Antimicrobial peptides in black soldier fly larvae: Opportunities and challenges

Although ongoing research on AMPs shows promising in vitro antimicrobial properties, its applications in the insect industry and commercial products face several key challenges. First, the understanding of insect immune systems and the mechanisms involved in AMPs production, particularly in Hermetia illucens, remains limited. This lack of knowledge hampers treatments to efficiently enhance AMPs levels in the BSFL stage, posing challenges in both trial-scale and large-scale production.

In recent years, the Black Soldier Fly Larvae (BSFL) and its derived products have seen significant market growth. This expansion is particularly significant in the Animal Feed sector, where BSFL meal is becoming a key alternative protein source, offering both nutritional value and additional functional benefits. BSFL-derived products, such as those produced by Entobel, are closely aligned with Sustainable Development Goals (SDGs) providing an eco-friendly alternative by utilizing food industry by-products and helping to reduce carbon emissions of the feed industry. Entobel’s BSFL meal is notable for its high protein content – with an average of 55% crude protein – and its well-balanced amino acid profile, including Methionine (2.11%), Cystine (1.36%), Proline (5.87%), and Tyrosine (6.68%). These amino acids not only support immune function but also acts as natural attractants for animals. In addition to its protein content, BSFL meal naturally contains antimicrobial peptides (AMPs), which gained interest in both research and the feed industry for their potential ability to enhance animal health. As a result, BSFL-derived proteins future innovations while addressing environmental concerns and promoting more sustainable practices in the feed industry.

Antimicrobial peptides (AMPs) are small peptides that contain about 10–100 amino acids, known for their broad-spectrum activity against bacteria, fungi, viruses and parasites. Recent research has identified key AMPs families in the BSF genome. Jian Peng et al (2023) discovered 33 cecropin genes, while Vogel Heiko et al (2018) reported 26 genes coding for defensin AMPs and 6 genes coding for attacin1, 2. These AMPs families play a crucial role in the fly’s immune defense system and exhibit significant potential for antimicrobial applications.

The cecropin family in BSFL consists of four types of peptides, each containing approximately 69 amino acids and having a molecular mass of 7 kDa. Cecropins are particularly effective against a range of bacteria, including Gram-negative species such as Escherichia coli, Pseudomonas aeruginosa, and Vibrio parahaemolyticus, as well as Gram-positive bacteria like Bacillus subtilis (2). Besides that, the defensin family, comprises six mature peptides, each with a molecular weight of around 4 kDa. These defensins are potent against E. coli, Staphylococcus aureus, and Salmonella species, as noted by Jingjing Zhang et al (2021)3.

The antimicrobial effects of AMPs operate through diverse mechanisms, with the most well-studied being membrane disruption. These peptides interact with bacterial cell membranes, causing structural destabilization and increased permeability, which leads to cell lysis and death. Also, the interaction and binding with intracellular targets as well as immune modulation has been reported as antibacterial mode of actions of AMPs. Due to this broad spectrum potential antibacterial effect, it is challenging for bacteria to develop resistance to AMPs, unlike traditional antibiotics that target specific bacterial processes.

AMPs offer several advantages, including their rapid action against bacteria and a low risk of inducing bacterial resistance, as it is difficult for bacteria to alter their membranes to resist AMP-induced disruption. Furthermore, AMPs have a short half-life, reducing their environmental persistence. These properties make AMPs promising candidates for use in animal feed applications to support pathogenic bacteria control, enhance animal health, and improve productivity in the livestock and aquaculture industries.

With a mission to develop functional insect-based ingredients for animal feed and health, Entobel started with an in-house research project to investigate the AMPs properties found in its BSFL products. By exploring alternatives to antibiotics, Entobel aims to promote healthier fish and shrimp farming practices. One key area of research involves assessing the antimicrobial activity of extracts from fresh BSFL against bacterial infections in aquaculture, including Vibrio parahaemolyticus, a pathogen responsible for Acute Hepatopancreatic Necrosis Disease (AHPND) in shrimp and Streptococcus agalactiae, known to cause infections in freshwater fish. In a recent study, Entobel carried out preliminary research to assess the antimicrobial activity of whole, fresh BSFL which were reared on a diet of brewer’s spent grain, with no additional treatments applied. The crude AMPs were extracted, followed by purification and fractionation based on molecular mass. To confirm the antimicrobial activity of each fraction, the Agar Inhibition Zone method was used. Additionally, SDS-PAGE, a technique for separating proteins based on their molecular mass, was used to determine the size of the peptides in each fraction.

Two key fractions from the study demonstrated promising results. Fraction F3, which contained peptides ranging from 4 kDa to 7 kDa, showed antimicrobial activity against S. agalactiae, with a zone of inhibition measuring 13.5 mm at a 10.8 mg/mL concentration. Meanwhile, Fraction F4, accounting for the highest content in the crude extract and containing peptides smaller than 14 kDa, displayed antimicrobial activity against V. parahaemolyticus, and S. agalactiae. This fraction produced a zone of inhibition measuring 10.3 mm at a concentration of 21.5 mg/mL. (Figure 1 & 2)

These initial findings suggest that the BSFL-derived AMPs possess an antimicrobial potential, particularly against some pathogenic bacteria in aquaculture.

Although ongoing research on AMPs shows promising in vitro antimicrobial properties, its applications in the insect industry and commercial products face several key challenges. First, the understanding of insect immune systems and the mechanisms involved in AMPs production, particularly in Hermetia illucens, remains limited. This lack of knowledge hampers treatments to efficiently enhance AMPs levels in the BSFL stage, posing challenges in both trial-scale and large-scale production. Additionally, AMPs share the same characteristics of a protein, such as thermal sensitivity, active pH, and enzyme digestion. These put special requirements on scale-up processing and storage methods which need to be carefully optimized to preserve the antimicrobial activity of these peptides, as improper extraction and purification can degrade their effectiveness. Another challenge is ensuring the stability of AMPs in digestive tract systems. As proteins, AMPs can be broken down during digestion, potentially losing their bioactivity before they can act against pathogens, making it difficult to maintain their effectiveness when used as feed additives.

The potential of BSFL meal, combined with the antimicrobial properties of its peptides, offers a promising solution for sustainable protein sources and natural disease management in livestock and aquaculture. However, understanding is needed, such as enriching the amount of AMPs, improving their bioactivity, and overcoming limitations in processing and digestion. To drive sustainable development, collaboration between researchers, the BSF industry, and the feed industry is essential. By working together, they can unlock the full potential of BSFL products, improve efficiency, and address the growing need for alternatives to antibiotics in animal farming.

References
1. Jian Peng,Lu Li,Yan Wan,Yifan Yang,Xiaoqin An,Kexin Yuan,Zhilang Qiu,Yinhui Jiang,Guo Guo,Feng Shen,Guiyou Liang Molecular characterization and antimicrobial activity of cecropin family in Hermetia illucens, 2024.
DOI: 10.1016/j.dci.2023.105111
2. Vogel H, Müller A, Heckel DG, Gutzeit H, Vilcinskas A, Diversification and diet-dependent expression of antimicrobial peptides in the black soldier fly Hermetia illucens, 2018.
DOI: 10.1016/j.dci.2017.09.008.
3. Zhang J, Li J, Peng Y, Gao X, Song Q, Zhang H, Elhag O, Cai M, Zheng L, Yu Z, Zhang J. Structural and functional characterizations and heterogenous expression of the antimicrobial peptides, Hidefensins, from black soldier fly, Hermetia illucens, 2021.
DOI: 10.1016/j.pep.2021.106032