Potential applications of antimicrobial peptides from edible insects in the food supply chain: Uses in agriculture, packaging, and human nutrition

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in: Food Bioscience on December 2024, available at: https://doi.org/10.1016/j.fbio.2024.105396”. 1 Potential applications of antimicrobial peptides from edible insects in the food 1 supply chain: uses in agriculture, packaging, and human nutrition 2 Fernando Rivero-Pino1,2,3*, Maria J. Leon4, Sergio Montserrat-de la Paz1,2 3 1 Department of Medical Biochemistry, Molecular Biology, and Immunology, School of 4 Medicine, University of Seville, Av. Sanchez Pizjuan s/n, 41009, Seville, Spain. 5 2 Instituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del 6 Rocio/CSIC/Universidad de Sevilla, Seville, 41013, Spain. 7 3 European Food Safety Authority, Nutrition and Food Innovation Unit, Novel Foods 8 Team, Parma, Italy 9 Disclaimer: The author F. Rivero-Pino is carrying out a temporal secondment at European Food Safety Authority – EFSA- from June 10 1st, 2024, to May 31st, 2025. The work described in this article is not related to his current work at EFSA, it is published under the 11 sole responsibility of the author and may not be considered as an EFSA scientific output. The views and opinions expressed in this 12 manuscript do not represent those of EFSA. 13 4 Department of Microbiology and Parasitology, School of Pharmacy, University of 14 Seville, C. Profesor Garcia Gonzalez, 41012, Seville, Spain. 15 16 * Corresponding author: 17 Fernando Rivero-Pino ([email protected]) 18 Department of Medical Biochemistry, Molecular Biology, and Immunology, School of 19 Medicine, University of Seville, Av. Sanchez Pizjuan s/n, 41009, Seville, Spain. 20 21 22 23 24 25 26 27 2 ABSTRACT 28 The search for ingredients from novel sources is gaining interest in the framework of 29 promoting a more sustainable food system. The exploration of insects as source of 30 antimicrobial peptides could be useful to find compounds able to inhibit the growth of 31 several microorganisms in different food applications. The purpose of this review is to 32 recapitulate the literature on edible insect protein hydrolysates as a source of 33 antimicrobial peptides, their mechanisms, and their use in the food industry by producing 34 fortified products or edible coatings, and their use in agriculture. Scarce investigation has 35 been carried out in the framework of their incorporation into the food system regarding 36 their antimicrobial properties. Locusta migratoria, Hermetia illucens, and Tenebrio 37 molitor are some of the most studied species. The literature available suggests promising 38 results in areas such as fortification of foods or active packaging, based on the 39 antimicrobial properties they might exert. 40 Keywords: antibacterial, antifungal, antivirus, bioactive peptides, edible insects, food 41 safety, functional ingredients. 42 43 44 45 46 47 48 49 50 51 52 53 54 55 3 1. INTRODUCTION 56 More than ever, a re-evaluation of our present food system by strengthening the resilience 57 and sustainability of the whole food chain in order to address the pressing need to rebuild 58 our relationship with the environment is required. The usage of current dietary sources as 59 well as the exploration of new ones that might contribute to the safe and secure provision 60 of both macro and micronutrients to humans are among the variables that need to be 61 reviewed. Edible insects provide the possibility for a wide variety of meals and food 62 components among the alternative dietary options. Especially in their dried forms, several 63 bug species include notable quantities of proteins, unsaturated fatty acids, minerals, 64 vitamins, and fibre (van Huis et al., 2021; Ververis et al., 2022), which vary depending 65 on feed, growth conditions, insect’s developmental stage, and processing methods, among 66 other factors (Ojha et al., 2021; Ververis et al., 2022). The consumption of insects by 67 humans is widely occurring in countries from Africa, Asia or South America, but it is 68 only being introduced to the European market recently (Boehm et al., 2021; van Huis and 69 Rumpold, 2023). The high protein content and quality of edible insects have gained the 70 attention of the food industry, and in this line, the production of concentrates and isolates 71 as ingredients is starting to be one of the main applications of edible insects. These 72 powders can be used, subsequently, to obtain protein hydrolysates (mixture of peptides 73 produced by hydrolysing a protein) potentially containing bioactive sequences (i.e., 74 bioactive peptides), which are released due to the enzymatic hydrolysis or fermentation 75 process applied and might exert improved techno functional and biological properties 76 compared to the native protein (Aguilar-Toalá et al., 2022; Mendoza-Salazar et al., 2021). 77 Bioactive peptides are short sequences of amino acids that exert a positive impact on 78 different functions or conditions and may ultimately influence on health or in foodstuff, 79 depending on their use (Rivero-Pino et al., 2023a; Rivero-Pino, 2023). Edible insects have 80 been demonstrated to contain bioactive sequences exerting different activities in vitro and 81 in vivo such as antihypertensive, anti-inflammatory, or antidiabetic (Rivero-Pino et al., 82 2021; Teixeira et al., 2023). Among these, antimicrobial peptides (AMPs) have attracted 83 significant attention due to their potent ability to combat bacterial, viral, and fungal 84 infections. Additionally, peptides that have been reported to have antimicrobial action 85 may be employed to stop the spread of food-borne illnesses and food deterioration caused 86 by microorganisms, if those are inhibited (Sharma et al., 2022). This is relevant, in light 87 of the current economic crisis and rising population, as it is imperative to also find 88 4 innovative solutions to the worldwide problem of food waste. Around 1,300 million tons 89 of food are lost or wasted annually, which is about one-third of the food output that is 90 meant for human use (Munesue et al., 2015). Together with this situation, bacterial 91 antimicrobial resistance (AMR) is also considered a major health problem in the current 92 society, especially with pathogens such as Escherichia coli, Staphylococcus aureus, 93 Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and 94 Pseudomonas aeruginosa which were estimated to cause almost 5 million deaths in 2019 95 (Collaborators, 2022). In the food industry, AMPs offer a promising solution for 96 enhancing food safety and extending shelf life by preventing the growth of spoilage and 97 pathogenic microorganisms. Their natural origin and effectiveness at low concentrations 98 make them an attractive alternative to synthetic preservatives, which are often associated 99 with adverse health effects and consumer resistance. Additionally, AMPs can contribute 100 to the development of functional foods that not only provide nutrition but also deliver 101 health benefits such as improved gut health and enhanced immune function. The 102 exploration of insect-derived AMPs, therefore, presents a valuable opportunity for the 103 food industry to innovate in the areas of food preservation and health promotion. 104 Edible insects have been proposed to be an adequate source of bioactive compounds of 105 diverse nature. Their low negative impact on the environment, and the socio-economic 106 impact associated with the consumption of insects and product thereof is gaining interest 107 (Varela-Ortega et al., 2021). Nonetheless, no insect peptides are currently used in the food 108 industry for these purposes. Bioactive peptides from edible insects could be introduced 109 in the food industry to overcome the food spoilage of fresh products, if added as part of 110 the foodstuff, or as coating, avoiding microbial spoilage and/or lipid oxidation. Similarly, 111 the identification of antimicrobial peptides is interesting in the fields of agriculture and 112 livestock, promoting a healthier growth of organisms. On top of that, contrary to most 113 conventional antibiotics, it is widely known that antimicrobial peptides action does not 114 cause the emergence of natural bacterial resistance, or at least the frequency of resistance 115 is thought to be minimal (Dho et al., 2023). Insect-derived antimicrobial peptides (AMPs) 116 are gaining relevance in the scientific community due to their advantages over those from 117 other sources, such as plants or marine organisms. One key reason for their utility is the 118 diversity of insects, which contributes to a broader range of AMPs with distinct 119 mechanisms of action, making them highly adaptable to different microbial threats. In 120 addition, insects have evolved strong immune defenses due to their constant exposure to 121 5 pathogens in various environments, leading to AMPs with potent antimicrobial 122 properties. Comparatively, insect AMPs are often easier to extract and produce, with 123 insect farming and biotechnological processes being more cost-effective and scalable than 124 those for plants or marine organisms. 125 The purpose of this revision is to recapitulate studies on the production of antimicrobial 126 peptides (including antibacterial, antifungal, and antiviral compounds) from edible 127 insects. In this regard, the analytical methods to assess the bioactivity, the mechanisms 128 by which these compounds exert the activity, together with the potential uses of these 129 peptides in the food industry, as functional ingredients, preservatives, or agricultural aids 130 were described. 131 132 2. PRODUCTION AND ANALYSIS OF PEPTIDES 133 2.1. Pre-treatments 134 Generally, protein hydrolysates are obtained from flours, which can be directly obtained 135 by grinding the insect until it becomes a powder, or these can be processed to obtain 136 protein concentrates or isolates. These samples of more or less concentrated protein 137 represent the raw material containing the native proteins of insects. Pre-treatment of 138 native protein prior to the release of bioactive peptides is a common practice being 139 evaluated by researchers. The application of non-thermal technologies such as ultrasound, 140 microwave, or high pressures is gaining interest because it modifies the structure of the 141 proteins, the surface hydrophobicity and the solubility, among others, facilitating the 142 action of proteases and microorganisms to exert their proteolytic activity. 143 Ultrasound works by generating high-frequency sound waves that cause cavitation, where 144 the rapid formation and collapse of microbubbles create localized high temperatures and 145 shear forces. This disrupts the tertiary and secondary structures of proteins, exposing 146 cleavage sites and enhancing the efficiency of enzymatic hydrolysis. Similarly, HPP 147 exerts its effects by applying high hydrostatic pressure (typically between 100 and 600 148 MPa) to disrupt non-covalent bonds within the protein. This pressure-induced unfolding 149 increases protein solubility and exposes hydrophobic regions, promoting enzyme access 150 and peptide release. Other technologies, such as pulsed electric field (PEF) and thermal 151 treatments, also modify protein structures by inducing conformational changes or partial 152 6 denaturation, making them more susceptible to hydrolysis. Collectively, these pre- 153 treatment techniques not only improve the yield of bioactive peptides but also enhance 154 their bioactivity by facilitating the release of peptides with bioactive properties (Dash et 155 al., 2024; Fadimu et al., 2022) A broader understanding of how these pre-treatments 156 influence protein structure and functionality is essential for optimising peptide production 157 and their subsequent health benefits. 158 In this regard, the effect of ultrasound (20 kHz, 15 min, 500 W, pulse duration of 5/5 s on 159 and off) and microwave (10 min at 90 ºC) has been evaluated by Lone et al., (2023a, 160 2023b), as an effective manner to enhance the release of bioactive peptides from crickets 161 (Acheta domesticus) with antioxidant and antimicrobial activity. In the same line, Singh 162 et al., (2023) employed the same pre-treatment to obtain a protein hydrolysate from locust 163 protein (Locusta migratoria), meant to be used as antimicrobial and antioxidant agent in 164 fortified products, as will be described in the corresponding section. In all cases, the 165 improved bioactivity obtained for the hydrolysates when pre-treatment was applied was 166 attributed to conformational changes in the structure of the protein, facilitating the action 167 of the proteases afterwards. 168 However, it has to be noted that research on this topic is only being carried out nowadays, 169 and there are many parameters in these processing techniques to be considered, which 170 should be optimized depending on the properties desired to be optimized. One of the 171 problems for instance that these pre-treatments might overcome is solubility, as insects’ 172 powders have been reported to have poor solubility (Stone et al., 2019), or to improve 173 bioavailability (Hussain et al., 2023). In Figure 1, a graphic representation of the pre- 174 treatment of samples and the production of bioactive is depicted. 175 176 177 178 179 180 181 182 7 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 Figure 1. Schematic representation of the production of bioactive peptides from insect 198 proteins. GID, gastrointestinal digestion. 199 2.2. Release of peptides 200 The release of peptides can be carried out by the action of (food-grade) proteases, which 201 would act on the peptide bonds, specifically, based on their active site; or by the action 202 of microorganisms, whose activity is related to their activity and the proteases that these 203 are able to secrete. Proteases most commonly used in research and in the industry are 204 subtilisin (i.e., Alcalase, (EC 3.4.21.62), trypsin (EC 3.4.21.4), or Flavourzyme 205 (3.4.11.1). Differences in their specificity would imply different profiles of peptides 206 released after their action (Tavano et al., 2018). Particularly, it has been described that 207 trypsin, which acts towards residues of arginine and lysine mainly at its optimal 208 conditions, is an appropriate protease to release antimicrobial peptides (Rivero-Pino et 209 8 al., 2023a). In the case of fermentation, the main advantage is the more economical cost 210 compared to the use of isolated proteases (Nasri et al., 2022), whereas on the contrary, 211 the possibility of generating undesirable compounds (e.g., bacteriocins) is a disavantage. 212 The microorganisms widely used is research to obtain protein hydrolysates are lactic acid 213 bacteria (Lactobacilli species). Generally, fermentation is considered less specific than 214 proteases (Cruz-Casas et al., 2021). A decrease in pH is anticipated due to microbial 215 activity during fermentation, and this lower pH level acts as a barrier against the majority 216 of foodborne pathogens (Kewuyemi et al., 2020). In this latter reference, a descriptive 217 revision of how fermented edible insects contribute to food security can be consulted. In 218 section 3, release of edible-insect AMPs according to the target evaluated are described. 219 2.3. Methods of analysis 220 The in vitro antimicrobial properties of a sample can be assessed using several established 221 techniques. When evaluating insect-derived products such as protein hydrolysates, it is 222 crucial to account for the presence of other nutrients, like proteins and carbohydrates, as 223 these may serve as substrates for microbial growth and could interfere with the 224 antimicrobial evaluation (Moscoso-Mujica et al., 2021). Therefore, assessing the activity 225 of individual peptides alone may not be sufficient if the goal is to use them within a 226 complete food system, where interactions with other food components could either 227 enhance or inhibit their antimicrobial properties. 228 Among the most commonly used methods for assessing antimicrobial activity are disk 229 diffusion, well diffusion, and broth or agar dilution. Disk diffusion involves placing a 230 filter paper disk impregnated with the antimicrobial substance on an agar plate seeded 231 with the test microorganism. The substance diffuses from the disk, creating an inhibition 232 zone around the disk where microbial growth is suppressed. Well diffusion follows a 233 similar principle, where the antimicrobial agent is placed in wells made on an agar plate, 234 and diffusion leads to inhibition zones. These methods are simple and cost-effective, 235 making them useful for preliminary screening. However, they are subject to limitations 236 such as poor reproducibility and limited diffusion of certain antimicrobial agents in the 237 agar medium. 238 Broth and agar dilution methods, on the other hand, allow for a more precise 239 determination of the minimum inhibitory concentration (MIC)—the lowest concentration 240 15 glycoprotein (Ong et al., 2021). Similarly, Gonzalez de-la Rosa et al., (2024), as 435 previously mentioned, identified the peptidome of a T. molitor protein hydrolysate, and 436 based on several in silico analyses, proposed several peptides as antiviral components 437 towards the COVID-19 main protease in the apo state. 438 439 The study of antiviral peptides, particularly anti-COVID peptides, is becoming more 440 popular among researchers. Nonetheless, the majority of the studies done thus far have 441 been done in silico, thus, there is little scientific value in extending the conclusions to 442 actual societal applications. The next stage would include a thorough examination of these 443 peptides' performance in animal tests in order to comprehend the processes by which they 444 may (or cannot) exhibit certain bioactivity. In this sense, carefully planned human trials 445 are required to demonstrate the antiviral effects of these insect-derived peptides. 446 3.4. Mechanisms 447 The mechanisms by which insect peptides obtained by enzymatic hydrolysis or 448 fermentation is still a challenge since every sequence might exert the activity by specific 449 mechanisms. Overall, certain residues at certain positions are associated with bioactivity. 450 On top of that, the membrane of bacteria/fungi has negative charge because of the 451 presence of phospholipids, lipopolysaccharides, or teichoic acids, and it is the main target 452 by which peptides can exert their activity. 453 The efficacy of AMPs is intrinsically linked to their cationic and amphipathic properties, 454 which play crucial roles in their mechanism of action. The cationic nature of these 455 peptides facilitates electrostatic interactions with negatively charged microbial 456 membranes, allowing them to target pathogens. Once bound, the amphipathic structure, 457 characterized by distinct hydrophobic and hydrophilic regions, promotes membrane 458 disruption. This dual nature enables the peptides to insert into the lipid bilayer and form 459 pores, leading to membrane destabilization and cell lysis. Additionally, these properties 460 can enhance the peptides' ability to penetrate bacterial biofilms and interfere with 461 intracellular targets. In this regard, the balance of charge and amphipathicity is critical in 462 defining the bioactivity and specificity of AMPs against a wide range of pathogens. 463 Gonzalez de la Rosa et al., (2024) reported, for antimicrobial peptides obtained from 464 Tenebrio molitor, the correlation between specific molecular characteristics and the 465 16 efficacy of these peptides against various pathogens. One of the main conclusions was 466 that net hydrogen had a significant effect on antifungal capacity. 467 Consequently, the interaction of cationic and amphipathic peptides is more likely to occur 468 towards these structures, because of electrostatic attraction. Some of the hypotheses 469 proposed are membrane thinning or thickening, oxidised lipid targeting, electroporation, 470 or non-lytic membrane depolarization, among others (Magana et al., 2020; Silveira et al., 471 2021). In the case of virus, the mechanisms proposed are related to the inhibition of 472 transcription, viral attachment, and binding with key proteins. Further descriptions of the 473 underlying mechanisms can be consulted elsewhere (Sharma et al., 2022; Shwaiki et al., 474 2021; Silveira et al., 2021; Struyfs et al., 2021; Sultana et al., 2021; Tyagi et al., 2021). 475 Current investigations intend to shorten forms of the peptide, adjust the structure, and 476 assess the effects on the growth of the target microorganisms in order to investigate the 477 impact of the peptide’s spatial structure on its bioactivity. According to current research, 478 surface charge, hydrophobic characteristics, and secondary structural integrity are all 479 crucial for antimicrobial activity (Barashkova et al., 2022). 480 4. USE IN THE FOOD INDUSTRY 481 4.1. Agriculture and livestock 482 Insect production yields can be reduced because of diseases caused by viruses, bacteria, 483 or fungi, as this could adversely affect the production, leading to economic losses. 484 Therefore, antimicrobial peptides could be employed, by over-expressing them in the 485 insects (transformation of the insects by genes of the peptides), to prevent their 486 susceptibility to bacteria and increase their resistance to infectious diseases and control 487 the development of these pathogens (Yashwant et al., 2023; Patyra et al., 2023). In order 488 to address bacterial and fungal diseases, transgenic plants expressing insect peptides can 489 be also used in agriculture (Quah et al., 2023). In this case, it mainly refers to peptides 490 used as defensive agents by insects, as described in section 3.1 (Azmiera et al., 2022; 491 Eleftherianos et al., 2021). Additionally, there are neuropeptides obtained from insects 492 that may provide plants the ability to repel herbivorous insects. These neuropeptides often 493 alter the physiology and behaviour of insects by obstructing their development, which 494 includes growth, reproduction, and energy metabolism (Gäde and Goldsworthy, 2003). 495 17 Nonetheless, the use of antimicrobial peptides obtained by the hydrolysis of edible insect 496 proteins through processing to release low molecular weight peptides is currently scarcely 497 studied to be employed in agriculture as antimicrobial, even when the use of hydrolysates 498 as biostimulats has been reported, improving the productivity of cultivars from insects 499 (Szopa et al., 2023) and from other sources (Rouphael et al., 2021). In this regard, it was 500 shown that a protein hydrolysate from T. molitor could lead to improvements in plant 501 growth (cucumber germination), concerning steam length and root length (Szopa et al., 502 2023). 503 The use of feed additives in animal nutrition, on the other hand, has grown recently in an 504 effort to enhance nutrient utilization, health indicators, and animal performance. In the 505 European Union, eight insect species have already been authorized to be used as feed 506 ingredients for aquaculture organisms, pets, poultry, and pigs (Lourenço et al., 2022). It 507 has been highlighted that the use of antibiotics as feed additives has aided in the 508 development of antimicrobial resistance, which raises rates of morbidity and death among 509 illnesses that were previously treated with antibiotics. Thus, antimicrobial peptides, 510 which contain a variety of biological functions, are an effective way to stop the emergence 511 of resistant microorganisms. Despite the few in vivo studies that have been done, 512 antimicrobial peptides have been proven to be a powerful alternative to antibiotics in 513 animal nutrition since they improve feed efficiency and prevents/treats several animal 514 diseases (Józefiak and Engberg, 2017; Silveira et al., 2021). It has been proposed that 515 using antimicrobial peptides with diverse modes of action could help diminish the risk of 516 the development of resistant bacteria (Quah et al., 2023). 517 The effectiveness of mealworm and super mealworm (Zophobas morio larvae) as oral 518 antibiotics against Salmonella and E. coli infection in broiler chicks was examined by 519 Islam and Yang (2017). After a week, numerous physiological indicators were examined 520 and according to the authors, lower counts of the two bacteria were found in the caecum, 521 although it was not described if those effects were due to the peptides. In the same line, 522 broiler chick survival rate increased in the presence of black soldier fly (H. illucens) 523 larvae (added at 1 to 3% in feed) following experimental Salmonella Gallinarum 524 infection, having measured the viable counts in faecal, liver, spleen, cecum, and bursa of 525 Fabricius (Lee et al., 2018). 526 18 Regarding the impact on microorganisms, several studies have investigated the effects of 527 the ingestion of insect-derived protein and peptides on microbiota (Benzertiha et al., 528 2020; Meyer et al., 2020; Terova et al., 2019). The anti-microbial potential of black 529 soldier fly (H. illucens) in rats, rabbits, and reptiles as a model for humans has also been 530 recently reviewed (Shah and Cetingul, 2022). Similarly, Gasco et al., (2021) summarized 531 the information regarding the impact bioactive components from insects would have on 532 the immune system, microbiota, and resistance against diseases in several animal species, 533 including fish, crustaceans, poultry, pigs, and rabbits. Recently, Kim et al., (2024) 534 reported the potential effects of T. molitor hydrolysates in relation to the immune function 535 and gut microbiota, in cyclophosphamide-induced mice. According to the authors, the 536 administration of the test item led to increased natural killer cell activity and cytokine 537 levels, among other changes, as well as a modification of the gut microbiota, implying a 538 higher relative abundance of Akkermansia and Corynebacterium. 539 Given the variety of the evidence, authors were unable to draw definitive conclusions, but 540 they came to the opinion that the summarized data may be used as a starting point for 541 human studies to study certain health impacts. On top of that, these animal studies are 542 relevant in the framework of employing insects-derived products as feed additives, with 543 potential antimicrobial activity which would contribute to avoiding microbial infections. 544 545 4.2. Functional ingredient 546 Extending the shelf life of food products is one usage for peptides having 547 antibacterial/antifungal action. The proliferation of microorganisms, particularly fungus, 548 inhibits the long-term preservation of fresh goods like bread or necessitates the 549 application of preservatives to improve the product's quality. Antimicrobial peptides 550 might thus be employed to enhance the functioning of these products by limiting their 551 proliferation. In addition to profiling peptides with antimicrobial action in order to 552 maximize the release from the insects, it is critical to assess how stable they are under 553 various environmental factors. The activity of the bioactive peptides can be influenced by 554 a number of variables, such as temperature, pH fluctuations, or salt concentration, as 555 previously stated for Maillard products. Their stability and, thus, bioactivity, as well as 556 whether or not this is maintained when the product is stored, would be determined by how 557 these peptides interacted with the various food matrix components (Rivero-Pino, 2023). 558 19 The antimicrobial effects of fermenting composite meal of sorghum flour substituted with 559 10% or 20% powdered roasted mealworm larvae were reported by Klunder et al., (2012), 560 and the resulting product effectively inactivated the pathogenicity of Enterobacteriaceae 561 while controlling spore-forming bacteria proliferation in the acidic medium. Many studies 562 have been recently published concerning how the incorporation of insect-derived 563 products affects the quality or properties of foodstuffs, but usually microbiological 564 analyses are not included (Kowalski et al., 2022). Other reports include microbiological 565 analysis and have highlighted potential safety issues such as the formation of spore- 566 forming bacteria in cricket-based breads (Osimani et al., 2017) and in doughs and breads 567 fortified with mealworm powder (Roncolini et al., 2019). 568 Dang et al., (Dang et al., 2020) employed peptides (obtained after infection with Musca 569 domestica pupae, not reporting the use of enzymatic hydrolysis of the sample) showing 570 antibacterial properties at concentrations from 0.4 to 0.8 mg/mL as preservative agent in 571 chilled pork. According to the authors, by inhibiting bacterial growth, the shelf life could 572 be improved by up to six days. Similarly, microbiological quality analyses were carried 573 out on cooked meat products fortified with protein from T. molitor and H. illucens, 574 showing no significant differences among the different groups up to 3 weeks of storage. 575 In this case, no processing to release potentially bioactive peptides was carried out 576 (Lemke et al., 2023). 577 In the scope of evaluating antibacterial and antifungal activity of an insect-derived extract 578 as preservative, this was evaluated against E. coli, B. cereus, and S. aureus – leading to 579 MIC of 1 mg/mL), together with Aspergillus flavus, Aspergillus parasiticus, and Pichia 580 anomala. On the other hand, the samples were also able to inhibit beneficial 581 microorganisms (B. subtilis and A. oryzae). In addition, subjecting the extract to diverse 582 conditions of temperature (40 ºC up to 72 h or at 100 ºC for 15 s), salinity (up to 25 %) 583 and pH (4-6) did not affect the activity. However, after hydrolysing the extract with 584 trypsin and chymotrypsin, the antibacterial activity was lost. In the food matrix Ganjang, 585 which was inoculated with harmful bacteria, the original sample was able to inhibit 586 bacterial growth (Hwang et al., 2022). It might be interesting to increase the resistance of 587 the extract, to avoid loss of bioactivity during digestion, if intended to be used as food 588 ingredient. 589 Lone et al., (2023a) employed cricket protein to produce hydrolysates with preservative 590 activity due to their antioxidant and antimicrobial activity. In this regard, after different 591 20 pre-treatments, the hydrolysis was carried out with Alcalase, and the peptides were 592 assessed in a goat meat emulsion as a food-model system (at 2 %). MIC values for the 593 hydrolysates ranged from 90 to 100 mg/mL for E. coli and 30-40 mg/mL for S. aureus. 594 The samples were evaluated during two weeks of storage at 4 ºC for several parameters, 595 including the growth of several microorganisms, leading to an improvement in storage 596 stability according to the authors. In this regard, on days 7 and 14, all hydrolysate- 597 containing emulsion samples had considerably lower total plate counts than control 598 samples. On day 14, the other counts (psychrophilic, coliform, yeast, and mould) were 599 found and followed a similar pattern, with the greatest numbers being found in control 600 samples. These authors evaluated in a similar way, the effect of these cricket protein 601 hydrolysates on microbial and lipid oxidative stability of cheddar cheese, in this case, for 602 a storage period of 3 months (4 ºC), also leading to a positive impact of the inclusion of 603 the peptides on the microbiological count associated with food spoilage, thus, improving 604 the quality of the product (Lone et al., 2023b). In the same line, Singh et al., (2023) 605 employed a protein hydrolysate obtained with Alcalase from locust, as fraction with 606 potential preservative properties in a meat emulsion. The antimicrobial properties of the 607 emulsion, containing the hydrolysate at 1.5 % were assessed by enumerating total plate, 608 psychrophilic, yeast, mould, and coliform by the pour plate technique. Compared to the 609 control, the inclusion of peptides led to an enhanced quality of the product, including a 610 decrease in microbiological spoilage, even after two weeks of storage at 4 ºC. Recently, 611 Gantner et al., (2024) reported the use of cricket powder as a substitute for wheat flour in 612 the production of bread, at 5, 10 anf 15 % of level of replacement. All breads had good 613 microbiological quality after baking and during 7 days of storage, without substantial 614 differences among samples. 615 616 Although no microbiological analyses were carried out, recently the advantages of 617 fermented insects in elaborating bread were also reported (Pérez-Rodríguez et al., 2023). 618 These authors reported that solid-sated fermentation with A. oryzae increased the protein 619 content of T. molitor and S. purpurascens, while also improving texture and rheological 620 and sensorial properties in the case of S. purpurascens. 621 622 21 However, because peptides are highly reactive substances, they may change after 623 interacting with proteins or lipids, which might impact their stability and, in turn, their 624 bioactivity. Encapsulation is seen to be a suitable strategy for overcoming these 625 constraints (Berraquero-García et al., 2023). Additionally, the finished product that 626 contains peptides might exhibit altered behaviour (such as rheology, appearance, texture, 627 etc.). A model of how protein hydrolysates influence the growth of various bacteria and 628 the potential of these substances as preservatives might be created by in-depth 629 investigations examining how these compounds work and, more specifically, the exact 630 sequences responsible for the action. The combination of these antimicrobial peptides 631 with non-thermal techniques offers up new avenues for study that will spur commercial 632 innovation (Rivero-Pino et al., 2023a). When ingredients are added directly to food, the 633 chemical nature of the meal may alter, the client may become dissatisfied due to 634 perception, and there may be health risks if more than the suggested quantity is ingested. 635 The incorporation of peptides into packaging only serves to release active ingredients 636 onto the surface of food or into the headspace (Sultana et al., 2021). 637 4.3. Packaging 638 Directly incorporating antimicrobials into food products can improve food preservation 639 by thwarting microbial contamination (Becerril et al., 2020). However, in light of the 640 quick diffusion of antimicrobial compounds from the surface to the bulk of a product, this 641 technique proved to be of limited efficiency, therefore the food sector sought new and 642 novel approaches. Antimicrobial packaging decreases, delays, or even suppresses 643 microbial development through direct or indirect contact with the packed food. Then, the 644 antimicrobial properties of peptides can also be used to build food packaging, with the 645 aim of introducing them into the food matrix to increase the shelf-life. 646 Weng et al., (2023) recently reviewed about insect-derived materials for food packaging, 647 but no specific information about their production in relation to peptides was discussed. 648 Similarly, a specific review of antimicrobial peptides from H. illucens to be used as food 649 packaging is also available (Sultana et al., 2021). Overall, most of the reports are focused 650 on the use of chitin or chitosan, rather than peptides, for the production of the films. On 651 top of that, the use of the protein fraction has also been investigated, but usually the 652 antimicrobial activity has not been investigated, but the focus was on the mechanical 653 properties (Weng et al., 2023). In fact, protein hydrolysates cannot be used as the sole 654 22 material for film formation by self-crosslinking due to their low molecular weight (Wang 655 et al., 2020). 656 A food packaging might be considered as (bio)active when one or more of the compounds 657 in its composition exert one or more activities to improve their performance, such as 658 antimicrobial or antioxidant properties. These compounds might also be encapsulated, 659 facilitating a gradual release of the active molecules responsible for the activity 660 maintained for longer times (Becerril et al., 2020; Jamróz et al., 2021). There are three 661 main mechanisms by which antimicrobial peptides are understood in the context of an 662 active packaging, including their incorporation, their release and the interaction and 663 inhibition system (Sultana et al., 2021). 664 Zhang et al., (2022a) aimed to manufacture an edible film by solvent casting, using 665 Locusta migratoria, evaluating the influence of the protein content as well as the glycerol 666 and pH. These authors reported the stability of the product due to hydrogen bonds but did 667 not carry out an antimicrobial test. However, these same authors lately reported the 668 production of a composite material obtained with this same source of protein, plus a soy 669 protein isolate, which as a blend was studied by solution casting using xylose as a 670 crosslinker and cinnamaldehyde as an antimicrobial agent. In fact, on top of the objective 671 of enhancing mechanical properties, it was reported the resistance to microorganisms 672 such as E. coli and S. aureus, activity attributed to the content of cinnamaldehyde, 673 although it might be possible that the protein fraction is also contributing to this 674 bioactivity (Zhang et al., 2022b). In the same line, these authors also developed a 675 grasshopper (Locusta migratoria) protein/soy protein isolate/pullulan ternary mixture, 676 together with a hesperidin derivative aimed to be used as antimicrobial edible film. The 677 authors characterized physic-chemically the product obtained and reported antibacterial 678 activity against S. aureus (ATCC 6538) and E. coli (ATCC 25922), using the inhibition 679 zone method, which was higher in the case of S. aureus (Zhang et al., 2023). 680 The available literature suggests a comprehension of the mechanisms by which these 681 protein-derived sequences might exert the antimicrobial activity as edible coating, 682 including the inhibition of the microorganism’ growth and the interaction with the 683 nutrients in the food (Figure 2). 684 In the formulation of an active packaging, some of the challenges to be encountered are 685 the compatibility of the peptides within the packaging, considering the other components, 686 23 and the mechanical properties they would have. In addition to that, the compatibility with 687 the food, as these peptides might interact on the surface with the components (Sultana et 688 al., 2021). Also, depending on the mechanisms, whether the peptide is intended to be 689 released, it has to be characterized and ensured, even after the potential treatments the 690 complete packaging with the food would have to go through. To sum up, different 691 biological and technical features would be impacted by the effects of various components, 692 including these peptides, used in the formulation of edible films, which would then have 693 an impact on the product that would be packaged. A bioactive edible package must take 694 into account a wide range of factors, which makes it now one of the most difficult tasks 695 facing the food business. 696 697 Figure 2. Schematic representation of antimicrobial mechanisms of packaging containing 698 bioactive peptides from insect proteins for food applications. 699 700 4.4. Human nutrition 701 Edible insect AMPs could also emerge as a promising frontier in human nutrition, 702 offering a novel solution to the growing challenges of food security and antibiotic 703 resistance. These peptides could potentially enhance immune functions and improve gut 704 24 health. As the global population continues to rise, exploring the integration of insect 705 AMPs in food systems could revolutionize our approach to nutrition and health, providing 706 an innovative strategy to address some of the pressing issues in contemporary food 707 science and medicine (Patyra et al., 2023) However, the number of human studies dealing 708 with edible insect consumption reported is scarce (Rivero-Pino et al., 2024). 709 However, other studies have evaluated for instance, the biological activity of gluten-free 710 bread enriched with cricket powder, in relation to the gut microbiota, which was isolated 711 from the faecalis of a mature person was introduced into the experimental model. These 712 authors found that low concentrations of insect flour did not inhibit the growth of 713 microflora, but as the amount of cricket flour in the bread increased, so did the levels of 714 antimicrobial compounds (Kowalczewski et al., 2021). In the same line, antimicrobial 715 peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by 716 suppressing viral gene expression (Patyra et al,, 2023). 717 Although numerous insect AMPs have been synthesized and tested against bacterial 718 strains, there are few clinical studies, particularly regarding their anticancer properties. 719 For instance, two peptides from the rhinoceros beetle (Oryctes rhinoceros) have shown 720 promising results: the defensin ALYLAIRRR-NH2 significantly inhibited S. aureus 721 methicillin-resistant in both in vivo and in vitro studies, while the D-peptide B, an 722 anticancer peptide, disrupted mouse myeloma cells in vitro without affecting normal 723 leukocytes. Additionally, Pierisin-1, an AMP from Pieris rapae, has demonstrated 724 anticancer activity by inducing apoptosis and cytotoxicity in various mammalian cancer 725 cell lines, including lung, renal, colorectal, bladder, cervical, and liver cancer, through 726 mono-ADP-ribosylation of DNA (Manniello et al., 2021). 727 Then, although not directly linked to insect peptides, it is interesting to mention the 728 double-blind, randomised crossover trial in healthy adults (n=20, age: 26.45 ± 6.33 years 729 old, 45% males) carried out by Stull et al., (2018) to assess whether ingesting for 6 weeks, 730 25 g/day of whole cricket powder could affect human gut microbiota. It was indicated an 731 enhancement in the growth of the probiotic bacterium, Bifidobacterium animalis and a 732 reduction of plasma Tumour Necrosis Factor α (TNF-α), which is an indicator of 733 improved gut health. 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