A bee exosome-based compound with antibiotic properties against biofilm formation, and a method for preparing this compound.

Abstract

The invention relates to a composition with antibacterial and antibiotic properties that can be used as a washing solution in wounds or infected areas, is easy to spray, and prevents biofilm formation on wounds, and the method of producing this composition. The composition in question contains at least one antibiotic for use against bacteria, bee exosome obtained from bee venom, and an isotonic liquid to facilitate spraying.

Description

1 TARIFF BEE EXOSOME WITH ANTIBIOTIC PROPERTIES AGAINST BIOFILM FORMATION. A BASE-BASED COMPOSITION AND ITS PREPARATION METHOD Technical Field to Which the Invention Relates 5 The invention relates to a washing solution or toothpaste for wounds, burns, or infections. biofilm on the wound that can be used as a tooth cleaning solution in operations a compound with antibacterial and antibiotic properties that inhibits its production, and this compound It relates to a method for its preparation. State of the Art Biofilms adhere to living or non-living surfaces, forming a protective extracellular polymerization. The substance (EPS) is a community of bacteria structured within a matrix. This gel-like layer contains bacteria. In terminology, it is called "extracellular polymeric structure" or "exopolysaccharide", produced by cells. It is a polysaccharide-based network structure called "exopolymer (EPS)". 15 The formation of biofilms by microorganisms allows them to survive in unfavorable environments. It is a complex issue. All infections, especially instrument-related infections, skin and 65% or more of soft tissue infections and chronic infections The majority of these infections are caused by biofilm infections. The bacteria within a biofilm are quite heterogeneous. A small group are slow reproducers known as persistents, while some reproduce quickly. These 20 Due to heterogeneity and the EPS matrix, bacteria within the biofilm pose a host defense risk. They are resistant to the mechanisms and effects of antibiotics. Therefore, biofilms are damaged. It delays the healing process of tissues or wounds [1]. Biofilm formation can occur on living cells or non-living surfaces, and other The presence of bacteria is affected by factors such as nutrient availability, temperature, oxygen levels, and pH. 25 Biofilms can be formed by a single type of microorganism or by multiple types. It can harbor different types of microcolony in its structure. In biofilms composed of different species, each species forms its own microcolony. It forms and these microcolonies are separated from each other by water channels. Water 2 The continuous flow of water within the channels facilitates the diffusion of nutrients and oxygen. The biofilm consists of 98% water, and the other substances in its composition are... They are held together by an extracellular matrix structure. EPSs are morphological components of biofilms. It is responsible for its structural and physicochemical properties. The composition of EPS includes polysaccharides, proteins, It consists of nucleic acid, teichoic acid, phospholipids, and other polymeric substances, and 5 It constitutes 75-90% of biofilms. The water found in the biofilm structure is the bacteria's It binds to the EPS it forms. EPSs form a biofilm, allowing microorganisms to adhere to it. They help the cells to form colonies and attach to surfaces. Multiple cells the penetration of EPS antimicrobials and their reaching the inner layers They can form a complex structure that makes things difficult. Thus, they expose the organism to osmotic stress, phage 10 resistant to contamination, toxic compounds, disinfectants, and antibiotics. They are able to provide protection. Microorganisms in biofilms are resistant to antimicrobial components. They are 10-1000 times more resistant than planktonic forms [2]. Around the world, antibiotics or drugs with antibiotic properties are used against various bacterial infections. Washing solutions have been developed. Antibiotics, which are antimicrobial chemical agents, and 15 Surfactants, despite the risks of being harmful to the environment, can be used when necessary. using the smallest possible amount to control biofilms It is a conventional method. Exopolysaccharide found in biofilm structure. The structure reduces the penetration of antibiotics, preventing them from reaching the bacteria, and The number of infections caused by antibiotic-resistant microorganisms is 20 With the increasing prevalence of biofilm, these traditional approaches are proving insufficient in eliminating biofilm structures, and It does not prevent biofilm formation. It fails to remove biofilms. The current methods are also not financially advantageous [3]. Current techniques involve antibiotics or antibiotics against bacterial infections. a vesicle obtained from cells in specialized washing solutions and antibacterial 25 Specialized exosomes are used. A vesicle is a closed system that stores or transports substances. It is a relatively small intracellular sac formed by a membrane. Exosomes, on the other hand, are 30-150 nm in diameter. They are vesicles. These structures carry bioactive substances. Exosomes, antibiofilms. Thanks to its properties, it reduces bacterial infection and biofilm formation. It prevents. In addition, exosomes contribute to strengthening the immune system. 30 3 and plays an active role in reducing bacterial populations. In addition, exosomes It also prevents the development of antibacterial resistance [4]. Apitoxin, also known as bee venom, is a bitter, colorless, liquid chemical produced by honey bees. It is found inside the venom sacs and is released into the human body when exposed to a bee sting. It is the ingested substance. The active ingredient of apitoxin poison is proteins, and this substance is ethanol 5 It can be neutralized by. It is an acidic chemical substance with a pH value between 4.5 and 5.5. Bee venom contains hyaluronic acid, phospholipase A2 molecule, dopamine, seretamine, and others. It is very rich in methamine and norepinephrine. The venom contains these substances. Hyaluronic acid, a substance of particular importance for skin health, combines two healthy cells. It binds them together, delays the signs of aging, rejuvenates and beautifies the skin. Phospholipase 10 The A2 molecule, on the other hand, supports cell formation and destroys bacteria. Bee venom is a powerful agent. It is anti-inflammatory. It reduces inflammation in the body; therefore, it reduces the risk of autoimmune diseases. It eliminates the contributing factors. Additionally, bee venom is effective against skin disorders and... It heals scars quickly. It also soothes the skin in cases of psoriasis and eczema. [5]. 15 Patent application number US2012088671A1, which is included in the prior art, It relates to plant-derived polyphenolic compounds that inhibit biofilm formation. The same at least one phenolic phytochemical that inhibits the growth of an established biofilm, and Combinations containing at least one antimicrobial agent are also provided. Patent application number US2012088671A1 describes the formation and growth of biofilms. It provides methods for inhibiting a polyphenolic compound in a regulation. is provided. The method of producing the polyphenolic compound is “(a) a water and alcohol extract of a plant and to form a primary water section and a hexane section with a hexane mixture (b) partitioning the first water to form a second water partition and an ethyl acetate partition. (c) a third compartment of water and a 25 To create the butanol compartment, the second water compartment is filled with a mixture of water and butanol. (d) the division of butanol into a mobile containing a mixture of methanol and dichloromethane. Fractionation of phases by column chromatography, where polyphenolic composition, "Separation of methanol with dichloromethane using a mobile phase where the volume ratio is approximately 40:60" It includes the process steps. However, the composition in question allows biofilm formation in 30 seconds. 4 Because it does not contain any components that would obstruct open or closed wounds It does not provide an effective treatment for recovery either. Current technology involves antibiotics, solutions, or disinfectants that are bacterial. It does not provide an effective treatment for infections or diabetic wounds, biofilm. not preventing its formation, not contributing to the immune system, the 5 that form the biofilm the low effectiveness of antibacterial agents in reducing bacterial populations and antibacterial resistance disadvantages such as its insufficient effectiveness in preventing its formation Therefore, it has become necessary to make improvements in the relevant field. Brief Description and Objectives of the Invention 10 The invention relates to a washing solution or toothpaste for wounds, burns, or infections. It can be used as a dental cleaning solution in operations, is easy to spray, Biofilm on wound containing bee exosomes obtained by isolating exosomes a compound with antibacterial and antibiotic properties that prevents its formation, and this compound It relates to a method for its preparation. The composition that is the subject of the invention is to be used against bacteria. 15 at least one antibiotic, bee exosome, and isotonic solution (serum) to facilitate spraying. (physiology) is included. The main purpose of the invention is to provide a washing solution for wounds, burns, or infected areas. The goal is to provide an antibacterial compound that can be used to prevent biofilm formation. The compound described in the invention consists of at least one antibiotic used against bacteria and 20 derived from bee venom. It contains bee exosomes. Hyaluronic acid, phospholipase A2 molecule, dopamine, Bee venom, which is very rich in seretamine, hitamine, and norepinephrine, affects cells. It supports its formation and destroys bacteria. Bee venom is a powerful anti-inflammatory. Furthermore, Bee venom is effective against skin disorders and heals scars quickly. In the invention... 25 using bee exosomes obtained from bee venom to prevent biofilm formation. An antibacterial compound is provided. Another purpose of the invention is to provide a washing solution for wounds, burns, or infected areas. a compound that can be used and contributes to strengthening the immune system The purpose is to provide. The compound that is the subject of the invention contains bee exosome obtained from bee venom. Bee The toxin helps reduce inflammation in the body; therefore, it helps with autoimmune diseases. It eliminates the factors that pave the way for [the development of a problem]. Thus, in the invention, bee venom is obtained... The use of bee exosomes contributes to strengthening the immune system. The combination is achieved. Another purpose of the invention is to reduce the bacterial population in wounds, burns, or infected areas. The aim is to provide a compound that reduces or inhibits the growth of these bacteria. This is the subject of the invention. The compound contains at least one antibiotic used against bacteria. Antibiotics effectively kill bacteria and even prevent their reproduction. Thus, the bacterial population is reduced in wound or infection areas, and this A compound is provided to inhibit bacterial growth. 10 Another purpose of the invention is to eliminate bacteria found in wounds, burns, or infected areas. The aim is to provide a compound that prevents the development of antibacterial resistance. This is the subject of the invention. the synergistic effect of the antibiotic and bee exosome derived from bee venom included in the composition Its antibacterial effect targets bacteria found in wounds, burns, or infected areas. The creation of resistance is prevented. 15 Another purpose of the invention is to provide a washing solution for wounds, burns, or infected areas. The aim is to provide an easily sprayable compound that can be used as such. The compound subject to the invention It contains an isotonic liquid. This isotonic liquid allows the aforementioned composition to be easily dissolved. It enables spraying. The invention describes 20 washing solutions for wounds, burns, or infected areas. available, prevents biofilm formation, contributes to strengthening the immune system. providing, or reducing the bacterial population in wounds, burns or infected areas. these bacteria are prevented from growing in wounds, burns, or infected areas. a sprayable product that prevents bacteria from developing antibacterial resistance The composition is provided. 25 Explanation of the Figures 6 Figure 1. Scanning electron microscope (SEM) of a bee exosome obtained from bee venom. image. Detailed Description of the Invention The invention relates to a washing solution or toothpaste for wounds, burns, or infections. an easy-to-spray solution that can be used as a dental cleaning solution in operations, for bees Exosome-containing, antibacterial, and prevents biofilm formation on wounds. It relates to a compound with antibiotic properties and a method for preparing this compound. The bee exosome mentioned here is obtained from bee venom in its liquid form. The composition described in the invention contains at least one antibiotic or 10 antibiotics intended for use against bacteria. antibiotic mixture, bee exosome derived from bee venom, and a substance that facilitates spraying. It contains isotonic solution (physiological saline). The antibiotic in question is gentamicin or amoxicillin, or a mixture that has bactericidal or bacteriostatic effects, may be the case with amoxicillin. It is preferred as a mixture of gentamicin and amoxicillin. It is a bactericidal or... The antibiotic mixture with bacteriostatic effects constitutes 1-5% by weight of the composition subject to the invention. It is used in proportion to the ratio of the aforementioned antibiotic mixture to the composition that is the subject of the invention. The use of this compound broadens its antibacterial spectrum. The composition contains 0.1-2% bee exosome by weight. Bee exosome is in liquid form. It is a component found in bee venom. Bee exosomes are extracted from bee venom by centrifugation. It is separated using a method or a kit. 20 bee exosomes obtained from bee venom The minimum inhibitory concentration (MIC) is 400 mg / mg. Containing bee exosome, it prevents biofilm formation on wounds and is antibacterial. The antibiotic-containing compound that is the subject of this invention contains 93-98.8% isotonic liquid by weight. The isotonic solution prevents the breakdown of the bee exosome included in the composition, thus preserving the composition. It facilitates easy spraying. In addition, the isotonic water in the composition contains 25 bees. It balances the pressure of the exosome; thus preventing exosome degradation. It prevents. Isotonic solution is a simple salt solution. 1 ml of isotonic solution contains 0.9 milligrams It contains sodium chloride (NaCl). The pH of the isotonic liquid is 7.2; its temperature is 20-40˚C. It must be so. Thanks to the use of isototic liquid in the composition subject to the invention at a rate of 93-98.8%. 7 It stabilizes the internal pressure of the exosome, which has a vesicle and internal pressure, preventing the exosome from bursting. or prevents shrinkage. Isototic fluid is used for storing exosomes. It is used. The production method of the compound subject to the invention shall be carried out in the following order or in a mixed order: The following procedures are being implemented. 5  Extraction of bee exosomes from bee venom by ultracentrifugation or with a kit separation and obtaining of bee exosome,  At least one, and preferably at least two, antibiotics in isotonic solution in a separate location dissolving it in and obtaining an antibiotic solution,  Bee exosomes isolated from bee venom, dissolved in isotonic solution 10 added to an antibiotic solution and an antibiotic solution containing bee exosome obtaining the solution,  Ultrasonic vibration of a solution containing bee exosome and antibiotics and This way, it contains bee exosome and prevents biofilm formation on the wound, The goal is to obtain a compound with antibacterial and antibiotic properties. 15 The antibiotic mixture used in the production of the compound whose processing details are given above. It is derived from gentamicin and amoxicillin. A mixture of gentamicin and amoxicillin... a mixture of 1-5% by weight in isotonic liquid of 93-98.8% by weight It is dissolved, and 0.1-2% by weight of bee exosome is added to this solution. The resulting solution is then subjected to ultrasonic vibration at 20-40˚C for 30-45 minutes. bee exosomes are captured and spun at 100,000-200,000 rpm for 1 hour. containing, preventing biofilm formation on wounds, and having antibacterial and antibiotic properties. a compound is obtained. The production method of the compound that is the subject of the invention;  Bee exosomes can be extracted from bee venom using the ultracentrifugation method or a kit. separation,  Antibiotics containing gentamicin and amoxicillin, available in dry and powder form in a separate location Preparation of the mixture and 1-5% by weight of the mixture, 93-98.8% by weight. Dissolving in isotonic liquid at that ratio, 8  Adding 0.1-2% by weight of bee exosome to the resulting solution,  Ultrasound treatment of the solution containing bee exosomes at 20-40˚C for 30-45 minutes. subjected to vibration,  Solution containing bee exosomes subjected to vibration at 100,000-200,000 rpm for 1 hour biofilm containing bee exosome, formed on the wound as a result of being turned over along its length 5 a compound with antibacterial and antibiotic properties that prevents its formation to be done It includes the steps of the process either sequentially or in a mixed order. Ultrasonic treatment of the solution containing bee exosome, which is involved in one of the above process steps. During the vibration process step, the shaker of the ultrasonic vibration device is 1000 10 It operates at rpm, which allows the device to vibrate at the desired level. It provides. One application of the invention involves producing the aforementioned compound using a method derived from bee venom. If the ultracentrifugation method is used to separate the exosome of bee venom; Bee exosomes are obtained by centrifuging them at 100,000-200,000 rpm for 1 hour. It is separated from its venom. Another application of the invention involves producing the aforementioned compound from bee venom. If a commercial kit is used for the isolation of bee exosomes; the kit in question must be suitable for the bee. In the separation of the exosome, high centrifugation speeds are not required, as it remains in liquid form. It breaks down the structure of bee venom, causing the exosomes to degrade. The kit used breaks down 20 bee venoms. It breaks down the layers of water molecules inside and holds onto the surface of the exosome, making it lower. The kit ensures the separation of pure exosomes at a specific centrifugation speed and time (5 minutes at 10,000 rpm). The purity level of bee exosomes obtained using high-speed ultracentrifuges It is lower than bee exosomes obtained by centrifugation (other proteins and (It can also retain residues). Therefore, the ultracentrifugation method is preferred over the kit method. 25 is being done. Table 1. Bee venom, bee venom exosome, and a mixture of gentamicin and amoxicillin antibiotics. minimum inhibitory concentration (MIC) values ​​and antibacterial efficacy 9 Bacterial Strains Bee Venom MIC µg / ml Bee venom exosome MIC µg / ml Antibiotics MIC mg / L Escherichia coli ATCC 25922 125 1.95 4 Klebsiella pneumoniae ATCC 700603 1.95 1.95 16 Methicillin resistant Staphylococcus aureus (MRSA) ATCC 43300 250 7.81 16 Pseudomonas aureginosa ATCC 27853 3.9 1.95 3 Growing in blood cultures methicillin-resistant Staphylococcus aureus (MRKNS) ATCC 29213 1000 62.5 1 Staphylococcus hemolyticus ATCC JCSC1435 15.62 1.95 1 Enterobacter aerogenes ATCC 13048 1.95 1.95 No data available Enterococcus feacalis ATCC 29212 250 31.25 2 Streptococcus mitis ATCC 12261 1000 250 2 As shown in Table 1, the minimum amount of antibiotic that stops bacterial growth. The term minimum inhibitory concentration (MIC) is used to determine this. MIC suppressor It is the dose, and the smaller the MIC value, the stronger the effect. The purpose of treatment. After antibiotic administration, the antibiotic concentration reaches the MIC value and 5 If it treats the condition, the microorganism is considered susceptible to this antibiotic. Invention The subject is bee exosome, antibacterial, which prevents biofilm formation on wounds. and identified components of bee venom and exosomes for a compound with antibiotic properties MIC values ​​against pathogens were determined using the microdilution method. Bee venom The dosage range for exosome and antibiotic was determined to be 1028-32 µg / ml. 11 REFERENCES [1] Gün, İ. & Ekinci, FY (2009). Biofilms: Microbial Life on Surfaces. Food, 34 (3), 165-173. Retrieved from https: / / dergipark.org.tr / tr / pub / gida / issue / 6849 / 91833 5 [2] Ünal Turhan, E., & Erginkaya, Z. (2019). Antimicrobial in Bacterial Biofilms Resistance Mechanism. Academic Food. https: / / doi.org / 10.24323 / akademik- food.544978 [3] Nagela Bernadelli Sousa, S., Lara De Andrade, M., & Denise Von Dolinger De Brito, R. (2020). Antibiofilm Activity of Natural Products: Promising Strategies for 10 Combating Microbial Biofilms. Annals of Public Health Reports, 4(1). https: / / doi.org / 10.36959 / 856 / 502 [4] INANIR, C., & EKİCİ, L. (2020). Exosomes: Composition, Biological Functions and their Potential in the Transport of Bioactive Compounds. Academic Food, 421–432. https: / / doi.org / 10.24323 / akademik-gida.850939 15 [5] Kubala, M. J. S. (2019, June 24). Bee Venom: Uses, Benefits, and Side Effects. Healthline. https: / / www.healthline.com / nutrition / bee- venom#:%7E:text=May%20benefit%20skin%20health,antibacterial%20effects%2 C%20and%20reducing%20wrinkles. 25

Claims

12 REQUESTS 1. It prevents biofilm formation on wounds and has antibacterial and antibiotic properties. It is a compound characterized by containing at least one antibiotic in an amount of 1-5% by weight, and 0.1-2% bee exosomes obtained from bee venom at a certain percentage and isotonic water at a rate of 93-98.8% 5 It includes.

2. According to claim 1, it is a combination characterized by the fact that the antibiotic in question is gentamicin and It contains amoxicillin.

3. It is a compound according to claim 2, characterized by containing the aforementioned antibiotic in a 1:10 ratio. Gentamicin: It contains amoxicillin. 10 4. Washing solution or toothpaste on wounds, burns, or infected areas. For use as a dental cleaning solution in operations, from Claim 1-3. A combination according to someone.

5. A method for preparing a compound according to any of claims 1-3. Feature; 15  Extraction of bee exosomes from bee venom by ultracentrifugation or with a kit separation and obtaining of bee exosome,  At least one, and preferably at least two, isotonic antibiotics in a separate location dissolving it in liquid and obtaining an antibiotic solution,  Bee exosomes isolated from bee venom, dissolved in isotonic solution 20 added to the antibiotic solution and antibiotic containing bee exosome obtaining a solution, • Ultrasonic vibration of a solution containing bee exosome and antibiotic and in this way, biofilm formation on the wound containing bee exosome. Obtaining a compound that inhibits, antibacterial and antibiotic properties 25 It includes the steps of the process.

6. A method according to claim 5, characterized by the fact that the antibiotic mixture used is an antibiotic. It is a mixture obtained from gentamicin and amoxicillin.

7. A method according to claim 5, characterized by a mixture of gentamicin and amoxicillin. a mixture of 1-5% by weight, or 93-98.8% by weight isotonic liquid 30 13 It is dissolved in it and is being dissolved, and 0.1-2% by weight of this solution is added. It involves adding bee exosomes in a specific ratio.

8. A method according to claim 5, characterized by containing a mixture of bee exosome and antibiotic. The isotonic solution in question is subjected to ultrasonic vibration at 20-40˚C for 30-45 minutes. It is to be subjected to. 5 9. A method according to claim 8, characterized by its ultrasonic vibration process. The shaker of the vibrating device operates at 100,000-200,000 rpm. and it needs to be turned over for 1 hour.

10. A method for preparing a compound according to any of claims 1-3. Feature; 10  Extraction of bee exosomes from bee venom by ultracentrifugation or with a kit separation,  containing gentamicin and amoxicillin in dry and powder form in a separate location Preparation of the antibiotic mixture and 1-5% of the mixture by weight 15 Dissolving in an isotonic solution at a rate of 93-98.8% by weight.  Adding 0.1-2% by weight of bee exosome to the resulting solution being done,  Ultrasound treatment of the solution containing bee exosomes at 20-40˚C for 30-45 minutes. subjected to vibration, 20  Solution containing bee exosome subjected to vibration at 100,000-200,000 rpm After being turned over for 1 hour, a wound containing bee exosome was treated. a biofilm-inhibiting, antibacterial and antibiotic-like substance obtaining the compound It includes the steps of the process. 25 11. A compound produced by a method according to any of claims 5-10.

12. It prevents biofilm formation on wounds and has antibacterial and antibiotic properties. A combination according to request11. 14 13. Washing solution or toothpaste on wounds, burns, or infected areas. For use as a dental cleaning solution in operations, Claim 11 or Composition according to 12. 10

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