Unraveling the Microbiota of Natural Black cv. Kalamata Fermented Olives through 16S and ITS Metataxonomic Analysis

Abstract

Kalamata natural black olives are one of the most economically important Greek varieties. The microbial ecology of table olives is highly influenced by the co-existence of bacteria and yeasts/fungi, as well as the physicochemical parameters throughout the fermentation. Therefore, the aim of this study was the identification of bacterial and yeast/fungal microbiota of both olives and brines obtained from 29 cv. Kalamata olive samples industrially fermented in the two main producing geographical regions of Greece, namely Aitoloakarnania and Messinia/Lakonia. The potential microbial biogeography association between certain taxa and geographical area was also assessed. The dominant bacterial family identified in olive and brine samples from both regions was Lactobacillaceae, presenting, however, higher average abundances in the samples from Aitoloakarnania compared to Messinia/Lakonia. At the genus level, Lactobacillus, Celerinatantimonas, Propionibacterium and Pseudomonas were the most abundant. In addition, the yeasts/fungal communities were less diverse compared to those of bacteria, with Pichiaceae being the dominant family and Pichia, Ogataea, and Saccharomyces being the most abundant genera. To the best of our knowledge, this is the first report on the microbiota of both olives and brines of cv. Kalamata black olives fermented on an industrial scale between two geographical regions of Greece using metagenomics analysis.

Keywords: 16S metagenomics analysis; Greek style fermentation; ITS metagenomics analysis; Kalamata olives; high-throughput sequencing; microbiological analysis; microbiota; table olives.

Figure 1

Geographical origin of fermented cv. Kalamata natural black olives from (A) Aitoloakarnania (western Greece) and (B) Messinia/Lakonia (southern Peloponnese).

Figure 2

Boxplots of lactic acid bacteria (LAB) (A) and yeast (B) counts as well as pH values (C) from the olives and brines, along with salt concentration (D) from the brines of natural black cv. Kalamata olives from the two geographical regions. Different letters indicate statistically significant differences (p < 0.05).

Figure 3

Boxplots of alpha-diversity indices, namely observed, Shannon and inverse Simpson for bacterial communities in olive (A) and brine (B) samples, as well as for yeast/fungal microbiota in olives (C) and brines (D). Samples are colored according to the two geographical regions, i.e., pink for Aitoloakarnania and light blue for Messinia/Lakonia.

Figure 4

Average relative abundance (%) of dominant bacterial families obtained by 16S metagenomics analysis of olive (A) and brine (B) samples from the Messinia/Lakonia and Aitoloakarnania regions.

Figure 5

Average relative abundance (%) of dominant bacterial genera obtained by 16S metagenomics analysis of olive (A) and brine (B) samples from the Messinia/Lakonia and Aitoloakarnania regions. Venn diagram showing the number of unique and shared bacterial genera among olives (C) and brines (D) from the two geographical regions.

Figure 6

Average relative abundance (%) of dominant yeast/fungal families obtained by internal transcribed spacer (ITS) metagenomics analysis of olive (A) and brine (B) samples from the Messinia/Lakonia and Aitoloakarnania regions.

Figure 7

Average relative abundance (%) of dominant yeast/fungal genera obtained by ITS metagenomics analysis of olive (A) and brine (B) samples from Messinia/Lakonia and Aitoloakarnania regions. Venn diagram showing the number of unique and shared yeast/fungal genera among olives (C) and brines (D) from the two geographical regions.

Figure 8

Hierarchically clustered heatmap of the bacterial community from cv. Kalamata natural black olives from the brine (A) and olives (B). Classes A and M correspond to the samples from the region of Aitoloakarnania (A) and Messinia/Lakonia (M), respectively. The sample codes are indicated in Table 1.

Figure 9

Hierarchically clustered heatmap of the yeasts/fungal community from cv. Kalamata natural black olives from the brine (A) and olives (B). Class A and M corresponds to the samples from the region of Aitoloakarnania (A) and Messinia/Lakonia (M), respectively. The sample codes are indicated in Table 1.

Figure 10

Plot of scores (A) and loadings (B) of the first two latent variables of the PLS-DA model of the bacterial community build on the brine samples from the region of Aitoloakarnania (A) and Messinia/Lakonia (M).

Figure 11

Most influential bacterial genera of the brine samples from the region of Aitoloakarnania (A) and Messinia/Lakonia (M) based on the Variable Importance in Projection (VIP) scores from the Partial Least Squares Discriminant Analysis (PLS-DA) analysis. The color bars indicate the intensity of thevariable in the respective group.

Figure 12

Plot of scores (A) and loadings (B) of the first two latent variables of the PLS-DA model of the yeasts/fungal community build on the brine samples from the region of Aitoloakarnania (A) and Messinia/Lakonia (M).

Figure 13

Most influential yeasts/fungal genera of the brine samples from the region of Aitoloakarnania (A) and Messinia/Lakonia (M) based on the VIP scores from the PLS-DA analysis. The color bars indicate the intensity of the variable in the respective group.