Amylase is one of three main enzymes involved in the breakdown and utilization of macromolecules. In honey bees (Apis mellifera), amylase was traditionally believed to originate only as secretion of the bee. However, other sources of amylase production, such as microbes in the guts of bees remained to be studied. In our research, we compared the differences in the amylase values in rape flower nectar and honey in the stomachs of bees. Then, culture-dependent and culture-independent (16sDNA) methods were used to isolate and identify bacteria in rape flower nectar and bee stomach honey. The dominant bacteria were added to nectar, and the amylase values of nectar with added gut microbes and nectar without added microbes were compared. Our findings indicate that the amylase value was significantly increased, which correlates with the increase in number of bacteria that occurred from nectar to honey in the stomachs of bees. The dominant bacteria in honey bee stomachs were mainly Bacillus during the flowering season of rape. Experiments confirmed that the amylase levels in nectar increased significantly when bacteria were added. These results indicate that bacteria in the foregut of bees help in the processing floral nectar into honey.

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.