NaOH-Debittering Induces Changes in Bacterial Ecology during Table Olives Fermentation Luca Cocolin 1 *, Valentina Alessandria 1 , Cristian Botta 1 , Roberta Gorra 1 , Francesca De Filippis 2 , Danilo Ercolini 2 , Kalliopi Rantsiou 1 1 Dipartimento di Scienze Agrarie, Forestali e Alimentari, Universita ` degli Studi di Torino, Grugliasco, Italy, 2 Dipartimento di Agraria, Universita ` degli Studi di Napoli Federico II, Portici, Italy Abstract Limited information is available on the impact of the NaOH treatment on table olive fermentations, and for this reason a polyphasic approach has been adopted here to investigate its effect on the fermentation dynamics and bacterial biodiversity. The microbial counts of the main groups involved in the transformation have not shown any differences, apart from a more prompt start of the fermentation when the olives were subjected to the NaOH treatment. The data produced by culture-independent analyses highlighted that the fermentation of table olives not treated with NaOH is the result of the coexistence of two different ecosystems: the surface of the olives and the brines. A sodium hydroxide treatment not only eliminates this difference, but also affects the bacterial ecology of the olives to a great extent. As proved by high- throughput sequencing, the fermentation of the olives not treated with NaOH was characterized by the presence of halophilic bacteria, which were substituted by Lactobacillus at the later stages of the fermentation, while enterobacteria were dominant when the olives were treated with sodium hydroxide. Higher biodiversity was found for Lactobacillus plantarum isolated during untreated fermentation. Different biotypes were found on the olive surface and in the brines. When the debittering process was carried out, a decrease in the number of L. plantarum biotypes were observed and those originating from the surface of the olive did not differentiate from the ones present in the brines. Citation: Cocolin L, Alessandria V, Botta C, Gorra R, De Filippis F, et al. (2013) NaOH-Debittering Induces Changes in Bacterial Ecology during Table Olives Fermentation. PLoS ONE 8(7): e69074. doi:10.1371/journal.pone.0069074 Editor: Paul D. Cotter, Teagasc Food Research Centre, Ireland Received February 4, 2013; Accepted June 5, 2013; Published July 31, 2013 Copyright: ß 2013 Cocolin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research conducted to obtain these results received funding from the EU (FP7/2007–2013), under grant agreement no. 243471- PROBIOLIVES (www.probiolives.eu). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lucasimone.cocolin@unito.it Introduction Table olives are an important fermented food in Mediterranean countries and they constitute a fundamental food production sector. Olives are the fruit that is produced by the olive tree (Olea europea), a species that is successfully cultivated in all Mediterra- nean countries. Estimations made by the International Olive Council for the 2011/2012 campaign indicate a world production of 3.1 million tons, with a 3% increase compared to the previous year. About 3 quarters of this production is from the European Union (EU), with a production of 2.18 million tons. Spain is the first producing country in the EU, producing 1.35 million tons (62% of the production), and this is followed by Italy (20%), Greece (14%), Portugal (3%) and other EU countries (1%) [1]. The olive fruit, which has a strong bitter taste that comes from glucoside oleuropein, is subjected to a number of transformations, through fermentation, which make them edible, stable and safe. Most table olive fermentation processes start spontaneously and are influenced to a great extent by the olive cultivar itself, its indigenous microbiota [2] and methodological factors, such as fermentation temperature and the salt concentration in the brines [3]. It is widely accepted that the main microbiota responsible for table olive fermentations are lactic acid bacteria (LAB) members, namely Lactobacillus spp., and yeasts [4,5]. There are several ways of treating table olives. However, the green Spanish type and the Greek type are those that account for most of the world’s production, with the Spanish type covering about 60% [6]. The first method consists of treating the fruit with a diluted NaOH solution (2–3%) to reduce the bitterness, through the degradation of oleuropein and polyphenols, but also to increase the permeability of the olive pericarp. The debittering treatment is followed by a water wash to remove the excess alkali. The olives are then placed under brine (initial concentration of 8– 12%), where they undergo lactic acid fermentation [7]. Instead, in the Greek production, the natural or untreated olives (green or naturally black) are directly brined after picking. In brine, the olives undergo a mixed-acid fermentation until they at least partially lose their bitterness. The fermentation period therefore depends on the physico-chemical conditions, such as the type of cultivar, the salt content and the temperature [3,4,6]. The scientific community recognizes that the use of methods that rely on the cultivation of microorganisms (culture-dependent techniques) do not offer a complete profile of the microbial diversity that is present in a specific ecosystem [8]. Culture- independent techniques have attracted the attention of many scientists from different investigation domains, ranging from environmental microbiology to food fermentation. At present, high-throughput sequencing has emerged as a new culture- independent tool to quantitatively investigate the structure of PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e69074