BIOTECHNOLOGICAL PROCESS FOR PRODUCING NOVEL ANTIMICROBIAL PEPTIDES FROM MARINE MICROORGANISMS FOR ANIMAL HEALTH APPLICATIONS

Abstract

The present invention provides a biotechnological process for producing antimicrobial peptides (AMPs) derived from marine microorganisms, intended for animal health applications. The process involves identifying and isolating marine microorganisms, such as bacteria, actinomycetes, and fungi, from marine environments. The selected microorganisms are cultured under optimized fermentation conditions to enhance AMP yield. The antimicrobial peptides are then extracted and purified using techniques including solvent extraction, chromatography, and lyophilization. The purified peptides are tested for antimicrobial efficacy against animal pathogens, such as Escherichia coli, Staphylococcus aureus, Salmonella spp., and Candida albicans. Based on their bioactivity, the peptides are formulated for various animal health applications, such as feed additives, topical creams, and injectable solutions. This invention offers a sustainable alternative to traditional antibiotics in veterinary settings, addressing issues related to antimicrobial resistance while promoting improved animal health through the use of bioactive compounds from natural sources.

Specification

Description:In the following description, for the purposes of explanation, various specific details are set forth in order to provide a thorough understanding of embodiments of the present disclosure. It will be apparent, however, that embodiments of the present disclosure may be practiced without these specific details. Several features described hereafter can each be used independently of one another or with any combination of other features. An individual feature may not address all of the problems discussed above or might address only some of the problems discussed above. Some of the problems discussed above might not be fully addressed by any of the features described herein.

The ensuing description provides exemplary embodiments only and is not intended to limit the scope, applicability, or configuration of the disclosure. Rather, the ensuing description of the exemplary embodiments will provide those skilled in the art with an enabling description for implementing an exemplary embodiment. It should be understood that various changes may be made in the function and arrangement of elements without departing from the spirit and scope of the disclosure as set forth.

Specific details are given in the following description to provide a thorough understanding of the embodiments. However, it will be understood by one of ordinary skill in the art that the embodiments may be practiced without these specific details. For example, circuits, systems, networks, processes, and other components may be shown as components in block diagram form in order not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, algorithms, structures, and techniques may be shown without unnecessary detail to avoid obscuring the embodiments.

Also, it is noted that individual embodiments may be described as a process that is depicted as a flowchart, a flow diagram, a data flow diagram, a structure diagram, or a block diagram. Although a flowchart may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be re-arranged. A process is terminated when its operations are completed but could have additional steps not included in a figure. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a function, its termination can correspond to a return of the function to the calling function or the main function.

The word "exemplary" and/or "demonstrative" is used herein to mean serving as an example, instance, or illustration. For the avoidance of doubt, the subject matter disclosed herein is not limited by such examples. In addition, any aspect or design described herein as "exemplary" and/or "demonstrative" is not necessarily to be construed as preferred or advantageous over other aspects or designs, nor is it meant to preclude equivalent exemplary structures and techniques known to those of ordinary skill in the art. Furthermore, to the extent that the terms "includes," "has," "contains," and other similar words are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising" as an open transition word without precluding any additional or other elements.

Reference throughout this specification to "one embodiment" or "an embodiment" or "an instance" or "one instance" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The invention provides a comprehensive biotechnological process for producing antimicrobial peptides (AMPs) derived from marine microorganisms, optimized for application in animal health. The process involves several stages, including the identification of suitable microorganisms, culture and fermentation to maximize AMP yield, extraction and purification of the peptides, and formulation for effective administration in animals. The invention is illustrated with three exemplary embodiments, each demonstrating a unique approach to AMP production and application.

In first embodiment, marine bacteria are isolated from sediment samples collected from various ocean depths. Suitable bacterial strains are identified based on their genetic profiles, particularly focusing on strains known to produce bioactive peptides. After identification, selected strains are cultured in a nutrient-rich medium under controlled temperature (25-30°C), pH (7-8), and oxygenation (aeration rate of 0.5-1.0 vvm) conditions to maximize AMP production. The fermentation process is carried out over several days, during which metabolic activities are closely monitored to ensure optimal peptide synthesis.

Once fermentation is complete, the bacterial culture is harvested, and AMPs are extracted using organic solvent extraction, followed by lyophilization. Purification is achieved through high-performance liquid chromatography (HPLC), ensuring a high purity level of the target peptides. The antimicrobial activity of the isolated peptides is tested against common animal pathogens, including Escherichia coli and Staphylococcus aureus. The peptides demonstrate significant efficacy, making them suitable for use in animal feed formulations to support gastrointestinal health. The purified AMPs are incorporated into animal feed as a powder additive, offering a preventive measure against bacterial infections without the risks associated with traditional antibiotics.

Second embodiment focuses on the production of AMPs from marine actinomycetes, a group of filamentous bacteria known for their capacity to produce secondary metabolites with antimicrobial properties. Actinomycetes are isolated from marine sponges collected in coastal regions. After initial identification and screening, promising actinomycete strains are cultured in a specialized medium containing salts and minerals that mimic marine conditions, facilitating the optimal growth of these microorganisms and the production of AMPs.

Fermentation is performed in a bioreactor with continuous monitoring and control of parameters such as pH, temperature, and nutrient concentration. After fermentation, the culture broth is subjected to sequential filtration to remove unwanted debris, followed by solvent extraction of peptides. The AMPs are then purified through column chromatography to isolate bioactive fractions. These peptides are tested in vitro against fungal pathogens that commonly affect animals, such as Candida albicans. The peptides exhibit broad-spectrum antifungal activity, making them suitable for use as topical applications in animals to treat fungal infections. The peptides are formulated into a topical cream, allowing for easy application on affected areas to treat infections and promote healing.

In third embodiment, marine fungi are used as the source of antimicrobial peptides. Marine fungi are collected from coral reefs and are isolated using selective growth media that favor fungal proliferation. Genetic analysis and preliminary screening are conducted to identify fungi with a high potential for AMP production. Selected fungal strains are then cultured in a fermenter under aerobic conditions at a pH range of 6-7 and a temperature of 20-25°C.

The fungi are subjected to a two-stage fermentation process. In the initial stage, biomass growth is prioritized with high-nutrient media, and in the secondary stage, nutrient limitations are introduced to induce stress conditions, which stimulate AMP production. Following fermentation, the fungal biomass is separated from the culture medium, and peptides are extracted using aqueous extraction followed by ultrafiltration. Purification is performed using ion-exchange chromatography, targeting peptides with high antimicrobial potency.

The extracted AMPs show strong activity against Salmonella spp., a common cause of infections in animals. These peptides are formulated into an injectable solution for targeted therapeutic use. The injectable formulation ensures high bioavailability and rapid action, making it effective for treating systemic infections in livestock. This embodiment offers a robust solution for controlling bacterial infections in animals while reducing the need for traditional antibiotics.

While considerable emphasis has been placed herein on the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the invention. These and other changes in the preferred embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter to be implemented merely as illustrative of the invention and not as limitation. , Claims:1.A biotechnological process for producing antimicrobial peptides from marine microorganisms for animal health applications, comprising:
identifying and collecting marine microorganisms from a marine environment, wherein the marine microorganisms are selected from the group consisting of bacteria, actinomycetes, and fungi;
culturing the marine microorganisms under optimized fermentation conditions to enhance the production of antimicrobial peptides;
extracting and purifying the antimicrobial peptides from the cultured microorganisms using solvent extraction, chromatography, and/or lyophilization techniques;
testing the antimicrobial activity of the purified peptides against bacterial and/or fungal pathogens relevant to animal health; and
formulating the antimicrobial peptides into a composition suitable for administration to animals.

2.The process of Claim 1, wherein the optimized fermentation conditions include a temperature range of 25-30°C, a pH of 7-8, and an aeration rate of 0.5-1.0 vvm.

3.The process of Claim 1, wherein the marine microorganisms are isolated from sediment samples collected at ocean depths of 200-1000 meters.

4.The process of Claim 1, wherein the extracted antimicrobial peptides are purified using high-performance liquid chromatography (HPLC).

5.The process of Claim 1, wherein the antimicrobial peptides are tested against pathogens selected from Escherichia coli, Staphylococcus aureus, Salmonella spp., and Candida albicans.

6.The process of Claim 1, wherein the formulation of the antimicrobial peptides is designed as a feed additive for administration to animals to support gastrointestinal health.
7.The process of Claim 1, wherein the antimicrobial peptides are formulated as a topical cream for external application to treat fungal infections in animals.

8.The process of Claim 1, wherein the antimicrobial peptides are formulated as an injectable solution for treating systemic bacterial infections in animals.

9.The process of Claim 1, wherein the antimicrobial peptides exhibit a minimum inhibitory concentration (MIC) of 10 µg/mL or lower against target pathogens, demonstrating high efficacy in pathogen inhibition.

Documents