Exploring microbial diversity in Kermanshah province's Kermanshahi oil through DGGE and sequencing analysis

Abstract

Background: Ghee, known as "roghane heiwâni," or "Kermanshahi oil" is a traditional fermented butter-like product highly esteemed for its nutritional value. Ghee is prepared using traditional methods and has substantial potential as a reservoir of probiotic microorganisms. Previous research delved into isolating and identifying lactic acid bacteria (LAB) in Kermanshahi through culture and PCR sequencing. This study seeks to elucidate the microbial profiles and diversity within Kermanshahi using culture, Denaturing Gradient Gel Electrophoresis (DGGE), and sequencing methodologies.

Methods: Twenty samples of Kermanshahi oil were meticulously gathered from diverse locales across Kermanshah province. These samples were cultivated under specialized conditions in MRS and M17 environments spanning 24 to 72 h. Following DNA extraction, amplification of the 16SrRNA gene sequences was performed, culminating in sequencing for conclusive identification of the isolates. Furthermore, the DGGE technique was directly employed to separate and identify various species present in the oil samples utilizing bioinformatics software.

Results: Sequencing outcomes revealed a diverse array of microorganisms among the isolates, with Lactobacillus constituting 43%, Streptococcus comprising 27.6%, Enterococcus at 4.61%, and yeasts at 7.6%. Other species exhibited lower frequencies, encompassing Rhizobium, Bacillus coagulans, and Staphylococcus hominis.

Conclusions: The isolation of a diverse spectrum of probiotic microorganisms underscores their potential utility in the realm of industrial dairy product production. These findings allude to the possibility of integrating these valuable microorganisms, which have historically been associated with traditional products, into the contemporary dairy industry. As consumer interest in probiotic-enriched products surges, the insights gained from this study pave the way for harnessing the benefits of Kermanshahi-derived probiotics.

Keywords: Lactobacillus; Streptococcus; Denaturing gradient gel electrophoresis; Kermanshahi oil; Probiotic; Yeast.

Schematic Fig. 1

Collecting oil from different parts of Kermanshah province. 1 A: Culture on both media, 1B: Aerobic and anaerobic incubation, 1 C: Early diagnosis using biochemical tests and staining, 1D: DNA extraction from pure colony. 2 A: Direct extraction of DNA from oil, 2B: PCR using 16s-DGGE, 2 C: Running on a DGGE, 2D: DNA extraction from the gel. E: Sequencing, F: Edit and BLAST, G: Identification of bacterial species

Fig. 1

Electrophoresis results of PCR product for 16SrRNA gene; L: Ladder, C: negative control, 1–10: samples. The desired band for this gene was shown about 450 bp

Fig. 2

Electrophoresis results of PCR product for ITS gene; L: Ladder, C: negative control, 1–8: samples. The desired band for this gene was shown in the range of 270 bp

Fig. 3

Polyacrylamide gels in DGGE method; directly extracted DNA from oil samples after amplified by PCR for 16SrRNA gene were Electrophoresed