**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.

**Figure 1.** *L. plantarum* strain detected by fluorescence microscopy.

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).

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

starter cultures of *Lactobacillus plantarum* and *Debaryomyces hansenii*.

**2.3. LAB starter cultures** 

preventing microbial spoilage, and improve flavour.

**Figure 1.** *L. plantarum* strain detected by fluorescence microscopy.

stage and ensuring the safety of the final product (Chorianopoulos et al., 2005).

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

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

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 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 heterofermentative 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).

Microbiological Aspects of Table Olives 329

**3. Enumeration of microorganisms in fermented olives:** 

Each microorganism should grow and form a separate colony when the sample is plated in a solid medium during plate count procedures. Unfortunately, some organisms may not be capable to grow under the conditions used. Moreover, some chains of organisms could

Up to now, methods available for detection and identification of microbial population involved in table olive fermentation have been very limited and generally culturedependent, not providing reliable information on the composition of the entire microbial community (Randazzo et al., 2012). A culture-independent method, such as the denaturing gradient gel electrophoresis (DGGE), has the potential to study microbial population quickly and economically, avoiding the use of selective cultivation and isolation of bacteria (Rantsiou et al., 2005). So, plate count techniques are still the major but not the most representative method. Using aseptic techniques, brine samples may be taken and used directly to prepare serial dilutions. In order to analyse the whole olive, for example dry salted olives, the containers should be shaken and after mixing the olives, a sample should be taken aseptically with a sterile spoon, weighed and transferred to a sterile container such as plastic bag. After adding a sterile volume of quarter-strength Ringer's solution or 0.9% NaCl, the bag is then pummelled in a stomacher to prepare dilutions. Each mL collected by this bag represents 1 g of olive sample. The decimal dilutions are usually used for the calculation of results. Different ranges to obtain a readable number of colonies on plates may be necessary depending on the microorganism, procedures and initial contamination of olives. After pipetting the diluted sample into the petri plate, about 15 mL of each liquefied medium is added (the temperature must not exceed 45°C) into the plate. The medium is mixed with inoculum by carefully rotating. Several dilutions or replicate plates for each dilution should be prepared for each sample, making sure that the sample is tested, at least, in triplicates. After solidification, the petri plates should be inverted and placed in the incubator at specific temperature shown in the next sections. After incubation, the plates should contain between 25 and 250 colonies for the best count accuracy (Swanson et al., 2001). If microbial changes during fermentation are to be followed, the sampling must start at time zero, when the olives are salted or brined, and samples should be collected at regular intervals up to the end of fermentation. The sampling intervals may be of 1-3 days during the first month of fermentation, then these intervals may be extended up to 7 days. The brine samples, such as olives, should be examined as soon as possible, but if it is temporarily impossible, the samples must be refrigerated and analysed within 24 hours.

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

**Methods of analysis** 

**3.1. Lactic acid bacteria** 

appear as a single colony (Swanson et al., 2001).


**Table 1.** LAB tested as starter cultures in table olives (references are shown for the last twelve years)
