Accumulation of soluble menaquinones MK-7 in honey coincides with death of Bacillus spp. present in honey

Long-chain menaquinones (MK) are of bacterial origin. We investigated the possibility that MKs observed in honey are also the products of bacteria present in honey. The bacterial composition of honey was analyzed using culture-dependent methods. 16S rRNA gene sequencing and MALDI-TOF showed prevalence of the members of Bacillus subtilis and Bacillus cereus groups. The dominant menaquinones in both bacteria and honey were menaquinones MK-7 and MK-8 as indicated by UHPLC-ESI-MS/MS coupled to quadrupole orbitrap. The EICs showed alignment of mass ions of MK-7 and MK-8 from culture supernatants with that of honey. The unique MS/MS fragmentation pattern indicated that fragment ions were arising from the same menaquinone present in both samples. During Bacillus growth, the accumulation of MK-7 in supernatants occurred in a stationary phase and coincided with cell death. These novel findings suggest that the soluble MKs in honey originate from shedding of cell membranes of dead vegetative cells.