Antimicrobial compositions with hermetia extract and uses thereof

Abstract

The present invention relates to compositions comprising a Hermetia illucens extract for use as antimicrobial, antifungal, antibacterial, antiseptic or disinfecting agent, methods of preparation of such extracts and uses thereof. The composition of the invention shows efficient growth inhibitory activity against both gram negative and gram positive bacteria, and fungi.

Description

[0001] TITLE

[0002] ANTIMICROBIAL COMPOSITIONS WITH HERMETIA EXTRACT AND USES THEREOF

[0003] DESCRIPTION

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to compositions comprising a Hermetia illucens extract for use as an antimicrobial, antifungal, antibacterial, antiseptic or disinfecting agent, methods of production of such extract and uses thereof.

[0006] BACKGROUND

[0007] Drug resistance and especially antibiotic resistance has been identified as one of the most challenging problems facing medical research today. This is due to the rapid rate at which microorganisms, affecting humans, animals, insects and plants are evolving in order to survive the onslaught of antibiotics since the discovery of penicillin in 1928. It has been estimated that every year in Europe over 25,000 people die due to a multidrug resistant microorganism infection and the European Union spends over 1.5 billion euro annually to combat this issue which is progressively increasing. Moreover, worldwide honeybee population is in steady decline due to many hazards such as pesticides, climate change and infections like the one caused by Paenibacillus larvae (Mahdi and Fisher, 2018). Since antibiotics have been prohibited from use in apiculture, there are no registered substances to protect the bee larvae from the above worldwide spread endospore forming bacterium in the European Union. Similarly, other pathogens like Salmonella spp., Listeria monocytogenes, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and many other bacteria, constitute threats for the global health.

[0008] Escherichia coli (E. coli) is a gram negative, rod-shaped anaerobic bacterium with a rapid reproductive capacity. E coli is commonly found in the human gastrointestinal tract and can cause a variety of health problems in the event of overpopulation. E. coli is most commonly responsible for urinary tract infections and is the bacterium commonly responsible for diarrhoea in people travelling long distances. E. coli 'is a common pathogen in foodborne infections, which can have severe health consequences particularly on young children.

[0009] Pseudomonas aeruginosa (P. aeruginosa) is an environmental gram negative bacterium very often identified in lung infections of patients with cystic fibrosis. Chronic infection occurs in over 60% of adults with cystic fibrosis and it is associated with accelerated disease progression and increased mortality. Staphylococcus aureus (Staph, aureus) is a gram positive bacterium that can be found in the 25% of humans and it can commonly cause illness through direct infection or through toxins produced by the bacterial cells. Staph, aureus can cause a variety of community and hospital-associated pathologies, such as bacteraemia, sepsis, endocarditis, pneumonia, osteomyelitis, arthritis and skin diseases. Staph, aureus has also become a much more threating for human health as the antibiotic resistant strain known as MRSA (methicillin resistant Staphylococcus aureus) is extremely difficult to treat. MRSA can produce high rates of morbidity and mortality and can cause metastatic or complicated infections such as infective endocarditis or sepsis. MRSA is responsible for most global Staph, aureus bacteraemia cases and compared with methicillin-sensitive Staph, aureus, MRSA infection is associated with poorer clinical outcomes. Staph, aureus virulence is affected by the unique combination of toxin and immune-modulatory gene products.

[0010] Similarly, some species of fungi are naturally resistant to certain types of antifungal drugs. For example, the drug fluconazole does not work against infections caused by the fungus Aspergillus, a type of mould found throughout the environment. Resistance can also develop over time when fungi are exposed to antifungal drugs. This resistance can occur when antifungal drugs are used to treat sick people, especially if the drugs are used improperly (for example, when dosages are too low or when treatment courses are not long enough). Additionally, use of fungicides in agriculture to prevent and treat fungal diseases in crops can also contribute to resistant disease in people. For example, when Aspergillus found in the environment is exposed to fungicides, which are similar to medical antifungal drugs, the Aspergillus can become resistant to the drugs used to treat infections in people. Taking into consideration the fact that all organisms are affected by life threatening, microorganisms- related infections and the drawbacks of currently used antimicrobial agents, there is an urgent need for new antimicrobial, antifungal, antiseptic or disinfecting agents that can overcome such drawbacks.

[0011] SUMMARY

[0012] The present invention relates to compositions comprising a Hermetia illucens extract for use as antimicrobial, antifungal, antibacterial, antiseptic or disinfecting agent, methods of production of such extracts and uses thereof.

[0013] In one aspect the current invention relates to a composition comprising a Hermetia illucens larval extract having a concentration of about 0.3 to 4% w / v.

[0014] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total nitrogen concentration of about 6%.

[0015] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total fatty acid concentration of about 525 mg / g of drv extract In one embodiment said composition comprising a Hermetia illucens larval extract contains a total fatty acid concentration of about 525 mg / g of dry extract, a monounsaturated fatty acids concentration of about 76 mg / g of dry extract, a polysaturated fatty acids concentration of about 90 mg / g of dry extract, and a saturated fatty acids concentration of about 359 mg / g of dry extract.

[0016] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total nitrogen concentration of about 6% and a total fatty acid concentration of about 525 mg / g of dry extract.

[0017] In another aspect the present invention relates to a method of preparation of a Hermetia illucens larval extract comprising the steps of

[0018] -washing and drying preferably by lyophilization of Hermetia illucens larvae

[0019] -freezing and powdering of said dried larvae

[0020] -mixing of said frozen and powdered larvae with a 9:1 ethanol: water mixture

[0021] -filtering of said larvae and ethanol:water mixture under vacuum

[0022] -collection of the filtrate

[0023] -mixing the solid residue with a water- ethanol mixture as previously

[0024] -filtering as previously

[0025] -collection of the second flitrate as previoulsy

[0026] -mixing of the two flitrates

[0027] -concentrating said mixture of titrates preferably using rotary vacuum evaporation

[0028] -cooling of the resulting concentrate

[0029] -drying of said concentrated liquid preferably by lyophilization until it is solidified

[0030] -homogenization of said solid larval extract and suspension in Tween 80 or dimethyl sulfoxide (DMSO).

[0031] In one embodiment said method comprises the following steps

[0032] -washing of Hermetia illucens larvae twice with tap water and once with deionized water,

[0033] -drying preferably by lyophilization of said washed larvae and freezing at -20 °C

[0034] -powdering of said frozen larvae

[0035] -mixing of said powdered larvae with a 9:1 ethanol: water mixture and stirring at ambient temperature for 24 hours

[0036] -filtering of said larvae and ethanol:water mixture under vacuum

[0037] -collection of the filtrate and cooling at 4°C

[0038] -mixing the solid residue with water- ethanol mixture as previously

[0039] -filtering as previously

[0040] -collection of the second filtrate as previously

[0041] -mixing of the two filtrates

[0042] -concentrating said mixture of filtrates preferably using rotary vacuum evaporation at 25-35 °C. -cooling the resulting concentrate to -20°C

[0043] -drying of said concentrated liquid preferably by lyophilization until it is solidified

[0044] -homogenization of said solid larval extract and suspension in Tween 80 or dimethyl sulfoxide (DMSO)

[0045] In one embodiment said method comprises the following steps

[0046] -wasing of 800 g of Hermetia illucens larvae twice with tap water and once with deionized water

[0047] -drying preferably by lyophilization of said washed larvae and freezing at -20°C until used.

[0048] -powdering of 20 g of said frozen larvae

[0049] -mixing of said powdered and frozen larvae with 200 mL of a 9:1 ethanol: water mixture and stirring at ambient temperature for 24 hours

[0050] -filtering of said larvae and ethanol:water mixture under vacuum

[0051] -collection of the filtrate and cooling at 4°C

[0052] -mixing the solid residue with 200 mL of water- ethanol mixture for 24 hours as previously

[0053] -filtering as previously

[0054] -collection of the second filtrate as previously

[0055] -mixing of the two filtates

[0056] -concentrating said mixture of filtrates preferably using rotary vacuum evaporation at 25-35 °C

[0057] -cooling of the resulting concentrate to -20°C

[0058] -drying of said concentrated liquid preferably by lyophilization until it is solidified

[0059] -homogenization of said solid larval extract and suspension in Tween 80 1 1 .86 %w / w or Dimethyl sulfoxide (DMSO) 12.46 % w / w.

[0060] In a further aspect the present invention relates to a composition comprisning a Hermetia illucens larval extract directly obtainable or obtained by a method of the invention.

[0061] In an even further aspect the present invention relates to a use of a composition of the invention as an antimicrobial, or antibacterial or antifungal or antiseptic or disinfecting agent.

[0062] In one embodiment the present invention relates to a use of an extract of the invention for the preparation of an antimicrobial, or antibacterial or antifungal or antiseptic or disinfecting composition.

[0063] BRIEF DESCRIPTION OF THE DRAWINGS iqure 1 Changes in optical density (600 nm) of Salmonella typhimurium population in the presence of a BSF larval extract. A1 represents the highest concentration in the BSF larval extract which is successively diluted to A11 . A12 represents the control growth of S. typhimurium. iqure 2 Changes in optical density (600 nm) of Staphylococcus aureus MRSA population in the presence of a BSF larval extract. A1 represents the highest concentration in the BSF larval extract which is successively diluted to A11 . A12 represents the control growth of Staph, aureus. iqure 3 Paenibacillus larvae grown on MYPGP agar and disk diffusion assay applied. T80 corresponds to Tween 80 and BSFT refers to BSF extract diluted to Tween 80.

[0064] Figure 4 Changes in optical density (600 nm) of Paenibacillus larvae population in the presence of a BSF larval extract. A1 represents the highest concentration in the BSF larval extract which is successively diluted to A11 . A12 represents the control growth of P. larvae. ure 5 Inhibition of fungal growth in PDA comprising dilution A1 of the BSF extract over 12 days in comparison to control plates which comprising Tween 80. The measurements were performed on the 3rd, 5th, 7th and 12th day after the fungus initial inoculation. Data shown represent mean values (SD) of the diameter length of the mycelium (comparing to the diameter of the plate, %) of 3 Petri dishes and letters indicate statistically significant differences among treatments, based on Tukey’s test at p = 0.05. For every bacterium tested, 1stcolumn represents 3 days of inoculation, 2ndcolumn 5 days of inoculation, 3rdcolumn 7 days of inoculation, 4thcolumn 9 days of inoculation and 5thcolumn 12 days of inoculation. Fungi that were tested: Botrytis cinerea (Botrytis) Fusarium oxysporum (Fox), Rhizoctonia solani (Rhizoct), Alternaria alternata (Alt), Thielaviosis basicola (Thiel), Verticillum dahliae (Vd), and Pythium sp. iqure 6 Exemplary Petri dishes with Fusarium oxysporum culture at one side and 3MM filter paper discs placed on top, subsequently inoculated with 10 pL of BSF larval extracts (dilution A1 , A2, A3) and with Tween 80 as a control.

[0065] DETAILED DESCRIPTION

[0066] It is a subject of the present disclosure to provide compositions comprising a Hermetia illucens extract for use as antimicrobial, antifungal, antibacterial, antiseptic or disinfecting agents, methods of production of such extracts and uses thereof.

[0067] Hermetia illucens (Black Soldier Fly - BSF) is a fly belonging to Stratiomyidae family (Diptera order). As all the members of Diptera, it is an holometabolous insect, with egg, larva, prepupa, pupa, and adult as the developmental cycle stages. Adults can feed with liquids, but they mainly consume reserves accumulated exclusively in the larval stage. Larvae is the only stage of the insect that can consume food, such as various rotten organic material.

[0068] The diverse range of research activity associated with this insect has revealed that its utility extends beyond feed production (Kaczor et al. 2023). BSF possesses numerous attributes and qualities that hold potential interest across various industries. These include its applicability in biomass utilization, as a source of chitin and chitosan, for biogas and biodiesel production, in entomoremediation, as well as its waste products show promise as fertilizers. The production of new generation antimicrobial agents is crucial, since many chemical compounds, antibiotics, and other substances, which are used as therapeutical and disinfecting agents are being withdrawn the last few years due to dangerous side effects. To add on this, microbes that have been extensively exposed to biocides very often show high resistance.

[0069] The problem becomes more crucial in the plant protective microbicide industry, due to the European Commission Farm to Fork and Biodiversity Strategies, which set two key targets for pesticides: Target 1 : to reduce by 50% the use and risk of chemical pesticides by 2030 and Target 2: to reduce by 50% the use of more hazardous pesticides by 2030. Therefore, the development of antimicrobial agents having highly reduced or no adverse effect to organisms and are enviromental friendly has become of utmost importance.

[0070] This problem is solved by the composition comprising a Hermetia illucens extract of the present invantion. The inventors of the present invention have surprisingly observed that a composition comprising extract of larvae of Hermetia illucens shows growth inhibition for Gram-positive and Gram-negative bacteria along with pathogenic fungi.

[0071] Notably, it was experimantally shown that the BSF larval extract of the invention has minimal growth inhibitory activity against beneficial plant bacterial strain and thus does not disrupt the beneficial microbiome of the plant.

[0072] The utilization of BSF extracts aligns with the growing consumer demand for organic and sustainable products, as these extracts offer a natural alternative to synthetic chemicals. This trend supports environmentally conscious practices in agriculture and apiculture, fostering healthier ecosystems and contributing to food security.

[0073] In the present invention, for the preparation of the Hermetia illucens larval extract a 9:1 ethanol:water mixture was chosen. The choice of polar solvents, such as ethanol, can lead to the recovery of both lipids / and polar organic compounds, while the addition of water favors the recovery of water-soluble bioactive molecules and lipids via emulsification (Feng et al., 2020). The use of polar solvents, such as methanol, was avoided due to its high toxicity. Preliminary experiments on the use of isopropanol as an extraction solvent showed high selectivity of the extract only on Gram-negative bacteria.

[0074] In one aspect the current invention relates to a composition comprising a Hermetia illucens larval extract.

[0075] In one embodiment said composition comprising a Hermetia illucens larval extract has a concentration of about 0.3 to 4 w / v%.

[0076] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total nitrogen concentration of about 6%.

[0077] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total nitrogen concentration of about 6 %, a non-protein nitrogen concentration of about 1.9%, a higher protein nitrogen concentration of about 5.1 %, a crude protein concentration of about 31 .1 %, a true protein concentration of about 22.3% and an ash concentration of about 18 %.

[0078] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total fatty acid concentration of about 525 mg / g of dry extract.

[0079] In one embodiment said composition comprising a Hermetia illucens larval extract contains a monounsaturated fatty acids concentration of about 76 mg / g of dry extract.

[0080] In one embodiment said composition comprising a Hermetia illucens larval extract contains a polysaturated fatty acids concentration of about 90 mg / g of dry extract.

[0081] In one embodiment said composition comprising a Hermetia illucens larval extract contains a saturated fatty acids concentration of about 359 mg / g of dry extract.

[0082] In one embodiment said composition comprising a Hermetia illucens larval extract contains a lauric acid concentration of about 245 mg / g of dry extract.

[0083] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total fatty acid concentration of about 525 mg / g of dry extract, a monounsaturated fatty acids concentration of about 76 mg / g of dry extract, a polysaturated fatty acids concentration of about 90 mg / g of dry extract, and a saturated fatty acids concentration of about 359 mg / g of dry extract.

[0084] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total nitrogen concentration of about 6% and a total fatty acid concentration of about 525 mg / g of dry extract.

[0085] In one embodiment said composition comprising a Hermetia illucens larval extract contains a total nitrogen concentration of about 6 %, a non-protein nitrogen concentration of about 1 .8%, a higher protein nitrogen concentration of about 4.5%, a crude protein concentration of about 29.9%, a true protein concentration of about 21.5% an ash concntration of about 19 %, a total fatty acid concentration of about 525 mg / g of dry extract, a monounsaturated fatty acids concentration of about 76 mg / g of dry extract, a polysaturated fatty acids concentration of about 90 mg / g of dry extract, and a saturated fatty acids concentration of about 359 mg / g of dry extract.

[0086] In another aspect the present invention relates to a method of preparation of a Hermetia illucens larval extract comprising the steps of

[0087] -washing and drying preferably by lyophilization of Hermetia illucens larvae

[0088] -freezing and powdering of said dried larvae

[0089] -mixing of said frozen and powdered larvae with a 9:1 ethanol: water mixture

[0090] -filtering of said larvae and ethanol:water mixture under vacuum -collection of the filtrate

[0091] -mixing the solid residue with a water- ethanol mixture as previously

[0092] -filtering as previously -collection of the second flitrate as previoulsy

[0093] -mixing of the two flitrates

[0094] -concentrating said mixture of titrates preferably using rotary vacuum evaporation

[0095] -cooling of the resulting concentrate

[0096] -drying of said concentrated liquid preferably by lyophilization until it is solidified

[0097] -homogenization of said solid larval extract and suspension in Tween 80 or dimethyl sulfoxide (DMSO).

[0098] In one embodiment said method comprises the following steps

[0099] -washing of Hermetia illucens larvae twice with tap water and once with deionized water,

[0100] -drying preferably by lyophylisation of said washed larvae for and freezing at -20°C

[0101] -powdering of said frozen larvae

[0102] -mixing of said powdered larvae with a 9:1 ethanol: water mixture and stirring at ambient temperature for 24 hours

[0103] -filtering of said larvae and ethanol:water mixture under vacuum

[0104] -collection of the filtrate and cooling at 4°C

[0105] -mixing the solid residue with water- ethanol mixture as previously

[0106] -filtering as previously

[0107] -collection of the second filtrate as previously

[0108] -mixing of the two filtrates

[0109] -concentrating said mixture of filtrates, preferably using rotary vacuum evaporation, at 25-35 °C.

[0110] -cooling the resulting concentrate to -20°C

[0111] -drying of said concentrated liquid preferably by lyophilization until it is solidified

[0112] -homogenization of said solid larval extract and suspension in Tween 80 or dimethyl sulfoxide (DMSO)

[0113] In one embodiment said method comprises the following steps

[0114] -wasing of 800 g of Hermetia illucens larvae twice with tap water and once with deionized water

[0115] -drying preferably by lyophilization of said washed larvae and freezing at -20°C until used.

[0116] -powdering of 20 g of said frozen larvae

[0117] -mixing of said powdered and frozen larvae with 200 mL of a 9:1 ethanol: water mixture and stirring at ambient temperature for 24 hours

[0118] -filtering of said larvae and ethanol:water mixture under vacuum

[0119] -collection of the filtrate and cooling at 4°C

[0120] -mixing the solid residue with 200 mL of water- ethanol mixture for 24 hours as previously

[0121] -filtering as previously

[0122] -collection of the second filtrate as previously

[0123] -mixing of the two filtates -concentrating said mixture of filtrates, preferably using rotary vacuum evaporation, at 25-35 °C -cooling of the resulting concentrate to -20°C

[0124] -drying preferably by lyophilization of said concentrated liquid until it is solidified -homogenization of said solid larval extract and suspension in Tween 80 11 .86 %w / w or dimethyl sulfoxide (DMSO) 12.46 % w / w.

[0125] In a further aspect the present invention relates to a composition comprisning a Hermetia illucens larval extract directly obtainable or obtained by a method of the invention.

[0126] In an even further aspect the present invention relates to a use of a composition of the invention as an antimicrobial, or antibacterial or antifungal or antiseptic or disinfecting agent.

[0127] In one embodiment the present invention relates to a use of an extract of the invention for the preparation of an antimicrobial, or antibacterial or antifungal or antiseptic or disinfecting composition.

[0128] In the above description, an embodiment is an example or implementation of the invention. The various appearances of "one embodiment”, "an embodiment", "certain embodiments" or "some embodiments" do not necessarily all refer to the same embodiments. Although various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be implemented in a single embodiment. Certain embodiments of the invention may include features from different embodiments disclosed above, and certain embodiments may incorporate elements from other embodiments disclosed above. The disclosure of elements of the invention in the context of a specific embodiment is not to be taken as limiting their use in the specific embodiment alone. Furthermore, it is to be understood that the invention can be carried out or practiced in various ways and that the invention can be implemented in certain embodiments other than the ones outlined in the description above.

[0129] Definitions

[0130] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.

[0131] The term “antimicrobial” in the context of the present disclosure shall refer to any agent, compound, molecule or composition that shows inhibition or elimination of growth against microrganisms including bacteria, algae, fungi and archaea,

[0132] The term “antifungal” in the context of the present disclosure shall refer to any agent, compound, molecule or composition that shows inhibition or elimination of growth of any species of fungi.

[0133] The term “antibacterial” in the context of the present disclosure shall refer to any agent, compound, molecule or composition that shows inhibition or elimination of growth of any species of bacteria. The terms “microbicide” and “antimicrobial” in the context of the present disclosure are used interchangeably.

[0134] The terms “bactericide” and “antibacterial in the context of the present disclosure are used interchangeably.

[0135] The terms “fungicide” and “antifungal” in the context of the present disclosure are used interchangeably.

[0136] The term “lyophilization” refers to a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; freezing, primary drying (sublimation), and secondary drying (desorption).

[0137] The term “plant beneficial bacteria” in the context of the present disclosure refers to endophytic bacteria that thrive inside plants and can improve plant growth under normal and challenging conditions. They can benefit host plants directly by improving plant nutrient uptake and by modulating growth and stress related phytohormones.

[0138] The term “extraction” in the context of the present disclosure refers to the process of selectively removing a compound of interest from a mixture using a solvent.

[0139] The term “extract” in the context of the present disclosre refers to a product of an extraction process. The term “about” or “approximately” means the mentioned value + / -10%, for example about 10 shall mean 9 to 11 .

[0140] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0141] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention using this disclosure as a guide. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

[0142] EXAMPLES

[0143] Starting materials and reagents disclosed below are known to those skilled in the art, are commercially available and / or can be prepared using well-known techniques.

[0144] Materials

[0145] Larvae- Insect rearing Insects for the present study were obtained from a BSF stock colony established in the Laboratory of Agricultural Zoology and Entomology of the Agricultural University of Athens, Greece. Initial insects were purchased from “lllucens GmbH” (Ahaus, Germany) in March 2019. BSF larvae diets consisted of chicken feed Fyrko No3 (Fyrko S.A., Corinth, Greece) with a 70% moisture content achieved by addition of water. The experimental procedure and calculations were based on established protocols described in detail by Bosch et al. (2019). Baseline larvae production was initiated by placing eggs, which were obtained from the maintained colony after a 6 h egg laying interval, in plastic containers with 1 Kg moistened chicken feed (reference feed) and transferring them in a controlled climate chamber at 30°C, 70% R.H. and 24 D photoperiod. Visual observation for pre-pupae and 6th instar larvae started from sixth day and continued in 1-2 day intervals. Pre-pupae and larvae were harvested and washed thoroughly under running lukewarm water to remove feed and fecal residues from their bodies, while their integument was dried with paper tissue. Then, samples were kept under refrigeration until chemical analyses.

[0146] Chemicals and Standards

[0147] Analytical grade ethanol, used for extraction of samples, and HPLC grade dimethyl sulfoxide (DMSO) were obtained from Fisher Chemicals (Hampton, NH, USA). Tween 80 was purchased from Merck (Oakville, ON, Canada). For the quantification of fatty acids, a mixture of their methyl ester standards (i.e., Supelco 37 Component FAME Mix) was obtained from Sigma-Aldrich. Potassium hydroxide (KOH), hydrochloric acid (HCI) and boron trifluoride (BF3, 14% in methanol) were also purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0148] Animal pathogenic and potential pathogenic bacteria

[0149] Several microorganisms, all collected by Laboratory of Microbiology and Biotechnology of Foods (LMBF) culture collection were used; in particular, Staphylococcus aureus ATCC 6538, Staphylococcus aureus MRSA B410 strain COL, Salmonella enterica DT 193 multi drug resistant, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa HER 1018, Listeria monocytogenes NCTC 10527, Escherichia coll O157:H7 VT1 and VT2 vero cytoxin producer NCTC 12079, Vibrio campbellii BB 120, Yersinia enterocolitica CITY 650, Serratia marcescens B152, Paenibacillus larvae LMG 9820, Bacillus cereus LMG 06910, and Staphylococcus epidermidis ACA- DC 4057.

[0150] The culture media used were Luria Bertani (Invitrogen 12780052), Tryptone Soya (Oxoid CM 0129R), MYPGP (Graaf et al. 2013), both agar and broth. All bacterial broth cultures were carried out at respectively optimum conditions that is, temperature 37 °C for 18-24 h, with the exception of P. larvae, and V. campbelli that were cultured at 25-28 °C for 24-48 h and re-cultured for 18h on broth media at the above temperatures. 1 mL of each culture serial diluted on 9 mL Ringer tubes and 0.1 mL of diluted bacterial cells were spread on culture media agar and incubated at optimal conditions. The population was expressed as CFU*mL1and the final concentration estimated to be 5*105CFU*mL-1.

[0151] Plant pathogenic potential pathogenic and beneficial microbes

[0152] Several phytopathogenic fungi were used in this study. All these fungal strains have been isolated from infected plant tissues and belong at the Culture Collection of Laboratory of Plant Pathology, Agricultural University of Athens, and stored at 4 °C until later use. These fungi are the following: Fusarium oxysporum, Verticillum dahliae, Rhizoctonia solani, Alternaria alternata, Thielaviosis basicola, Botrytis cinerea and an oomycetal strain Pythium sp.

[0153] The culture media used for fungi growth was Potato Dextroze Agar (PDA). All fungi cultures were carried out at respectively optimum conditions. Fresh Petri dishes with PDA with the fungi inoculum were incubating approximately 7 days at 25°C before used.

[0154] Phytopathogenic bacteria and beneficial bacterial strains

[0155] Phytopathogenic bacteria: Pseudomonas syringae pv tomato DC3000 (Gizjen, 2008), Pseudomonas syringae pv lachrymans 312, Pseudomonas syringae pv lachrymans 355, Pseudomonas syringae pv savastanoi 455 (Venieraki et al, 2014).

[0156] Beneficial bacterial strains with antifungal activity: Bacillus halotolerance Cal.1.30, Bacillus halotolerance Hil4, Bacillus velezensis BveH (Thomloudi et al., 2021 ; Nifakos et al., 2021 ; Tsalgatidou et al., 2022; Tsalgatidou et al., 2023), and a non-phytopathogenic bacterial strain Escherichia coli BW25113 (Datsenko and Wanner, 2000; Baba et al., 2006).

[0157] Strains Cal.1.30, Hil4, BveH have been isolated from infected or healthy plant tissues and all stored at -80 °C. The culture media that used in this study were Nutrient agar (NA, 1 %) and broth. All strains cultured for 24-48 h and re cultured for 18h on broth media at 28°C. The population was expressed as CFU*mL1and the final concentration estimated to 5*105CFU*mL1.

[0158] Data Analysis

[0159] Data were statistically analyzed using IBM SPSS Statistics for Windows, version 25 (IBM Corp., Armonk, NY, USA). Statistical analysis was performed with ANOVA followed by Tukey’s honestly significant difference (HSD) test (p < 0.05) to allow for comparisons among all means. Data obtained by optical density further fitted with Barany and Roberts (1994) model to estimate the kinetic parameters of the bacteria growth with or without BFS extract effect.

[0160] Optical density data fitted with Barany and Roberts (1994) model using DMFit 3.5, to estimate the microbial kinetic parameters under BFS extract effect. Example 1. Preparation and chemical analysis of Hermetia illucens extract

[0161] Sample preparation

[0162] 800 g of Hermetia illucens larvae were washed twice with tap water and once with deionized water, then they were dried (e.g., lyophilization). The dried larvae were kept at -20 °C until used. Before extraction, 20 g of frozen, unthawed larvae were powdered.

[0163] Extraction process

[0164] 20 g of ground larvae were placed in a 500 mL screw-capped flask and 200 mL of an ethanol-water mixture containing 80% to 95% v / v ethanol (preferably 90:10) was added. The mixture was allowed to stir at ambient temperature for 24 h (overnight), was filtered under vacuum and the filtrate was collected and kept at 4 °C. The solid residue was placed back into the flask, re-extracted with 200 mL of water- ethanol mixture for 24 h and filtered. The resulting filtrates were combined and concentrated using rotary vacuum evaporation at 25-35 °C. The aqueous residue was dried (e.g. freezing and lyophilization), resulting in the formation of a yellow sticky solid.

[0165] The extract was homogenized and suspended in Tween 80 (11.86 %w / w) or dimethyl sulfoxide- DMSO (12.46 % w / w). Dissolution was assisted by agitation and ultrasound.

[0166] Chemical analyses

[0167] All larvae samples were oven dried at 70°C until constant weight. Total nitrogen content was determined using the Kjeldahl method and converted to crude protein (CP) content by multiplication with factor 4.76 (Mariotti et al. 2008; Janssen et al. 2017). Non-protein nitrogen (NPN) was determined using the trichloroacetic acid method (TCA) (Licitra et al. 1996) and calculated after subtracting the protein nitrogen from total nitrogen. Protein nitrogen determined using the Kjeldahl method was converted to true protein by multiplication with factor 4.76. Crude fat (CF) was analyzed according to the Soxhlet method. Ash was determined by incineration at 550 °C for 4 h in a combustion oven.

[0168] Nutrient composition of the BSF larvae fed on chicken feed showed levels of total nitrogen 6.29 ± 0.01 %, non-protein nitrogen 1.77 ± 0.03%, higher protein nitrogen 4.52 ± 0.04%, crude protein 29.95 ± 0.04%, true protein 21.53 ± 0.18% and ash 19.10%. The only PUFA found in high concentration was linoleic acid.

[0169] Determination of Fatty Acids Content

[0170] Esterification of the fatty acids is necessary before analysis. For this purpose, the method described in reference (Myrtsi et al., 2023) with some modifications was employed. Experiments were performed in triplicate.

[0171] 50 mg of larvae extract was suspended in 1 mL of 1 M KOH solution in 70% v / v ethanol and the mixture was heated at 90 °C in a block heater for 1 h to undergo hydrolysis. Subsequently, acidification was carried out by adding 1 .2 mL of 1 M HCI and stirring vigorously for 1 min. 1 .5 mL of hexane was added to the mixture and stirred for 1 min. 1.0 mL of the organic phase was collected and evaporated under vacuum at a temperature below 25 °C . To the residue was added 1 mL of a 14 % BF3 solution in methanol and the mixture was stirred for 20 minutes at 37 °C. The reaction was quenched by the addition of 1 mL aqueous NaCI 4 M and 1 mL of hexane. The organic phase was separated and analyzed.

[0172] Fatty acid analyses were performed employing a 7820A GC-FID system (Agilent Technologies, Inc., Santa Clara, CA, USA). Hydrogen gas (H2) was produced by a hydrogen generator (Peak Scientific, Glasgow, UK) and used as fuel gas. High purity nitrogen (N2) was used as a carrier gas and synthetic air as oxidizer. The column that was used was DB-WAX Ultra Inert 30 m, 0.25 mm, 0.25 pm column (Agilent Technologies, Inc., CA, USA).

[0173] Samples were injected manually at a volume of 1 pL and a 10:1 separation mode. The oven operating program started at 40 °C for 0.5 minutes, the temperature was increased to 195 °C at a rate of 25 °C / min, then the temperature reached 205 °C in steps of 3 °C / min and 230 °C in steps of 8 °C / min, at that point it was maintained for 4 minutes. Finally, the temperature increased to 240 °C for 10 minutes and to 250 °C for 5 minutes. The larvae extract fatty acid content is shown in detail in Table 1.

[0174] As it can be seen in Table 1 below, the BSF larval extract contains a high percentage of saturated fatty acids (SFAs 68.40%), while monounsaturated fatty acids (MUFAs, 14.45%) and polyunsaturated fatty acids (PUFAs, 17.15%) found at similarly low levels. Among the SFAs, lauric acid (LA) (C12:0) was the dominant constituent (244.51 mg / g Dry Extract - DE). Additionally, palmitic (C16:0), oleic (C18:1 n-9) and myristic (C14:0) acid were remarkable with concentrations of 54.07 mg / g DE, 53.26 mg / g DE and 44.50 mg / g DE, respectively.

[0175] Table 1. Fatty acid content and profile of larval extract. Each fatty acid and the fatty acid totals (SFA, MUFA, PUFA) contents are presented on a Dry Extract basis (mg / g DE). Values are expressed as mean ± standard deviation (SD; n = 3). Fatty acid profile is presented on g / 100g fatty acid ( / %). g / 100g fatty acid

[0176] Fatty acids mg / g DE

[0177] (%)

[0178] Capric acid 7.09±0.05 1.35

[0179] Undecanoic acid 0.05±0.00 0.01

[0180] Lauric acid 244.51 ±0.06 46.53

[0181] Tridecanoic acid 0.04±0.00 0.01

[0182] Myristic acid 44.50±0.34 8.47

[0183] Myristoleic acid 1.35±0.01 0.26

[0184] Pentadecanoic acid 0.54±0.00 0.10 cis-10-Pentadecenoic acid 0.68±0.00 0.13

[0185] Palmitic acid 54.07±0.35 10.29

[0186] Palmitoleic acid 18.28±0.10 3.48 Heptadecanoic acid 0.49±0.01 0.09 c / s-10-Heptadecenoic acid 0.54±0.01 0.10

[0187] Stearic acid 6.44±0.45 1.23

[0188] Oleic acid 53.26±0.83 10.14

[0189] Linoleic acid 83.70±1.07 15.93 y-Linolenic acid 0.05±0.00 0.01

[0190] Linolenic acid 6.37±0.36 1.21

[0191] Arachidic acid 0.19±0.04 0.04 cis-11 -Eicosenoic acid 0.38±0.06 0.07 cis-13,16-Docosadienoic acid 1.44±0.14 0.27

[0192] Tricosanoic acid 1.47±0.17 0.28

[0193] Total Content 525.46

[0194] PUFA 90.12 17.15

[0195] MUFA 75.94 14.45

[0196] SFA 359.39 68.40

[0197] SFA = sum of all saturated fatty acids; MUFA = sum of all monounsaturated fatty acids; PUFA sum of all polyunsaturated fatty acids

[0198] Example 2: In vitro assessment of antibacterial activity of Hermetia illucens extracts

[0199] The antibacterial activity of BSF larvae extracts prepared as described in Example 1 was assessed and the Inhibition zone was measured using the agar disk diffusion assay, in accordance to CLSI (Clinical & Laboratories Science Institute; https: / / clsi )rg / media / 1462 / m26a sample.pdf). Every Petri dish was inoculated with 0.1 mL of the dilution with final concentration 5*105CFU*mL-1 of the re -cultured bacterial culture (using the spread technique). Filter paper discs, 5 mm in diameter, were placed on to surface and subsequently inoculated with 5 and 10 pL of the studied larval extracts. The dishes were incubated at the above conditions (i.e. 37 °C for 24 H and 25 °C for 2 Days, for all the microbes with the exception of both P. larvae, and V. campbelli, respectively. The inhibition zone diameter (IZD) was expressed in mm.

[0200] The minimum inhibitory concentration (MIC) was measured with broth dilution method (Wiegand et al. 2008). 96-well plate inoculated with serial twofold dilutions of the antibacterial agent- Each well was inoculated with the same amount of 50 pL with the prepared microbial inoculum with final concentration 5*105 CFU*mL-1.

[0201] Eight repeats for every case were studied. The plate incubated at the optimum conditions for 24 h and interval optical density measurements were observed with Microplate Reader at 600 and 620 nm. Subsequently, samples transferred to petri dishes to estimate the minimum bactericidal concentration (MBC) for each microbe. According to the guidelines of the method standardized by CLSI (1999), all parameters such as inoculum size, growth medium, incubation time and inoculum preparation were taken in account. The Figures 1 to 4 show the antimicrobial activity of BSF extract of the invention. Figure 1 shows changes on optical density (600 nm) of Salmonella typhimurium population after addition of several concentrations of a BSF extract. As it can be ssen, the growth of Salmonella typhimurium was inhibited only at A1 wells whereas the BSF extract was at the highest concentration. The MBC also estimated at the same concentration. Similarly, Figure 2 shows changes on optical density (600 nm) of a Staphylococcus aureus population after addition of several concentrations of a BSF extract. Measurement of the optical density showed no growth for S. aureus MRSA at the A1 , A2, A3, and A4 wells. MBC was also estimated on the A3 wells of BSF concentration. Additionally, the antimicrobial activity of the BFS extract of the invention on Paenibacillus larvae was is shown in Figures 3 and 4. More specifically, Figure 3 shows P. larvae grown on MYPGP agar and application of the disk diffusion assay. Table shows that BSF concentration affects lag phase, ymax and rate of the microbe. Based on the Table’s results, up to A3 wells BSF concentrations, the P. larvae had no rate or up to 0.02 (nm*h-1), and the lag phase was the highest. According to the optical density obtained data, the minimum inhibitory concentration was estimated to be less than 0.48 % w / v and the minimum bactericidal concentration was assessed to be 0.96 % w / v.

[0202] Table 2. Kinetic parameters of P. larvae grown on 96 wells plate with different BSFT concentrations. As A1 is referred to the highest concentration which is successively diluted to A11. A12 represents no BSFT addition. In conclusion, BSF extract inhibited both Gram negative and positive bacteria that can cause food poisoning, zoonosis, human diseases, and incest death. The product was determined as human and animal friendly and specified as a natural antimicrobial agent. The MBC volume range of the BFS extract was estimated between 4-14 g*L-1and 10-38 g*L-1, for Gram positive and negative bacteria, respectively, depended on the bacteria species.

[0203] Example 3: In vitro assessment of antifungal activity of Hermetia illucens extracts

[0204] Antifungal activity against plant pathogenic fungi was estimated by dual culture assay. Phytopathogenic fungi Fusarium oxysporum, Verticillum dahliae, Rhizoctonia solani, Alternaria alternata, Thielaviosis basicola, Botrytis cinerea and Pythium sp. were tested in vitro for growth inhibition after exposure to different dilutions of the Larvae extract. More particularly, the antifungal activity was estimated by growing the fungi in nutrient medium (PDA medium) comprising the BSF. A fungal disc of 5 mm diameter was placed onto the center of PDA (1 .5% (w / v) agar) plates including a dilution of the BSF extract. Plates were incubated at 25 °C in darkness. Fungal growth was observed for 12 days with daily observation and measurement of their mycelium growth (diameter). BSF larval extract solution was incorporated into the liquid PDA medium before solidification. Every plate was supplemented with the 25 pl of A1 , A2, A3 dilutions of the BSF extract , A1 : 10-fold dilution, A2: 20-fold dilution, A3: 100-fold dilution in the plate. Control plates were supplemented with the equal volume of Tween 80 . It is obvious that each fungus had its own control, due to the different rate of growth.

[0205] Preliminary experiments using the three different dilutions showed that there was inhibition of growth of all the fungi used by dilution A1. For this reason, we continued our experiments by incorporating into the nutrient medium, liquid from dilution A1 .

[0206] As it can be seen in Figure 5, inhibition of fungal growth was observed in the plates were the BSF extract was added for all tested bacteria species.

[0207] In the case of Botrytis cinerea, the effect of extract on the growth and sporulation was further studied, after monitoring was continued for another 12 days, when the formation of sclerotia was observed under microscopic observation (Huang and Hull, 2017).

[0208] The sporulation response changed as Botrytis culture aged. Control culture (without comprising BSF dilution A1) maximum response occurred after 4-6 days' growth in the dark, while Botrytis- BSF cultures delayed their sporulation by 3-4 more days.

[0209] Screening for BSF Volatile-Mediated Fusarium oxysporum (Fox) growth

[0210] In this experiment, Sterile Petri dishes were divided in 2 chambers. One chamber had the PDA medium. A mycelial plug collected from a fungal culture was placed at the center of the medium. In the other chamber a Whatman Grade 3MM filter paper discs were placed and subsequently inoculated with 10 pL of the studied BSF larval extracts and with Tween 80 as a control (Fig. 6). The dishes were incubated at 25 °C.

[0211] BSF larval extracts solution was diluted in the corresponding broth into three dilutions to reduce the concentration of the initial solvent. A1 : 10-fold dilution, A2: 20-fold dilution, A3: 100-fold dilution.

[0212] The fungal response to BSF volatiles was studied by comparing the phenotypic responses of fungus development in the presence of the three dilutions daily.

[0213] As it can be seen in Figure 6, volatiles of all dilutions of the BSF larval extract can inhibit the growth of Fusarium oxysporum, with increased inhibition observed in the volatiles of dilution A1.

[0214] Example 4: In vitro assessment of antibacterial activity of Hermetia illucens extracts against phytopathogenic and beneficial bacteria strains.

[0215] The bacterial Inhibition zone measured using the agar disk diffusion assay as described in Eaxample 2. In particular, 1 mL of each culture serial diluted on 9 mL Ringer tubes and 0.1 mL of diluted bacterial cells were incorporated aseptically into the nutrient medium (NA) before solidification. The population was expressed as CFU*mL-1 and the final concentration estimated to 5*105CFU*ml_-1. Every Petri dish was inoculated with 0.1 mL of the recultured bacterial culture. Then, 5 mm in diameter filter paper discs were placed on to surface and subsequently inoculated with 5 and 10 pL of the studied larval extracts. The dishes were incubated at 28 °C . The inhibition zone diameter (IZD) expressed to mm.

[0216] The highest BSF extract antibacterial potency was observed for phytopathogenic bacterial strains, P. syringae pv lachrymans 312, P. syringae pv lachrymans 355, P. syringae pv savastanoi 455 (Venieraki et al, 2014) and Pseudomonas syringae pv tomato DC3000 and the laboratory strain E. coli BW BW25113.

[0217] BSF extract bacterial inhibition assessment against beneficial bacterial strains, which are natural endophytes isolated from medicinal plants and they have positive reaction to different plant growth promoting (PGP) traits, survival rate and inhibition of phytopathogenic fungi in vitro, ex vivo and in planta (Thomloudi et al., 2021 ; Nifakos et al., 2021 ; Tsalgatidou et al., 2022; Tsalgatidou et al., 2023), has notably shown that the antibacterial activity of the BSF larval extract on the beneficial bacteria was minimal.

[0218] On the other side, the laboratory Gram negative strain E. coli BW BW25113 appears to be affected by the presence of BSF extracts in the culture media leading to the conclusion that this E. coli strain behaves similarly to the tested the phytopathogenic bacterial strains and this behaviour maybe due to common secreted metabolites with pathogenic E. coli strains e.g. E. coli O157:H7.

[0219] In previous studies, it has been found that these beneficial bacterial strains secrete different metabolites based on the analysis of the genome and the metabolic profiling (Thomloudi et al., 2021 ; Nifakos et al., 2021 ; Tsalgatidou et al., 2022) compared to phytopathogenic bacteria.

Claims

TITLE ANTIMICROBIAL COMPOSITIONS AND USES THEREOFCLAIMS1 . A composition comprising a Hermetia illucens larval extract characterised in that said extract contains a total nitrogen concentration of about 6 %.

2. The composition of claim 1 , wherein said Hermetia illucens larval extract characterised in that said extract contains a total nitrogen concentration of about 6 %, a non-protein nitrogen concentration of about 1.8%, a higher protein nitrogen concentration of about 4.5%, a crude protein concentration of about 29.9%, a true protein concentration of about 21.5% and an ash concntration of about 19 %.

3. The composition of any one of claims 1 or 2, wherein said Hermetia illucens larval extract contains a total fatty acid concentration of about 525 mg / g of dry extract.

4. The composition of anyone of claims 1 to 3, wherein said Hermetia illucens larval extract contains a lauric acid concentration of about 245 mg / g of dry extract5. A method of preparing of a Hermetia illucens larval extract comprising the steps of-washing and drying preferably by lyophilization of Hermetia illucens larvae-freezing and powdering of said dried larvae-mixing of said frozen and powdered larvae with a 9:1 ethanol: water mixture-filtering of said larvae and ethanol:water mixture under vacuum-collection of the filtrate-mixing the solid residue with a water- ethanol mixture as previously-filtering as previously-collection of the second flitrate as previoulsy-mixing of the two flitrates-concentrating said mixture of titrates preferably using rotary vacuum evaporation-cooling of the resulting concentrate-drying of said concentrated liquid preferably by lyophilization until it is solidified -homogenization of said solid larval extract and suspension in Tween 80 or dimethyl sulfoxide (DMSO).

6. The method of claim 5, wherein said method comprises the following steps-washing of Hermetia illucens larvae twice with tap water and once with deionized water, -drying preferably by lyophylization of said washed larvae and freezing at -20°C -powdering of said frozen larvae-mixing of said frozen and powdered larvae with a 9:1 ethanol: water mixture and stirring at ambient temperature for 24 hours-filtering of said larvae and ethanol:water mixture under vacuum-collection of the filtrate and cooling at 4°C-mixing the solid residue with water- ethanol mixture as previously-filtering as previously-collection of the second filtrate as previously-mixing of the two filtrates-concentrating said mixture of filtrates, preferably using rotary vacuum evaporation, at 25-35 °C.-cooling the resulting concentrate to -20°C-drying of said concentrated liquid preferably by lyophilization until it is solidified -homogenization of said solid larval extract and suspension in Tween 80 or dimethyl sulfoxide(DMSO)7. The method of any one of claims 5 or 6, wherein said method comprises the following steps -wasing of 800 g of Hermetia illucens larvae twice with tap water and once with deionized water -drying preferably by lyophilization of said washed larvae and freezing at -20°C until used, -powdering of 20 g of said frozen larvae-mixing of said powdered and frozen larvae with 200 mL of a 9:1 ethanol: water mixture and stirring at ambient temperature for 24 hours-filtering of said larvae and ethanol:water mixture under vacuum-collection of the filtrate and cooling at 4°C-mixing the solid residue with 200 mL of water- ethanol mixture for 24 hours as previously-filtering as previously-collection of the second filtrate as previously-mixing of the two filtates-concentrating said mixture of filtrates, preferably using rotary vacuum evaporation, at 25-35 °C -cooling of the resulting concentrate to -20°C-drying of said concentrated liquid preferably by lyophilization until it is solidified -homogenization of said solid larval extract and suspension in Tween 80 11 .86 %w / w or dimethyl sulfoxide (DMSO) 12.46 % w / w.

8. A composition comprisning a Hermetia illucens larval extract directly obtainable or obtained by the method of anyone of claims 5 to 7.

9. Use of a composition of anyone of claims 1 to 4 or 8 as an antimicrobial, or antibacterial or antifungal or antiseptic or disinfecting agent.

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