Identification of Lactobacillus species in honey using 16S rRNA gene sequencing

Abstract: Honey is a natural product composed of numeral substances and microorganisms. Although honey presents physicochemical properties that confer antimicrobial characteristics, certain microorganisms are still able to survive and multiply in honey. Some of these microorganisms are important to human health, such as Lactobacillus bacteria, which can be used as probiotic to improve human gastrointestinal condition. However, there is still a lack of knowledge about the microorganisms found in honey, and further research is necessary to better identify and understand these microorganisms. The aim of this study was to detect and identify Lactobacillus species from honey by sequencing the 16S ribosomal RNA gene with the universal primers 27F and 1492R. This was partly obtained by searching for Lactobacillus spp. directly in pure honey and after growing the unknown viable Lactobacillus spp. from honey in selective media. The microorganisms were isolated by centrifugation, had their DNA extracted, and only the bacterial 16S rRNA gene was amplified using Polymerase Chain Reaction and sequenced using the Oxford MinION Nanopore sequencing technology. EPI2ME, Oxford Nanopore Technology data analysis platform, was used to identify the bacteria. Results showed the presence of numeral Lactobacillus species in honey such as L. melliventris, L. mellifer, L. mellis, and L. helsinbgborgen, but in very low amounts. The analysis of honey samples was quite difficult. Low PCR product yield during the entire study, microscopic visualization and microbial analysis using fungicides supported the presence of fungi. In conclusion, a diversity of Lactobacillus spp. can be found in honey but in low quantities.