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

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

*Lactobacillus*

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).

**Genus Species Authors & year**

Randazzo et al., 2011 Ruiz-Barba et al., 2010 Perricone et al., 2010 Hurtado et al., 2010 Kumral et al., 2009 Sabatini et al., 2008 Panagou et al., 2008 Saravanos et al., 2008 Romeo & Poiana, 2007 Marsilio et al., 2005 Chorianopoulos et al., 2005 Lamzira et al., 2005 Caggia et al., 2004 Leal-Sánchez et al., 2003 Sánchez et al., 2001

Aponte et al., 2012 Hurtado et al., 2010 Medina et al., 2009; 2008 Panagou et al., 2008 Peres et al., 2008 Romeo & Poiana, 2007 Servili et al., 2006 Caggia et al., 2004 De Castro et al., 2002 Sánchez et al., 2001

Caggia et al., 2004

Saravanos et al., 2008

Romeo & Poiana, 2007

*casei* Randazzo et al., 2011

*paracasei* De Bellis et al., 2010

*paraplantarum* Romeo & Poiana, 2007 *brevis* Kumral et al., 2009

*coryniformis* Aponte et al., 2012

*paramesenteroides* Kumral et al., 2009

*casseliflavus* De Castro et al., 2002

*Leuconostoc cremoris* Kumral et al., 2009

*Pediococcus pentosaceus* Ruiz-Barba et al., 2010 *Enterococcus faecium* Ruiz-Barba et al., 2010

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

*plantarum* 

*pentosus* 

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

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.

### **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.

Microbiological Aspects of Table Olives 331

**3.4.** *Enterobacteriaceae* **and coliform bacteria** 

**Figure 2.** *E. coli* strain detected by fluorescence microscopy.

*E. coli* cells present in raw material.

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

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.

differential media based on a chromatic response were developed.

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
