State of the art

Human DigestiveIn the last two decades, probiotic health-promoting microorgansims have been increasingly included into commercial products in a response to the consumer demand for healthy food products. With the market success and the growing popularity of these fortified foods, global food companies have begun researching possible probiotic enhanced line extensions. Many of the probiotic microorganisms are lactic acid bacteria and, among them, lactobacilli represent certainly one of the fundamental microbial groups which have been introduced in a wide range of food products. Many studies have reported that the best matrices to deliver probiotics are dairy fermented products (fermented milks, yogurt, cheese, ice creams, and desserts). However, these foods cannot be consumed by certain groups of the population who suffer from lactose intolerance, high cholesterol levels or need a diet based on non-milk derived products. For this reason, the food industry has the great challenge to produce novel and non-dairy probiotics, whereas recent research reveals that other traditional foods may constitute a good working base for the development of probiotic-type functional foods. Given the awareness of consumers for health-promoting food products, innovation should be targeted to the identification of other health-proactive food matrices for use as vectors of delivery of probiotic microorganisms to the human body, while keeping their traditional character. Fermented foods of plant origin have been increasingly considered as vectors for incorporation of probiotic cultures (Soccol et al., 2010) following the well-established worldwide know-how on vegetable processing via lactic acid bacteria. A shift of interest has been recently experienced with table olives using strains preferred for their probiotic features. A new frontier tentatively named ‘probiotic vegetable gastronomy’ has disclosed olives as a promising candidate (Perricone et al., 2010). An attempt was made to use table olives as a vehicle to incorporate probiotic bacterial species of human origin, namely Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium bifidum, and Bifidobacterium longum (Lavermicocca et al., 2005; de Bellis et al., 2010) with promising results and a relevant patent application has been claimed (USPTO Patent Application 20070086990). Recently, the EU has funded a relevant project (PROBIOLIVES, contract no 243471: Table olive fermentation with selected strains of probiotic lactic acid bacteria. Towards a new functional food) having as a final goal the isolation of lactic acid bacteria from the indigenous microbiota of fermented olives and their in vitro probiotic characterization to be used as starters. In this way, the EU is sensitized with the table olive industry in an attempt to provide the necessary know-how to increase the technological level and competitiveness of the sector. The results of the project showed that olive brines were interesting source of indigenous lactic acid bacteria with probiotic potential. However, the contribution of yeasts, a major microbial group co-existing with lactic acid bacteria in many vegetable fermentation, was totally unexplored and previous results obtained from our group suggest that there is a strong association and synergy between lactic acid bacteria and yeasts, opening new perspectives in the application of mixed starter cultures with both technological and probiotic features.

An important characteristic of the putative starter cultures should be the ability to colonize the olive surface and form polymicrobial aggregates called biofilms. This is particularly important to consider in the case of probiotic strains given that the consumers ingest the olives and do not drink the brine, so in order to get any potential benefit from the functional properties of the selected starters it must be ensured that the microorganisms are adhered to the surface of olives and the dynamic growth of their population reaches high numbers to exert a beneficial effect on human health. The first observations of how microorganisms associate in directly brined black olives were reported a few years ago (Nychas et al., 2002), employing scanning electron microscopy to show that epicuticular waxes appearing as a bloom on the surface of olives provide a substrate for the development of biofilms containing both yeasts and lactic acid bacteria. Recent research has focused on the exploitation of the micro-architecture of the olive surface in an attempt to use it as a carrier of probiotic strains of lactic acid bacteria confirming for the first time the suitability of the olive surface for this purpose (Lavermicocca et al., 2005; de Bellis et al., 2010). It has also been demonstrated that anchoring of microorganisms on the surface of vegetables contributes to microbial cell integrity and stability during transit in the gastrointestinal tract until their release in the colon (Lavermicocca, 2006). Finally, the development of biofilms on the surface of green olives processed by the Spanish method using lactic starter cultures has been recently confirmed (Arroyo-López et al., 2012; Domínguez-Manzano et al., 2012). The biofilm consisted of the inoculated starter cultures both of lactic acid bacteria and yeasts embedded in polymeric matrix, the population of which was retained in high levels enough to exert beneficial effects. Hence, the green olives surface provides an appropriate environment for the suitable development and formation of complex biofilms during controlled table olive processing. It is still unknown how these biofilms are formed.

Advances of our group in the matter

In the WebPage of ProBiolives can be found a complete list of publications related to identification of microorganisms with probiotic potential isolated from table olive processing, their ability to form biofilm and their use as starters to conduct  fermentation. Below, you can see a selection of the Spanish publications:

– Arroyo-López, F.N., Romero-Gil, V., Bautista-Gallego, J., Rodríguez-Gómez, F., Jiménez-Díaz, R., García-García, P., Querol, A., Garrido-Fernández, A. 2012. Potential benefits of the application of yeast starters in table olive processing. Frontiers in Microbiology, 3, 1-4.

– Domínguez-Manzano, J., Olmo-Ruiz, C., Bautista-Gallego, J., Arroyo-López, F.N., Garrido-Fernández, A., Jiménez-Díaz, R. 2012. Biofilm formation on abiotic and biotic surfaces during Spanish style green table olive fermentation. International Journal of Food Microbiology, 157, 230-238.

– Arroyo-López, F.N., Bautista-Gallego, J., Domínguez-Manzano, J., Romero-Gil, V., Rodríguez-Gómez, F., García-García, P., Garrido-Fernández, A., Jiménez-Díaz, R. 2012. Formation of lactic acid bacteria-yeasts communities on the olive surface during Spanish-style Manzanilla fermentations, 32, 295-301.

– Bautista-Gallego, J., Arroyo-López, F.N., Rantsiou,K., Jiménez-Díaz, R., Garrido-Fernández, A., Cocolin, L. 2013. Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential, 50, 135-142.

– Rodríguez-Gómez, F., Bautista-Gallego, J., Arroyo-López, F.N., Romero-Gil, V., Jiménez-Díaz, R., Garrido-Fernández, A., García-García, P. 2013. Table olive fermentation with multifunctional Lactobacillus pentosus strains. Food Control 34, 96-105.

– Rodríguez-Gómez, F., Romero-Gil, V.,  Bautista-Gallego, J., García-García, P., Garrido-Fernández, A., Arroyo-López, F.N. 2014. Production of potential probiotic Spanish-style green table olives at pilot plant scale using multifunctional starters. Food Microbiology 44, 278-287.

– Rodríguez-Gómez, F., Romero-Gil, V., García-García, P., Garrido-Fernández, A., Arroyo-López, F.N. 2014. Fortification of table olive packing with the potential probiotic bacteria Lactobacillus pentosus TOMC-LAB2. Frontiers in Microbiology, 5, 1-9.

– Bonatsou, S., Benítez, A., Rodríguez-Gómez, F., Panagou, E.Z., Arroyo-López, F.N. 2015. Selection of yeasts with multifuncional features for application as starter in natural black table olive processing. Food Microbiology 46, 66-73.

 Innovative parts of Olifilm project

Respect to previous studies, the present project is totally innovative in the following aspects:

· Microorganisms with multifunctional features will be exclusively isolated and identified from biofilm matrix adhered to olive epidermis instead brines.

· Special attention will be put to the study of the probiotic potential of yeasts and their interacion with lactic acid bacteria.

· More clinical studies will be performed to determine the real probiotic potential of the mixed yeasts-lactic acid bacteria associations.

· Pyrosequencing and volatile compund determination will be applied.

· Study of the genes and process leading to the formation of mixed yeasts-lactic acid bacteria biofilm will be determined.

Scientific achievements obtained in the present project at this moment

TFM Portada AB

Item 1: Antonio Benítez’s thesis of Master. Master’s degree in Food and Beverage Technology from University Pablo Olavide (Seville, Spain). Title. Biofilm formation during fermentation of Gordal directly brined olives: Assessment of different methodologies for dettachment and counts of microorganisms associated to olive epidermis. Directors: Francisco Noé Arroyo López and Rufino Jiménez Díaz.

Foto 3

Item 2: Conferences in the V International Table Olive Symposium. Please, watch the video in https://www.youtube.com/watch?v=IiLPBiNQdy0&list=UUsWzoUiCm1tfxagG3GEdMlg.