Rational design of hyperstable antibacterial peptides for food preservation

Microbial spoilage accounts for significant post-harvest food loss globally (up to 25%), leading to economic losses and food safety concerns. While chemical preservatives are widely used, their potential side effects necessitate safer alternatives. Antimicrobial peptides (AMPs) are promising candidates due to their inhibitory activity and biocompatibility. However, challenges such as poor solubility, toxicity, low stability, and high production costs limit their applications. Nisin, a commercially used AMP, exemplifies these limitations, exhibiting limited effectiveness at higher pH levels and temperatures, and inactivity against Gram-negative bacteria, molds, and yeasts. This study focuses on the rational design of novel AMPs to overcome these limitations, targeting improved stability and broader antimicrobial activity for food preservation applications. AMPs, typically less than 50 amino acid residues, are highly selective and exhibit diverse mechanisms of action, including membrane destabilization, pore formation, and targeting intracellular biosynthetic processes. However, their clinical use is often hampered by low *in vivo* stability, toxicity, and susceptibility to proteolytic degradation. This study aims to address these challenges by rationally designing peptides with multiple desirable properties—stability across a broad pH range, thermostability, anti-trypsin activity, and low cytotoxicity—making them suitable for food preservation applications. The design strategy is inspired by Bowman-Birk inhibitors (BBIs), a class of serine protease inhibitors known for their high stability.

The literature review section discusses existing antimicrobial peptides (AMPs) and their limitations in food preservation. The review emphasizes the need for AMPs with improved stability (thermostability and pH stability), broad-spectrum activity (against Gram-positive and Gram-negative bacteria, molds, and yeasts), and low cytotoxicity. The authors review existing AMPs like Nisin and highlight its limitations in terms of pH and temperature sensitivity. The review explores various classes of AMPs and their mechanisms of action, focusing on membrane destabilization and intracellular target inhibition. The authors also discuss the use of Bowman-Birk inhibitors (BBIs) as a source of inspiration for designing stable and effective AMPs. The review supports the rationale for designing a new class of AMPs with multiple desirable properties to overcome the shortcomings of existing AMPs for food preservation.

The study employed a rational design approach using two natural peptides, HVBBI and SFTI, as templates. These peptides share a trypsin inhibitory loop flanked by cysteine residues forming a disulfide bridge, contributing to thermostability. Differences in their tail segments, including the number of cationic and hydrophobic residues, were analyzed to understand their impact on antibacterial activity. Gram-positive bacterium *Micrococcus luteus* served as a model organism. Minimum inhibitory concentration (MIC) assays determined antibacterial activity. Trypsin inhibition assays evaluated protease resistance. Mutational studies explored the structure-function relationship by altering the loop and tail segments of the peptides, leading to the design of HSEP1, HSEP2, and HSEP3. The thermostability and pH stability of the engineered peptides were assessed via trypsin inhibition assays. Confocal laser-scanning microscopy, live/dead staining, propidium iodide (PI) uptake assays, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) elucidated the peptides' mode of action, focusing on membrane destabilization and intracellular trypsin inhibition. All-atom molecular dynamics (MD) simulations investigated the peptide-membrane interactions. Cytotoxicity assays (using human retinal pigment epithelial cells and intestinal epithelial cells) and hemolytic assays assessed peptide safety. Finally, food preservation assays using cooked rice inoculated with various bacterial species evaluated the peptides' efficacy in preventing food spoilage. Statistical analyses were performed to determine significance.

Analysis of HVBBI and SFTI revealed that the tail segment, containing cationic and hydrophobic residues, is crucial for antibacterial activity. The engineered peptides, particularly HSEP3, showed significantly enhanced antibacterial activity compared to the natural templates. HSEP3 exhibited high thermostability, retaining 50% of its trypsin inhibitory activity after 200 min at 95 °C, and broad pH stability (retaining >80% activity at pH 2.5, 5, and 9). Confocal microscopy and live/dead staining demonstrated membrane localization and increased membrane permeability. SEM and TEM imaging revealed membrane damage and pore formation. MD simulations supported the carpet model of membrane disruption, showing bilayer thinning and increased water permeation. Mutational studies revealed that both membrane destabilization and intracellular trypsin inhibition contribute to the peptides' antibacterial activity. The loop region's trypsin inhibitory activity and the tail's membrane-destabilizing properties act synergistically. Cytotoxicity and hemolytic assays confirmed the peptides' low toxicity. Food preservation assays using cooked rice demonstrated the efficacy of HSEP3 in preventing bacterial growth for up to six days.

The findings demonstrate the successful rational design of hyperstable AMPs with broad-spectrum antibacterial activity. The study highlights the synergistic effect of membrane destabilization and intracellular trypsin inhibition in the overall antimicrobial mechanism. This approach allows for tailoring the peptide properties to optimize specific applications. The low toxicity and efficacy in food preservation assays demonstrate the potential of these peptides as safe and effective food preservatives. The success of the rational design strategy, compared to purely combinatorial approaches, emphasizes the importance of understanding fundamental structure-function relationships in designing novel antimicrobials. The results pave the way for further development of peptide cocktails with even broader spectrum activity and enhanced efficacy.

This study successfully demonstrated the rational design of hyperstable antibacterial peptides, exemplified by HSEP3. HSEP3 exhibits superior stability, broad-spectrum activity, and low toxicity, making it a potential alternative to existing food preservatives. The findings highlight the synergistic roles of membrane destabilization and intracellular trypsin inhibition, providing insights for future antimicrobial peptide design. Future research could focus on exploring synergistic combinations of peptides and investigating the molecular details underlying varying antibacterial efficacy against different bacteria to achieve broader spectrum activity.

The study primarily focused on a limited set of bacterial strains. Further investigation is needed to determine the effectiveness against a wider range of foodborne pathogens and spoilage organisms. While the study demonstrated efficacy in rice preservation, additional studies are necessary to evaluate the peptides' performance in other food matrices. The long-term stability of the peptides under various storage conditions also warrants further investigation. The safety assessment was conducted in vitro; in vivo studies are needed to fully confirm the peptides' safety for human consumption.