Royal jelly (RJ) is a well-known functional and medicinal food for human health promotion. Major royal jelly proteins (MRJPs), which are the major protein components in RJ, exhibit antimicrobial activities. However, the identities of the MRJPs of RJ responsible for its antioxidant effects have remained unclear. Here, we report that honeybee (Apis cerana) MRJP 2 (AcMRJP2) acts as an antimicrobial and antioxidant agent in RJ. Using recombinant AcMRJP2, which was produced in baculovirus-infected insect cells, we established the antimicrobial and antioxidant roles of MRJP 2. AcMRJP2 bound to the surfaces of bacteria, fungi, and yeast, which then induced structural damage in the microbial cell walls and led to a broad spectrum of antimicrobial activities. AcMRJP2 protected mammalian and insect cells via the direct shielding of the cell against oxidative stress, which led to reduced levels of caspase-3 activity and oxidative stress-induced cell apoptosis, followed by increased cell viability. Moreover, AcMRJP2 exhibited DNA protection activity against reactive oxygen species (ROS). Our data indicate that AcMRJP2 could play a crucial role as an antimicrobial agent and antioxidant in RJ, suggesting that MRJP 2 is a component responsible for the antimicrobial and antioxidant activities of RJ.

The honeybee inhibitor cysteine knot (ICK) peptide acts as an antifungal peptide and insecticidal venom toxin. However, the ICK peptide from bumblebees has not been characterized. Here, we report the molecular cloning and antifungal activity of a bumblebee (Bombus ignitus) ICK peptide (BiICK). We identified a BiICK that contains an ICK fold. The BiICK was expressed in the epidermis, fat body, and venom gland of B. ignitus worker bees. A 6.7-kDa recombinant BiICK peptide was expressed in baculovirus-infected insect cells. Recombinant BiICK peptides directly bound to Beauveria bassiana, Ascosphaera apis, and Fusarium graminearum, but they did not bind to Escherichia coli, Paenibacillus larvae, or Bacillus thuringiensis. Consistent with this finding, BiICK exhibited antifungal activity against fungi. These results demonstrate that BiICK acts as an antifungal peptide.