Identificationtion of Phenolic Compounds in Table Olives and Samples of Brines during Table Olives Fermentation: Biological and Antimicrobial Activity

Table olives are one of the most important traditional fermented vegetables in Southern European (Italy, Greece and Spain) countries. In the Greek-style production system, the fruits are placed directly into the brine, thus allowing the natural fermentation to take place. The spontaneous fermentations, that can last 8–12 months, are driven by mixed populations of microorganisms, mainly the epiphytic microbial population of yeasts and lactic acid bacteria (LAB) (Romero et al., 2004). At present, the industrial table olive process is not predictable and depends on the empirical experience of the producers. In order to avoid the unpredictability of the olive spontaneous fermentation, to improve the productive process and to constantly produce high-quality final products, the use of strains of LAB as starter cultures for olive production has been proposed (Sabatini and Marsilioet al., 2008; Panagou et al., 2008; Blana et al., 2014). However, in the last years, the importance and the potential applications of yeasts as starters for table olive processing has been recognized (Arroyo-López et al., 2008, 2012; Bevilacqua et al., 2012). Objectives: In the present work, we have studied the main physical, chemical and aromatic parameters of natural fermentations of Cellina di Nardò, Leccino, Kalamata and Conservolea table olives in order to determine chemical descriptors correlated to microbiological activities and the dynamics of microorganisms in order to select LAB and yeast strains as candidate autochthonous starter cultures. Conclusions: The identified chemical descriptors can be suitable to follow the trend and to control the outcome of the fermentation and a new protocol aimed to the selection of LAB and yeast strains as candidates autochthonous starters has been developed and applied (Bleve et al. 2014 a, b). Selected microbial starters have been successfully used to ferment olives in pilot and industrial-scale and a new method for table olive production has been set up (Bleve et al. 2103). The use of selected autochthonous starter cultures produced fermented table olives with improved organoleptic, sensorial and nutritional characteristics. Objectives: Proceedings of the Bio-Olea Conference (Extended Abstracts) Corfu, Greece, 21-22 February 2014 Table Olives, Virgin Olive Oil and Olive Mill Wastewater: Developments and Potential Solutions Organized by: Region of Ionian Islands – University of Ioannina 11 The objectives were 1. the selection of autochthonous LAB and yeasts from naturally fermented Italian cultivar of Cellina di Nardò and Leccino table olives and from two naturally fermented Greek cultivar Kalamata and Conservolea of table olives; 2. the evaluation of organoleptic and sensory characteristics of table olives fermented by autochthonous selected LAB and yeasts, for the maintenance and / or improvement of the nutritional benefits of the product; 3. the study and characterization of chemical profiles associated to olives and brines during the fermentation process in order to identify chemical descriptors suitable to monitor the process Methodology Cellina and Leccino Kalamata and Conservolea ripe olives were kindly supplied by Italian ad Greek producers. All olives fermentations were performed following the Italian and Greek producer’s practices (NaCl concentrations in brines, temperature, pH). The olives were allowed to ferment at external temperature adopting producer’s practices (air inflation, correction of salinity by addition of salt, removal of superficial mold layer). During spontaneous industrial olive fermentations a number of different isolates was randomly selected from either yeast and LAB populations, , in order to study the dynamics of the dominating yeasts and LAB. Selected yeast and LAB, to be used as starters, were grown in 5 L and in 30 L fermentor in appropriate culture medium (Bleve et al., 2014) and used, respectively, to inoculate pilot or industrial fermentations The biochemical parameters were determined by HPLC-DAD, sensorial and metabolic analyses were carried out by HPLC-DAD and headspace solid-phase microextraction (HS-SPME) and GC–MS. Identification of yeast and LAB isolates was performed by DNA extraction, PCR amplification of 16S rRNA gene and of ITS1-5,8S-ITS2 region, (for LAB and yeast, respectively) and sequencing of produced amplicons. Results Selection of autochthonous LAB and yeasts from naturally fermented table olives Black olive samples of the Italian cultivar (Cellina di Nardò and Leccino) and of the two Greek cultivars (Conservolea and Kalamàta) were collected at the black stage of ripening. Spontaneous fermentations of these two Italian and two Greek table olive cultivars were performed in collaboration with Agr. Nuova Generazione of Martano (Le) and the Department of Chemistry, Section of Food Chemistry, University of Ioannina (Figure 1) pH, temperature and NaCl concentration were followed throughout the whole fermentation process. The microbial count values obtained by analyzing microflora associated to each of the four olive varieties indicated a similar growth behavior for yeast and LAB Proceedings of the Bio-Olea Conference (Extended Abstracts) Corfu, Greece, 21-22 February 2014 Table Olives, Virgin Olive Oil and Olive Mill Wastewater: Developments and Potential Solutions Organized by: Region of Ionian Islands – University of Ioannina 12 during spontaneous fermentations. In particular, for all the tested table olive varieties the fermentation process can be divided into two main phases: the first phase characterized by the presence of yeasts that can reach counts about 10 3 -10 5 CFU/ml and by the absence of LAB and a second phase driven by LAB (that can reach counts about 10 4 -10 6 CFU/ml). For all the fermentations temperature, NaCl concentration and pH variations were recorded (Figure 2 and3) and pH declined during the process reaching the minimum values (4,0-4,3). In order to establish a screening procedure of the yeast and LAB isolates obtained from all the sampling times and from all the fermentations, a selective medium was formulated by preparing two model brines, each one specific for yeasts and LAB. Each model brine was formulated on the basis of the main phenolic compounds and the average concentrations of sugars, organic acids and alcohols identified and quantified in olive brines from natural fermentations (Bleve et al, 2014 a, b). A new procedure has been assessed for the isolation and characterization of yeasts and bacteria from fermented table olives and for the preparation of starter cultures suitable for industrial production (Figure 6). In addition to the classical technological parameters (resistance to high salt concentration, low temperature and low pH; production of extracellular hydrolytic enzymes) new criteria have been added to the developed procedure. By these screening conditions, more than 2000 yeast and about 540 bacteria colonies were assayed for their resistance to NaCl, low temperature, low pH, oleuropein and verbascoside, the last being a potent inhibitors of microbial growth. After this first selection step, a yeast population being resistant to the above constraints was isolated and it consisted of 897 isolates, whereas 184 LAB isolates resulted resistant to the above constraints. Isolates have been then evaluated for the absence of negative traits, such as the production of biogenic amines as well as, for positive traits such as the beta-glucosidase activity against oleuropein, that is responsible of the bitter taste of olives. After this second selection step, yeast and LAB populations (278 and 43 isolates, respectively) which satisfied the above parameters were isolated (examples are reported in Figure 5). This last yeast and LAB isolates were identified at the genus and species level by PCR analysis of their rDNA region in order to identify the isolates belonging to GRAS species (Bleve et al, 2014 a, b). Use of selected autochthonous LAB and yeasts as starters in pilot fermentations At the end of the selection protocol, several yeast-LAB pairs selected from each table olive cultivar were tested for their ability to grow together and for possible inhibition effects in co-inoculation experiments. One autochthonous yeast-LAB pair was the chosen for each table olive cultivar and grown to produce a suitable quantity in order to inoculate table olives in 200 Kg tanks (Figure 6) . Five different strategies of inculum were tested: 1) co-inoculum yeast and LAB 2) inoculum of yeast firstly and then (after 45-60 days) LAB, 3) inoculum of LAB firstly and then (after 15-30 days) yeast, Proceedings of the Bio-Olea Conference (Extended Abstracts) Corfu, Greece, 21-22 February 2014 Table Olives, Virgin Olive Oil and Olive Mill Wastewater: Developments and Potential Solutions Organized by: Region of Ionian Islands – University of Ioannina 13 4) only yeast, 5) only LAB The fermentations were followed for a period of about 3-4 months. The best strategy of inoculum resulted n. 2 (use of yeast firstly and then LAB). During fermentations, microorganisms were isolated.DNA was extracted and used to perform strain-specific PCR profile. The inoculated autochthonous yeast and LAB strains were able to dominate the fermentations against wild microflora. In fact, for each of the four tested table olive cultivars, the starter microorganisms were able to dominate and drive the fermentations with a percentage ranging from 60 to 80% of the total population. Metabolic profile of fermentations were followed analyzing (at different time points) samples of brines and drupes in order to detect and quantify sugars, organic acids and alcohols and to monitor the quali-quantitative changes of mono and polyphenols. Following metabolites during fermentations, as expected a decrease of sugar content, a corresponding increase in alcohols and an increase in organic acids due to the fermentations within the drupes was observed. Analogously, for mono and polyphenol compounds, it was observed a decrease of oleuropein, and an increase of hydroxytyrosol levels. Moreover, for each cultivar differences in phenolic contents and in the profile of each phenolic compound were observed (Figure 7 and Bleve et al., 2014 a, b). The use of the autochthonous yeast and LAB starters mimics natural fermentation and produced similar results but in shorter time period (3-4 months). Industrial scale fermentations A new procedure was set up to produce yeast and LAB as starter cultures in a 30 L fermentor to be used to control table olive fermentation. Yeast and LAB biomasses were produced in fermentor in a quantity suitable to inoculate industrial-scale fermentations of olives (3000 Kg) in several companies of Apulia Region (Figure 8). The strategy of inoculum was the same set up in pilot scale fermentations. The industrial-scale fermentations are now in progress (Figure 9). In order to optimize the extraction of volatile compounds from olive and brine samples, different protocols were carried out testing different SPME fibers, pre-treatment of olives and brine samples, time necessary for an adequate equilibration of the samples before sampling, time of exposition of fibers to the samples. The optimized extractive procedure using headspace solid-phase microextraction (HS-SPME) and the subsequent analysis using GC–MS were used for monitoring in olives and brines the presence of volatile compound and providing a snapshot of the olive’s status at different step of fermentation. The volatile compounds present in the four table olive cultivars were analyzed by gas chromatography and mass spectrometry (GC-MS). The different profiles differed in amount of esters, terpenes, alcohols and fatty acids. Others volatile compounds found in table olives were hydrocarbons. In spontaneous fermentations it was observed the presence of molecule classes representative of flavor Proceedings of the Bio-Olea Conference (Extended Abstracts) Corfu, Greece, 21-22 February 2014 Table Olives, Virgin Olive Oil and Olive Mill Wastewater: Developments and Potential Solutions Organized by: Region of Ionian Islands – University of Ioannina 14 and aroma notes like green sweet-winery, fruity, floral, fatty. It could be also interesting to investigate for the presence of high levels of hydrocarbons and volatile phenols. By statistical analyses of chemical results, it was possible to determine the class of volatile compounds most representative of each fermentation phase. For all the four table olive cultivars, at the first stage aldehydes dominated, then the most representative compounds belonged to alcohols and terpenes and finally, the fermentation was dominated by the presence of fatty acids, esters and alcohols. These classes of compounds can be followed by quali-quantitave assay to monitor the whole fermentation process. Using the spontaneous fermentations as model for the description of the process, in pilot-scale fermentations inoculated with autochthonous yeast-LAB pairs, the fermentation phases were determined by the sequential appearance of the above described classes of molecules. The autochthonous yeast-LAB pairs were considered efficient to perform good fermentations when the fermentations followed the above described behavior. Independently from the cultivar, olives inoculated with the microbial starter (following the strategy of inoculum n.2) resulted richer in alcohols, esters and aldehydes than noninoculated spontaneous controls. The use of starters resulted in the production of several classes of volatile compounds with a positive role in the organoleptic profile of the olives, whereas the volatile profile observed in the spontaneous fermentations, used as control, resulted very poor. Moreover, the volatile profile obtained by the use of the starters was comparable or more complex than that produced by an industrial spontaneous fermentation and resulted even better when compared to that obtained from different top quality commercial products. It is also important to note that a considerable reduction of negative molecules such as hydrocarbons and volatile phenols was also observed. Conclusions In this study, it was produced the description of the microbial population dynamics associated to spontaneous fermentation by the Greek method of two Italian and two Greek traditional table olive cultivars. Moreover, a new protocol for selection of starter cultures was set up and authochtonous selected microorganisms was used as starter cultures to drive and control fermentations. The use of selected autochthonous microbial starters allowed the improvement of the process by the standardization as well as the reduction of the time necessary to obtain a final product. Chemical descriptors suitable to monitor and eventually correct the evolution of the process were for the first time identified and experimental data were produced about their application to pilot scale fermentations of table olives. The use of selected autochthonous microbial starters produced an improvement of the organoleptic and sensorial characteristics and of the nutritional traits of the final product. Proceedings of the Bio-Olea Conference (Extended Abstracts) Corfu, Greece, 21-22 February 2014 Table Olives, Virgin Olive Oil and Olive Mill Wastewater: Developments and Potential Solutions Organized by: Region of Ionian Islands – University of Ioannina 15 References Arroyo-López,F.N.,Querol,A., Bautista-Gallego,J.,andGarrido-Fernández,A.(2008). Role of yeasts in table olive production. Int.J. Food Microbiol. 128, 189–196. Arroyo-López, F.N., Romero-Gil,V., Bautista-Gallego,J.,RodriguezGómez,F.,Jiménez-Díaz,R., GarcíaGarcía,P., etal. (2012). Yeasts in table olive processing: desirable or spoilage microorganisms. Int. J. Food Microbiol. 160, 42–49. Bevilacqua A, Corbo MR, Sinigaglia M. (2012) Selection of Yeasts as Starter Cultures for Table Olives: A Stepby-Step Procedure. Front Microbiol. 31;3:194 Blana VA, Grounta A, Tassou CC, Nychas GJ, Panagou EZ. 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Food Chemistry 107 (2008) 1522–1528 Proceedings of the Bio-Olea Conference (Extended Abstracts) Corfu, Greece, 21-22 February 2014 Table Olives, Virgin Olive Oil and Olive Mill Wastewater: Developments and Potential Solutions Organized by: Region of Ionian Islands – University of Ioannina 16 IDENTIFICATIONTION OF PHENOLIC COMPOUNDS IN TABLE OLIVES AND SAMPLES OF BRINES DURING TABLE OLIVES FERMENTATION: BIOLOGICAL AND ANTIMICROBIAL ACTIVITY CHYTIRI AGATHI*, KONTOGIANNI VASILIKI G., KALLIMANIS ARISTEIDIS, TASIOULAMARGARI MARIA Department of Chemistry, Sector of Industrial Chemistry and Food Chemistry, University of Ioannina, 45110, Ioannina, 45110, Greece