Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds

Abstract

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

Keywords: Bacillus; DGGE; fermentation; iru; safety.

Figure 1

Non-metric Multidimensional Scaling analysis of DGGE data Group (a): iru Abeokuta 2 southwest Nigeria (); iru Abeokuta 3 southwest Nigeria (); iru Lagos 1 southwest Nigeria (); iru Lagos 2 southwest Nigeria (•); iru Ilorin 1 northcentral Nigeria (); iru Ilori n 2 northcentral Nigeria (); iru Ibadan 1 southwest Nigeria (). Group (b): iru Abeokuta 4 southwest Nigeria (). Group (c): iru Abeokuta 1 southwest Nigeria (); iru Abeokuta 5 southwest Nigeria (); iru Abeokuta 8 southwest Nigeria (); iru Oyo southwest Nigeria (); iru Ado-Ekiti southwest Nigeria (). Group (d): iru Abeokuta 6 southwest Nigeria (); iru lbadan 2 southwest Nigeria (). Group (e): iru Abeokuta 7 southwest Nigeria ().

Figure 2

DGGE profiles of PCR-amplified 16S rRNA gene fragments of sixteen (16) fermented iru samples showing major bacterial amplicons. Samples A: iru Oyo; B: iru Lagos 1; C: iru Ilorin 2; D: iru Ilorin 1; E: iru Ibadan 1; F: iru Ado-Ekiti; G: iru Abeokuta 2; H: iru Abeokuta 1; I: iru Ibadan 2; J: iru Abeokuta 3; K: iru Abeokuta 4; L: iru Abeokuta 5; M: iru Abeokuta 6; N: iru Lagos 2; Î: iru Abeokuta 7; P: iru Abeokuta 8; “m” is DGGE reference DNA ladder. Bands 1: Staphylococcus vitulinus; 2: Morganella morganii; 3: Bacillus thuringiensis; 4: B. licheniformis; 5: S. saprophyticus; 6: Uncultured bacterium clone; 7: Tetragenococcus halophilus; 8: Ureibacillus thermosphaericus; 9: Bacillus sp.; 10: B. subtilis; 11: Brevibacillus parabrevis; 12: Salinicoccus jeotgali; 13: Brevibacterium sp.