**2.2. The microbiological hazards in table olives**

Fermented vegetable technology is based on lactic acid and alcoholic fermentations, that convert sugars to different end-products, and the obtained food products take on new and different characteristics (Hutkins, 2006). To enhance the quality of final product is one of the main scientific and technological challenges for table olive production together with the reduction of cost of harvesting, of spoilage occurrence, and of environmental pollution (Brenes, 2004).

The olive-ecosystem is influenced by the indigenous microbial population, by the intrinsic factors related to the olives (pH, aw, phenols, sugar content, etc.) and extrinsic factors (temperature, oxygen and salt levels). The microbial population characterizing the first days of fermentation seem to be always the same: *Enterobacteriaceae*, lactic cocci, *Bacillaceae* and yeasts, whose evolution is strongly related to the pH value. In addition to these populations, Nychas and co-workers (2002) also reported the presence of *Pseudomonas* spp. at the start of fermentation, that decreased as other Gram-negative bacteria, within the first two weeks of fermentation.

Among the three main commercial preparations of table olives, there are some processing parameters affecting the fermentation process. The most important is the pH. The fermentation process in Spanish-style olives (treated olives) begins at an alkaline pH, higher than 9–10, because the fruits are previously treated with NaOH to hydrolyse the oleuropein (Medina et al., 2010). In this case, the microbial population is mainly composed of *Enterobacteriaceae*, lactic cocci and other epiphytic microorganisms which are able to drop the pH value below 7.0, creating the optimal conditions for LAB growth. Regarding the other two main preparations, Greek-style and California-style, the fermentation is influenced since the first steps by the processing conditions. In this case, the microbial population starts to grow at an acidic pH because organic acid (acetic, citric, lactic) are added to prevent the growth of Gram-negative bacteria.

No official microbiological criteria for table olives are available. However, the Standards of the Codex Alimentarius prescribes the minimum requirements related to hygiene for table olives. The final product shall be free from microorganisms and parasites in amounts which 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 agriculture (GAP), hygiene (GHP) and manufacturing (GMP) must be applied.

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

yeasts to be used as starters, alone or in combination with LAB.

**2.2. The microbiological hazards in table olives** 

Gallego et al., 2011).

(Brenes, 2004).

fermentation.

growth of Gram-negative bacteria.

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

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-

Fermented vegetable technology is based on lactic acid and alcoholic fermentations, that convert sugars to different end-products, and the obtained food products take on new and different characteristics (Hutkins, 2006). To enhance the quality of final product is one of the main scientific and technological challenges for table olive production together with the reduction of cost of harvesting, of spoilage occurrence, and of environmental pollution

The olive-ecosystem is influenced by the indigenous microbial population, by the intrinsic factors related to the olives (pH, aw, phenols, sugar content, etc.) and extrinsic factors (temperature, oxygen and salt levels). The microbial population characterizing the first days of fermentation seem to be always the same: *Enterobacteriaceae*, lactic cocci, *Bacillaceae* and yeasts, whose evolution is strongly related to the pH value. In addition to these populations, Nychas and co-workers (2002) also reported the presence of *Pseudomonas* spp. at the start of fermentation, that decreased as other Gram-negative bacteria, within the first two weeks of

Among the three main commercial preparations of table olives, there are some processing parameters affecting the fermentation process. The most important is the pH. The fermentation process in Spanish-style olives (treated olives) begins at an alkaline pH, higher than 9–10, because the fruits are previously treated with NaOH to hydrolyse the oleuropein (Medina et al., 2010). In this case, the microbial population is mainly composed of *Enterobacteriaceae*, lactic cocci and other epiphytic microorganisms which are able to drop the pH value below 7.0, creating the optimal conditions for LAB growth. Regarding the other two main preparations, Greek-style and California-style, the fermentation is influenced since the first steps by the processing conditions. In this case, the microbial population starts to grow at an acidic pH because organic acid (acetic, citric, lactic) are added to prevent the

No official microbiological criteria for table olives are available. However, the Standards of the Codex Alimentarius prescribes the minimum requirements related to hygiene for table olives. The final product shall be free from microorganisms and parasites in amounts which 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 or post-sterilisation contamination.

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 Control (ECDC), issued on 21 February 2012.

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

usual fermentation profiles and producing good sensorial characteristics. In particular, a mixture of NaCl, CaCl2 showed the ability to reduce both bacterial and yeast growth, while KCl showed similar effect of NaCl. Moreover, using different mixed salts, Tsapatsaris and Kotzekidou (2004) showed that the replacement of NaCl by KCl in Kalamon olives resulted in a strong synergy between calcium lactate and calcium acetate with higher growth rates of starter cultures of *Lactobacillus plantarum* and *Debaryomyces hansenii*.

Microbiological Aspects of Table Olives 327

In table olive processing, starter cultures must have some properties such as good resistance to the inhibitory effect of polyphenols, good survival against wild strains of related species, rapid acid production, complete utilization of fermentable sugars, good tolerance against high levels of salt and low pH, and a possibly inhibitory effect against undesirable organisms. The latter effect is due to the production of bacteriocin, peptides that were found to be active against a number of natural competitors of *L. plantarum* in the fermentation brines and also against bacteria that can cause olive spoilage (Leal-Sánchez et al., 2003). This property is considered of importance in the development of new preservation technologies

Moreover, lactobacilli are important members of the healthy human microbiota and exert several beneficial physiological effects, such as antimicrobial and antitumorigenic activities (Nguyen et al., 2007; Bevilacqua et al., 2010b). The reduction of cholesterol by LAB has been demonstrated in human, mouse, and pig studies (Nguyen et al., 2007). Nowadays, foods fortified with health-promoting probiotic bacteria are mainly produced with milk derivatives, so functional food industries are focusing on new non-dairy foods that can

The species of the genus *Lactobacillus* are widely occurring in many natural environments often playing important roles in fermentation processes and in the regulation of relationships among species of complex ecosystems. In particular, *L. plantarum* and *L. pentosus* are regarded as the main species leading this process (Table 1) often being used as a starter in guided olive fermentation (Sánchez et al., 2001; Leal-Sánchez et al., 2003; Hurtado et al., 2009). *L. pentosus* and *L. plantarum* are also the most frequently isolated species in table olives; the other species used as inocula, with little exception, have always been studied in

However, a significant occurrence of *Leuconostoc* spp. on olive fruits and leaves was highlighted in the study of Ercolini and co-workers (2006), suggesting that *Lactobacillus* spp. may also originate from the environment or tools of production and not exclusively from the olives. Lavermicocca and others (2005) used table olives as a vehicle for delivering probiotic bacterial species, such as *Lactobacillus rhamnosus*, *L. paracasei*, *Bifidobacterium longum* and *B. bifidum*, but these strains are not involved in spontaneous fermentation and so they are not

Isolation from olive brines of *Enterococcus* strains has been reported by several authors, so mixed starters of *E. faecium* and *L. plantarum* (Lavermicocca et al., 1998) or *E. casseliflavus* and *L. pentosus* have been studied. The suggestion to inoculate *E. casseliflavus,* isolated from fermenting olives, is due to its good tolerance to the initial high pH (in case of lye treatment), without the drawback of transmissible antibiotic resistance shown by *E. faecium*

The selection of starters is based on diverse criteria including homo- and heterofermentative metabolism, acid production, salt tolerance, flavour development, temperature range growth, oleuropein-splitting capability and bacteriocin production (Panagou et al.,

well adapted to the environmental conditions of table olives (Perricone et al., 2010).

contribute to a regular assumption of probiotics (Lavermicocca et al., 2005).

of foods (Devlieghere el al., 2004).

conjunction with them (Hurtado et al., 2012).

(de Castro et al., 2002).

The replacement of NaCl with other chlorides could be important in those productions traditionally processed in a high salt concentration, such as Greek-style olives, because this action could lower the NaCl concentration without reaching the lowest limits necessary to obtain a safe product. Therefore, besides the pH decrease and the NaCl concentration, several actions have been proposed in order to overcome all the fermentation problems: pasteurization, addition of sugars (glucose and sucrose), extra salt addition and use of starter cultures. Sugar supplements increase the pH drop rate reducing the dangerous early stage and ensuring the safety of the final product (Chorianopoulos et al., 2005).
