Engineered Fly-Larva Peptide KK12 Curbs Salmonella on Cooked Chicken in Lab Test

A peptide engineered from a molecule found in black soldier fly larvae has shown strong antibacterial activity against Salmonella Typhimurium in laboratory tests and a modest but measurable effect on the surface of cooked chicken meat, according to a study published on 1 September 2026 in the journal Food Production, Processing and Nutrition. The modified peptide, named KK12, carries the sequence KWWSKLKKFWKK and was designed by systematically altering the charge and hydrophobicity of a naturally occurring peptide, KGNSELEEAKKK, that had previously been identified in black soldier fly larvae extract. The work was carried out by a research team led by Ali Hamzeh and Jirawat Yongsawatdigul, with collaborators from institutions in Thailand, China, Canada and the United States.

The starting peptide had a net charge of +1 and a hydrophobic residue fraction of 16.67 percent, features that limited its antibacterial potency. Using helical wheel projections generated with the online tool HeliQuest, the researchers visualized how charged and hydrophobic residues were distributed along the peptide’s projected alpha-helical structure. They then created four modified analogues by substituting lysine, tryptophan and phenylalanine residues at selected positions. Replacing glutamic acid with lysine raised the positive charge, while substituting glycine and alanine with tryptophan and phenylalanine increased hydrophobicity. The final analogue, MP4, which the team renamed KK12, combined a net charge of +6 with a hydrophobic residue ratio of 41.67 percent, the highest values among all the designed variants.

Screening against Salmonella enterica serovar Typhimurium TISTR 292 revealed that KK12 was by far the most active of the modified peptides. It achieved a minimum inhibitory concentration, or MIC, of 2 micromolar in a microbroth dilution assay following Clinical and Laboratory Standards Institute protocols, whereas the other analogues inhibited bacterial growth by only 28 to 53 percent at a concentration of 2 millimolar. The original peptide required a concentration of 2 millimolar to inhibit growth, meaning the rational redesign improved potency by roughly a thousandfold. The researchers attribute this gain to the simultaneous expansion of both the cationic face and the hydrophobic face of the projected helix, a spatial coordination they note has been largely overlooked in previous peptide engineering efforts that targeted only one property at a time.

KK12 was then tested against a broader panel of Salmonella serotypes associated with salmonellosis, including S. Typhimurium ATCC 14028, S. Enteritidis ATCC 13076, S. Enteritidis DMST 15679 and S. Newport ATCC 6962. Across these strains, MIC values ranged from 2 to 4 micromolar and minimum bactericidal concentrations ranged from 4 to 32 micromolar. The peptide outperformed an egg yolk-derived peptide with MIC values in the millimolar range and also showed greater potency than a previously reported engineered peptide with MIC values of 7.3 to 58.9 micromolar. These comparisons suggest that KK12’s balanced physicochemical profile, rather than extreme charge or hydrophobicity alone, underlies its activity.

Time-kill experiments revealed that the peptide’s bactericidal effect depends strongly on concentration. At 0.5 times and 1 times the MIC, KK12 reduced viable counts of S. Typhimurium TISTR 292 for up to 4 hours, but bacterial numbers rebounded thereafter, reaching 9 and 3 log CFU/mL respectively after 24 hours compared with 11 log CFU/mL in untreated controls. The authors interpret this regrowth as evidence that sublethal peptide levels may only partially disrupt membranes, allowing surviving cells to recover. At 2 times the MIC, corresponding to 4 micromolar, viable counts fell below the assay’s detection limit of 1 log CFU/mL within 2 hours and remained suppressed through 24 hours, indicating irreversible damage at higher concentrations.

Microscopic and spectroscopic analyses provided insight into how KK12 damages bacterial cells. Confocal laser scanning microscopy using a fluorescein-labeled peptide and propidium iodide staining showed that KK12 at 1 times the MIC compromised membrane integrity in treated cells. Measurements of nucleic acid release at 260 nanometers confirmed dose-dependent membrane permeabilization, mainly within the first hour of exposure. Scanning and transmission electron microscopy revealed flattened and ruptured cells with leakage of intracellular material, in contrast to the regular rod shape and smooth surface of untreated bacteria. Synchrotron-based Fourier-transform infrared spectroscopy detected changes in spectral regions associated with lipid stretching vibrations, protein amide bands and phosphate-containing compounds, suggesting perturbation of membrane lipids and possible downstream effects on intracellular constituents, although the authors caution that FTIR data do not provide direct mechanistic evidence.

Safety testing showed that KK12 displayed limited toxicity toward mammalian cells at concentrations well above its antibacterial dose. Using human red blood cells, the peptide produced a hemolytic rate below the 5 percent safety threshold up to 16 micromolar, eight times its MIC, and its concentration producing 50 percent hemolysis exceeded 250 micromolar. In cytotoxicity assays using the MTT method, Vero kidney cells and HepG2 liver cells maintained viability comparable to untreated controls at concentrations up to 36 and 18 micromolar respectively, approximately 18 and 9 times the MIC. The authors suggest that the peptide’s positive charge contributes to selectivity because bacterial membranes carry a higher negative surface charge than mammalian cell membranes.

The critical test came in a cooked chicken meat challenge model. Boneless chicken breast pieces were autoclaved at 121 degrees Celsius for 15 minutes to eliminate indigenous microorganisms, then inoculated on the surface with approximately 10 to the power 4 CFU of S. Typhimurium TISTR 292 and air-dried. Peptide solutions were applied at concentrations ranging from 0 to 1.6 nanomoles, equivalent to 0 to 2.71 micrograms, and the meat was held at room temperature for 6 hours to simulate temperature abuse. Bacterial counts decreased during the first 2 hours, particularly at the highest peptide dose, but increased thereafter. After 6 hours, the control samples had grown by 1.65 log CFU while the 1.6-nanomole treatment limited growth to 0.65 log CFU, a statistically significant difference that represented a maximum reduction of 1.09 log CFU relative to the untreated control.

The authors are candid that this effect is modest compared with the peptide’s strong performance in laboratory broth. They note that complex food matrices can substantially reduce antimicrobial peptide activity through interactions with muscle proteins and lipids, through proteolytic degradation by enzymes naturally present in tissue or produced by contaminating microbes, and through chemical reactions between food constituents and peptide side chains. Prior studies support this pattern: a peptide called P1R3 with an MIC of 72.8 micromolar against S. Typhimurium failed to reduce bacterial counts on raw chicken breast, while a related peptide achieved roughly a 1-log reduction only at 8 times its MIC over 24 hours at 4 degrees Celsius. Nisin, the most widely used food bacteriocin, likewise showed no effect against S. Enteritidis in sheep meat mince when applied alone.

The researchers frame KK12 as a proof-of-concept candidate rather than a ready food additive. They emphasize that the autoclaved chicken system does not fully represent commercial poultry products, which contain native microbiota and varied surface properties that could affect peptide performance. They call for further evaluation in raw meat models and across different food matrices, ionic strengths, pH values and temperature ranges before practical applications can be developed. The study was published as open access under volume 8, article number 62 of the journal, and the authors report that no datasets were generated or analysed during the study beyond those presented in the paper itself.

Subject of Research: Rational design and food-matrix evaluation of the antimicrobial peptide KK12 derived from black soldier fly larvae against Salmonella Typhimurium

Article Title: Antimicrobial activity of the rationally modified peptide KK12 and its evaluation in a cooked chicken challenge model inoculated with Salmonella enterica serovar Typhimurium

Article References: Hamzeh, A., Pimchan, T., Tian, F., Thumanu, K., Aluko, R. E., Park, S. H., Yahuafai, J., Onsrisawat, P., Riyachan, I., Thongjit, S., & Yongsawatdigul, J. (2026). Antimicrobial activity of the rationally modified peptide KK12 and its evaluation in a cooked chicken challenge model inoculated with Salmonella enterica serovar Typhimurium. Food Production, Processing and Nutrition, 8(1), Article 62. https://doi.org/10.1186/s43014-026-00417-5

Image Credits: AI Generated

DOI: 10.1186/s43014-026-00417-5

Keywords: antimicrobial peptides, KK12, Salmonella Typhimurium, black soldier fly larvae, peptide engineering, food safety, cooked chicken model, membrane disruption, minimum inhibitory concentration, cytotoxicity, hemolysis, chicken meat preservation