Antimicrobial Peptides Designed in silico: Safeguarding Against Botrytis cinerea and Food spoilage

Antimicrobial Peptides Designed in silico: Safeguarding Against Botrytis cinerea and Food spoilage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antimicrobial Peptides Designed in silico: Safeguarding Against Botrytis cinerea and Food spoilage Shayma A. Osman, Nahla O. Eltai, Nura A. Mohamed, Muhammad Suleman, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6060973/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Botrytis cinerea, a fungal pathogen known for causing grey mold diseases in post-harvest plant products, presents a serious global concern due to its impact on food safety and economic stability. To address this challenge, various studies have explored the use of safe and effective fungicides to curb fungal growth associated with food spoilage. In this study, antimicrobial peptides (AMPs) designed through computational methods were tested for their antifungal effects on Botrytis cinerea ( B. cinerea ). The tested peptide sequences showed strong antifungal properties, with complete inhibition observed at the highest concentrations tested (2500, 1250, and 625 µg/ml). Additionally, AMP cytotoxicity was assessed through Alamar Blue absorbance readings to evaluate the viability of treated neonatal fibroblast cells. Results showed that the computationally modelled AMPs are effective at a concentration of 312 µg/ml, indicating their potential as an alternative to conventional fungicides in preventing food spoilage. This study emphasizes the potential of bioengineered AMPs to support a safer, more sustainable agricultural practice and address the rising demand for effective biocontrol agents in food preservation. Botrytis cinerea food spoilage antimicrobial peptides fungicides cytotoxicity. Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Food spoilage, a significant public health concern, predominantly results from microbial contamination, posing substantial risks to food safety and economic stability. Mechanical damages often occur during the harvesting, handling, transporting, and storing of fruits and vegetables, which can facilitate microbial invasions leading to spoilage[ 8 ]. Such spoilage affects the texture and color of the produce and renders it unsafe for consumption. Particularly detrimental are spoilage microorganisms, including various bacteria and fungi, which can degrade food quality significantly. Fungi, as a significant concern, produce mycotoxins—metabolic byproducts that are carcinogenic and mutagenic, accumulating in the human body and presenting severe health risks[ 1 ]. Ingesting foods contaminated with mycotoxin-producing fungi is a critical source of foodborne illnesses [ 13 ]. Consequently, robust food preservation techniques are essential not only to prolong shelf life but also to ensure the safety and quality of food. Traditional preservation methods such as heating, freezing, and the application of chemical preservatives are prevalent; however, these methods may diminish the nutritional value of food and pose health risks due to chemical additives [ 7 , 17 ]. Antimicrobial Peptides (AMPs), naturally occurring small bioactive proteins in the immune system, present a promising alternative. AMPs play a critical role in defending against bacterial, fungal, and viral infections through their ability to selectively inhibit the growth of specific microorganisms, thereby preventing the development of bacterial resistance [ 4 , 6 ]. Their mechanisms include penetrating microbial cell membranes, disrupting cellular processes, and inducing cell death through interactions with DNA and mitochondrial functions [ 19 ], [ 9 ]. Despite the potential of natural AMPs as food preservatives, their commercial application has been limited by high purification costs and associated toxicity. In contrast, synthetic AMPs have gained interest due to their ease of design and synthesis, which reduces the need for extensive purification and minimizes cellular toxicity. These synthetic variants effectively target microbial cell membranes and organelles, causing irreversible damage and cell death[ 16 ]. Within the pathogens involved in the food spoilage, Botrytis cinerea (B. cinerea), a pathogenic fungus known for its rapid development of fungicide resistance, commonly affects soft fruits like raspberries and strawberries by causing grey mold disease through airborne conidia spores [ 3 ]. Our study aims to critically assess the effectiveness of in silico-designed Antimicrobial Peptides (AMPs) against Botrytis cinerea ( B. cinerea ) to mitigate food spoilage in agricultural products; here, we will use strawberries as a crop product to analyze AMPs' efficacy in reducing fungal infection. B. cinerea , commonly associated with the spoilage of high-value crops like berries, poses significant challenges due to its rapid adaptability and resistance to traditional fungicides. The innovative use of AMPs could help to preserving perishable goods, enhancing food safety, and reducing economic losses. The comprehensive approach of this study is of utmost importance as it addresses the urgent need for innovative and effective food preservation strategies and contributes significantly to the broader field of agricultural biotechnology. By exploring the application of synthetically designed AMPs, this research could pave the way for new methods to combat fungal pathogens resistant to current treatments. Moreover, the findings could inform future guidelines on the using AMPs in food systems, ensuring safety, efficacy, and sustainability. Additionally, insights gained from the cytotoxicity assessment will help design peptides that are effective antimicrobials and safe for use in food products and therapeutic applications. This dual focus on efficacy and safety is expected to facilitate the development of regulatory frameworks for AMPs in agricultural and food industries. The first phase of our study involves determining the Minimum Inhibitory Concentration (MIC) of AMPs designed through computational models. The MIC is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. We will employ the microdilution method, a standardized technique used widely in microbiology to assess the susceptibility of pathogens to antimicrobials. This method involves serially diluting the peptide in a microtiter plate with a standardized number of fungal spores and then incubating the plates to allow spores to grow. Observations of fungal growth are recorded, allowing us to precisely determine the effective concentration of the AMPs necessary to prevent the proliferation of B. cinerea . To ensure the safety and feasibility of using these AMPs in food applications, evaluating their cytotoxic effects on human cells is crucial. The Alamar Blue Assay, a reliable and sensitive method, will be utilized to measure the viability of neonatal fibroblast cells in the presence of various concentrations of AMPs. This assay employs a fluorometric/colorimetric growth indicator based on detecting metabolic activity. Cells that are alive reduce the Alamar Blue dye in a quantifiable manner, thus providing insights into the cytotoxic nature of the peptides at different concentrations. 2 Materials and methods 2.1 Antimicrobial peptides in silico design 2.1.1 AMP Synthesis and Characterization We began our study by selecting antimicrobial peptide (AMP) sequences from public databases such as the Collection of Antimicrobial Peptides (CAMPs), CAMPSign, and ClassAMP. These sequences were used to identify homologous peptides from various eukaryotic species, provided their genetic information was available in online repositories. Following this, we designed AMP analogues based on the mature sequences obtained, focusing on peptides approximately 10–34 amino acids in length, with a cationic surface charge and potential antimicrobial and therapeutic properties. The in-silico pipeline has been developed according to dos Santos-Silva et al. [ 14 ]. 2.1.2 Molecular Modeling The structural modelling of the AMPs was performed using the ColabFold platform, which integrates AlphaFold2 within a Google Collaboratory environment. The AMP sequences were input into the tool, which employs an AI algorithm from DeepMind to predict protein structures. The resulting models were validated theoretically to ensure their reliability for future applications. Metrics such as the per-residue confidence score (pLDDT) and the global structural similarity score (TM-score) were used to assess the quality of the modelled structures [ 12 ]. 2.1.3 Molecular Dynamics Simulation Molecular dynamics simulations were conducted using GROMACS version 2019.4. Initially, the modelled AMPs were centered in a cubic box and solvated using the SPC (Simple Point Charge) water model. The system was then equilibrated with a NaCl solution at a physiological concentration of 0.15 M, followed by energy minimization. To maintain a temperature of 300 K, the NvT ensemble was used, with solute atoms restrained to their initial positions. The LINCS method was applied to constrain bonds involving hydrogen atoms, and atom movements were integrated using the leapfrog algorithm with a 2fs time step. An initial energy optimization was performed using 50,000 steps of the steepest descent algorithm. Simulations ran for 100 ns using the GROMOS 53A6 force field under constant pressure and temperature conditions, providing a realistic environment to observe the peptide's dynamic behavior. 2.1.4 Peptide Synthesis and Characterization By applying the pipeline described above two sequences showing in silico the best predicted antifungal activity according to PhytoAFP ( http://2nhojjq.257.cz/multi_pep.php ) as well as the Deep-AFPpred, namely Seq4 (SCRGGLCRGFRRRAFCTKRC; Molecular weight: 2333.8 g/mol, Iso-electric point, pH 11.47, Net charge at pH 7: 6.7) and Seq8 (RFTGGHCRGFRRRCFCTKHC; Molecular weight:2428.86 g/mol, Iso-electric point: pH 10.97, Net charge at pH 7:5.9) have undergone chemical synthesis [ 15 ]. The peptides were synthesized by NovoPro Bioscience Inc. (Shanghai, China). NovoPro Bioscience assessed peptide purity using reverse-phase high-performance liquid chromatography (HPLC, purity > 95%) and confirmed molecular weight through mass spectrometry. 2.2 Sample collection and fungus identification Strawberry samples were collected from Qatari stores and incubated at room temperature at 25°C in plastic bags. The hyphal growth of the fungi was monitored and observed visually. Then, mold sample was sub-cultured to grow on Potato Dextrose Agar (PDA) plates (HIMEDIA, USA) using a sterile needle. After 5 days of incubation, lactophenol cotton blue staining was used for fungal identification at 40x microscopic magnification (Micros, Austria). 2.3 Determination of Minimum Inhibitory Concentration (MIC) The activity of antimicrobial peptides (AMPs) was evaluated using a 96-well plate microdilution test to measure antifungal activity. Each well was inoculated with 100µl of RPMI 1640 broth (LIOFILCHEM, Italy), to determine the minimum inhibitory concentrations (MICs) of the antifungal agents and assessing fungal growth. Two peptides were tested: Seq4 (SCRGGLCRGFRRRAFCTKRC) and Seq8 (RFTGGHCRGFRRRCFCTKHC). Twelve different concentrations of each peptide were evaluated, from 2500, 1250, 625, 312, 156.25, 78.125, 39.1, 19.5, 9.8, 4.9, 2.4, to 1.2 µg/ml. A spore suspension containing 10² spores/ml was prepared by diluting fungal spores in sterile distilled water. Each well was inoculated with 10µl of this suspension. Four replicates of each concentration were tested, and each experiment was repeated three times. Negative controls included only the media, while positive controls contained 1% Clotrimazole antifungal solution. The plates were incubated at 25°C, and fungal growth was observed after 72 hours[ 13 ]. Alamar Blue (Invitrogen, U.S.) was added to all experimental wells and incubated for 3–4 hours to detect fungal growth. The FLUOstar Omega microplate reader was then used to measure the absorbance of the dye at wavelengths between 570 and 620 nm. Alamar Blue is a light-sensitive blue dye used in cell culture to detect cell proliferation and viability. It turns from blue to pink when cellular activity reduces resazurin to resorufin[ 2 ]. Data were collected and analyzed using Prism GraphPad. 2.4 Cell Culture Neonatal Fibroblast cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, UK), which contained 10% Fetal Bovine Serum (FBS). The cells were incubated at 37°C in a humidified incubator (Thermo Fisher Scientific, U.S.) with 95% air and 5% CO 2 . 2.5 Cell viability Assay Neonatal Fibroblast cells were seeded in a 96 well-plate where each well had 1.0 × 10 4 cells per well. The treatment of two peptide concentrations was added to the plate after 24 hours of the seeding. The following peptide concentrations were used (2500, 1250, 625, 312, 156.25, 78.125, 39.1,19.5 µg/ml). Then, the media was removed, and 10% of Alamar Blue (Invitrogen, U.S.) was added to the plate and incubated for 3–4 hours at 37°C. The FLUOstar Omega device was used to read the absorbance of the Alamar Blue at 570- 620nm wavelength and detect cell viability. The results were analyzed using one-way ANOVA. 3 Results 3.1 Antifungal activity of AMPs against B. cinerea growth using the MIC method The antifungal effect of AMP concentrations was evaluated by MIC method using B. cinerea fungi. Our findings show that the tested sequences of Seq-4 and Seq-8 antifungal peptides completely inhibited the growth of B. cinerea at concentrations ranging from 2500 to 625 µg/ml. The hyphal growth was hindered at a MIC value of 625 µg/ml (Figs. 1 and 2 ). The efficacy of the AMPs can be demonstrated by Alamar Blue Assay change in color, where the blue color indicates unviable spores, and the pink color indicates the viable ones (Figs. 1 a. 2a). 3.2 The effect of AMPs on Cell viability The cytotoxicity of the peptides was evaluated using the Alamar Blue Assay to observe the effect on neonatal fibroblast cell viability. AMPs significantly reduce cell viability at the higher concentrations (2500 to 625 µg/ml) compared to the negative control which contained untreated cells. However, we did not observe a difference in the cell viability at 312 µg/ml and lower concentrations compared to the negative control (Figs. 3 and 4 ). 4 Discussion Food spoilage has different implications for food security and economies; also caused by B. cinerea, one of the most common pathogens worldwide that can infect up to 500 plant species [ 5 ]. The formation of grey molds in Post-harvested fruits and vegetables is a primary concern for food safety and shelf life. Recently, AMPs are used alternatively to chemical fugicides in controlling infections. The use of AMPs is effectively preventing the growth of pathogens, including yeast and filamentous fungi species. They can help limit food spoilage while being less costly and less toxic to mammalian cells than other treatments [ 10 ]. In this study, the antifungal activity of two sequences of AMPs was evaluated by determining MIC values against B. cinerea, one of the most common spoilage microbes. This synthetic treatment of AMPs was effective in inhibiting the growth of B. cinerea. Both sequences gave MIC values of 625 µg/ml. According to Yang et al. (2023), the antifungal activity of Mytichitin-CB, an antimicrobial peptide, was tested against Botrytis cinerea and showed a minimum inhibitory concentration (MIC) of 2000 µg/ml, with a conidial survival rate of 19.2% [ 20 ]. This suggests that the currently tested AMPs (Seq-4 and Seq-8) are more effectively inhibited the growth of B. cinerea at lower concentrations, with an MIC of 625 µg/ml. The neonatal fibroblast cell line is commonly used for cytotoxicity screening and the evaluation of the biosafety of materials. Fibroblasts have defined phenotype and have the tendency of tissue repair and inflammatory responses, which makes them sensitive to any toxic exposure and suitable for testing material that could interact with human tissues [ 18 ]. Therefore, the peptides were tested on neonatal fibroblast cells to evaluate the cytotoxicity of AMPs to human cell lines. The results indicate that the lowest inhibitory concentrations of the fungal growth do not significantly affect human cells’ viability. Various studies have used plant extracts, rich in peptides, to address food spoilage where the efficacy depends on the extraction method and the plant type[ 12 ]. However, AMPs are considered safer alternatives for protecting fruits and vegetables from spoilage[ 11 ]. 5 Conclusions This study underscores the potential of antimicrobial peptides (AMPs), specifically Seq-4 and Seq-8, in combating Botrytis cinerea , a common spoilage fungus affecting high-value crops like strawberries. The in silico-designed AMPs demonstrated significant antifungal activity at lower concentrations than natural peptides such as Mytichitin-CB. The minimum inhibitory concentration (MIC) for Seq-4 and Seq-8 was determined to be 625 µg/ml, notably lower than the MIC of 2000 µg/ml reported for Mytichitin-CB. This highlights the enhanced efficacy of these synthetic peptides. Moreover, this study employed a comprehensive approach to assess the safety of Seq-4 and Seq-8 at their effective antifungal concentrations, a crucial factor for their potential use in food preservation. The Alamar Blue assay results were reassuring, showing that these peptides do not significantly affect cell viability. This finding suggests that Seq-4 and Seq-8 could be safer alternatives to traditional chemical preservatives, which often pose health risks and reduce nutritional value. Additionally, this research contributes to the broader field of agricultural biotechnology by providing insights into the application of synthetic AMPs in food systems. The findings suggest that these peptides could play a role in developing new methods to preserve perishable goods, enhancing food safety, and reducing economic losses due to spoilage. The successful inhibition of B. cinerea growth by Seq-4 and Seq-8 also opens avenues for further exploration into their application against other spoilage microorganisms. In conclusion, the study demonstrates the promise of in silico-designed AMPs in addressing food spoilage and highlights the importance of developing innovative and effective food preservation strategies. Future research should focus on expanding the application of these peptides to a broader range of food products and pathogens, ensuring their safety and efficacy in various food systems. The dual focus on antifungal efficacy and low cytotoxicity positions these AMPs as valuable tools in enhancing food preservation while maintaining safety and quality. Declarations Funding This study has been supported by the by Qatar University QUCG-LARC-22/23-499 Collaborative Grant, by the QUPD-CAS-23-24-491 and by the QNRF UREP28-115-1-025. Competing interests The authors have no competing interests to declare that are relevant to the content of this article. Author contributions Conceptualization: [Sergio Crovella]. Material preparation, data collection, and analysis by [Nura A. Mohamed] and [ Shayma A. Osman]. The first draft of the manuscript was written by [ Shayma A. Osman] and all authors commented on previous versions of the manuscript. Review and editing, by [Sergio Crovella], [Nahla Eltai], [Nura A. Mohamed], and [Muhammad Suleman]. Supervision: [Sergio Crovella] and [Nura A. Mohamed]. All authors have read and approved the final version of the manuscript. Competing interests The authors have no conflict of interest to declare that are relevant to the content of this article. Data availability statement The authors declare that all data are directly reported in the text of our manuscript. References Awuchi CG, Ondari EN, Nwozo S, Odongo GA, Eseoghene IJ, Twinomuhwezi H, Ogbonna CU, Upadhyay AK, Adeleye AO, Okpala COR (2022) Mycotoxins’ Toxicological Mechanisms Involving Humans, Livestock and Their Associated Health Concerns: A Review. Toxins (Basel). doi: 10.3390/toxins14030167 Barua P, You MP, Bayliss K, Lanoiselet V, Barbetti MJ (2017) A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity. Eur J Plant Pathol 148:139–150 Cheung N, Tian L, Liu X, Li X (2020) The Destructive Fungal Pathogen Botrytis cinerea—Insights from Genes Studied with Mutant Analysis. Pathogens 9:923 Fernández de Ullivarri M, Arbulu S, Garcia-Gutierrez E, Cotter PD (2020) Antifungal Peptides as Therapeutic Agents. Front Cell Infect Microbiol. doi: 10.3389/fcimb.2020.00105 Hua L, Yong C, Zhanquan Z, Boqiang L, Guozheng Q, Shiping T (2018) Pathogenic mechanisms and control strategies of Botrytis cinerea causing post-harvest decay in fruits and vegetables. Food Quality and Safety 2:111–119 Huan Y, Kong Q, Mou H, Yi H (2020) Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Front Microbiol. doi: 10.3389/fmicb.2020.582779 Kamal I, Ashfaq UA, Hayat S, Aslam B, Sarfraz MH, Yaseen H, Rajoka MSR, Shah AA, Khurshid M (2023) Prospects of antimicrobial peptides as an alternative to chemical preservatives for food safety. Biotechnol Lett 45:137–162 Karanth S, Feng S, Patra D, Pradhan AK (2023) Linking microbial contamination to food spoilage and food waste: the role of smart packaging, spoilage risk assessments, and date labeling. Front Microbiol. doi: 10.3389/fmicb.2023.1198124 Lei J, Sun L, Huang S, Zhu C, Li P, He J, Mackey V, Coy DH, He Q (2019) The antimicrobial peptides and their potential clinical applications. Am J Transl Res 11:3919–3931 Lima PG, Freitas CDT, Oliveira JTA, Neto NAS, Amaral JL, Silva AFB, Sousa JS, Franco OL, Souza PFN (2021) Synthetic antimicrobial peptides control Penicillium digitatum infection in orange fruits. Food Research International 147:110582 Mignone G, Shwaiki LN, Arendt EK, Coffey A (2022) Inhibitory activity of two synthetic Pharabitis nil (L.) antimicrobial peptides against common spoilage yeasts. Applied Food Research. doi: 10.1016/j.afres.2022.100168 Roca-Couso R, Flores-Félix JD, Rivas R (2021) Mechanisms of Action of Microbial Biocontrol Agents against Botrytis cinerea. Journal of Fungi 7:1045 Saleh I, Abu-Dieyeh MH (2021) Novel Prosopis juliflora leaf ethanolic extract as natural antimicrobial agent against food spoiling microorganisms. Sci Rep 11:7871 Santos-Silva CA dos, Tricarico PM, Vilela LMB, Roldan-Filho RS, Amador VC, d’Adamo AP, Rêgo M de S, Benko-Iseppon AM, Crovella S (2021) Plant Antimicrobial Peptides as Potential Tool for Topic Treatment of Hidradenitis Suppurativa. Front Microbiol. doi: 10.3389/fmicb.2021.795217 Sharma R, Shrivastava S, Kumar Singh S, Kumar A, Saxena S, Kumar Singh R (2022) Deep-AFPpred: identifying novel antifungal peptides using pretrained embeddings from seq2vec with 1DCNN-BiLSTM. Brief Bioinform. doi: 10.1093/bib/bbab422 Souza PF (2022) Synthetic antimicrobial peptides for controlling fungi in foods. Curr Opin Food Sci. doi: 10.1016/j.cofs.2022.100819 Sridhar A, Ponnuchamy M, Kumar PS, Kapoor A (2021) Food preservation techniques and nanotechnology for increased shelf life of fruits, vegetables, beverages and spices: a review. Environ Chem Lett 19:1715–1735 Wang X, Li S, Cao T, Fu X, Yu G (2012) Evaluating biotoxicity with fibroblasts derived from human embryonic stem cells. Toxicology in Vitro 26:1056–1063 Wang H, Zhang C, Li M, Liu C, Wang J, Ou X, Han Y (2022) Antimicrobial Peptides Mediate Apoptosis by Changing Mitochondrial Membrane Permeability. Int J Mol Sci 23:12732 Yang X, Wang Y, Jiang H, Song R, Liu Y, Guo H, Meng D (2023) Antimicrobial peptide CB-M exhibits direct antifungal activity against Botrytis cinerea and induces disease resistance to gray mold in cherry tomato fruit. Postharvest Biol Technol 196:112184 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6060973","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434248690,"identity":"90236647-5652-4d82-9114-668a5f6f461e","order_by":0,"name":"Shayma A. Osman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYFACxvYfiX9s6vnZG0A8ZmK0MDdIfGxIS5DsOUC0FvYGyZkNhxMMbiQQqUW+vbHBmHcHc57BzcfbJBgqrBMb2JMf4NVicOZgQzLvGbZiydtpZRIMZ9ITG3ieGeDXIpHYcJiHjYex73aOmQRj2+HEBokE/FrkZyQ2NvOwSTA23DwD1PIPpCX9A37P3EhsZpzZZpA44QYPUEsDSEsOAYedOdjG8OFMgrFkT1qxRcKxdOM2njcF+B3W3v6MIaHivxw/++GNNz7UWMv2s6dvwO8wZBsZEoAkG5gkWgsEkKBlFIyCUTAKRgQAAGYNS5ciM2ihAAAAAElFTkSuQmCC","orcid":"","institution":"Qatar University","correspondingAuthor":true,"prefix":"","firstName":"Shayma","middleName":"A.","lastName":"Osman","suffix":""},{"id":434248691,"identity":"f7f3505c-f6ba-4d82-aea1-574831163c36","order_by":1,"name":"Nahla O. Eltai","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Nahla","middleName":"O.","lastName":"Eltai","suffix":""},{"id":434248692,"identity":"192a15e7-27b3-4d44-8ce1-989c2388922d","order_by":2,"name":"Nura A. Mohamed","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Nura","middleName":"A.","lastName":"Mohamed","suffix":""},{"id":434248693,"identity":"9a79725f-086d-4911-85fb-8077fc4ae04c","order_by":3,"name":"Muhammad Suleman","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Suleman","suffix":""},{"id":434248694,"identity":"d11739aa-b0b7-461b-90fa-67762aabb238","order_by":4,"name":"Sergio Crovella","email":"","orcid":"","institution":"Qatar University","correspondingAuthor":false,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Crovella","suffix":""}],"badges":[],"createdAt":"2025-02-19 06:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6060973/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6060973/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79658650,"identity":"92e32c56-fc75-4e03-bb9e-a07e02965450","added_by":"auto","created_at":"2025-04-01 09:17:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94204,"visible":true,"origin":"","legend":"\u003cp\u003eThe MIC is determined for AMPs Seq-8 antifungal activity. (\u003cstrong\u003ea\u003c/strong\u003e) The 96-well plate with Alamar Blue represents the viable and nonviable spores of B. cinerea treated with AMP Seq-8. (\u003cstrong\u003eb\u003c/strong\u003e)Data are mean ± S.E.M for n=12 accomplished in three experiments. Statistical analysis for effect between different groups was done by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. Statistical significance was noted at *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6060973/v1/e032a7bae767cd57b91777cd.png"},{"id":79657783,"identity":"12598511-7b84-42a8-9c3d-238ee1babb61","added_by":"auto","created_at":"2025-04-01 09:09:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80474,"visible":true,"origin":"","legend":"\u003cp\u003eMIC is determined for AMPs Seq-4 antifungal activity. (\u003cstrong\u003ea\u003c/strong\u003e)An image of the 96-well plate containing B. cinerea treated with AMP Seq-4. (\u003cstrong\u003eb\u003c/strong\u003e) Data are mean ± S.E.M for n=12 done in three experiments. Statistical analysis for the effect between different groups using one-way ANOVA followed by Dunnett’s Multiple Comparison Test. Statistical significance was noted at *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6060973/v1/97e1ea4ee75880aedb4a2049.png"},{"id":79657781,"identity":"673e551f-2f63-4a80-b098-c9218ecd7e25","added_by":"auto","created_at":"2025-04-01 09:09:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":48449,"visible":true,"origin":"","legend":"\u003cp\u003eSeq-8 effect on the fibroblast cell viability. Data are mean ± S.E.M for n=9. Statistical analysis of the effect between different groups was done using one-way ANOVA followed by Dunnett’s Multiple Comparison Test. Statistical significance was noted at *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6060973/v1/22c0952a68ccfcf4e3d2fc11.png"},{"id":79657799,"identity":"3438a755-8cb2-4d6b-ab8e-9dec276ab8c3","added_by":"auto","created_at":"2025-04-01 09:09:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47667,"visible":true,"origin":"","legend":"\u003cp\u003eSeq-4 effect on fibroblast cell viability. Data are mean ± S.E.M for n=5. Statistical analysis of the effect between different groups was done using one-way ANOVA followed by Dunnett’s Multiple Comparison Test. Statistical significance was noted at *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6060973/v1/a1f364d11352642154f5df1c.png"},{"id":89943801,"identity":"96745677-e737-4587-b56c-3ab5ab5b8a44","added_by":"auto","created_at":"2025-08-26 16:38:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":913951,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6060973/v1/9319504b-a873-4ad8-917a-3b7c29c66c9e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimicrobial Peptides Designed in silico: Safeguarding Against Botrytis cinerea and Food spoilage","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eFood spoilage, a significant public health concern, predominantly results from microbial contamination, posing substantial risks to food safety and economic stability. Mechanical damages often occur during the harvesting, handling, transporting, and storing of fruits and vegetables, which can facilitate microbial invasions leading to spoilage[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Such spoilage affects the texture and color of the produce and renders it unsafe for consumption. Particularly detrimental are spoilage microorganisms, including various bacteria and fungi, which can degrade food quality significantly.\u003c/p\u003e \u003cp\u003eFungi, as a significant concern, produce mycotoxins\u0026mdash;metabolic byproducts that are carcinogenic and mutagenic, accumulating in the human body and presenting severe health risks[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Ingesting foods contaminated with mycotoxin-producing fungi is a critical source of foodborne illnesses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Consequently, robust food preservation techniques are essential not only to prolong shelf life but also to ensure the safety and quality of food. Traditional preservation methods such as heating, freezing, and the application of chemical preservatives are prevalent; however, these methods may diminish the nutritional value of food and pose health risks due to chemical additives [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Antimicrobial Peptides (AMPs), naturally occurring small bioactive proteins in the immune system, present a promising alternative. AMPs play a critical role in defending against bacterial, fungal, and viral infections through their ability to selectively inhibit the growth of specific microorganisms, thereby preventing the development of bacterial resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Their mechanisms include penetrating microbial cell membranes, disrupting cellular processes, and inducing cell death through interactions with DNA and mitochondrial functions [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite the potential of natural AMPs as food preservatives, their commercial application has been limited by high purification costs and associated toxicity. In contrast, synthetic AMPs have gained interest due to their ease of design and synthesis, which reduces the need for extensive purification and minimizes cellular toxicity. These synthetic variants effectively target microbial cell membranes and organelles, causing irreversible damage and cell death[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWithin the pathogens involved in the food spoilage, \u003cem\u003eBotrytis cinerea\u003c/em\u003e (B. cinerea), a pathogenic fungus known for its rapid development of fungicide resistance, commonly affects soft fruits like raspberries and strawberries by causing grey mold disease through airborne conidia spores [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study aims to critically assess the effectiveness of in silico-designed Antimicrobial Peptides (AMPs) against \u003cem\u003eBotrytis cinerea\u003c/em\u003e (\u003cem\u003eB. cinerea\u003c/em\u003e) to mitigate food spoilage in agricultural products; here, we will use strawberries as a crop product to analyze AMPs' efficacy in reducing fungal infection. \u003cem\u003eB. cinerea\u003c/em\u003e, commonly associated with the spoilage of high-value crops like berries, poses significant challenges due to its rapid adaptability and resistance to traditional fungicides. The innovative use of AMPs could help to preserving perishable goods, enhancing food safety, and reducing economic losses.\u003c/p\u003e \u003cp\u003eThe comprehensive approach of this study is of utmost importance as it addresses the urgent need for innovative and effective food preservation strategies and contributes significantly to the broader field of agricultural biotechnology. By exploring the application of synthetically designed AMPs, this research could pave the way for new methods to combat fungal pathogens resistant to current treatments. Moreover, the findings could inform future guidelines on the using AMPs in food systems, ensuring safety, efficacy, and sustainability. Additionally, insights gained from the cytotoxicity assessment will help design peptides that are effective antimicrobials and safe for use in food products and therapeutic applications. This dual focus on efficacy and safety is expected to facilitate the development of regulatory frameworks for AMPs in agricultural and food industries.\u003c/p\u003e \u003cp\u003eThe first phase of our study involves determining the Minimum Inhibitory Concentration (MIC) of AMPs designed through computational models. The MIC is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation. We will employ the microdilution method, a standardized technique used widely in microbiology to assess the susceptibility of pathogens to antimicrobials. This method involves serially diluting the peptide in a microtiter plate with a standardized number of fungal spores and then incubating the plates to allow spores to grow. Observations of fungal growth are recorded, allowing us to precisely determine the effective concentration of the AMPs necessary to prevent the proliferation of \u003cem\u003eB. cinerea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo ensure the safety and feasibility of using these AMPs in food applications, evaluating their cytotoxic effects on human cells is crucial. The Alamar Blue Assay, a reliable and sensitive method, will be utilized to measure the viability of neonatal fibroblast cells in the presence of various concentrations of AMPs. This assay employs a fluorometric/colorimetric growth indicator based on detecting metabolic activity. Cells that are alive reduce the Alamar Blue dye in a quantifiable manner, thus providing insights into the cytotoxic nature of the peptides at different concentrations.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Antimicrobial peptides in silico design\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 AMP Synthesis and Characterization\u003c/h2\u003e \u003cp\u003eWe began our study by selecting antimicrobial peptide (AMP) sequences from public databases such as the Collection of Antimicrobial Peptides (CAMPs), CAMPSign, and ClassAMP. These sequences were used to identify homologous peptides from various eukaryotic species, provided their genetic information was available in online repositories. Following this, we designed AMP analogues based on the mature sequences obtained, focusing on peptides approximately 10\u0026ndash;34 amino acids in length, with a cationic surface charge and potential antimicrobial and therapeutic properties. The in-silico pipeline has been developed according to dos Santos-Silva et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Molecular Modeling\u003c/h2\u003e \u003cp\u003eThe structural modelling of the AMPs was performed using the ColabFold platform, which integrates AlphaFold2 within a Google Collaboratory environment. The AMP sequences were input into the tool, which employs an AI algorithm from DeepMind to predict protein structures. The resulting models were validated theoretically to ensure their reliability for future applications. Metrics such as the per-residue confidence score (pLDDT) and the global structural similarity score (TM-score) were used to assess the quality of the modelled structures [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3 Molecular Dynamics Simulation\u003c/h2\u003e \u003cp\u003eMolecular dynamics simulations were conducted using GROMACS version 2019.4. Initially, the modelled AMPs were centered in a cubic box and solvated using the SPC (Simple Point Charge) water model. The system was then equilibrated with a NaCl solution at a physiological concentration of 0.15 M, followed by energy minimization. To maintain a temperature of 300 K, the NvT ensemble was used, with solute atoms restrained to their initial positions. The LINCS method was applied to constrain bonds involving hydrogen atoms, and atom movements were integrated using the leapfrog algorithm with a 2fs time step. An initial energy optimization was performed using 50,000 steps of the steepest descent algorithm. Simulations ran for 100 ns using the GROMOS 53A6 force field under constant pressure and temperature conditions, providing a realistic environment to observe the peptide's dynamic behavior.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.1.4 Peptide Synthesis and Characterization\u003c/h2\u003e \u003cp\u003eBy applying the pipeline described above two sequences showing in silico the best predicted antifungal activity according to PhytoAFP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://2nhojjq.257.cz/multi_pep.php\u003c/span\u003e\u003cspan address=\"http://2nhojjq.257.cz/multi_pep.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as well as the Deep-AFPpred, namely Seq4 (SCRGGLCRGFRRRAFCTKRC; Molecular weight: 2333.8 g/mol, Iso-electric point, pH 11.47, Net charge at pH 7: 6.7) and Seq8 (RFTGGHCRGFRRRCFCTKHC; Molecular weight:2428.86 g/mol, Iso-electric point: pH 10.97, Net charge at pH 7:5.9) have undergone chemical synthesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The peptides were synthesized by NovoPro Bioscience Inc. (Shanghai, China). NovoPro Bioscience assessed peptide purity using reverse-phase high-performance liquid chromatography (HPLC, purity \u0026gt; 95%) and confirmed molecular weight through mass spectrometry.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample collection and fungus identification\u003c/h2\u003e \u003cp\u003eStrawberry samples were collected from Qatari stores and incubated at room temperature at 25\u0026deg;C in plastic bags. The hyphal growth of the fungi was monitored and observed visually. Then, mold sample was sub-cultured to grow on Potato Dextrose Agar (PDA) plates (HIMEDIA, USA) using a sterile needle. After 5 days of incubation, lactophenol cotton blue staining was used for fungal identification at 40x microscopic magnification (Micros, Austria).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of Minimum Inhibitory Concentration (MIC)\u003c/h2\u003e \u003cp\u003eThe activity of antimicrobial peptides (AMPs) was evaluated using a 96-well plate microdilution test to measure antifungal activity. Each well was inoculated with 100\u0026micro;l of RPMI 1640 broth (LIOFILCHEM, Italy), to determine the minimum inhibitory concentrations (MICs) of the antifungal agents and assessing fungal growth. Two peptides were tested: Seq4 (SCRGGLCRGFRRRAFCTKRC) and Seq8 (RFTGGHCRGFRRRCFCTKHC). Twelve different concentrations of each peptide were evaluated, from 2500, 1250, 625, 312, 156.25, 78.125, 39.1, 19.5, 9.8, 4.9, 2.4, to 1.2 \u0026micro;g/ml. A spore suspension containing 10\u0026sup2; spores/ml was prepared by diluting fungal spores in sterile distilled water. Each well was inoculated with 10\u0026micro;l of this suspension. Four replicates of each concentration were tested, and each experiment was repeated three times. Negative controls included only the media, while positive controls contained 1% Clotrimazole antifungal solution. The plates were incubated at 25\u0026deg;C, and fungal growth was observed after 72 hours[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Alamar Blue (Invitrogen, U.S.) was added to all experimental wells and incubated for 3\u0026ndash;4 hours to detect fungal growth. The FLUOstar Omega microplate reader was then used to measure the absorbance of the dye at wavelengths between 570 and 620 nm. Alamar Blue is a light-sensitive blue dye used in cell culture to detect cell proliferation and viability. It turns from blue to pink when cellular activity reduces resazurin to resorufin[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Data were collected and analyzed using Prism GraphPad.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell Culture\u003c/h2\u003e \u003cp\u003eNeonatal Fibroblast cell line was cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco, UK), which contained 10% Fetal Bovine Serum (FBS). The cells were incubated at 37\u0026deg;C in a humidified incubator (Thermo Fisher Scientific, U.S.) with 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell viability Assay\u003c/h2\u003e \u003cp\u003eNeonatal Fibroblast cells were seeded in a 96 well-plate where each well had 1.0 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. The treatment of two peptide concentrations was added to the plate after 24 hours of the seeding. The following peptide concentrations were used (2500, 1250, 625, 312, 156.25, 78.125, 39.1,19.5 \u0026micro;g/ml). Then, the media was removed, and 10% of Alamar Blue (Invitrogen, U.S.) was added to the plate and incubated for 3\u0026ndash;4 hours at 37\u0026deg;C. The FLUOstar Omega device was used to read the absorbance of the Alamar Blue at 570- 620nm wavelength and detect cell viability. The results were analyzed using one-way ANOVA.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Antifungal activity of AMPs against B. cinerea growth using the MIC method\u003c/h2\u003e \u003cp\u003eThe antifungal effect of AMP concentrations was evaluated by MIC method using \u003cem\u003eB. cinerea\u003c/em\u003e fungi. Our findings show that the tested sequences of Seq-4 and Seq-8 antifungal peptides completely inhibited the growth of \u003cem\u003eB. cinerea\u003c/em\u003e at concentrations ranging from 2500 to 625 \u0026micro;g/ml. The hyphal growth was hindered at a MIC value of 625 \u0026micro;g/ml (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The efficacy of the AMPs can be demonstrated by Alamar Blue Assay change in color, where the blue color indicates unviable spores, and the pink color indicates the viable ones (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. 2a).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The effect of AMPs on Cell viability\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of the peptides was evaluated using the Alamar Blue Assay to observe the effect on neonatal fibroblast cell viability. AMPs significantly reduce cell viability at the higher concentrations (2500 to 625 \u0026micro;g/ml) compared to the negative control which contained untreated cells. However, we did not observe a difference in the cell viability at 312 \u0026micro;g/ml and lower concentrations compared to the negative control (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eFood spoilage has different implications for food security and economies; also caused by B. cinerea, one of the most common pathogens worldwide that can infect up to 500 plant species [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The formation of grey molds in Post-harvested fruits and vegetables is a primary concern for food safety and shelf life. Recently, AMPs are used alternatively to chemical fugicides in controlling infections. The use of AMPs is effectively preventing the growth of pathogens, including yeast and filamentous fungi species. They can help limit food spoilage while being less costly and less toxic to mammalian cells than other treatments [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the antifungal activity of two sequences of AMPs was evaluated by determining MIC values against B. cinerea, one of the most common spoilage microbes. This synthetic treatment of AMPs was effective in inhibiting the growth of B. cinerea. Both sequences gave MIC values of 625 \u0026micro;g/ml. According to Yang et al. (2023), the antifungal activity of Mytichitin-CB, an antimicrobial peptide, was tested against \u003cem\u003eBotrytis cinerea\u003c/em\u003e and showed a minimum inhibitory concentration (MIC) of 2000 \u0026micro;g/ml, with a conidial survival rate of 19.2% [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This suggests that the currently tested AMPs (Seq-4 and Seq-8) are more effectively inhibited the growth of B. cinerea at lower concentrations, with an MIC of 625 \u0026micro;g/ml.\u003c/p\u003e \u003cp\u003eThe neonatal fibroblast cell line is commonly used for cytotoxicity screening and the evaluation of the biosafety of materials. Fibroblasts have defined phenotype and have the tendency of tissue repair and inflammatory responses, which makes them sensitive to any toxic exposure and suitable for testing material that could interact with human tissues [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, the peptides were tested on neonatal fibroblast cells to evaluate the cytotoxicity of AMPs to human cell lines. The results indicate that the lowest inhibitory concentrations of the fungal growth do not significantly affect human cells\u0026rsquo; viability. Various studies have used plant extracts, rich in peptides, to address food spoilage where the efficacy depends on the extraction method and the plant type[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, AMPs are considered safer alternatives for protecting fruits and vegetables from spoilage[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThis study underscores the potential of antimicrobial peptides (AMPs), specifically Seq-4 and Seq-8, in combating \u003cem\u003eBotrytis cinerea\u003c/em\u003e, a common spoilage fungus affecting high-value crops like strawberries. The in silico-designed AMPs demonstrated significant antifungal activity at lower concentrations than natural peptides such as Mytichitin-CB. The minimum inhibitory concentration (MIC) for Seq-4 and Seq-8 was determined to be 625 \u0026micro;g/ml, notably lower than the MIC of 2000 \u0026micro;g/ml reported for Mytichitin-CB. This highlights the enhanced efficacy of these synthetic peptides.\u003c/p\u003e \u003cp\u003eMoreover, this study employed a comprehensive approach to assess the safety of Seq-4 and Seq-8 at their effective antifungal concentrations, a crucial factor for their potential use in food preservation. The Alamar Blue assay results were reassuring, showing that these peptides do not significantly affect cell viability. This finding suggests that Seq-4 and Seq-8 could be safer alternatives to traditional chemical preservatives, which often pose health risks and reduce nutritional value.\u003c/p\u003e \u003cp\u003eAdditionally, this research contributes to the broader field of agricultural biotechnology by providing insights into the application of synthetic AMPs in food systems. The findings suggest that these peptides could play a role in developing new methods to preserve perishable goods, enhancing food safety, and reducing economic losses due to spoilage. The successful inhibition of \u003cem\u003eB. cinerea\u003c/em\u003e growth by Seq-4 and Seq-8 also opens avenues for further exploration into their application against other spoilage microorganisms.\u003c/p\u003e \u003cp\u003eIn conclusion, the study demonstrates the promise of in silico-designed AMPs in addressing food spoilage and highlights the importance of developing innovative and effective food preservation strategies. Future research should focus on expanding the application of these peptides to a broader range of food products and pathogens, ensuring their safety and efficacy in various food systems. The dual focus on antifungal efficacy and low cytotoxicity positions these AMPs as valuable tools in enhancing food preservation while maintaining safety and quality.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been supported by the by Qatar University QUCG-LARC-22/23-499 Collaborative Grant, by the QUPD-CAS-23-24-491 and by the QNRF UREP28-115-1-025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: [Sergio Crovella]. Material preparation, data collection, and analysis by [Nura A. Mohamed] and [ Shayma A. Osman]. The first draft of the manuscript was written by [ Shayma A. Osman] and all authors commented on previous versions of the manuscript. Review and editing, by [Sergio Crovella], [Nahla Eltai], [Nura A. Mohamed], and [Muhammad Suleman]. Supervision: [Sergio Crovella] and [Nura A. Mohamed]. All authors have read and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that all data are directly reported in the text of our manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAwuchi CG, Ondari EN, Nwozo S, Odongo GA, Eseoghene IJ, Twinomuhwezi H, Ogbonna CU, Upadhyay AK, Adeleye AO, Okpala COR (2022) Mycotoxins\u0026rsquo; Toxicological Mechanisms Involving Humans, Livestock and Their Associated Health Concerns: A Review. Toxins (Basel). doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/toxins14030167\u003c/span\u003e\u003cspan address=\"10.3390/toxins14030167\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarua P, You MP, Bayliss K, Lanoiselet V, Barbetti MJ (2017) A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity. Eur J Plant Pathol 148:139\u0026ndash;150\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheung N, Tian L, Liu X, Li X (2020) The Destructive Fungal Pathogen Botrytis cinerea\u0026mdash;Insights from Genes Studied with Mutant Analysis. Pathogens 9:923\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez de Ullivarri M, Arbulu S, Garcia-Gutierrez E, Cotter PD (2020) Antifungal Peptides as Therapeutic Agents. Front Cell Infect Microbiol. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcimb.2020.00105\u003c/span\u003e\u003cspan address=\"10.3389/fcimb.2020.00105\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua L, Yong C, Zhanquan Z, Boqiang L, Guozheng Q, Shiping T (2018) Pathogenic mechanisms and control strategies of Botrytis cinerea causing post-harvest decay in fruits and vegetables. Food Quality and Safety 2:111\u0026ndash;119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuan Y, Kong Q, Mou H, Yi H (2020) Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Front Microbiol. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2020.582779\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2020.582779\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamal I, Ashfaq UA, Hayat S, Aslam B, Sarfraz MH, Yaseen H, Rajoka MSR, Shah AA, Khurshid M (2023) Prospects of antimicrobial peptides as an alternative to chemical preservatives for food safety. Biotechnol Lett 45:137\u0026ndash;162\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaranth S, Feng S, Patra D, Pradhan AK (2023) Linking microbial contamination to food spoilage and food waste: the role of smart packaging, spoilage risk assessments, and date labeling. Front Microbiol. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2023.1198124\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2023.1198124\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei J, Sun L, Huang S, Zhu C, Li P, He J, Mackey V, Coy DH, He Q (2019) The antimicrobial peptides and their potential clinical applications. Am J Transl Res 11:3919\u0026ndash;3931\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLima PG, Freitas CDT, Oliveira JTA, Neto NAS, Amaral JL, Silva AFB, Sousa JS, Franco OL, Souza PFN (2021) Synthetic antimicrobial peptides control Penicillium digitatum infection in orange fruits. Food Research International 147:110582\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMignone G, Shwaiki LN, Arendt EK, Coffey A (2022) Inhibitory activity of two synthetic Pharabitis nil (L.) antimicrobial peptides against common spoilage yeasts. Applied Food Research. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.afres.2022.100168\u003c/span\u003e\u003cspan address=\"10.1016/j.afres.2022.100168\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoca-Couso R, Flores-F\u0026eacute;lix JD, Rivas R (2021) Mechanisms of Action of Microbial Biocontrol Agents against Botrytis cinerea. Journal of Fungi 7:1045\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaleh I, Abu-Dieyeh MH (2021) Novel Prosopis juliflora leaf ethanolic extract as natural antimicrobial agent against food spoiling microorganisms. Sci Rep 11:7871\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantos-Silva CA dos, Tricarico PM, Vilela LMB, Roldan-Filho RS, Amador VC, d\u0026rsquo;Adamo AP, R\u0026ecirc;go M de S, Benko-Iseppon AM, Crovella S (2021) Plant Antimicrobial Peptides as Potential Tool for Topic Treatment of Hidradenitis Suppurativa. Front Microbiol. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2021.795217\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2021.795217\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma R, Shrivastava S, Kumar Singh S, Kumar A, Saxena S, Kumar Singh R (2022) Deep-AFPpred: identifying novel antifungal peptides using pretrained embeddings from seq2vec with 1DCNN-BiLSTM. Brief Bioinform. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bib/bbab422\u003c/span\u003e\u003cspan address=\"10.1093/bib/bbab422\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSouza PF (2022) Synthetic antimicrobial peptides for controlling fungi in foods. Curr Opin Food Sci. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cofs.2022.100819\u003c/span\u003e\u003cspan address=\"10.1016/j.cofs.2022.100819\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSridhar A, Ponnuchamy M, Kumar PS, Kapoor A (2021) Food preservation techniques and nanotechnology for increased shelf life of fruits, vegetables, beverages and spices: a review. Environ Chem Lett 19:1715\u0026ndash;1735\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Li S, Cao T, Fu X, Yu G (2012) Evaluating biotoxicity with fibroblasts derived from human embryonic stem cells. Toxicology in Vitro 26:1056\u0026ndash;1063\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang H, Zhang C, Li M, Liu C, Wang J, Ou X, Han Y (2022) Antimicrobial Peptides Mediate Apoptosis by Changing Mitochondrial Membrane Permeability. Int J Mol Sci 23:12732\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Wang Y, Jiang H, Song R, Liu Y, Guo H, Meng D (2023) Antimicrobial peptide CB-M exhibits direct antifungal activity against Botrytis cinerea and induces disease resistance to gray mold in cherry tomato fruit. Postharvest Biol Technol 196:112184\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Botrytis cinerea, food spoilage, antimicrobial peptides, fungicides, cytotoxicity.","lastPublishedDoi":"10.21203/rs.3.rs-6060973/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6060973/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBotrytis cinerea, a fungal pathogen known for causing grey mold diseases in post-harvest plant products, presents a serious global concern due to its impact on food safety and economic stability. To address this challenge, various studies have explored the use of safe and effective fungicides to curb fungal growth associated with food spoilage. In this study, antimicrobial peptides (AMPs) designed through computational methods were tested for their antifungal effects on \u003cem\u003eBotrytis cinerea\u003c/em\u003e (\u003cem\u003eB. cinerea\u003c/em\u003e). The tested peptide sequences showed strong antifungal properties, with complete inhibition observed at the highest concentrations tested (2500, 1250, and 625 \u0026micro;g/ml). Additionally, AMP cytotoxicity was assessed through Alamar Blue absorbance readings to evaluate the viability of treated neonatal fibroblast cells. Results showed that the computationally modelled AMPs are effective at a concentration of 312 \u0026micro;g/ml, indicating their potential as an alternative to conventional fungicides in preventing food spoilage. This study emphasizes the potential of bioengineered AMPs to support a safer, more sustainable agricultural practice and address the rising demand for effective biocontrol agents in food preservation.\u003c/p\u003e","manuscriptTitle":"Antimicrobial Peptides Designed in silico: Safeguarding Against Botrytis cinerea and Food spoilage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-01 09:09:28","doi":"10.21203/rs.3.rs-6060973/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cfa84cd6-cafb-4b4f-ac59-fa1d4fd73965","owner":[],"postedDate":"April 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-26T16:38:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-01 09:09:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6060973","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6060973","identity":"rs-6060973","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}