# Microbiota and Metabolite Profiling of Spoiled Spanish-Style Green Table Olives
## 1 Introduction  
Spanish-style green olives are one the most highly-valued fermented vegetables. Spain is the main producer of different types of table olives with around 550,000 tons produced annually [1]. Among them, approximately 250,000 tons correspond to the green Spanish-style, whose processing is characterized by an initial alkaline treatment with 20-35 g L\({}^{-1}\) sodium hydroxide (lye) for several hours; then a wash step with tap water is carried out in order to remove excess alkali, and finally the olives are covered with a 90-110 g L\({}^{-1}\) sodium chloride brine where microbial fermentation takes place [2]. Normally, the fermentation is carried out by environmental lactic acid bacteria and yeasts without the addition of starters [3]. The table olive industry occasionally reports the spoilage of fermented green olives associated with increases in brine pH and unpleasant odors. Olives may suffer different kinds of microbial spoilage if the microbial population is not well controlled during the different phases of fermentation [4]. Three different types of malodorous spoilage have been recognized in this product: zapatera, butyric, and putrid. The flavor of olives affected by the zapatera spoilage is hard to describe but is clearly abnormal and very distinctive. Although the characteristic off-odor of the zapatera samples is different from that of butyric or putrid fermentations (the butyricdeterioration is reminiscent of the smell of rancid butter, while the putrid type is reminiscent of the smell of decomposing organic matter), there is sometimes confusion over the term \"zapatera\", with a tendency to classify most olives with abnormal flavor as such. Since the finding of cyclohexanoic acid in appreciable amounts in zapatera olives [5], it is recognized that this acid is mainly responsible for the typical off-odor of this spoilage. In fact, aqueous solutions of cyclohexanoic acid are used as reference materials for taster and panel leader training in the sensory assessment of table olives [6]. The volatile metabolites produced by the microorganisms involved in olive spoilage are responsible for the off-odor. These microorganisms have been found to belong to the genus _Clostridium_ in all three of the above-mentioned types of spoilage, although _Propionibacterium_ can also be prominent, especially in the case of zapatera spoilage [4]. Although butyric and cyclohexanoic acids are the main volatile metabolites responsible for butyric and zapatera spoilages, respectively, other metabolites could be associated with each type of spoilage. Propionic, 3-methylbutyric, valeric, and caproic acids have been found at concentrations above their odor threshold in the case of zapatera olives [5]. However, other volatile compounds, which may be closely related to spoilage, have not been identified to date. A deeper knowledge of the microbial and metabolite compositions of spoiled table olives is necessary for a better characterization of each type of spoilage. All the above-mentioned investigations on microbial spoilage in table olives were carried out decades ago. DNA-based methods are nowadays the most commonly applied techniques for microbiota identification in fermented vegetables (including table olives). Among them, high-throughput sequencing (HTS) has been used to study different food matrices [7]. These techniques confer a more comprehensive identification of the different taxa than the classical culture-dependent methods. However, molecular methods have hardly been used in spoiled table olives [8]. Regarding volatile metabolites, headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) is currently one of the most popular techniques for the analysis of volatile compounds in food [9]. This technique has recently been used by our group to study the volatile composition of Spanish-style green table olives [10; 11; 12; 13; 14; 15], but so far it has not been applied to spoiled table olives.