Microbiological Aspects of Table Olives

2.1. Role of yeasts

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.

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).

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.

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.

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).

2.3. LAB starter cultures

At industrial level LAB play a positive role in the production of wines and beers, but therefore they represent major spoilage organisms for such products (Bamforth, 2005). Table olive processing is still based on empirical methods despite its growing economical value. However, interest in developing starter cultures to be used in table olives is increasing. LAB have long been employed in fermentation as a food preservation technique owing to their progressive acidification of the fermenting brine with a consequent pH decrease (Marsilio et al., 2005). In addition, the use of LAB could standardize olive fermentation and reduce the use of highly polluting chemicals as NaOH (lye solution), contribute significantly to storage preventing microbial spoilage, and improve flavour.

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 of foods (Devlieghere el al., 2004).

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

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

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

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 (de Castro et al., 2002).

The selection of starters is based on diverse criteria including homo- and hetero-fermentative metabolism, acid production, salt tolerance, flavour development, temperature range growth, oleuropein-splitting capability and bacteriocin production (Panagou et al., 2008). Furthermore, the ability to grow at low temperatures must be considered essential in cold regions, since heating brine is complex and expensive (Durán Quintana et al., 1999).

3.1. Lactic acid bacteria

The simply enumeration of LAB may be carried out on MRS agar. It is probably the most commonly used medium for the cultivation of lactobacilli and other LAB (Schillinger and Holzapfel, 2003). This medium must be supplemented with nystatin (50 mg/L) or cycloheximide (100 mg/L) as inhibitors of eukaryotic organisms (to prevent yeasts and molds growth) or with sodium azide (200 mg/L) as Gram-negative bacteria inhibitor. A number of differential and selective media were created for the isolation and characterization of certain groups of LAB. HHD medium is the most used for the differential enumeration of homofermentative and heterofermentative LAB. This medium contains fructose which is reduced to mannitol by heterofermentative but not by homofermentative LAB. Differences in the colour of the colonies are based on differences in the amounts of acids produced by these bacterial groups (Schillinger & Holzapfel, 2003). So in this medium, homofermentative LAB are blue to green, while heterofermentative LAB colonies are white (Fleming et al., 2001). M17 medium should be used for lactococci isolation, but isolation of these LAB is more frequent when analysing dairy products rather than fermented vegetables. All LAB isolation requires anaerobic conditions and incubation at 30-32°C for 48-72 hours, depending on the medium used.

3.2. Mesophilic aerobic bacteria

The mesophilic count, or standard plate count, is generally obtained on Plate Count agar (PCA) which is a generic medium for aerobic microorganisms that grow at mesophilic temperatures. Aerobic plate counts are poor indicators of safety in some products such as those fermented which commonly show a high aerobic count. However, this count gives information about the hygienic and sensorial quality, about the adherence to good manufacturing practice and shelf life of the product (Morton, 2001).

Alternatively to PCA, similar generic media are available as Nutrient agar. The growth conditions are 25-30°C for 24-48 hours, aerobically.

3.3. Yeasts and molds

Yeasts and molds are widely diffused eukaryotic microorganisms because of their adaptation to different environmental conditions. Yeasts and molds can cause various degrees of food decomposition. Invasion and growth may occur on virtually any type of food (Beuchat & Cousin, 2001). Their contamination of food can lead to product losses but, in particular, the highest risk is due to the mycotoxin production by the molds. Several yeasts genera may be important for the sensorial properties of fermented products and for their interrelation with Lactobacillus bacteria (see section 2.1) in fermented olives. However, film-forming yeasts as Debaryomyces, Candida, Pichia and Endomycopsis, are often associated with pickled products and vegetable brines (Fleming et al., 2001), representing the cause of olive defects and consequent product losses.

Available media for yeasts and molds count are several: Rose Bengal Chloramphenicol agar, YM agar, Oxytetracycline Glucose Yeast Extract agar, Sabouraud agar and others. When not included in the medium, the addition of 100 mg/L chloramphenicol is recommended to inhibit the growth of bacteria. For simple enumeration of yeasts and molds, the plates should be incubated aerobically at 25°C for 48 hours.

3.4. Enterobacteriaceae and coliform bacteria

The Enterobacteriaceae family consists of Gram-negative, facultative anaerobic rods widely distributed in the environment, but which are usually associated with intestinal infections.

Numerous studies have determined that Escherichia coli, coliforms, faecal coliforms and Enterobacteriaceae are unreliable when used as index of pathogen contamination of food. So National and International advisory committees invalidated the prediction of food safety based on levels of Enterobacteriaceae, coliforms, faecal coliforms and E. coli (Kornacki & Johnson, 2001). The most important application of Enterobacteriaceae and coliforms is their enumeration to assess if pasteurization has been adequately performed for example in pasteurized milk, because a proper pasteurization under appropriate conditions inactivates E. coli cells present in raw material.

Total coliform bacteria belong to the Enterobacteriaceae family, and include E. coli as well as various members of the genera Enterobacter, Klebsiella and Citrobacter. All ferment lactose with gas and acid production in 48 hours at 35-37°C. While faecal coliforms are coliforms which can ferment lactose to acid and gas within 48 hours at 45°C and they are so called because are more closely associated with faecal pollution (Manafi, 2003). The term coliform is based only on lactose hydrolysis and has no taxonomy validity. In order to perform the best examination of olive samples, it is preferable enumerate both Enterobacteriaceae and coliforms, because some lactose-negative bacteria, such as Salmonella, are pathogens.

The most probable number (MPN) is a method which indicates the most likely number of microorganisms present in the analysed sample. This method, used for several years, has now been replaced with techniques on agar media because the MPN is based on a statistical approximation. So, in addition to Violet Red Bile agar or Violet Red Bile Glucose agar, other differential media based on a chromatic response were developed.

Mac Conkey's MUG agar and Eosin Methylene Blue agar (EMB) are differential and selective media, suitable to obtain at the same time the isolation of Salmonella, Shigella and coliform bacteria, in particular E. coli. In the first medium, lactose-negative colonies are colourless, lactose-positive colonies are red and often surrounded by a turbid zone due to the precipitation of bile acids. E. coli can be identified by fluorescence in UV due to its β-D-glucoronidase production. In EMB agar, lactose-fermenters form colonies with dark-blue centres (E. coli may also have a green metallic sheen) while the non-lactose fermenters form completely colourless colonies.

3.5. Staphylococcus aureus

The foods most associated with staphylococcal poisoning are meat products, dairy products and cream filled bakery products. In processed foods in which S. aureus is destroyed by processing, its presence usually indicates post-processing contamination from human skin, mouth, nose or food handlers (Lancette & Bennett, 2003). Due to the high salt tolerance of S. aureus, it can grow in table olives even though the low pH and the olive phenols may represent natural inhibitors (Tassou & Nychas, 1994). However, it may be isolated and enumerated in table olives for the same above mentioned reason (as contamination index).

A variety of coagulase-positive and coagulase-negative staphylococci are able to produce enterotoxins, but S. aureus still plays a predominant role in staphylococcal food poisoning.

Baird-Parker agar and Rabbit Plasma Fibrinogen agar are the media recommended by the International Organisation for Standardisation (ISO). Moreover, Baird-Parker agar is also used in the official AOAC method in the United States (Zangerl &Asperger, 2003). Tellurite reduction, egg yolk reaction and a high level of sodium chloride are the most applied selective chemicals added in media for S. aureus isolation and enumeration.

S. aureus colonies in Baird-Parker agar are black, with an opaque zone around the colony because of the egg yolk reaction. Another medium frequently used is the Mannitol Salt agar, which contains mannitol. Coagulase-positive staphylococci grow and produce acid from mannitol showing a yellow colony and halo in a red medium. For all these media, the chemical inhibitors usually used are not completely selective, so additional diagnostic tests may be necessary to identify S. aureus colonies. Microscopic examination, catalase test and coagulase test may be rapidly executed to identify S. aureus from isolates. Common MPN procedures may be used also for enumeration of S. aureus. In most cases, the methodologies need a liquid enrichment procedure to detect low numbers of staphylococci (<100 UFC/g).

Culture conditions are usually 37°C for 48 hours aerobically.

3.6. Sulphite reducing clostridia

Clostridia are a widespread heterogeneous group of bacteria showing metabolic and nutritional differences. They easily contaminate foods because they produce resistant spores which can survive under mild processing conditions. The group of sulphite reducing clostridia may be used as marker of raw material quality and hygiene of manufacturing practices. Of particular concern for public health are C. botulinum, which forms a deadly toxin in foods, and C. perfringens which causes enteritis when present in high numbers. Other species or strains are also known to be toxinogenic or neurotoxinogenic (Bredius & Ree, 2003). C. perfringens is the agent of a food poisoning usually associated with consumption of cooked meats or poultry products. Sporulating cells of C. perfringens produce a heat-labile enterotoxin which appears to be released in vivo in the intestine (Labbe, 2001).

Most isolation media include sulphite and an appropriate iron salt. Sulphite is reduced to sulphide by the clostridial enzyme sulphite reductase; the sulphide will then precipitate as a black deposit in the presence of iron salt. This causes a blackening of the liquid media and clostridia will appear as black colonies. To obtain a higher selectivity for C. perfringens isolation, the Oleandomycin Polymixin Sulphadiazine Perfringens (OPSP) agar medium was performed. It contains selective antibiotics and utilises sodium metabisulphite and liver extract as sources of H₂S with ferric ammonium citrate as the indicator. In any case, subsequent confirmation tests such as motility, reduction of nitrate, lactose fermentation, gelatin liquefaction (Labbe, 2001) should be necessary. The plates are incubated anaerobically at 37°C for 18-48 hours.

3.7. Listeria monocytogenes

Listeria species are ubiquitous organisms widely distributed in the environment, especially in plant matter and soil. The microorganism is established as an important foodborne pathogen, which can grow at high salt concentration (up to 10% of NaCl) and at refrigerated temperatures. A study carried out by the U.S. Food and Drug Administration, U.S. Department of Agriculture (USDA, 2001), indicated that vegetables are able to support L. monocytogenes growth and, after a few years, Caggia and co-workers (2004) demonstrated that L. monocytogenes can survive and grow in green table olives. As indicated by the most recent scientific report of the European Food Safety Authority (2012), the number of listeriosis cases in humans in the EU slightly decreased, and 1,601 confirmed human cases were reported in 2010.

The minimum infective dose of L. monocytogenes has not yet been established and many authorities require that the organism must be absent in 25 g of product. This has led to the development of methods following the sequence of pre-enrichment, selective enrichment and diagnostic plating. The whole procedure may take about five days. Then, presumptive positive results need to be confirmed adding further time to complete the examination (Beumer & Curtis, 2003).

Most of the isolation media differentiate Listeria spp. by means of aesculin hydrolysis which, in the presence of iron, forms a black phenolic compound. According to the USDA method (McClain & Lee, 1988) 25 mL of brine sample are added to 225 mL of Listeria Enrichment broth base for the resuscitation of stressed cells. After blending, the bag is incubated at 30°C and then, at intervals from 4 hours to 7 days, an aliquot is plated for enumeration in Listeria Selective agar base and incubated at 37°C for 24 h. Black colonies on selective agar base medium were considered presumptive Listeria colonies.

The presumptive Listeria isolates are to be tested for sugar fermentation, tumbling motility, hemolytic reaction and growth at different salt concentrations (Caggia et al., 2004). Alternatively to this medium, ALOA agar contains the chromogenic compound for the detection of β-glucosidase, common to all Listeria, which appear as blue coloured colonies. While L. monocytogenes, which possesses a specific phospholipase, hydrolyses the specific substrate added to the medium producing an opaque halo around the colonies (Beumer & Curtis, 2003). Another chromogenic medium used is the Rapid’ L. mono agar.

Where counts < 100 CFU/g are expected, it is necessary to use the Most Probable Number (MPN) technique (USDA, 2001).

In order to rapidly perform additional confirmation tests, systems of strips are commercially available for Listeria (API Listeria) as for other pathogens.

3.8. Propionibacteria

Propionibacteria are often associated with food spoilage. The “zapateria” spoilage, which can occur in brined olives, is characterized by a malodorous fermentation due to propionic acid produced by certain species of Propionibacterium (Jay, 2000), alone or together with Clostridium spp.. The propionibacteria may be considered as marker of the end of shelf life of table olives, because the growth of these bacteria cause an increase in pH values creating conditions for the growth of spoilage or pathogen bacteria and the arising of off-odors (Plastourgos & Vaughn, 1957).

Unfortunately, propionibacteria are difficult to isolate because they grow very slowly on solid media. The complex medium usually used is not able to suppress competing organisms. This medium is Sodium Lactate agar (1% typticase, 1% yeast extract, 1% sodium lactate, 0.025% dipotassium phosphate, 1.5% agar) in which the propionibacteria growth appears in 5-7 days at 32°C under anaerobic or microaerophilic conditions (Richter & Vedamuthu, 2001).