Composition of Olea europaea variety silvestris with antibacterial activity

Abstract

The present invention relates to a method for preparing an olive (Olea europaea subsp. europaea), preferably acebuche (Olea europaea subsp. europaea var. sylvestris) composition, the prepared olive or acebuche compositions, and their use in the prevention or treatment of bacterial or fungal infections in mammals, preferably infections caused by antibiotic-resistant or multi-resistant bacteria. Other uses relate to functional food additives, especially animal feed additives, or in cosmetics.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing an olive (Olea europaea subspecies europaea), preferably an acebuche (Olea europaea subspecies europaea variety sylvestris) composition, the prepared olive or acebuche composition, and their use in the prevention or treatment of bacterial or fungal infections in mammals, preferably infections caused by antibiotic-resistant or multi-resistant bacteria. Other uses relate to functional food additives, particularly animal feed additives, or cosmetics. [Background technology]

[0002] The olive tree (Olea europaea L.) has been cultivated since ancient times and is a characteristic plant of the Oleaceae family. This family includes 30 genera and 600 different species. The genus Olea includes 50 species that can be found in various continents and regions, including Africa, China, India, the United States, and Australia (Non-Patent Literature 1). Olea europaea has several subspecies: among them, the European olive tree is divided into the subspecies Olea europaea 'europaea', and the subspecies Olea europaea 'africana', which grows in Africa, Madagascar, India, and China. Both species have wild and cultivated forms (Non-Patent Literature 1). Olive trees can grow up to 1000 years and require very little water, which explains their presence in arid regions (Non-Patent Literature 1). Most of the world's olive harvest comes from the Mediterranean coast (Non-Patent Literature 2).

[0003] The wild olive, Olea europaea varietal sylvestris, the ancestor of all olive plants, is commonly known in Spain as "acebuche." For example, the distribution of acebuche trees on the Spanish peninsula is irregular, and they grow naturally and occasionally as isolated shrubs or clusters of shrubs, mainly along the Mediterranean coast.

[0004] Olive tree leaves are quite different from those of fruit trees, and are sometimes used as a natural remedy. Olive trees are also highly resistant to fungal and bacterial attacks. These antibiotic and protective properties are thought to be due to the numerous active compounds produced by olives, such as oleuropein, one of the most studied compounds today (Non-Patent Literature 1).

[0005] The active compounds in olive trees are present in various concentrations in the leaves, fruits, buds, stems, branches, and roots (Non-Patent Literature 3). It is hypothesized that olive leaves may have immune-enhancing, anti-inflammatory, and blood pressure-lowering effects (Non-Patent Literature 1). Nevertheless, little is known about the actual medical effects involved.

[0006] Numerous bioactive substances have been characterized in the olive tree, and these can be assigned to the following substance classes, for example, secoiridoid glycosides, phenolic compounds, flavonoids, monoterpenes, triterpenes, steroids, quinoline alkaloids, carotenoids, chlorophyll, phenolic carboxylic acids, tannins, and vitamins (Non-Patent Literature 3).

[0007] Olive tree leaves accumulate during the olive harvest, providing an easily usable by-product that can be used for other purposes (Non-Patent Literature 4). Developing new uses for by-products of olive oil production is of great importance, especially for olive groves. Research has increasingly focused on the chemical composition of olive fruit, and little is known about the chemical composition of olive leaves, particularly the wild olive tree, acebuche.

[0008] Since their discovery more than 70 years ago, antibiotics have been the primary weapon in treating bacterial infections, including life-threatening hospital-acquired infections. However, antibiotics are often prescribed routinely and taken inappropriately. Antibiotics are also used in livestock farming for treatment, disease prevention, and growth promotion. Antibiotics are also found in the environment, for example, in some water supply systems (Non-Patent Literature 5).

[0009] Antibiotic resistance is a predictable natural mechanism that occurs when antibiotics that would normally stop the growth of a particular bacterial species no longer have any effect (Non-Patent Literature 6). Globally, approximately 700,000 people die each year due to antimicrobial resistance (AMR). In the EU, the figure is about 25,000. There are concerns that by 2050, more deaths may be caused by antimicrobial resistance than by cancer (Non-Patent Literature 7).

[0010] Infections caused by antibiotic-resistant bacteria are often difficult to cure, sometimes even impossible, and their numbers are increasing. However, research into new and effective antibiotics is extremely costly and time-consuming, and antibiotic resistance often emerges even after new antibiotics are introduced to the market. Currently, only a small number of new antibiotics are under development. If effective new antibiotics are not discovered and resistance continues to spread, society risks returning to a situation where children were often involved in deaths from simple pneumonia and doctors were powerless against meningitis, as was the case before the discovery of antibiotics. Furthermore, if effective antibiotics are no longer available for prevention, several complex medical interventions and diagnoses will become impossible.

[0011] The emergence of antibiotic-resistant bacteria is a major problem in healthcare facilities, leading to life-threatening bloodborne infections, wound infections, and pneumonia. Antibiotic resistance leads to increased healthcare costs due to longer hospital stays, higher spending on antibiotics and treatments, and indirect costs to families and society. In many European countries, antibiotics do not require a prescription. Data on antibiotic-resistant infections is often not collected, and as a result, the extent of the problem is not documented, despite a high level of awareness among physicians. Hospital-acquired infections are among the most common infections in Germany. The problem of antibiotic resistance and its spread, which is precisely linked to these infections, is one of the greatest challenges for modern medicine (Non-Patent Literature 8).

[0012] Ahmed et al. (Non-Patent Literature 9) evaluated the effect of olive leaf extract derived from Olea europaea L. on the microbial load of raw, unveined, peeled shrimp and discussed the potential use of olive leaf extract formulations for improving microbial quality and as a natural preservative.

[0013] Paudel et al. (Non-Patent Document 10) disclosed a screening of crude wild olive extracts for antibacterial activity against five different bacterial human pathogens, in which the extract obtained using methanol appeared to be the most effective against all pathogenic bacteria.

[0014] Patent Document 1 describes a transparent liquid formulation for lipophilic substances that are very poorly soluble in water, which comprises exactly one solubilizing agent and can be produced simply by stirring without any further complex process steps.

[0015] Further studies (e.g., Non-Patent Document 11) have shown that olive leaf extract inhibits the growth of several bacteria and fungi, such as Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Aspergillus flavus, and Aspergillus parasiticus. Aqueous extracts of olive leaves showed no antibacterial effect against the test microorganisms, but acetone extracts showed inhibitory effects against Salmonella enteritidis, Bacillus cereus, Klebsiella pneumoniae, Escherichia coli, Enterococcus faecalis, Streptococcus thermophilus, and Lactobacillus bulgaricus. Furthermore, the antibacterial activity of several phenolic compounds against microorganisms was tested. Oleuropein was found to be the most effective compound, while syringic acid was found to be ineffective. This type of experiment has not yet been performed with the wild olive variety "Acebuche".

[0016] Wang et al. (Non-Patent Literature 12) disclose five known triterpenoids, namely beta-amyrin, oleanolic acid, erythrodiol, urus-2-beta,3-beta-dihydroxy-12-ene-28-euic acid, and beta-massic acid, along with two secoiridoid glycosides, oleuricines A(1) and B(2), extracted from the ethyl acetate (siRNA) soluble portion of an ethanol (EtOH) extract of Olea europaea L. leaves. The structures of these compounds were elucidated by various spectroscopic methods, including intensive 1D and 2D NMR and HR-ESI-MS techniques. [Prior art documents] [Chartered documents]

[0017]

Patent Document 1

Non-licensed literature

[0018]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0019] Considering the above, one object of the present invention is to provide novel and effective antibacterial compounds and compositions derived from olive or acebuche. Other objects and advantages of the present invention will become apparent to those skilled in the art by carefully reading the following more detailed description of the invention. [Means for solving the problem]

[0020] In their first embodiment, the present invention relates to a method for preparing an olive (Olea europaea subspecies europaea), preferably an acebuche (Olea europaea subspecies europaea sylvestris) composition, a) The process of preparing parts of the olive or acebuche plant, b) A step of removing moisture from the olive or acebuche portion by dehydrating or drying the olive or acebuche portion, c) A step of crushing the olive or acebuche portion of step b) by shearing, grinding, milling, pulverizing or other methods to obtain pulverized olive or acebuche, d) A step of appropriately sterilizing the above-mentioned finely ground olive or acebuche at a temperature of preferably 121°C or 134°C to obtain sterilized finely ground olive or acebuche, and optionally, e) A step of blending the above-mentioned sterilized, finely ground olive or acebuche with water and a thickener to obtain an antibiotic or antifungal active olive or acebuche gel, or d') Extraction, preferably by CO2 extraction, of an antibiotic or antifungal active agent from the pulverized olive or acebuche of step c), and suspension of the active agent in a non-toxic solubilizer and emulsifier to obtain an antibiotic or antifungal active olive or acebuche suspension. The above objective is solved by providing a method that includes [the above].

[0021] Surprisingly, the pharmacological differences were found to be particularly pronounced between cultivated olives and acebuche (see, for example, Table 2 below).

[0022] In a second embodiment thereof, the present invention solves the above object by providing antibiotic or antifungal active sterilized pulverized olive or acebuche obtained from step a), b), c), or d) of the method according to the present invention described above.

[0023] In a third embodiment of these embodiments, the present invention solves the above object by providing an olive or asebuche composition produced by the method according to the present invention, which optionally includes a pharmaceutically or cosmetically acceptable carrier, diluent, or excipient.

[0024] In a fourth embodiment thereof, the present invention solves the above object by providing sterilized pulverized olive or acebuche as described herein, or an olive composition or acebuche composition as described herein, which is used for the prevention or treatment of bacterial or fungal infections in mammals, preferably for the prevention or treatment of infections caused by antibiotic-resistant or multi-resistant bacteria.

[0025] In a fifth embodiment thereof, the present invention solves the above object by providing the use of sterilized pulverized olive or acebuche as described herein, or the olive composition or acebuche composition as described herein, as a functional food additive, in particular as an animal feed additive.

[0026] In a sixth embodiment thereof, the present invention solves the above object by providing the use of the sterilized, finely ground olive or acebuche described herein, or the olive or acebuche composition described herein, as a cosmetic product and / or in a cosmetic method. [Brief explanation of the drawing]

[0027] [Figure 1] This figure shows a schematic method and steps for preparing an antibiotic / antifungal gel or an antibiotic / antifungal suspension according to the present invention, including each intermediate product. [Figure 2] This figure shows the principle of the apparatus used in the process of removing moisture from olive or acebuche portion by dehydration or drying, and the process of grinding the olive or acebuche portion by shearing, grinding, milling, and fine grinding to obtain finely ground olive or acebuche. The process is carried out simultaneously by subjecting the olive or acebuche portion to a force generated by a dynamic airflow, preferably by passing the olive or acebuche through a venturi nozzle together with air, preferably preheated air. [Figure 3] This figure shows a comparison of the active substances (iridoids) in various olive varieties. [Figure 4]This diagram also shows a comparison of the active substances (flavonoids) of various olive varieties. [Figure 5] This figure shows another comparison of the active substances (terpenes) of various olive varieties. [Figure 6] This figure shows yet another comparison of the active substances (tyrosol and hydroxytyrosol) in various olive varieties. [Modes for carrying out the invention]

[0028] As described above, in a first embodiment of the present invention, a method for preparing an olive (Olea europaea subspecies europaea), preferably acebuche (Olea europaea subspecies europaea sylvestris) composition, comprising: a) a step of preparing a part of the olive or acebuche plant; b) a step of removing moisture from the olive or acebuche part by dehydration and / or drying; c) a step of grinding the olive or acebuche part from step b) by shearing, grinding, milling, pulverizing and / or grinding, or other suitable method, in order to obtain pulverized olive or acebuche; and d) a step of obtaining sterilized pulverized olive or acebuche, the fine powder The above problem is solved by providing a method comprising the steps of: appropriately sterilizing crushed olive or acebuche at a temperature preferably about 120°C to about 140°C, preferably about 121°C or about 134°C; optionally, e) blending the sterilized pulverized olive or acebuche with water and a thickener to obtain an antibiotic or antifungal active olive or acebuche gel; or d') extracting an antibiotic or antifungal active agent from the pulverized olive or acebuche of step c) by extraction, preferably CO2 extraction, and suspending the active agent in at least one non-toxic solubilizer and / or emulsifier to obtain an antibiotic or antifungal active olive or acebuche suspension (see Figure 1).

[0029] Accordingly, the present invention relates to two particularly important compositions having desirable properties, namely, i) an antibiotic or antifungal active olive or acebuche gel, and ii) an antibiotic or antifungal active olive or acebuche suspension in at least one non-toxic solubilizer and / or emulsifier.

[0030] The most well-studied secoiridoid glycoside in the olive plant is oleuropein. Oleuropein is present in all parts of the olive plant and is responsible for a variety of beneficial effects, including antibacterial, antiviral, anti-inflammatory, anti-rheumatic, antioxidant, and cardioprotective activities (Fleming, 1973). Olive tree leaves contain 60 mg / g to 90 mg / g of oleuropein in dry weight (Khan, 2007).

[0031] In addition to oleuropein, olive leaves contain other phenolic compounds, such as dimethyloleuropein, ligstroside, verbacoside, oleoside dimethyl ester, and oleuroside (Cayuela, 2006). Olive leaves also contain flavonoids, including rutin, luteolin, and apigenin. Flavonoids reduce oxidative damage by absorbing UV light and prevent oxidation by free radicals (Cayuela, 2006).

[0032] Guinda et al. have described olive trees as a suitable raw material for the production of oleanolic acid and other five-membered triterpenes (Guinda, 2010). These include ursolic acid, betulinic acid, maslic acid, erythrodiol, and ubaol. Guinda et al. have already described that the content of five-membered triterpenes in olive leaves is higher than that in the fruit itself. Oleanolic acid and ursolic acid are most strongly present (Guinda, 2010, Bianchi, 1992). The concentrations of various triterpenes are strongly dependent on the developmental stage of the fruit and plant, as well as the variety (Guinda, 2010, Stiti, 2007).

[0033] Oleanolic acid and maslic acid, which can be extracted from olive leaves, are desirable raw materials for the pharmaceutical and cosmetic industries (Guinda, 2010). For this reason, the leaves of the wild olive 'Acebuche' were investigated for their five-membered triterpene profile, which may potentially influence their antimicrobial activity.

[0034] In this invention, the inventors have developed an optimal processing method for olive or acebuche compositions having antimicrobial activity. One particular advantage of the method according to the present invention is that two critical steps, namely water extraction and grinding, are performed in a single apparatus, thereby significantly reducing investment and saving on operating and energy costs.

[0035] Furthermore, it was found that using a venturi nozzle in this method significantly reduces the required energy consumption.

[0036] Furthermore, and most importantly, it was found that the olive or acebuche processed using the Venturi apparatus yielded finely ground olive or acebuche with significantly higher antibiotic and antifungal effects than materials processed using conventional mills, i.e., without a Venturi apparatus. It was also found that the finely ground olive or acebuche produced using the above Venturi apparatus had a particularly long shelf life.

[0037] Table 1 below shows the reduction in CFU of Pseudomonas aeruginosa bacteria when treated with olive substrate processed by a conventional milling method, compared to olive substrate processed by a Venturi apparatus.

[0038] [Table 1]

[0039] Olive leaf extract, a natural antibiotic or antifungal agent with broad antibacterial activity, represents an innovative step in the fight against infection, particularly in the case of antibiotic-resistant microorganisms, and therefore particularly problematic microorganisms.

[0040] Active, sterilized, pulverized olive or acebuche, or olive or acebuche compositions, can, for example, serve as a base for interactive wound dressings, but can also be used for antiseptic topical disinfection of the body, medical cleaning of sites prone to pathogenic colonization, and in particular for reducing and / or eliminating dominant pathogens and their multi-resistance variants in wound treatment.

[0041] In the present invention, the term “prepare” means preparing the olive and acebuche plants as feedstock for the methods described herein. For example, it may be necessary to clean the olive and acebuche plants from any surface impurities present by a water bath, rinsing or spraying or by further methods known to those skilled in the art. It may also be necessary to cut the olive and acebuche plants to a size that is considered suitable for handling. Each of these methods is also known to those skilled in the art.

[0042] As used herein, “dehydrating” or “drying” usually and preferably refers to direct drying achieved by simple exposure of the material to air (also called natural air drying) or to hot air of airflow drying enhanced by, for example, a ventilation system, blower or other device known in the prior art; or indirect drying achieved by contact drying or drum drying; or dielectric drying by, for example, microwaves; or infrared drying; vacuum drying; or freeze-drying, where these methods may be suitable for reducing drying time or, if necessary, enabling higher sensitivity. Methods using high temperatures to shorten the time of moisture extraction have limitations, as temperatures that are too high and the duration of high-temperature exposure can lead to degradation or destruction of antibacterial / antifungal active components of the raw material. To avoid any contamination or spoilage processes during handling and storage of dried materials, the material is preferably dried to a degree of 95% dry matter (DS) or higher. The moisture content can be optimized to a lower or higher percentage (%-age) of the dry material, which may be recommended for subsequent grinding and pulverization processes.

[0043] As used herein, “grinding” is typically and preferably carried out in one or more steps to achieve a desired degree of fine grinding. The most desirable degree of fine grinding is determined by considering, on the one hand, the cost of grinding, and on the other hand, the effectiveness of the antibacterial or antifungal effects of the resulting pulverized material. According to the invention described herein, the desired particle size of the olive or acebuche portion is in the range of 1 μm to 1000 μm, more preferably 40 μm to 500 μm, and most preferably 125 μm to 250 μm. The desired degree of fine grinding is achieved by processing the olive or acebuche portion several times in a grinding apparatus, or by using different types of grinding apparatus that allow for handling of the desired particle size. Typical grinding apparatuses are selected from, but are not limited to, crushers, shredders, choppers, cutters, grinders and mills, such as hammer mills, ball mills, impact mills, rotary mills, centrifugal mills, jet mills, bark mills, planetary mills, mortars, pulverizers, micronizers, ultrasonic micromills or microgrinders, etc. Depending on the specifications, the process can include the classification of the pulverized material to separate the plant material into the required size classes. This can also be useful for further pulverizing particles classified as too large to reach the desired size class.

[0044] As used herein, the term “sterilization” means removing natural contamination of finely ground olive / acebuche by, for example, aerobic spore-forming bacteria using a variety of sterilization processes. Different types of sterilization can be selected from thermal sterilization, in which microorganisms are killed by heat, for example, heating in a wet state (steam sterilization) or heating in a dry state (dry heat sterilization), or intermittent sterilization, in which heating is repeated continuously, or by physical sterilization techniques, or from other sterilization methods known in the prior art. In this disclosure, sterilization at 121°C and 134°C in a vacuum sterilizer is preferred, where these temperatures are suitable for preserving pharmaceutically active components, and sterilization may be carried out by steam sterilization.

[0045] As used herein, the term “blend” means to mix or homogenize sterile, finely ground olive or acebuche with water for injection and a thickener to obtain an antibiotic or antifungal active olive or acebuche gel, as described below herein, until a uniform gel is formed. There are various methods known to those skilled in the art that can be used to achieve the desired results.

[0046] As used herein, “extract” refers to the isolation of an antibiotic or antifungal active agent from pulverized olive or acebuche obtained by the method described herein. According to the present invention, extraction is preferably achieved by CO2 extraction. Extraction can be carried out in batch mode, as the extractor can be emptied and refilled only at atmospheric pressure. During extraction, supercritical carbon dioxide (CO2) permeates the raw materials under high pressure, extracting soluble substances (corresponding to extracts) from the pulverized olive or acebuche. By gradually reducing the pressure, the dissolved substances are separated into fractions having different compositions. In the first fraction (separator 1), less soluble substances accumulate, while more soluble substances are collected in the next separator 2. Without limiting these, further types of solid-liquid extraction, such as cold maceration and decantation, can be selected, as long as the active agent remains sufficiently effective.

[0047] In a preferred embodiment of the method according to the present invention, steps b) and c) are performed simultaneously by subjecting the olive or acebuche portion to a force generated by a dynamic airflow, preferably by passing the olive or acebuche through a venturi nozzle together with air or another suitable gas, preferably preheated air. U.S. Patent Nos. 7,429,008, 7,500,830, and 7,909,577 disclose the pulverization of a material that is subjected to moisture extraction and drying during pulverization, for example, by an airflow generator connected to a venturi nozzle, generally using polymers or waste as the feed material. Furthermore, International Publication No. 2013 / 052583 generally discloses the dehydration, pulverization, and pyrolysis of biomass such as sewage sludge. International Publication No. 2013 / 075003 provides a method for producing eggshell powder for use in the manufacture of bio-based products. This method involves high airflow velocity at room temperature to finely grind the eggshell and separate the eggshell components from the inner membrane components. Nevertheless, it is not yet possible to develop a method, such as the one provided by the present invention, that can simultaneously dehydrate and dry the olive or acebuche portions and grind them simultaneously. The active agents of the olive or acebuche are retained, and the powder has a long shelf life.

[0048] Therefore, the olive and / or asebuche portion is exposed to a dynamic airflow, for example, by passing the olive or asebuche portion through the venturi nozzle together with the air. In doing so, the airflow draws in the feed material through the venturi nozzle positioned in connection with the apparatus. The movement of air through the venturi nozzle then accelerates both the air and the olive or asebuche contained in the airflow. The airflow is generated, for example, by the rotation of the blades of a turbomachinery or by another suitable device for drawing in the airflow. The airflow and the dried, finely ground olive or asebuche exit the apparatus through an outlet nozzle provided in the apparatus, where the finely ground olive or asebuche can be separated from the airflow by a sieve, cyclone or similar method known to those skilled in the art. Simultaneously with grinding, the airflow is heated by the energy loss (heat) due to the operation of the turbomachinery. The accelerated airflow (and the finely ground olive or asebuche) then absorbs this heat, raising its temperature. Physically, air at a higher temperature can absorb relatively more moisture than air at a lower temperature. Due to this natural phenomenon, in the principle of a Venturi apparatus, grinding and pulverization and the utilization of heat loss are complementary. Grinding of the feed material drawn in through the apparatus including the Venturi facilitates the expansion of the surface of the feed material for moisture transfer into the airflow, and the increased air temperature due to the operation of the turbomachinery increases the transfer of moisture from the material to the airflow. Using a Venturi apparatus, steps b) and c) of the method according to the present invention can be carried out completely or partially. Preferably, the feed point of the olive or acebuche portion may be before the inlet pipe connected to the apparatus (see Figure 2).

[0049] In a more preferred embodiment of the method according to the present invention, the thickening agent used is selected from gelling agents, such as gellan gum, alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propylene glycol alginate, agar, carrageenan, fercereran, locust bean gum, guar gum, tragacanth, acacia gum, xanthan gum, karaya, tara gum, pectin, cellulose, gelatin, and modified starch.

[0050] In yet another preferred embodiment of the method according to the present invention, the non-toxic solubilizer and / or emulsifier is selected from surfactants, where the surfactant is preferably selected from Kolliphor®, most preferably from Kolliphor® RH40. Further surfactants can be selected from nonoxynol-9, octoxynol-9 (Triton X-100®), polysorbate 20 (Tween 20®), octyl-β-glucoside, and N-octyl-β-D-1-thioglucopyranoside. Other non-toxic solubilizers and / or emulsifiers can be selected from ethanol, acetone, hexane, chloroform, and methyl tert-butyl ether (MTBE).

[0051] The olive or asebuche parts for the methods according to the present invention can be selected from leaves, twigs, root bark, stem bark, fruit or oil-producing residues and combinations thereof. As used herein, the term “part” refers to all suitable components of the olive and asebuche plants, for example, in the form of leaves, twigs, stem or root bark, olives or products and residues derived from the processing of olives. Parts may also refer to, for example, plants growing in the soil, harvested parts of plants, discarded parts of plants, already processed parts of plants and withered parts of plants.

[0052] A preferred method according to the present invention is one in which at least one active agent selected from antibiotic or antifungal compounds is included in a prepared composition. The preferred agent is selected from the iridoid or phenol class, and preferably at least one organic compound is selected from the group of oleuropein, oleacein, oleocanthal, tyrosol, hydroxytyrosol, ubaol, and erythrodiol. Furthermore, in this disclosure, the active agent may further include oleiroside, ligstroside, beruvascoside, flavonoid, maslinic acid, apigenin, luteolin, and oleanolic acid. Oleuropein, oleacein, oleocanthal, tyrosol, hydroxytyrosol, ubaol, and erythrodiol are most preferred.

[0053] In a second embodiment, the present invention solves the above-mentioned problems by providing an antibiotic or antifungal active sterile pulverized olive or acebuche product / composition obtained by the method according to the present invention.

[0054] Sterilized, finely ground olive or acebuche that is antibiotic or antifungal active according to the present invention, obtained from steps a), b), c), and d) of the method according to the present invention, is particularly preferred.

[0055] As used herein, the term “antibiotically active” generally refers to activity that acts against infections caused by microorganisms. These microorganisms are primarily bacteria, but also include, for example, protozoa. The effect may include killing the microorganisms and / or inhibiting their growth and / or reproduction. In the present invention, the antibacterial agent is particularly active and effective against microorganisms resistant to commercially available antibiotics, and especially against multi-resistant microorganisms (i.e., bacteria or fungi resistant to at least two different antibiotics, for example, commercially available antibiotics). As used herein, “antifungally active” describes activity that acts against infections caused by fungi, such as molds. The effect may include killing the fungi or inhibiting their growth or reproduction. As used herein, the term “resistance” refers to a characteristic of a microorganism that allows it to weaken or completely neutralize the effects of commercially available antibiotics. As used herein, the term “multi-resistance” refers to a characteristic of a microorganism that allows it to weaken or completely neutralize the effects of at least two different commercially available antibiotics.

[0056] The present invention prefers sterile, finely ground acebuche that is antibiotic or antifungal active, wherein the finely ground olive or acebuche is a powder and consists of more than 95% by weight of dry biomass. As used herein, the term “dry biomass” refers to the actual dry amount of substance mass excluding water or other components, e.g., other liquids. As used herein, “weight%” refers to the mass ratio of the mixture, in particular the finely ground olive or acebuche. The composition of the finely ground olive or acebuche depends on the ratio of a single component in 100 grams of the mixture. In the present invention, it is preferable that more than about 95% of 100 g consists of dry biomass.

[0057] In a third embodiment, the present invention solves the above-mentioned problems by providing an olive or acebuche composition produced by the method according to the present invention. The composition may, and preferably, include a pharmaceutically or cosmetically acceptable carrier, diluent, or excipient.

[0058] In another preferred embodiment, the present invention relates to an antibacterial or antifungal composition comprising at least one organic compound selected from oleacein, hydroxytyrosol, oleocanthal, ubaol, and erythrodiol. The composition is preferably a pharmaceutical or cosmetic composition as described herein.

[0059] In another preferred embodiment, the present invention relates to the above-mentioned compositions used for the prevention or treatment of bacterial or fungal infections in mammals, preferably for infections caused by antibiotic-resistant or multi-resistant bacteria.

[0060] As used herein, “pharmaceutically acceptable carrier, diluent, or excipient” refers to a component in a pharmaceutical formulation or composition other than the active ingredient that is non-toxic to the subject. Examples of pharmaceutically acceptable carriers, diluents, or excipients include any suitable physiologically compatible solvent, dispersion medium, coating agent, further antibacterial and antifungal agents, isotonic and absorption retarders. Examples of carriers include various preservatives, antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the composition of the present invention. Furthermore, sustained absorption of injectable dosage forms can be achieved by using absorption retarders, such as aluminum monostearate and gelatin.

[0061] Regardless of the chosen route of administration, the compositions and / or pharmaceutical compositions of the present invention, which can be used in an appropriate hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The actual dose level of the active ingredient of the pharmaceutical compositions of the present invention may vary. The chosen dose level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health and medical history of the patient being treated, and similar factors known in the medical field.

[0062] The composition must be sterile and fluid enough to be delivered by syringe. Often, the composition contains isotonic agents, such as sugars, polyalcohols, such as mannitol or sorbitol, and sodium chloride.

[0063] The compositions of the present invention can be administered locally or systemically. Administration is generally parenteral, for example, intravenously. Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and organic esters for injection, such as ethyl oleate. Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions, or suspensions, including physiological saline and buffering media. Parenteral vehicles include sodium chloride solutions, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or non-volatile oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte replacement agents (e.g., those based on ringer's dextrose), etc. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0064] As used herein, “cosmetically acceptable carriers, diluents, or excipients” refers to suitable components in a cosmetic formulation other than the active ingredient. Generally, such components must be suitable for use in topical contact with tissues, such as skin, without excessive toxicity, incompatibility, instability, irritation, allergic reactions, etc. Examples of cosmetically acceptable carriers, diluents, or excipients include water, liquid or solid emollients, solvents, water-retaining agents, and thickeners, while powders facilitate the distribution of the composition when applied, for example, to skin, hair, and / or nails.

[0065] A more particularly preferred embodiment of the present invention is an olive or acebuche composition according to the present invention, which is a gel, preferably a homogeneous colloidal gel. In the present invention, “colloidal gel” means that the lipophilic pulverized olive or acebuche obtained by the method according to the present invention is present in colloidal size particles, i.e., between 1 μm and 1000 μm, at a loading amount of up to 100%, such that the poorly soluble lipophilic pulverized olive or acebuche becomes soluble and a uniform distribution of the pulverized olive or acebuche is ensured.

[0066] Other particularly preferred embodiments relate to olive or acebuche suspensions according to the present invention, which are aqueous suspensions. As used herein, this term particularly includes olive or acebuche suspensions that are water-soluble using a surfactant, and aqueous solutions of each which become conveniently available by the above use.

[0067] A further preferred olive or acebuche composition according to the present invention is a suspension in which the amount of a non-toxic solubilizer, particularly a surfactant, is less than 10%, preferably less than 5%, and more preferably less than 3%, of the total volume of the olive or acebuche suspension. As described above, the surfactant is preferably selected from Kolliphor®, most preferably Kolliphor® RH40.

[0068] In another preferred embodiment of the present invention, the compositions described herein are in the form of ointments, lotions, creams, sprays, gels, liquids, drops, capsules, or suppositories. The compositions can be administered systemically, i.e., by mucosal or transdermal means. For mucosal or transdermal administration, a penetrating agent suitable for the barrier to be penetrated is used in the formulation. Such penetrating agents are generally known in the art, and examples of penetrating agents for mucosal administration include detergents, bile salts, and fusidic acid derivatives. For mucosal administration, the compositions according to the present invention are formulated into liquids, drops, capsules, or suppositories. For topical (transdermal) administration, the compositions are formulated into ointments, lotions, creams, sprays, or gels, as is generally known in the art.

[0069] In a fourth embodiment, the present invention solves the above-mentioned problems by providing a sterile, finely ground olive or acebuche or an olive composition or acebuche composition described herein for use in pharmaceuticals. The sterile, finely ground olive or acebuche or an olive composition or acebuche composition described herein is more preferably used for the prevention or treatment of microbial infections in mammals, such as bacterial or fungal infections, preferably for use against infections caused by antibiotic-resistant bacteria or even more resistant bacteria. Another embodiment relates to the use of a sterile, finely ground olive or acebuche or an olive composition or acebuche composition described herein for the manufacture of a pharmaceutical for the prevention or treatment of microbial infections in mammals, such as bacterial or fungal infections, preferably for use against infections caused by antibiotic-resistant bacteria or even more resistant bacteria.

[0070] As used herein, “mammal” may refer to livestock (e.g., horses, cattle, sheep, or pigs), pets (e.g., cats, dogs, rabbits, or guinea pigs), rodents, or especially humans. Accordingly, the compositions according to the present invention can be used to treat any of these mammals.

[0071] As used herein, the term “treatment” preferably includes the administration of the composition to the mammal in a therapeutically effective dose to alleviate the disease or the progression of the disease. Thus, the effective dose is the amount of the composition or pharmaceutical composition described herein above that normalizes the infectious condition in the mammal. This amount alleviates the symptoms seen for the infection and / or condition without being toxic to the subject. The drug regimen is determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any one mammal depends on many factors, including the mammal’s size, body surface area, age, the specific compound administered, sex, time and route of administration, overall health status, and other drugs administered concurrently.

[0072] As used herein, the term “prevention” preferably includes the administration of the composition to the mammal in a prophylactically effective amount such that it relates to reducing the predisposition or risk of developing an infection by antibiotic-resistant or multi-resistant bacteria, no matter how small the amount. For prevention, the mammal is preferably one that is at risk of or prone to developing an infection by antibiotic-resistant or multi-resistant bacteria, and the composition is preferably administered by injection. Furthermore, enteral and topical administration may also be included in the context of this disclosure, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injections and infusions.

[0073] In yet another preferred embodiment of the present invention, the sterilized pulverized olive or acebuche according to the present invention, or the olive or acebuche composition according to the present invention, is for use in the prevention or treatment of bacterial or fungal infections in mammals, preferably infections caused by antibiotic-resistant or multi-resistant bacteria (wherein the bacteria are Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus hirae, Enterococcus faecalis, Enterobacter, Mycobacterium tuberculosis). (Selected from tuberculosis, Serratia, Proteus, Providencia, Morganella, Enterococcus faecium, Heliocobacter pylori, Campylobacter, Salmonella, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, Shigella, Acinetobacter baumannii, and resistant and multi-resistant strains thereof), and for use in the prevention or treatment of fungal infections in mammals (where the above fungi are Candida albicans). It is selected from *Aspergillus albicans* and *Aspergillus brasiliensis*.

[0074] In a fifth embodiment, the present invention solves the above-mentioned problems by providing a functional food additive comprising the sterilized pulverized olive or acebuche described herein or the olive composition or acebuche composition described herein. The above-mentioned problems are further solved by providing the use of the sterilized pulverized olive or acebuche described herein or the olive composition or acebuche composition described herein as a functional food additive, in particular as an animal feed additive. As used herein, the term "animal feed additive" means the addition of a composition to animal feed.

[0075] Furthermore, as used herein, the term “functional food additive” describes the use of a composition to increase the supply of the composition’s active ingredients to mammals by adding it to a general diet. The composition may also be taken as a prescribed diet or nutritional supplement, in the form of a concentrate or in doses, particularly in the form of capsules, tablets, pills, effervescent tablets and other similar forms, powder sachets, liquid ampoules, droppers and similar forms of liquids and powders, for oral intake in appropriate amounts in addition to general nutrition. In particular, the term “animal feed additive” means the addition of the composition to livestock feed, and this does not exclude feed for other animal species.

[0076] In a sixth embodiment, the present invention solves the above-mentioned problems by providing a cosmetic product comprising sterilized pulverized olive or acebuche according to the present invention, a gel or suspension of olive or acebuche according to the present invention, or a pharmaceutical composition according to the present invention. Another embodiment then relates to the use of the sterilized pulverized olive or acebuche described herein, or the olive or acebuche composition described herein, in cosmetics or cosmetic methods.

[0077] As used herein, the term “cosmetics” encompasses the entire area of ​​body care, or the entire area of ​​helping to maintain, restore, or enhance the appearance of mammals. In particular, the areas of cleansing, care and protection, and especially dental and oral care applications are included within the scope of this disclosure.

[0078] In this invention, the terms “about” and “approximately” mean intervals of precision that a person skilled in the art would understand to still guarantee the technical effect of the feature being discussed. These terms typically indicate deviations from a given numerical value, such as ±20%, ±15%, ±10%, or, for example, ±5%. As recognized by a person skilled in the art, a particular deviation of a numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have larger deviations than artificial or engineered technical effects. When an indefinite or definite article, e.g., “a,” “an,” or “the,” is used when referring to a singular noun, this includes the plural form of that noun unless otherwise specified.

[0079] The present invention will now be further described by the following examples, but is not intended to be limited thereto. In the present invention, all references, patents and publications cited herein constitute an entire part of this specification by reference. [Examples]

[0080] Example 1: Extraction of olive tree components using supercritical carbon dioxide Dried and pre-ground olive wood components are placed in the extractor. Extraction is always carried out in batch mode, as the extractor can only be emptied and refilled at atmospheric pressure. During extraction, supercritical carbon dioxide (CO2) permeates the raw materials under high pressure, extracting soluble substances (i.e., extracts) from the raw materials. By gradually reducing the pressure, the dissolved substances can be separated into fractions having different compositions. In the first fraction (separator 1), the pressure may result in the accumulation of less soluble substances, while in the next separator 2, more readily soluble substances can be collected.

[0081] For the experiment to be conducted, an extraction variant having only one separation step was selected, thereby collecting all dissolved substances in separator 1. The amount of substance depends on the selected extraction conditions (pressure and temperature), as well as the solubility and amount of the substance contained in the raw materials.

[0082] Process Flow: A high-pressure experimental system (HDL 4) was used for the experiment. This is an experimental plant with one extractor and two separators. This plant is designed for extraction pressures up to 1000 bar and temperatures up to 95°C. The extraction pressure is less than 1000 bar. Liquid CO2 from the CO2 tank is pumped to the extraction pressure and heated to the extraction temperature by a heat exchanger. In the extractor, at this stage, supercritical CO2 flows through the raw materials, becoming rich in soluble substances. After depressurization, the mixture is separated into gaseous CO2 and extract. The extract can be collected in the separator and removed from the plant. The uncharged gaseous CO2 can then be liquefied again in the condenser and reused for the extraction cycle.

[0083] Procedure: For extraction, the raw materials were subjected to an HDL 4 extractor. Subsequently, an attempt was made to extract useful components (e.g., terpenes and terpenoids) from the natural substance. Only one separation step was used for the subsequent separation of the extracts, and the entire extract was collected in separator 1. Since the obtained extract is not fluid at -60°C, conventional extraction methods via the outlet valve of separator 1 are difficult or impossible. The extract must be removed after each test and further scraped off after opening the separator, which can wash away some of the solvent.

[0084] Example 2: Processing of olive wood material using a venturi dryer The initial olive wood material is subjected to a thermomechanical process in which the particle size and moisture content of the raw materials are clearly altered. The result of this technical application is a dehydrated, granular, and possibly powdery product for direct further use in the process chain, and possibly for intermediate storage.

[0085] Example 3: Sterilization Process Due to natural contamination of the starting product by aerobic spore-forming bacteria, various sterilization procedures were applied. Different sterilization temperatures were used to determine the optimal temperature for preserving the pharmaceutically active components: 121°C and 134°C. Sterilization was performed at different temperatures.

[0086] Example 4: Comparison of active substances in cultivated olive leaves and wild olive leaves, and grinding method. Sampling of plant parts from wild and cultivated olives was performed manually in Casa de Porros, Valdevaqueros, Tarifa, and Spain. The plant parts were processed as follows: fresh leaves, twigs, stem bark, and some root bark were dried in a Taprogge Venturi apparatus, while the rest were finely ground in a Thermomix. The sample materials were stored at ambient temperature in light-shielded, sealed containers. For comparison, both samples were sieved to obtain identical particle size spectra. Subsequently, Soxhlet extraction and chromatography were performed to analyze the chemical composition of active agents in both cultivated and wild olives. A positive effect was found in the oxygen radical absorption capacity (ORAC) test, and sampling of active substances after Soxhlet extraction revealed that higher extraction yields were achieved for materials ground in the Taprogge Venturi apparatus. A favorable effect on ORAC (oxygen radical absorption capacity) was detected in materials ground in the Taprogge Venturi apparatus, which in turn leads to positive storage characteristics, particularly a longer shelf life. This was demonstrated by measuring the antioxidant activity of leaves and twigs processed using the Taprogge Venturi apparatus over a 30-day period, comparing it to the antioxidant activity of fresh samples while keeping the values ​​of the Taprogge Venturi apparatus samples constant. The results are shown in Table 2.

[0087] Table 2: Comparison of antioxidant activity of fresh samples and samples processed using the Taprogge Venturi apparatus over a 30-day period. [Table 2]

[0088] By comparing the active substances in wild and cultivated olives, it was found that wild-type olive trees contain higher concentrations of active substances. The varieties Olea europaea and Olea silvestris differ in many respects. Cultivated olives have larger fruits of a certain shape and are therefore more suitable for olive oil production. However, the tendency to produce larger fruits clearly means that the rest of the olive plant contains fewer active substances. The comparison of the two plants was based on already published data on the three most common cultivated olive species in Andalusia: Arbequina, Hojiblanca, and Picual. The leaves of fresh Acebuche olives were examined for their active substance profile (Table 3).

[0089] Table 3: Concentration of a single substance (mg / g) [Table 3]

[0090] Figures 3-6 show a comparison of the active substances of various olive varieties. Figure 3 shows a comparison of iridoids, Figure 4 shows a comparison of flavonoids, Figure 5 shows a comparison of terpenes, and Figure 6 shows a comparison of tyrosol and hydroxytyrosol.

[0091] Table 4 below compares the percentage of active substances in acebuche versus cultivated olives.

[0092] Table 4: Amount of identified active substances in acebuche versus cultivated olives [Table 4]

[0093] Comparisons based on these nine substances show that acebuche olive leaves have the highest concentrations of phenols and monophenols, particularly flavonoids. From a pharmaceutical standpoint, wild olives (acebuche) and cultivated olives are entirely different, and therefore, wild olives are clearly a superior raw material for potentially therapeutic or cosmetic products.

[0094] Example 5: Study of the antibacterial efficacy of olive or acebuche suspension The purpose of this study was to test the antimicrobial efficacy of olive or acebuche suspensions suspended in Kolliphor RH40 and water for injection (WFI) against an extended spectrum of test pathogens (Table 5).

[0095] Table 5: Spectra of test pathogens in microbiological studies of this disclosure [Table 5]

[0096] Procedure: Preparation of test pathogen suspension: Test bacteria were cultured to a maximum passage number of 5. A test bacterial suspension was prepared in sterile 0.9% NaCl solution at a concentration of approximately 1000 CFU / 0.1 ml.

[0097] Sample preparation: 3.00 g of olive extract in 1.01 g of Kolliphor RH40 was heated in a water bath at 45°C for approximately 5 minutes, then heated in a water bath at 80°C for approximately 15 minutes, and shaken by hand. The olive extract and Kolliphor RH40 were mixed with 15 ml of WFI to obtain a very viscous, dark green to dark brown suspension that could not be pipetted. The following day, the preservation solution was heated again in a water bath at 80°C, and another 5 ml of WFI heated to 80°C was added. The preservation solution was heated again at 80°C for approximately 5 minutes. The water bath was warmed and shaken by hand for approximately 1 minute. As a result, a preservation solution containing 125 mg of olive extract and 4.2% Kolliphor RH40 per ml was obtained. After this processing, the olive extract was available as a uniform, pipettable suspension ranging from dark brown to dark green.

[0098] Evaluation of the antimicrobial efficacy of olive extract after preparation of preservation solutions: For each test pathogen, 1 ml of the preservation solution was replaced with 1 ml of double-concentrated CaSo broth and Sab broth (C. albicans). Then, 0.1 ml of each test pathogen suspension, adjusted to approximately 1000 CFU / 0.1 ml, was inoculated into the preparation. The bacterial count in the test pathogen suspension was confirmed on blood agar and Sab agar by the spatula method. All culture medium preparations were incubated at (30°C to 35°C) for up to 72 hours. After incubation for 24, 48, and 72 hours, all 0.1 ml batches were subcultured by streaking onto blood agar plates and Sab agar plates, respectively. The agar plates were incubated aerobically at 30°C to 35°C for 24 to 48 hours (yeast) and then tested. Positive control: 1 ml of WFI was added to 1 ml of double-concentrated CaSo or Sab broth (C. albicans), and then 0.1 ml of the test pathogen suspension, adjusted to approximately 1000 kBE / 0.1 ml in a single measurement, was inoculated into it. The positive control was used as a reference for the growth of the test bacteria in double-concentrated culture medium after a 1:2 dilution. The results are shown in the table below.

[0099] Table 6: Results of the antimicrobial efficacy of olive suspension using the test pathogen C. albicans ATCC10231; Inoculum: Effectively inoculated bacterial count per test batch: 1160 CFU = 580 CFU / ml culture medium batch [Table 6] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0100] Table 7: Results of the antimicrobial efficacy of olive suspension using the test bacterium E. faecium ATCC BAA2317; Inoculum: Effectively inoculated bacterial count per test batch: 1810 CFU = 905 CFU / ml culture medium batch [Table 7] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0101] Table 8: Results of the antimicrobial efficacy of olive suspension using the test bacterium E. hilae ATCC 10541; Inoculum: Effectively inoculated bacterial count per test batch: 760 CFU = 380 CFU / ml culture medium batch [Table 8] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0102] Table 9: Results of the antimicrobial efficacy of olive suspension using the test bacterium Klebsiella pneumoniae subspecies CCUG 56233; Inoculum: Effectively inoculated bacterial count per test batch: 1200 CFU = 600 CFU / ml culture medium batch [Table 9] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0103] Table 10: Results of the antimicrobial efficacy of olive suspension using the test bacterium Klebsiella pneumoniae subspecies pneumoniae ATCC 10031; Inoculum: Effectively inoculated bacterial count per test batch: 740 CFU = 370 CFU / ml culture medium batch [Table 10] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0104] Table 11: Results of the antimicrobial efficacy of olive suspension using the test bacterium Pseudomonas aeruginosa ATCC 9027; Inoculum: Number of bacteria effectively inoculated per test batch: 640 CFU = 320 CFU / ml culture medium batch [Table 11] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0105] Table 12: Results of the antimicrobial efficacy of olive suspension using the test bacterium Pseudomonas aeruginosa ESBL DSM 24600; Inoculum: Effectively inoculated bacterial count per test batch: 680 CFU = 340 CFU / ml culture medium batch [Table 12] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0106] Table 13: Results of the antimicrobial efficacy of olive suspension using the test bacterium Escherichia coli ATCC 8739; Inoculum: Effectively inoculated bacterial count per test batch: 860 CFU = 430 CFU / ml culture medium batch [Table 13] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0107] Table 14: Results of the antimicrobial efficacy of olive suspension using the test bacterium Escherichia coli DSM 22312; Inoculum: Number of bacteria effectively inoculated per test batch: 670 CFU = 435 CFU / ml culture medium batch [Table 14] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0108] Table 15: Results of the antimicrobial efficacy of olive suspension using the test bacterium Staphylococcus aureus ATCC 6538; Inoculum: Effectively inoculated bacterial count per test batch: 680 CFU = 340 CFU / ml culture medium batch [Table 15] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0109] Table 16: Results of the antimicrobial efficacy of olive suspension using the test bacterium Staphylococcus aureus subspecies Aureus ATCC 29213; Inoculum: Effectively inoculated bacterial count per test batch: 2020 CFU = 1010 CFU / ml culture medium batch [Table 16] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0110] For further study of two test pathogenic bacteria, Acinetobacter baumannii and Aspergillus brasiliensis, the Kolliphor RH40 content in the preservation solution was reduced by 50%, from 4.2% to 2.1%. In addition to this starting preservation solution A1, which contained olive extract at a concentration of 127.5 mg / ml, a 1:2 dilution with sterile water for injection was prepared so that the olive extract had a concentration of 63.8 mg / ml (preservation solution A2).

[0111] Table 17: Results of the antimicrobial efficacy of olive suspension using the test bacterium A. baumannii ATCC 19606; Inoculum: Effectively inoculated bacterial count per test batch: 4000 CFU = 2000 CFU / ml culture medium batch [Table 17] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0112] Table 18: Results of the antimicrobial efficacy of olive suspension using the test bacterium A. baumannii NCTC 13420; Inoculum: Effectively inoculated bacterial count per test batch: 890 CFU = 445 CFU / ml culture medium batch [Table 18] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0113] Table 19: Results of antimicrobial efficacy of olive suspension using the test pathogen A. brasiliensis ATCC 16404; Inoculum: Effectively inoculated bacterial count per test batch: 620 CFU = 310 CFU / ml culture medium batch [Table 19] +=Growth of test pathogens in the nutrient medium preparation in the form of visually apparent turbidity. P = Turbidity of the product; visual evaluation of the growth of the test pathogen is impossible. Subculturing (SC) by streaking each sample with 0.1 ml of nutrient medium preparation.

[0114] As the tests demonstrate, with the exception of the test pathogen A. brasiliensis ATCC 16404, the growth of the test pathogen could not be detected after 72 hours of incubation of the nutrient medium preparation following the corresponding subculturing. Therefore, the excellent antibacterial efficacy of olive extract at concentrations of 62.5 mg / ml or 63.8 mg / ml, respectively, can be demonstrated under the test conditions. For the test pathogen A. brasiliensis in the presence of olive suspensions from preservation solution A1 and preservation solution A2, a small number of isolated and remaining viable spores could still be detected throughout the tests, but the reduction in CFU is also significant here.

[0115] Example 6: Study of the antibacterial efficacy of olive or acebuche gel Procedure: A colloidal gel containing Kelcogel CG-HA was prepared from olive leaf powder. For this purpose, 0.1 g of Kelcogel CG-HA was dissolved in 100 ml of WFI by heating to 85°C-90°C, and then 15 g of olive leaf powder was added while stirring until a homogeneous suspension was visually formed. After cooling in an ice bath, a colloidal olive leaf powder gel was obtained. The prepared test pathogen suspension (10⁵ CFU / g-10⁶ CFU / g) was inoculated into 10 g aliquots of gel, and the number of pathogens was tested according to the test times listed in Table 15.

[0116] Table 20: Number of CFUs according to test duration [Table 20]

[0117] Escherichia coli already showed excellent efficacy at t0 time, and Staphylococcus aureus and Pseudomonas aeruginosa showed levels below the detection limit of 100 CFU / gram after 12 minutes, demonstrating overall excellent antibacterial efficacy with a short exposure period.

[0118] Example 7: Study of the antibacterial efficacy of olive or acebuche gel Procedure: A colloidal gel containing Kelcogel CG-HA was prepared from olive leaf powder. For this purpose, 0.1 g of Kelcogel CG-HA was dissolved in 100 ml of WFI by heating to 85°C-90°C, and then 15 g of olive leaf powder was added while stirring until a homogeneous suspension was visually formed. After cooling in an ice bath, a colloidal olive leaf powder gel was obtained. The prepared test pathogen suspension (10⁵ CFU / g-10⁶ CFU / g) was inoculated into 10 g aliquots of gel, and the number of pathogens was tested according to the test times listed in Table 21.

[0119] Table 21: Number of CFUs according to test duration [Table 21]

[0120] As a result, E. coli showed excellent efficacy after 6 minutes, and Staphylococcus aureus was below the detection limit at a maximum of 100 CFU / gram after 12 minutes. For Pseudomonas aeruginosa, the bacterial count decreased to below the detection limit after 30 minutes, demonstrating overall excellent antibacterial efficacy with a short exposure period.

[0121] Example 8: Study of the antibacterial efficacy of olive or acebuche composition containing dough pieces. Procedure: Dough samples were prepared at each test time by mixing 500 mg of the sample with 1 ml of WFI. After homogenization, 0.5 ml of the prepared pathogen suspension was added and mixed uniformly to form a dough. Subsequently, both test pathogens showed excellent efficacy after 6 hours of reaction at room temperature with untreated powdered olive leaf product and powdered olive leaf product steam-sterilized at 121°C for 15 minutes and 130°C for 30 minutes (Table 22).

[0122] Table 22: Number of CFUs according to test duration [Table 22]

[0123] Untreated olive leaf dough pieces were subjected to 1.4 × 10⁶ bacteria per 500 mg of dough piece by various aerobic spore-forming bacteria that did not die during the test periods of 6 hours and 24 hours, respectively. 3 CFU ~2.1 x 10 3 It shows natural contamination at a level of CFU. In test samples sterilized at 121°C for 15 minutes, the dough pieces showed 1.1 × 10⁶ per 500 mg. 3 CFU ~2.2 x 10 2 The samples showed natural contamination within the CFU range. Both sterile test specimens were sterilized at 130°C for 30 minutes, and no contaminating pathogens were detected.

[0124] Example 9: Study of the antibacterial efficacy of olive or acebuche compositions containing dough pieces. Procedure: For each test pathogen and test time, dough pieces were prepared by mixing 500 mg of olive substrate with 1 ml of WFI. After homogenization, 0.5 ml of the prepared pathogen suspension was added and mixed uniformly to form the dough. Subsequently, both test pathogens showed excellent efficacy after 24 hours of reaction at room temperature, both with untreated olive leaves and olive leaves steam-sterilized at 121°C for 15 minutes and 130°C for 30 minutes (Table 23).

[0125] Table 23: Number of CFUs according to test duration [Table 23]

[0126] Untreated olive leaf dough pieces were subjected to 2.9 × 10⁶ bacteria per 500 mg of dough, which did not die within a 24-hour test period. 4 It exhibits natural contamination at a CFU level. Sterilized test samples cannot detect contaminating pathogens.

[0127] Example 10: Study of the antibacterial efficacy of olive or acebuche composition containing dough pieces. Procedure: For each test pathogen and test time, dough pieces were prepared by mixing 500 mg of olive substrate with 1 ml of WFI. After homogenization, 0.5 ml of the prepared pathogen suspension was added and mixed uniformly to form a dough. Subsequently, both test pathogens showed excellent efficacy after a 24-hour reaction time at room temperature, both in the untreated powdered peel / strain product and the powdered peel / strain product steam-sterilized at 121°C for 15 minutes and 130°C for 30 minutes (Table 24).

[0128] Table 24: Number of CFUs according to test duration [Table 24]

[0129] Untreated powdered skin / strain was found to be 4.4 × 10⁶ per 500 mg of dough by various aerobic spore-forming bacteria that did not die within a 24-hour test period.2 showed natural contamination at the CFU level. 1.1×10 2 Contaminated pathogenic bacteria at the level of CFU / g of fabric piece could still be detected in the test specimens sterilized at 121°C. The test specimens were also sterilized at 121°C. Contaminated pathogenic bacteria could not be detected in the test specimens sterilized at 130°C.

[0130] Example 11: Study on the antibacterial efficacy of olive or acevich compositions containing fabric pieces Implementation: For each test pathogenic bacterium and test time, fabric pieces were prepared by mixing 500 mg of olive base material with 1 ml of WFI. After homogenization, 0.5 ml of the adjusted pathogenic bacterium suspension was added and uniformly mixed to form the fabric. Subsequently, both test pathogenic bacteria already showed very excellent efficacy after a reaction time of 24 hours at room temperature with both untreated powdery skin / root products and powdery skin / root products steam-sterilized at 121°C for 15 minutes and 130°C for 30 minutes (Table 25).

[0131] Table 25: Number of CFUs according to the test time

Table 25

[0132] The untreated powdery skin / root showed natural contamination in the range of 1.1×10 6 CFUs per 500 mg of fabric by various aerobic spore-forming bacteria that did not die within the test period after 24 hours. In the test samples sterilized at 121°C and 130°C, contaminated pathogenic bacteria at the levels of 1.8×10 3 CFU / g of fabric piece and 8.8×10 2 CFU / g of fabric piece could still be detected.

[0133] Example 12: Analysis of the antibacterial efficacy of hydroxytyrosol (single substance) against Staphylococcus aureus ATCC 6538 Hydroxytyrosol is found in base materials and extracts derived from olive trees.

[0134] A preservation solution was prepared by adding 0.1 ml of methanol to 10.48 mg of hydroxytyrosol. After dissolving the compound, the solution was quantitatively transferred to 4.9 ml of sterile water for injection (WFI). Effective concentration of hydroxytyrosol: 2.0 mg / ml.

[0135] Using a preservation solution, five concentrations of hydroxytyrosol ranging from 2 mg hydroxytyrosol / ml to 0.125 mg hydroxytyrosol / ml were tested using a quantitative suspension assay.

[0136] Approximately 2.0 × 10⁶ units were added to 2 ml each of the storage solution and diluent (V1-V4), as well as to a 2% methanol solution as a positive control. 5 A 0.1 ml test suspension of Staphylococcus aureus ATCC 6538, adjusted to CFU / 0.1 ml, was inoculated, yielding approximately 100,000 CFU / ml in each assay.

[0137] Next, samples (0.1 ml) were collected at t0, 12, 30, and 60 minutes after inoculation, and at t24, 48, and 72 hours. These were cultured at 20°C to 25°C and diluted in 0.9 ml of 0.9% NaCl solution. The samples were plated onto blood agar plates and incubated at 30°C to 35°C for up to 5 days.

[0138] The results are shown in Table 26 below.

[0139] Table 26 Number of CFUs; initial cell load: 1.7 × 10⁻⁶ 5 CFU / ml, Bacteria: Staphylococcus aureus ATCC 6538, Hyd=hydroxytyrosol [Table 26]

[0140] As shown in Table 26, 2% methanol did not show a significant antibacterial effect at the beginning of the test. The decrease at subsequent time points is due to the natural loss of bacterial viability. Hydroxytyrosol showed a significant effect only after 24 hours, but nevertheless, this effect was strong, i.e., below the detection limit of the assay at 100 CFU / ml. Further dilution further delayed the effect.

[0141] Example 13: Analysis of the antimicrobial efficacy of oleocanthal (single substance) against Staphylococcus aureus ATCC 6538 Oleocanthal is found in substrates and extracts derived from the olive tree.

[0142] A preservation solution was prepared with oleocanthal at a concentration of 2 mg / ml. This compound was dissolved in ethanol (99.8%) and WFI was added.

[0143] Using a preservation solution, the concentrations of oleocanthal in a geometric progression from 1 mg oleocanthal / ml to 0.125 mg oleocanthal / ml were tested using a quantitative suspension assay.

[0144] Each of the following was mixed with approximately 3.6 × 10⁶ of preservative solution and diluent (V1-V4) in 2 ml each, as well as a 2.85% ethanol solution as a positive control. 5 A 0.055 ml test suspension of Staphylococcus aureus ATCC 6538, adjusted to CFU / 0.1 ml, was inoculated, yielding approximately 99,000 CFU / ml in each assay.

[0145] Next, samples (0.1 ml) were collected at t0, 12, 30, and 60 minutes after inoculation, and at t24, 48, and 72 hours. These were cultured at 20°C to 25°C and diluted in 0.9 ml of 0.9% NaCl solution. The samples were plated onto blood agar plates and incubated at 30°C to 35°C for up to 5 days.

[0146] The results are shown in Table 27 below.

[0147] Table 27 Number of CFUs; initial cell load: 9.9 × 10 4 CFU / ml, Bacteria: Staphylococcus aureus ATCC 6538, Ole = Oleocanthal [Table 27]

[0148] As shown in Table 27, 3.84% ethanol did not show a significant antibacterial effect at the beginning of the test. The decrease at subsequent time points is due to the natural loss of bacterial viability. Oleocanthal shows an immediate effect at higher concentrations, below the detection limit of the assay at 100 CFU / ml. Dilution delays these effects, but the effect can still be confirmed.

[0149] Example 14: Analysis of the antibacterial efficacy of oleacin (a single substance) against Staphylococcus aureus ATCC 6538. Oleacin is found in substrates and extracts derived from the olive tree.

[0150] A preservation solution was prepared with oleacin at a concentration of 1 mg / ml. This compound was dissolved in ethanol (99.8%) and WFI was added.

[0151] Using a preservation solution, the concentrations of oleacin at 0.25 mg oleacin / ml and 0.125 mg oleacin / ml were tested using a quantitative suspension assay.

[0152] Approximately 3.5 × 10⁶ units were added to 2 ml each of the preservative solution and diluent (V1 and V2), as well as to a 0.44% ethanol solution as a positive control. 5 A 0.055 ml test suspension of Staphylococcus aureus ATCC 6538, adjusted to CFU / 0.1 ml, was inoculated, yielding approximately 99,000 CFU / ml in each assay.

[0153] Next, samples (0.1 ml) were collected at t0, 12, 30, and 60 minutes after inoculation, and at t24, 48, and 72 hours. These were cultured at 20°C to 25°C and diluted in 0.9 ml of 0.9% NaCl solution. The samples were plated onto blood agar plates and incubated at 30°C to 35°C for up to 5 days.

[0154] The results are shown in Table 28 below.

[0155] Table 28 Number of CFUs; initial cell load: 9.9 × 10 4 CFU / ml, Bacteria: Staphylococcus aureus ATCC 6538, Oci = oleacin [Table 28]

[0156] As shown in Table 28, 0.44% ethanol did not show a significant antibacterial effect at the beginning of the test. The decrease at subsequent time points is due to the natural loss of bacterial viability. Oleacin showed an immediate effect at the tested concentration, below the detection limit of the assay at 100 CFU / ml.

[0157] Example 15: Analysis of the antibacterial efficacy of ubaol (single substance) against Staphylococcus aureus ATCC 6538 Ubaol is found in base materials and extracts derived from the olive tree.

[0158] A preservation solution was prepared by adding 0.1 ml of chloroform to 25 mg of ubaol. After dissolving the compound, the solution was quantitatively transferred to 12.4 ml of sterile water for injection (WFI). Effective concentration of ubaol: 2.0 mg / ml.

[0159] Using a preservation solution, ubaol at five concentrations ranging from 2 mg ubaol / ml to 0.125 mg ubaol / ml in a geometric progression was tested using a quantitative suspension assay.

[0160] Each of the following samples was mixed with approximately 2.0 × 10⁶ of storage solution and diluent (V1-V4), as well as a 0.8% chloroform solution as a positive control. 5 A 0.1 ml test suspension of Staphylococcus aureus ATCC 6538, adjusted to CFU / 0.1 ml, was inoculated, yielding approximately 100,000 CFU / ml in each assay.

[0161] Next, samples (0.1 ml) were collected at t0, 12, 30, and 60 minutes after inoculation, and at t24, 48, and 72 hours. These were cultured at 20°C to 25°C and diluted in 0.9 ml of 0.9% NaCl solution. The samples were plated onto blood agar plates and incubated at 30°C to 35°C for up to 5 days.

[0162] The results are shown in Table 29 below.

[0163] Table 29 Number of CFUs; initial cell load: 2.0 × 10⁶ 5 CFU / ml, Bacteria: Staphylococcus aureus ATCC 6538, Uva = Uvaol [Table 29]

[0164] As shown in Table 29, 0.8% chloroform did not have a significant antibacterial effect during the test. Uvaol showed efficacy at concentrations of 0.25 Uva / ml or higher after incubation for more than 24 hours, but was below the detection limit of the 100 CFU / ml assay.

[0165] Example 16: Analysis of the antibacterial efficacy of erythrodiol (single substance) against Staphylococcus aureus ATCC 6538 Erythrodiol is found in substrates and extracts derived from the olive tree.

[0166] A preservation solution was prepared by adding 0.1 ml of chloroform to 10.45 mg of erythrodiol. After dissolving the compound, the solution was quantitatively transferred to 4.9 ml of sterile water for injection (WFI). Effective concentration of erythrodiol: 2.0 mg / ml.

[0167] Using a preservation solution, ubaol at five concentrations ranging from 2 mg ubaol / ml to 0.125 mg ubaol / ml in a geometric progression was tested using a quantitative suspension assay.

[0168] Each of the following was mixed with approximately 3.6 × 10⁶ of preservative solution and diluent (V1-V4) in 2 ml, as well as a 2% chloroform solution as a positive control. 5 A 0.1 ml test suspension of Staphylococcus aureus ATCC 6538, adjusted to CFU / 0.1 ml, was inoculated, yielding approximately 100,000 CFU / ml in each assay.

[0169] Next, samples (0.1 ml) were collected at t0, 12, 30, and 60 minutes after inoculation, and at t24, 48, and 72 hours. These were cultured at 20°C to 25°C and diluted in 0.9 ml of 0.9% NaCl solution. The samples were plated onto blood agar plates and incubated at 30°C to 35°C for up to 5 days.

[0170] The results are shown in Table 30 below.

[0171] Table 30 Number of CFUs; initial cell load: 9.5 × 10 4 CFU / ml, Bacteria: Staphylococcus aureus ATCC 6538, Ery = Erythrodiol [Table 30]

[0172] As shown in Table 30, 2% chloroform did not have a significant antibacterial effect at the beginning of the test. The decrease at subsequent time points is due to the natural loss of bacterial viability. Erythrodiol showed a strong effect after incubation at a concentration of 2.0 Ery / ml, while lower concentrations either showed subsequent bacterial reappearance or no effect. The lower effect compared to oleacin or oleocanthal appears to be at least in part due to the insufficient solubility of erythrodiol, as encountered during the assay.

[0173] References 1. AG, BM (2018)."Multiresistente Erreger." 2018, from https: / / www.bbraun.de / de / produkte-undtherapien / hyiene / multiresistente-erreger.html 2. Centers for Disease, Control and Prevention (2011). Surveillance for Foodborne Disease Outbreaks-United States, 2008, MMWR, Morbidity and Mortality Weekly Report. 60: 1197-1202. 3. Effect, BN (2017). "Olivenblattextrakt Forschung." Retrieved 10.10.2017, from http: / / www.best-natural-effect.c0m / info / 5 / olivenblattextrakt-forschung.html. 4. Fleming, HP, et al. (1973). "Antimicrobial properties of oleuropein and products of its hydrolysis from green olives." Appl Microbiol 26(5): 777-782. 5. Guinda, A., et al. (2010). "Pentacyclic triterpenoids from olive fruit and leaf." J Agric Food Chem 58(17): 9685-9691. 6. Iming, S. (2005). Olivenblattextrakte-Altbewahrte Heilmittel in der Praxis. Wien, Double-U GmbH. 7. Khan, M. Y., et al. (2007). "Olea europaea: A phyto-pharmacological review." Pharmacognosy Reviews 1(1): 114-118. 8. Kramer, J. (2007). Lebensmittel-Mikrobiologie, Ulmer. 9. Lieberei, R. R., C. (2007). Nutzpflanzenkunde. Stuttgart, Thieme Verlag. 10. Liu, Y., et al. (2017). "Assessment of the Antimicrobial Activity of Olive Leaf Extract Against Foodborne Bacterial Pathogens." Front Microbiol 8: 113. 11. Stiti, N., et al. (2007). "Formation of triterpenoids throughout Olea europaea fruit ontogeny." Lipids 42(1): 55-67. 12. WHO, W.H.O. (2018). "Antibiotikaresistenz." from http: / / www.euro.who.int / de / health-topics / disease-prevention / antimicrobial-resistance / antibiotic-resistance. 13. Ahmed et al. (2014); "Antibacterial effect of olive (Olea europaea L.) leaves extract in raw peeled undeveined shrimp (Penaeus semisulcatus) 14. US 7,429,008 B2; SYSTEM AND METHOD FOR PULVERIZING AND EXTRACTING MOISTURE; Graham et al.; 2008 15. US 7,500,830 B2 SYSTEM AND METHOD FOR PULVERIZING AND EXTRACTING MOISTURE; Graham et al.; 2009 16. US 7,909,577 B2 SYSTEM AND METHOD FOR PULVERIZING AND EXTRACTING MOISTURE; Graham et al.; 2011 17. WO 2013 / 052583 A2 SYSTEMS AND METHODS FOR CONVERTING SEWAGE SLUDGE WTO A COMBUSTIBLE FUEL; New et al.; 2013 18. WO 2013 / 075003 Ai; EGGSHELL POWDER COMPOSITIONS AND METHODS OF PRODUCING EGGSHELL POWDER COMPOSITIONS; Liu et al., 2013

[0174] Drawing translation Figure 1 PROCESS Preparing parts of an olive or acebuche plant Extracting moisture Extracting moisture + Commutation / Purification Composition / Purification: Grinding / Fine Grinding Sterilization Antibiotic / Antifungal Powder (sterilized) Blend with a thickening agent. Antibiotic / Antifungal Gel Antibiotic / Antifungal Powder (non-sterilized) Extraction Suspension in a non-toxic surfactant Antibiotic / Antifungal Suspension Figure 2 Air Feed material Venturi nozzle Pulverized material + Air Turbomachine Motor Figure 3 IRIDOIDS (mg / g) Sylvestris Arbequina Hojiblanca Picual Oleuropein Oleuroside Ligstroside Verbascoside Figure 4 FLAVONOIDS (mg / g) Sylvestris Arbequina Hojiblanca Picual Apigenin Luteolin Figure 5 TERPENES (mg / g) Sylvestris Arbequina Hojiblanca Picual Oleanolic acid. Maslinic ac. Figure 6 TRYOSOL + HYDROXYTYROSOL (mg / g) Sylvestris Arbequina Hojiblanca Picual Tyrsol + hydroxytyrosol

Claims

1. A pharmaceutical composition for use in the prevention or treatment of infections caused by antibiotic-resistant bacteria or fungi or multi-resistant bacteria or fungi in mammals, The aforementioned pharmaceutical composition comprises a sterilized, finely ground olive (Olea europaea subspecies europaea) or acebuche (Olea europaea subspecies europaea sylvestris) composition, The composition comprises olive or acebuche plant parts from which moisture has been removed by dehydrating or drying, and The part of the olive or acebuche plant is composed of the part of the olive or acebuche plant as a finely ground olive or acebuche powder, which is obtained by shearing, grinding, milling, pulverizing, or pulverizing, and A pharmaceutical composition wherein the finely ground olive or acebuche powder is properly sterilized, or the finely ground olive or acebuche powder is properly sterilized at a temperature of 121°C or 134°C, and the olive or acebuche plant portion is processed using a Venturi apparatus.

2. The pharmaceutical composition according to claim 1, wherein the finely ground olive or acebuche powder consists of more than 95% by weight of dry biomass.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises an antibiotic or antifungal agent appropriately extracted from the finely ground olive or acebuche powder or CO2 extracted.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is formulated as an interactive wound dressing, an antiseptic, a local body disinfectant, or a medical cleansing agent for sites prone to pathogen colonization, or is formulated as an ointment, lotion, cream, spray, or gel for local or transdermal administration.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is used to reduce and / or eliminate dominant pathogens in wound treatment.

6. a) Bacteria selected from Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Mycobacterium tuberculosis, Enterococcus hylae, Enterococcus faecalis, Enterobacter, Serratia, Proteus, Providencia, Morganella, Enterococcus faecium, Helicobacter pylori, Campylobacter, Salmonella, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, Shigella, Acinetobacter baumannii, and resistant and multi-resistant strains thereof, and b) The fungus is selected from Candida albicans and Aspergillus brasiliensis. A pharmaceutical composition according to any one of claims 1 to 5.

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