Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential

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Abstract

The aim of this work was to study the potential probiotic properties of lactobacilli associated with table olives. From a total of 111 isolates from spontaneously fermented green olive brines, 109 were identified at species level by multiplex PCR amplifications of the recA gene. One hundred and seven of these were identified as Lactobacillus pentosus, one as Lactobacillus plantarum, and another as Lactobacillus paraplantarum. Repetitive bacterial DNA element fingerprinting (rep-PCR) with GTG5 primer revealed a higher variability within the L. pentosus isolates and nine different clusters were obtained. Most of them showed high autoaggregation ability, low hydrophobicity properties, and lower survival to gastric than to pancreatic digestion; however, no isolate showed bacteriocin, haemolytic or bile salt hydrolase activities. A multivariate analysis based on results from phenotypic tests led to the segregation of some L. pentosus isolates with promising potential probiotic characteristics, which are even better than probiotic reference strains. Due to the autochthonous origin of the strains, their use as starter cultures may contribute to improving natural fermentation and the nutritional characteristics of table olives.

Highlights

► Table olives can be a new interesting probiotic source to consumers. ► Rep-PCR fingerprinting revealed a high variability within the L. pentosus isolates. ► As deduced by PCA, some L. pentosus isolates showed promising probiotic characteristics. ► GG2S-T2-168 and GM2F-T5-327 showed the best potential characteristics. ► Their use as starter culture in table olives could improve safety and sales.

Introduction

According to the latest consolidated statistics, worldwide table olive production for the 2007/2008 season was 2,153,500 tonnes (IOOC, 2008). Most table olives are prepared as Spanish-style or alkali treated green olives (IOOC, 2004), which represent about 60% of production, but there is also a general increasing interest for traditional and homemade products such as “seasoned” olives, usually prepared from directly brined fruits (Arroyo López et al., 2005).

Among the microorganisms involved in the Spanish style green table olive fermentation, lactic acid bacteria (LAB) is the most relevant group (Garrido-Fernández, Fernández Díaz, & Adams, 1997). The development of LAB in this process leads to a rapid acidification of brines and a drop in pH to below 4.5 (Delgado et al., 2007, Garrido-Fernández et al., 1997, Sánchez et al., 2001). However, the growth of LAB in directly brined olives is more difficult due to the high presence of phenolic compounds in the storage solutions. For this reason, LAB from non alkali-treated fruits have been scarcely studied (Garrido-Fernández et al., 1997).

According to the FAO/WHO (2001), probiotics are “live microorganisms which, when administered in adequate amounts, confer a health benefit to the host”. They “should be resistant to gastric juices and be able to grow in the presence of bile”. There is a series of in vitro tests such as acid and bile tolerance, antimicrobial production, etc., that, although requiring further refinement, are usually applied as a first approach for the selection of potential probiotic microorganisms (FAO/WHO, 2001).

Reviews on the importance of foods in the efficacy of probiotics have recently been published (Champagne and Gadner, 2005, Ranadheera et al., 2010). New sources of probiotic microorganisms such as Greek dry-fermented sausage (Papamanoli, Tzanetakis, Litopoulou-Tzanetaki, & Kotzekidou, 2003) cereal-based substrates (malt, barley and wheat media) (Charalampopoulos, Pandiella, & Webb, 2002) or kimchi (Chang, Shim, Cha, & Chee, 2010) are currently being investigated. The regular intake of probiotic preparations is considered useful to increase the bioavailability of trace minerals and proteins in the vegetarian diet (Famularo, De Simone, Pandey, Ranjan Sahu, & Minisola, 2005). Potentially probiotic strains of Lactobacillus plantarum and Lactobacillus paracasei survived in artichokes, which improved their survival in simulated gastrointestinal digestion (Valerio et al., 2006). L. paracasei IMPC2.1 (isolated from the human intestine) incorporated into an artichoke fed to a human was recovered from stools and also showed high viability for 3 months in ripe and green olive fermentations (Lavermicocca, Lonigro, et al., 2005). The use of table olives as a reliable food vehicle for this bacterium into the human gastrointestinal tract has been subjected to patents (Hurtado et al., 2012, Lavermicocca, Valerio, et al., 2005). Recently, L. paracasei IMPC2.1 has been used as starter culture for processing green table olives and its survival and dominance on the olive surface during fermentation was studied through molecular identification (De Bellis, Valerio, Sisto, Lonigro, & Lavermicocca, 2010). However, the probiotic potential of LAB naturally occurring in table olive fermentations still remains unexplored. The availability of LAB strains isolated from this product, the most common lactic acid fermented vegetable in western countries, could have a considerable value for developing starter cultures with the double role of being technologically relevant and able to enhance the health aspect of table olives.

Therefore, the present study focused on a) the identification and molecular characterization at strain level of the lactobacilli microbiota isolated from two types of table olive elaborations: lye-treated (Spanish style) and directly brined olives, and b) the study of certain in vitro phenotypic characteristics related to the probiotic potential of these microorganisms.

Section snippets

Olive cultivars and processing

Samples were directly obtained from three Spanish olive factories. In the case of Spanish style green olive fermentations, a single factory (plant A, located in Alcalá de Guadaira, Seville, Spain), which processes both Gordal and Manzanilla cultivars in underground, fiberglass containers with a 10-tonne capacity, was sampled. Briefly, Gordal olives were first treated with a 20 g ml 1 sodium hydroxide solution until reaching 2/3 of the flesh, washed with tap water for 18 h and then immersed in 110 g 

Physicochemical changes

The initial and final NaCl concentrations, combined and titratable acidities, and pH values for the different table olive elaborations studied in this work are shown in Table 1. The initial NaCl concentrations in directly brined olives ranged from 59.5 g l 1 (Manzanilla) to 91.0 g l 1 (Aloreña) and decreased to ~ 20.0 g l 1 at the end of the process. In Spanish style olives, the NaCl concentration was kept constant at around 64.0 g l 1 throughout the process. Combined acidity was significantly higher in

Discussion

LAB starter cultures are widely used in combination with probiotic bacteria to produce fermented dairy derivatives (Vinderola, Bailo, & Reinheimer, 2003). However, there are an increasing number of consumers who are allergic to milk, or simply, prefer more variation with respect to probiotic sources. Products traditionally subjected to lactic acid fermentation during their processing are good candidates. Among others, table olives, the most common fermented vegetable product in western

Conclusion

Certain L. pentosus isolates showed promising probiotic and differentiated characteristics as was proven by PCA. GG2S-T2-168 showed not only high survival rates but also high values for auto-aggregation and hydrophobicity. The GM2F-T5-327 isolate had a moderate auto-aggregation value although with a low level of hydrophobicity but similar to that of the reference probiotic strains used as the control. However, final selection would also depend on their real in vivo probiotic properties as well

Acknowledgments

The research leading to these results has received funding from the EU's Seventh Framework Program ([FP7/2007-2013] under grant agreement no. 243471 (PROBIOLIVES). We are also thankful for the financial support from the Spanish Government (projects AGL-2006-03540/ALI, AGL2009-07436/ALI, AGL2009-08016/ALI and AGL2010-15529/ALI partially financed by European regional development funds, ERDF), and Junta de Andalucía (through financial support to group AGR-125). J. Bautista-Gallego and F.N.

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