoen, 2006). However, in table olive processing yeasts can also produce spoilage such as off-flavour production, clouding of brines and softening of fruits (Arroyo-López et al., 2008a).

Recently, molecular methods have been applied for the identification of yeast associated with table olives. These techniques confer a higher degree of accuracy in the final identification than classical biochemical methods. Deiana et al. (1992) observed that the species found depended on the degree of maturation of the olive fruits. The genera *Candida, Pichia, Rhodotorula, Saccharomyces, Debaryomyces, Kluyveromyces, Kloeckera, Torulopsis, Trichosporon* and *Cryptococcus* were found by several authors (Arroyo-López et al., 2008a; Rodríguez-Gómez et al., 2010). While the main frequently isolated species, in both naturally

black and Spanish-style olive brines, are *Candida boidinii*, *Candida diddensiae*, *Pichia anomala*, *Pichia kluyveri*, *Pichia membranifaciens* and *Saccharomyces cerevisiae* (Oliveira et al., 2004; Coton et al., 2005; Arroyo-López et al., 2006). Recently Rodríguez-Gómez and co-workers (2012) drawn up a list of isolates representative of the yeasts of table olives, and the most suitable yeasts to be used as starters, alone or in combination with LAB.

Microbiological Aspects of Table Olives 325

may represent a hazard to health and shall not contain any substance originating from microorganisms in amounts which may represent a hazard to health (Pereira et al., 2008). To reduce the risk of food-borne illness and spoilage phenomenon, good practices in

Although heat treatments have some negative effects such as alterations in consistency and colour (Romeo et al., 2009), the correct use of temperature during pasteurization and/or sterilization is essential to ensure microbiological safety and stability, inactivating enzymes, and lessens the oxidizing processes. If heat sterilization is applied to olives, the treatment must be sufficient both in time and temperature, to destroy spores of *Clostridium botulinum* (COI, 2004). While olives preserved by salt and acidification or natural fermentation, are usually *C. botulinum* and its toxin free, only if the pH is constantly monitored and maintained below 4.6. Clostridial bacteria are relatively common in the environment because they are spore-forming. Spores of *C. botulinum* were detected both in pasteurized and sterilized olives (Pereira et al., 2008) indicating a poor attention to the application of sterilisation parameters. The occurrence of *C. botulinum* appears, however, to be rare. The sulphite reducing *Clostridium* spores are indicators of remote faecal contamination. Their presence in pasteurized olives is due to the occurrence of anaerobic fermentations or to the resistance of spores to pasteurization. However, the spores should be destroyed by sterilisation as its presence in a sterilised product indicates either inadequate heat treatment

The occurrence of *Listeria monocytogenes* in green table olives has been assessed, demonstrating that the product, despite its low pH and high salt concentration, can support *Listeria* survival for which an appropriate heat treatment must be applied (Caggia et al., 2004). Another hazard in table olives is *Escherichia coli* O157:H7, a pathogenic bacterium responsible for hemorrhagic colitis and hemolytic uremic syndrome. Its presence may be particularly associated to the Spanish-style method because the drop in the pH is slower than in natural fermented olive brine. The death rate of *E. coli* could be affected by using starter strain (Spyropoulou et al., 2001). More recently, the species *Enterobacter cloacae*, an opportunistic pathogen for humans, has been recovered in spontaneously fermented table olives (Bevilacqua et al., 2010a). The occurrence of *Listeria, Salmonella*, *Escherichia coli*, *Yersinia* pathogen strains and others are extensively reported in the scientific report of the European Food Safety Authority (EFSA) and European Centre for Disease Prevention and

Other than the pH value, a parameter which strongly influences the storage and quality of table olives is NaCl concentration. Its level is important for achieving stability of the products because it prevents spoilage and growth of pathogens. During recent years, consumers have developed an attitude on low sodium intake principally because a diet rich in sodium leads to higher blood pressure. So, several scientific studies (Arroyo-López et al., 2008b; Romeo et al., 2009; Bautista-Gallego et al., 2010; Bautista-Gallego et al., 2011; Panagou et al., 2011) have focalized on the viability, application and consequences of replacement of sodium with calcium or potassium in table olive fermentation. Apparently, NaCl may be substituted in diverse proportions with KCl or CaCl2 without substantially altering the

agriculture (GAP), hygiene (GHP) and manufacturing (GMP) must be applied.

or post-sterilisation contamination.

Control (ECDC), issued on 21 February 2012.

The interrelationships between *Lactobacillus* species and yeasts in table olives may also play an essential role in product preservation. Several authors have recently focused their attention on yeast biodiversity associated with the different types of olive processes with particular regard to their enzymatic activities, in order to propose yeast as starters (Bautista-Gallego et al., 2011).
