Background: Amylase activity is a critical biomarker for assessing the freshness of honey. Historically, bees have been considered the sole source of honey amylase. However, recent studies suggest that Bacillus subtilis may also contribute to amylase production in the honey sac of Apis mellifera. Methods: In this study, amylase levels were measured in samples of nectar, honey sac fluid, and honey. The identification of B. subtilis in nectar, honey sac, and honey was evaluated. An in vitro bacterial culture system and a feeding experiment were developed to simulate honey sac conditions. Results: Our results showed that B. subtilis was detected in all sample groups, with the highest concentration in honey sac samples. Amylase levels in honey sac and honey samples were significantly higher than those in nectar. In the simulation experiment, amylase activity was only observed in cultures containing both B. subtilis and sucrose/nectar; no activity was detected in cultures containing only H₂O or no B. subtilis. In the feeding experiment, bees fed sucrose or nectar showed higher amylase activity in their honey sacs than those fed water. Conclusions: Our data show that B. subtilis can produce amylase and offer potential for more standardized quality assessment of honey.

Background: The bacterial diversity of two bee species in the process of honey collection during the flowering season of three different floral sources in the winter was studied. The common bacterium in all samples was Bacillus subtilis.Methods: In the present study, we collected nectar, honey sacs, and fresh honey during the winter flowering season of Agastache rugosa, Prunus cerasoides, and Brassica rapa. The pure culture method was used to count and analyze the number of bacteria, they were identified using 16S rRNA sequencing, similarities were compared in NCBI, and the common dominant bacterial species B. subtilis in all samples using phylogenetic analysis and intersection analysis were determined to conduct further bacteriostatic experiments.Results: The results showed that the most abundant quantity of bacteria could be found in the honey sacs, compared to in nectar or fresh honey. At the same time, the highest abundance of bacteria could be found in the honey sacs of A. cerana when collected on Brassica rapa, while the highest abundance of bacteria could be found in the honey sacs of A. mellifera when collected on Prunus cerasoides and Agastache rugosa. A total of 33 bacterial species were isolated, with variations in their distribution across different sample types and sources. The inhibitory effect of 10^-1-10^-5 on Hafnia alvei by B. subtilis was very significant.Conclusions: B. subtilis was identified in all sample sources, indicating the potential importance of B. subtilis as a probiotic in the bee gut for honey production, and B. subtilis could promote the disease resistance and health of honeybees in winter.