Identification of Bulgarian Sourdough Microbiota by Metagenomic Approach Using Three Commercially Available DNA Extraction Protocols

Abstract

Research background. Sourdough is a spontaneously formed, complex microbial ecosystem of various lactic acid bacteria (LAB) and yeast which, by producing specific metabolites, determines the quality of the baked products. In order to design and control the sourdough with preferred nutritional characteristics, it is crucial that the LAB diversity of the product of interest be elucidated. Experimental approach. Using the opportunities of next-generation sequencing (NGS) of the V1-V3 hypervariable gene region of 16S rRNA, we studied the microbial ecosystem of a whole grain sourdough made of Triticum monococcum, originating from Southwestern Bulgaria. Since the DNA extraction method is considered crucial for the accuracy of the sequencing results, as it can introduce significant differences in the examined microbiota, we used three different commercial kits for DNA isolation and analyzed their impact on the observed bacterial diversity. Results and conclusions. All three DNA extraction kits provided bacterial DNA which passed quality control and was successfully sequenced on Illumina MiSeq platform. The results received from the different DNA protocols showed variations in the microbial profiles. Alpha diversity indices (ACE, Chao1, Shannon, and Simpson) were also different among the three groups of results. Nevertheless, a strong dominance of phylum Firmicutes, class Bacilli, order Lactobacillales, represented mostly by family Lactobacillaceae, genus Lactobacillus (relative abundance of 63.11–82.28%) and family Leuconostocaceae, genus Weissella (relative abundance of 3.67–36.31%) was observed. Lactiplantibacillus plantarum and Levilactobacillus brevis with relative abundance of 16.15–31.24% and 6.21−16.29% respectively, were the two dominant species identified in all three DNA isolates. Novelty and scientific contribution. The presented results give insight into the taxonomic composition of bacterial community of a specific Bulgarian sourdough. Having in mind that the sourdough is a difficult matrix for DNA isolation on the one hand, and that there is no standardized DNA extraction protocol for this matrix on the other hand, this pilot study aims to give a small contribution to the future establishment and validation of such a protocol, which will allow accurate assessment of the specific microbiota of sourdough samples.

Alternate abstract:

Pozadina istraživanja. Kiselo tijesto predstavlja spontano oblikovan složeni ekosustav različitih bakterija mliječno-kiselog vrenja i kvasaca koji stvaranjem specifičnih metabolita određuju kakvoću pekarskih proizvoda. Za kreiranje i kontroliranu pripremu kiselog tijesta sa željenim nutritivnim značajkama, neophodno je razumjeti raznolikost mliječno-kiselih bakterija. Eksperimentalni pristup. Sekvenciranjem nove generacije V1-V3 hipervarijabilnih regija 16S rRNA gena ispitali smo mikrobnu zajednicu kiselog tijesta proizvedenog od cjelovitih žitarica vrste Triticum monococcum, porijeklom iz jugozapadne Bugarske. Odabir metode ekstrakcije DNA je ključni korak za dobivanje točnih rezultata sekvenciranja, budući da ona može bitno utjecati na ispitanu mikrobnu zajednicu. Stoga smo odabrali tri komercijalna alata za izolaciju DNA i ispitali njihov učinak na bakterijsku raznolikost. Rezultati i zaključci. Pomoću sva tri alata izdvojen je bakterijski DNA materijal koji je zadovoljio kontrolu kvalitete, te je uspješno sekvenciran na platformi Illumina MiSeq. Rezultati dobiveni različitim DNA protokolima pokazali su razlike u profilima mikrobnih zajednica. Alfa-indeksi raznolikosti (ACE, Chao1, Shannon, i Simpson) također su bili različiti za svaku skupinu. Usprkos tome, opažena je izražena dominacija bakterija koljena Firmicutes, razreda Bacilli, reda Lactobacillales, i to najviše porodice Lactobacillaceae, roda Lactobacillus (relativna zastupljenost od 63,11-82,28 %) i porodice Leuconostocaceae, roda Weissella (relativna zastupljenost od 3,67–36,31 %). Dvije dominantne vrste koje su identificirane u sva tri DNA izolata bile su Lactiplantibacillus plantarum, čija je relativna zastupljenost bila 16,15-31,24 % i Levilactobacillus brevis, čija je relativna zastupljenost bila 6,21-16,29 %. Novina i znanstveni doprinos. Dobiveni rezultati daju uvid u taksonomski sastav bakterijske populacije kiselog tijesta porijeklom iz Bugarske. Imajući na umu da je kiselo tijesto zahtjevna podloga za izolaciju DNA, te da ne postoji standardizirani protokol za njegovu obradu, svrha je ove pilot studije bila dati mali doprinos uspostavljanju i validaciji budućih protokola, koji će omogućiti preciznu evaluaciju specifične mikrobne populacije kiselih tijesta.