European sweet clover olive forest variant composition with antibacterial effect

Abstract

The present application relates to a method for preparing a sweet olive (European sweet olive) composition, preferably a wild sweet olive (European sweet olive forest variant) composition, the prepared sweet olive or wild sweet olive composition, and their use in the prevention or treatment of bacterial or fungal infections in mammals, preferably infections caused by antibiotic-resistant bacteria or multidrug-resistant bacteria. Other uses relate to functional food additives, in particular animal feed additives, or in cosmetic products.

Description

[0001] This application is a divisional of the Chinese patent application with the filing date of October 8, 2020 and the application number 202080070806.1. TECHNICAL FIELD

[0002] The present application relates to a method for preparing Olea europaea, preferably Olea europaea subsp. europaea var. sylvestris, compositions, the prepared Olea europaea or Olea europaea subsp. europaea var. sylvestris compositions, and their use in the prevention or treatment of bacterial or fungal infections, preferably infections caused by antibiotic-resistant bacteria or multi-drug resistant bacteria, in mammals. Other uses relate to functional food additives, in particular animal feed additives, or in cosmetic products. BACKGROUND

[0003] Olive trees (Olea europaea L.) are a representative plant of the Oleaceae family with a long history of cultivation. It comprises 30 genera and 600 different species. The genus Olea comprises 50 species, distributed in different continents and regions, including Africa, China, India, the United States and Australia (Iming, 2005). Olea europaea is divided into different subspecies, including Olea europaea subsp. europaea, and Olea europaea subsp. africana, of which Olea europaea subsp. africana grows in Africa, Madagascar, India and China. Both species exist in the form of wild and cultivated varieties (Iming, 2005). Olive trees can grow for 1000 years and have very low water requirements, which also explains their presence in arid regions (Iming, 2005). Most of the olive harvest in the world comes from the Mediterranean (Lieberei, 2007).

[0004] Wild Olea europaea, i.e. Olea europaea subsp. europaea var. sylvestris, is the ancestor of all Olea plants and is commonly known as "acebuche" in Spain. Wild Olea trees are irregularly distributed in the Iberian Peninsula, for example; they grow spontaneously and accidentally as independent groves, or groups of groves, mainly in the Mediterranean region.

[0005] The leaves of the Olea tree are different from those of fruit trees and are sometimes used as natural medicines. The Olea tree is also very resistant to fungal and bacterial attacks. These antibiotic and protective properties seem to be based on the production of various active compounds by Olea, such as oleuropein, which is one of the most studied active compounds (Iming, 2005).

[0006] Olea europaea subsp. cuspidata has active compounds present in leaves, fruits, buds, stems, branches, and roots in different concentrations (Effect, 2017). It is speculated that O. europaea subsp. cuspidata leaves can have immune-enhancing, anti-inflammatory, and blood pressure-lowering effects (Iming, 2005). However, little is known about the actual medical effects involved.

[0007] A large number of bioactive substances have been identified in O. europaea subsp. cuspidata and can be classified, for example, in seco-iridoid glycosides, phenolic compounds, flavonoids, monoterpenes, triterpenes, steroids, quinoline alkaloids, carotenoids, chlorophyll, phenolic acids, tannins, and vitamins (Effect, 2017).

[0008] During the harvesting of O. europaea, the leaves of the O. europaea tree accumulate, thus providing an easily accessible by-product that can be used for other purposes (Cayuela, 2006). The development of new uses for by-products in olive oil production is particularly important, especially for O. europaea groves. More and more research is focusing on the chemical composition of O. europaea fruits, while little is known about the chemical composition of O. europaea leaves, especially the leaves of wild O. europaea trees.

[0009] Since their discovery more than 70 years ago, antibiotics have been our main tool in treating bacterial infections, including deadly hospital-acquired infections. However, antibiotics are often prescribed and taken inappropriately and routinely. Antibiotics are also used in livestock farming to treat, prevent disease, and promote growth. In the environment, for example in some water supply systems, antibiotics are also found (WHO, 2018).

[0010] Antibiotic resistance is a natural mechanism that occurs naturally, i.e. an antibiotic that usually prevents the growth of a particular bacterial species no longer has any effect (WHO, 2018). Worldwide, around 700,000 people die each year from antibiotic resistance (AMR). In the European Union, around 25,000 people. It is frightening that by 2050, antibiotic resistance could cause more deaths than cancer (Parliament, 2018).

[0011] Infections caused by drug resistant bacteria are often difficult to cure, and sometimes even impossible to cure, and the number of such cases is increasing. However, research into new effective antibiotics is very expensive and time consuming, and after a new antibiotic is brought to market, resistance to it often develops. Currently, there are very few new antibiotics in development. If effective new antibiotics cannot be found and resistance continues to spread, society will be threatened with a return to the pre-antibiotic era, when children often died from a small pneumonia and doctors were powerless against meningitis. At the same time, many complex medical interventions and diagnoses can not be made without effective antibiotics for prophylaxis.

[0012] The emergence of drug resistant bacteria is a serious problem in healthcare, including causing fatal blood and wound infections and pneumonia, among other problems. Antibiotic resistance leads to an increase in treatment costs due to longer hospital stays, higher antibiotic and treatment expenses, and indirect cost of family and society. In many countries in the European region, antibiotics do not require a doctor's prescription. Data on antibiotic resistant infections are often not collected, so there is no documentation of the severity of the problem, although there is a high degree of awareness among physicians. Hospital-acquired infections are one of the most common infections in Germany. The problem of antibiotic resistance associated with these infections and its spread is one of the greatest challenges to modern medicine (Braun AG, 2018).

[0013] Ahmed et al. (Ahmed, Ali & S. Rabii, Nancy & Garbajj, Aboubaker & Abolghait, Said. (2014). Antibacterial effect of olive (Olea europaea L.) leaves extract in raw peeled undeveined shrimp (Penaeus semisulcatus). International Journal of Veterinary Science and Medicine. 2. 10.1016 / j.ijvsm.2014.04.002) evaluated the effect of olive (Olea europaea L.) leaves extract on microbial load in raw peeled undeveined shrimp (Penaeus semisulcatus) and discussed the potential use of olive (Olea europaea L.) leaves extract formulation in improving microbial quality and as a natural preservative.

[0014] Paudel et al. (Paudel, Shambhu & Magrati, Thakur & Lamichhane, Jay Ram. (2011). Antimicrobial activity of wild olive crude extracts in vitro. International Journal of Pharma Sciences and Research) disclose that wild olive crude extracts were screened for antibacterial activity against 5 different human pathogenic bacteria, wherein the extract obtained with methanol appeared to be the most effective against all pathogenic bacteria.

[0015] DE 102011108948 A1 discloses a clear liquid formulation of a poorly water-soluble lipophilic substance which comprises only one solubilizer and can be prepared by stirring only, without further complex operational steps.

[0016] In further studies (e.g. Korukluoglu, Mihriban & Sahan, Yasemin & YIGIT, AYCAN & Tumay Elif & Gucer, Seref. (2010). Antibacterial activity and chemical constitutions of Olea europaea L. leaf extracts. Journal of Food Processing and Preservation. 34. 383396. 10.1111 / j.1745-4549.2008.00318.x), Olea europaea L. leaf extracts inhibited the growth of a number of bacteria and molds such as Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Aspergillus flavus, and Aspergillus parasiticus. The aqueous extract of O. europaea L. leaves had no antibacterial effect on the microorganisms tested, while the acetone extract showed inhibitory effect on Salmonella enteritidis, B. cereus, K. pneumoniae, E. coli, Enterococcus faecalis, Streptococcus thermophilus, and Lactobacillus bulgaricus. In addition, some phenolic compounds were tested for their antibacterial activity against microorganisms. The most effective compound was oleuropein, while syringic acid was not effective. This type of experiment has not been performed on wild O. europaea "wild olea".

[0017] Wang et al. (Wang, Xiao-Fei & Li, Chen & Shi, Yan-Ping & Di, Duo-Long. (2009). Two new secoiridoid glycosides from the leaves of Olea europaea L. Journal of Asian natural products research. 11. 940-4. 10.1080 / 10286020903310979) disclose the isolation of two secoiridoid glycosides, oleuricine A (1) and B (2), and five known triterpenes, β-amyrin, oleanolic acid, eburicolic acid, ursolic-2β,3β-dihydroxy-12-ene-28-oic acid, and β-maslinic acid, from the ethyl acetate-soluble fraction of the ethanol extract of the leaves of O. europaea. The structures of these compounds were identified by a variety of spectroscopic methods, including enhanced 1D, 2D NMR, and HR-ESI-MS techniques. SUMMARY

[0018] In view of the foregoing, it is an object of the present application to provide new effective antibacterial compounds and compositions from Olea europaea or wild Olea europaea. Other objects and advantages of the present application will become apparent to those skilled in the art upon reading the following more detailed description of the present application in conjunction with the included drawings.

[0019] In its first aspect, the present application achieves the above-mentioned objects by providing a method for preparing an Olea europaea, preferably wild Olea europaea (Olea europaea var. sylvestris) composition, comprising the steps of: a) providing a part of an Olea europaea or wild Olea europaea plant; and b) removing water from said Olea europaea or wild Olea europaea part by dehydrating or drying said Olea europaea or wild Olea europaea part; and c) breaking said Olea europaea or wild Olea europaea part of step b) by chopping, grinding, milling, pulverizing, or other method of breaking said Olea europaea or wild Olea europaea to obtain powdered Olea europaea or wild Olea europaea; d) optionally sterilizing said powdered Olea europaea or wild Olea europaea, preferably at a temperature of 121 °C or 134 °C, to obtain sterilized powdered Olea europaea or wild Olea europaea; and, optionally, e) mixing said sterilized powdered Olea europaea or wild Olea europaea with water and a thickening agent to obtain an Olea europaea or wild Olea europaea gel having antibiotic or antifungal activity; or d') extracting, preferably CO2 extraction, an antibiotic or antifungal active agent from said powdered Olea europaea or wild Olea europaea of step c); suspending said active agent in a non-toxic solubilizing and emulsifying agent to obtain an Olea europaea or wild Olea europaea suspension having antibiotic or antifungal activity.

[0020] Surprisingly, a particularly significant pharmacological difference was found between cultivated Olea europaea and wild Olea europaea (see below, e.g., Table 2).

[0021] In its second aspect, the present application achieves the above-mentioned objects by providing a sterilized powdered Olea europaea or wild Olea europaea having antibiotic or antifungal activity obtained from steps a), b), c), or d) of the method of the present application.

[0022] In its third aspect, the present application achieves the above-mentioned objects by providing an Olea europaea or wild Olea europaea composition prepared by the method of the present application, optionally comprising a pharmaceutically or cosmetically acceptable carrier, diluent, or excipient.

[0023] In its fourth aspect, the present application achieves the above-mentioned objects by providing the use of the sterilized powdered Olea europaea or wild Olea europaea, or the Olea europaea or wild Olea europaea composition described herein, in the prevention or treatment of bacterial or fungal infections in mammals, preferably in the treatment or therapy of infections caused by antibiotic-resistant bacteria or multi-drug resistant bacteria.

[0024] In its fifth aspect, the present application achieves the above-mentioned objects by providing the use of the sterilized powdered Olea europaea or wild Olea europaea, or the Olea europaea or wild Olea europaea composition described herein, as a functional food additive, in particular as an animal feed additive.

[0025] In its sixth aspect, the present application achieves the above-mentioned objects by providing the use of the sterilized powdered Olea europaea or wild Olea europaea, or the Olea europaea or wild Olea europaea composition described herein, as a cosmetic product and / or in a cosmetic method. BRIEF DESCRIPTION OF DRAWINGS

[0026] In the drawings, Figure 1 schematic method steps for the preparation of the antibiotic / antifungal gum of the present application, or for the preparation of the antibiotic / antifungal suspension of the present application, including the respective intermediate products, are shown.

[0027] Figure 2 schematic representation of the equipment used in the method steps for removing water from the Olea europaea or wild Olea europaea parts by dehydration or drying; breaking up the Olea europaea or wild Olea europaea parts to obtain powdered Olea europaea or wild Olea europaea by chopping, grinding, milling, crushing. These steps are performed simultaneously by subjecting the Olea europaea or wild Olea europaea parts to forces generated by a dynamic air flow, preferably by passing the Olea europaea or wild Olea europaea through a Venturi nozzle together with air, preferably preheated air.

[0028] Figure 3 comparison of the active substances (iridoids) of different Olea europaea cultivars is shown.

[0029] Figure 4 comparison of the active substances (flavonoids) of different Olea europaea cultivars is shown.

[0030] Figure 5 comparison of the active substances (terpenes) of another different Olea europaea cultivar is shown.

[0031] Figure 6 comparison of the active substances (tyrosol and hydroxytyrosol) of yet another different Olea europaea cultivar is shown. DETAILED DESCRIPTION

[0032] As explained above, in a first aspect of the application, the above problems are solved by providing a method for preparing Olea europaea, preferably wild Olea europaea (Olea europaea var. sylvestris), compositions, the method comprising the steps of: a) providing a part of an Olea europaea or wild Olea europaea plant; and b) removing water from the Olea europaea or wild Olea europaea part by subjecting the Olea europaea or wild Olea europaea part to dehydration and / or drying; and c) breaking up the Olea europaea or wild Olea europaea part of step b) by chopping, grinding, milling, pulverizing, and / or other methods of breaking up the Olea europaea or wild Olea europaea part to obtain powdered Olea europaea or wild Olea europaea; d) sterilizing the powdered Olea europaea or wild Olea europaea, as appropriate, preferably at a temperature of about 120 °C to about 140 °C, preferably at a temperature of about 121 °C or about 134 °C, to obtain sterilized powdered Olea europaea or wild Olea europaea; and, optionally, e) mixing the sterilized powdered Olea europaea or wild Olea europaea with water and a thickening agent to obtain an Olea europaea or wild Olea europaea gel that is antibiotic or antibacterial active; or d') extracting, preferably CO2 extraction, an antibiotic or antibacterial active agent from the powdered Olea europaea or wild Olea europaea of step c); suspending the antibiotic or antibacterial active agent in at least one non-toxic solubilizing and / or emulsifying agent to obtain an Olea europaea or wild Olea europaea suspension that is antibiotic or antibacterial active (see Figure 1 ).

[0033] Thus, the present application relates to two particularly important compositions having the desired properties, i) an Olea europaea or wild Olea europaea gel that is antibiotic or antibacterial active; ii) an Olea europaea or wild Olea europaea suspension in at least one non-toxic solubilizing and / or emulsifying agent that is antibiotic or antibacterial active.

[0034] The most studied secoiridoid glycoside in Olea europaea plants is oleuropein. Oleuropein is present in all parts of the Olea europaea plant and is responsible for a variety of beneficial effects, including antibacterial activity, antiviral activity, anti-inflammatory activity, anti-rheumatic activity, antioxidant activity, or cardioprotective activity (Fleming, 1973). The leaves of the Olea europaea tree contain 60-90 mg / g dry matter of oleuropein (Khan, 2007).

[0035] In addition to oleuropein, the leaves of the Olea europaea tree contain other phenolic ingredients, such as dimethyl oleuropein, ligstroside, verbacoside, oleuropein di-methyl ester, and oleuroside (Cayuela, 2006). The leaves of the Olea europaea tree also contain flavonoids. These include rutin, luteolin, and apigenin. Flavonoids reduce oxidative damage by absorbing UV light and prevent oxidation by free radicals (Cayuela, 2006).

[0036] Guinda et al. described that the Olea europaea L. is a suitable raw material for the production of oleanolic acid and other pentacyclic triterpenes (Guinda, 2010). It includes ursolic acid, betulinic acid, maslinic acid, erythrodiol, and uvaol. Guinda et al. have described that the content of pentacyclic triterpenes in the leaves of Olea europaea L. is higher than in the fruit itself. The most representative are oleanolic acid and ursolic acid (Guinda, 2010; Bianchi, 1992). The concentration of the various triterpenes is highly dependent on the stage of development of the fruit and the plant and the cultivar (Guinda, 2010; Stiti, 2007).

[0037] Oleanol and maslinic acid are desirable raw materials for the pharmaceutical and cosmetic industry that can be extracted from the leaves of Olea europaea L. (Guinda, 2010). For this reason, the leaves of the wild Olea europaea L. "wild olive" were tested for their pentacyclic triterpenes. It can have an impact on the antimicrobial efficacy.

[0038] In the context of the present application, the inventors developed a method for optimizing the processing of Olea europaea L. or wild olive compositions having antimicrobial efficacy. A particular advantage of the method of the present application is that the two decisive steps, i.e. the extraction of moisture and the comminution, are carried out in a single device, which considerably reduces the input, saves operating and energy expenditure.

[0039] Furthermore, in the context of the method, the required energy consumption is significantly lower when using a venturi nozzle.

[0040] Most importantly, and surprisingly, it was found that the Olea europaea L. or wild olive parts treated with the venturi device result in a powdered Olea europaea L. or wild olive having a significantly higher antibiotic and antifungal effect than the material treated in a conventional mill, i.e. without using a venturi device. It was also found that the powdered Olea europaea L. or wild olive prepared with the venturi device has a particularly long shelf life.

[0041] Table 1 below shows that the CFU of Pseudomonas aeruginosa bacteria is reduced when treated with Olea europaea L. material treated with a conventional grinding method than with Olea europaea L. material treated with a venturi device.

[0042]

[0043] Olea europaea L. leaf extract as a natural antibiotic or antifungal agent with broad-spectrum antibacterial activity is a pioneering step in the fight against infections, especially in the case of (multi-) antibiotic-resistant - thus particularly problematic - microorganisms.

[0044] Active powdered O. falcata or O. ferruginea, or O. falcata or O. ferruginea compositions, for example, can serve as the basis for interactive wound dressings, but also for antiseptics, body surface disinfection, medical cleaning of sites where microbial colonies thrive, and in particular for the reduction and / or elimination of the main pathogenic bacteria and their multi-resistant variants in wound treatment.

[0045] The term "provision" in the context of the present application means the O. falcata and O. ferruginea plant products as raw material for the methods described herein. It can be necessary to clean the O. falcata and O. ferruginea plants from the presence of surface impurities, for example by water bath, washing, or spraying, or other methods known to the person skilled in the art. It can also be necessary to size the O. falcata and O. ferruginea plants to make them suitable for handling the O. falcata and O. ferruginea plants. The methods are also known to the person skilled in the art.

[0046] "Dehydration" or "drying" as used herein generally and preferably relates to direct drying, or indirect drying, or high frequency drying, or vacuum drying, or freeze drying, wherein, for example, the direct drying is achieved by simply exposing the raw material to air (also known as natural air drying), or heated air that enhances the air flow drying, which is produced by means of, for example, a ventilation device, a blower, or other devices known in the art; wherein the indirect drying is achieved by contact drying or drum drying; the high frequency drying by means of, for example, microwaves, etc.; wherein these methods can be adapted to reduce the drying time, or to achieve a higher degree of sensitivity that can be required. The method of using elevated temperatures to shorten the water abstraction time has limitations, as too much elevated temperature and too long elevated temperature exposure time can cause deterioration or degradation of the antibacterial / antifungal active components in the raw material. To avoid any contamination or spoilage in the handling and storage of the dried material, the material is preferably dried to a degree of 95 dry substance (DS) % or more. The degree of moisture content can be optimized to recommend a lower or higher dry substance % for the subsequent breaking and pulverizing steps.

[0047] "Crushing" as used herein is typically and preferably performed in one or more steps to achieve the desired degree of comminution. The most desirable degree of comminution is determined taking into account the cost of crushing on the one hand and the efficacy of the antibacterial or antifungal action of the resulting pulverous material on the other hand. According to the application described herein, the desired particle size of the Olea europaea or parts of Olea ferruginea is in the range of 1 μm to 1000 μm, more preferably 40 μm to 500 μm, most preferably 125 to 250 μm. The desired degree of crushing is achieved by operating the Olea europaea or parts of Olea ferruginea in a crushing device multiple times, or by using different types of crushing devices that allow for the operation of the preferred particle size. Typical crushing devices are selected from the group consisting of presses, choppers, choppers, cutting tools, grinding appliances, and pulverizers, such as hammer mills, ball mills, impact mills, tumbling ball mills, centrifugal mills, jet mills, flaking mills, planetary mills, mortars, grinders, micropulverizers, ultrasonic fine grinders, or microgrinders, without being limited thereto. Depending on the specifications required, a classification of the pulverous material can be added to this step to separate the plant material into the desired size classes. It can also be useful to re-crush particles that are classified as too large to reach the desired size classes.

[0048] As used herein, the term "sterilization" refers to the removal of natural contaminants of the pulverous Olea europaea / Olea ferruginea having, for example, aerobic spore-forming bacteria, using various sterilization procedures. Different types of sterilization can be selected from heat sterilization, in which microorganisms are killed by heat, for example by heating in a moist state (steam sterilization), or in a dry state (hot air sterilization), or fractional sterilization, in which successive repeated heating, or by physical sterilization techniques, or other sterilization methods known in the art. In the context of the present application, sterilization is preferably performed in a vacuum sterilizer at 121 °C and 134 °C, wherein the temperature is suitable for preserving pharmaceutically active ingredients, wherein sterilization is optionally performed by steam sterilization.

[0049] As used herein, the term "mixing" refers to the mixing or homogenization of the sterilized pulverous Olea europaea or Olea ferruginea with water for injection and a thickening agent, as described herein below, to obtain a Olea europaea or Olea ferruginea gel that is active against bacteria or fungi, and wherein the mixing is continued until a homogeneous gel is formed. There are various methods known in the art that can be used to achieve the desired result.

[0050] As used herein, "extraction" refers to the separation of the antibiotic or antifungal active agent from powdered Olea europaea or wild Olea europaea obtained by the process described herein. According to the present application, extraction is preferably achieved by CO2 extraction. Extraction can be performed in a batch mode, as the extractor can only be emptied and refilled at atmospheric pressure. During extraction, supercritical carbon dioxide (CO2) penetrates the raw material at high pressure and extracts soluble substances from powdered Olea europaea or wild Olea europaea (aqueous extract). The dissolved substances are separated into fractions with different components by gradually reducing the pressure. In the first fraction (separator 1), substances with low solubility accumulate, while more soluble substances are collected in subsequent separator 2. Other solid-liquid extraction types can be selected, such as maceration and decantation, but are not limited to these, as long as the active agent remains fully effective.

[0051] In a preferred embodiment of the process according to the present application, steps b) and c) are performed simultaneously by subjecting the above-mentioned parts of Olea europaea or wild Olea europaea to the forces generated by a dynamic air flow, preferably by passing the above-mentioned parts of Olea europaea or wild Olea europaea through a venturi nozzle together with air or other suitable gas, preferably preheated air. The granted patents US 7,429,008 B2, US 7,500,830 B2 and US 7,909,577 B2 disclose comminution of materials, which are subjected to continuous moisture extraction and drying by means of an air flow generator coupled to the venturi nozzle during comminution, but generally use, for example, polymers or waste as raw material. Furthermore, the patent application WO 2013 / 052583 A2 generally discloses dewatering, comminution and pyrolysis of biomass, such as sludge. The patent application WO 2013 / 075003 A1 provides a method for the preparation of eggshell powder, which can be used for the preparation of biological products. The method involves comminution of eggshell at room temperature at high air flow rates, separating the eggshell component from the inner membrane component. However, no method has been developed which can simultaneously dewater and dry the parts of Olea europaea or wild Olea europaea and simultaneously comminute, as provided by the present application. The active agents of Olea europaea or wild Olea europaea are preserved and the powder has a long shelf life.

[0052] Therefore, for example, osmanthus or wild osmanthus segments are flowed through a Venturi nozzle along with air, exposing the osmanthus and / or wild osmanthus segments to a dynamic airflow. In doing so, the airflow drags the material through the Venturi nozzle connected to the equipment. The movement of the air through the Venturi nozzle accelerates the osmanthus or wild osmanthus contained in the airflow. The airflow is generated, for example, by the rotation of blades in a turbine or by another device suitable for dragging the airflow. The airflow and the dried powdered osmanthus or wild osmanthus exit the equipment through the outlet nozzle, where the crushed osmanthus or wild osmanthus can be separated from the airflow by a filter, cyclone, or similar methods known to those skilled in the art. Simultaneously, the airflow is heated by the energy loss (heat) of operating the turbine. The accelerated airflow (and the crushed osmanthus or wild osmanthus) then absorbs this heat, increasing its temperature. Physically, warmer air can absorb relatively more moisture than cooler air. Due to this natural phenomenon, the crushing and pulverizing, as well as the heat loss, are mutually compensated in the Venturi apparatus specification. The raw material, dragged through the equipment (including the Venturi apparatus), is pulverized, increasing the surface area of ​​the material for moisture transfer to the airflow, and the increased air temperature due to turbine operation causes more moisture to be transferred from the material to the airflow. The Venturi apparatus can be used to fully or partially implement steps b) and c) of the method of the invention. Preferably, the supply point for the osmanthus or wild osmanthus portion can be located before the inlet pipe connected to the equipment.

[0053] In other preferred embodiments of the method of this application, the thickener used is selected from gelling agents, such as gum arabic, alginate, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propylene glycol alginate, agar, carrageenan, red algae gum, locust bean gum, guanylic bean gum, amine yellow gum, gum arabic, xanthan gum, sycamore gum, pectin, cellulose, gelatin, and modified starch.

[0054] In another preferred embodiment of the method according to this application, the aforementioned non-toxic solubilizer and / or emulsifier is selected from surfactants, wherein the surfactant is preferably selected from... Optimal Selection RH40. Other surfactants can be selected from nonoxynol-9, octylbenzyl-9 (Triton... ), polysorbate 20 (Tween Octyl-β-glucoside and n-octyl-β-D-1-thiopyranoside. Other non-toxic solubilizers and / or emulsifiers may be selected from ethanol, acetone, hexane, chloroform, and methyl tert-butyl ether (MTBE).

[0055] The parts of Olea europaea or Olea ferruginea used in the process of the present application can be selected from the group consisting of leaves, twigs, barks, stems, fruits, or oil preparation residues, and combinations thereof. As used herein, the term "parts" refers to all suitable components of the Olea europaea and Olea ferruginea plants, such as leaves, twigs, barks of stems or roots, Olea europaea or products processed from Olea europaea and residues. Parts can also refer, for example, to plants growing in the soil, harvested plant parts, discarded plant parts, processed plant parts, and lifeless plant parts.

[0056] It is preferred according to the process of the present application, wherein at least one active agent is contained in the prepared composition, which is selected from the group consisting of compounds having antibiotic or antifungal activity. Preferred active agents are selected from the group consisting of iridoids or phenols, preferably at least one organic compound selected from the group consisting of oleuropein, oleuropein aglycone, oleocanthal, tyrosol, hydroxytyrosol, uvaol, and maslinic acid. Furthermore, in the context of the present application, active agents can also include oleuropein, oleuropein aglycone, oleuropein aglycone, oleocanthal, tyrosol, hydroxytyrosol, uvaol, and maslinic acid. Most preferred are oleuropein, oleuropein aglycone, oleocanthal, tyrosol, hydroxytyrosol, uvaol, and maslinic acid.

[0057] In its second aspect, the present application solves the above-mentioned problems by providing a sterilized powdered Olea europaea or Olea ferruginea product / composition having antibiotic or antifungal activity, which is obtained by the process of the present application.

[0058] It is particularly preferred according to the present application, a sterilized powdered Olea europaea or Olea ferruginea having antibiotic or antifungal activity, which is obtained by steps a), b), c) and d) of the process according to the present application.

[0059] The term "antibiotic activity" as used herein essentially refers to an activity acting on infections caused by microorganisms. These microorganisms are mainly bacteria, but they also include, for example, protozoa. The effect can include killing the microorganisms, and / or inhibiting growth and / or reproduction. In the context of the present application, the antibacterial agent is particularly active and effective against microorganisms resistant to commercially available antibiotic substances, in particular multiple resistant microorganisms (i.e. bacteria or fungi resistant to at least two antibiotic substances, for example commercially available antibiotic substances). The term "antifungal activity" as used herein refers to an activity acting on infections caused by fungi, for example molds. The effect can include killing the fungi, or inhibiting growth or reproduction. The term "resistant" as used herein refers to a characteristic of microorganisms that reduces or completely neutralizes the effect of commercially available antibiotic substances. The term "multiple resistant" as used herein refers to a characteristic of microorganisms that reduces or completely neutralizes the effect of at least two different commercially available antibiotic substances.

[0060] It is preferred that the sterilized powdered olea ferruginea according to the present application has antibiotic or antifungal activity, wherein the powdered olea ferruginea or wild olea ferruginea described above is a powder and consists of more than 95% by weight of dry biomass. The term "dry biomass" as used herein means a practically dry amount of a substance without water or other components such as other liquids. "Weight %" as used herein means the mass ratio of a mixture, in particular of the powdered olea ferruginea or wild olea ferruginea. The composition of the powdered olea ferruginea or wild olea ferruginea depends on the proportion of the individual components in 100 g of the mixture. In the context of the present application, it is preferred that more than 100 g consists of about 95% of dry biomass.

[0061] In its third aspect, the present application solves the above-mentioned problems by providing a composition of olea ferruginea or wild olea ferruginea prepared by the method according to the present application. Optionally and preferably, the composition described above comprises a pharmaceutically or cosmetically acceptable carrier, diluent or excipient.

[0062] In another preferred aspect, the present application relates to an antibacterial or antifungal composition comprising at least one organic compound selected from the group consisting of oleuropein, hydroxytyrosol, oleocanthal, oleanolic acid, and erythrodiol. The composition is preferably a pharmaceutical or cosmetic composition as described herein.

[0063] In another preferred aspect, the present application relates to the composition described above for use in the prevention or treatment of a bacterial or fungal infection in a mammal, preferably for use in the prevention or treatment of an infection caused by an antibiotic-resistant bacteria or a multi-drug resistant bacteria.

[0064] "Pharmaceutically acceptable carrier, diluent or excipient" as used herein means an ingredient in a pharmaceutical formulation or composition other than an active ingredient, which is nontoxic to the subject. A pharmaceutically acceptable carrier, diluent, or excipient includes any and all solvents, dispersion media, coatings, other antibacterial and antifungal agents, isotonic and absorption delaying agents, which are compatible with a physiologically acceptable delivery vehicle. Carriers include a variety of preservatives, antibacterial and antifungal agents such as p-hydroxybenzoic acid esters, chlorobutanol, phenol, sorbic acid and the like. It can be desirable to include isotonic agents, for example, sugars, sodium chloride and the like, in the compositions of the present application. In addition, sustained release of injectable formulations can be achieved by the use of absorbable

[0065] Regardless of the route of administration selected, the compositions of the present application, which can be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present application, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of active ingredients in the pharmaceutical compositions of the present application can be varied. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions of the present application employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0066] The compositions must be sterile and fluid enough for delivery by syringe. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride, are included in the compositions.

[0067] The compositions of the present application can be administered topically or systemically. Administration is generally parenteral, e.g., intravenous injection. Formulations 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 injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or non-volatile oils. Vehicles for intravenous injections include liquids and nutrient supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like.

[0068] As used herein, "cosmetically acceptable carrier, diluent, or excipient" means a suitable ingredient in a cosmetic formulation other than the active ingredient. Generally, such an ingredient must be compatible with the other ingredients in the formulation for local contact with the tissue (e.g., skin) without undue toxicity, incompatibility, instability, irritation, allergic response, and the like. A cosmetically acceptable carrier, diluent, or excipient can include, for example, water, liquid or solid emollients, solvents, humectants, thickening agents, and powders that facilitate distribution of the composition upon application to, for example, the skin, hair, and / or nails.

[0069] Other particularly preferred embodiments of the present application are Olea europaea or wild olive compositions according to the present application, wherein the above composition is a gel, preferably a homogeneous gel. In the context of the present application, "gel" means oleaginous powdered Olea europaea or wild olive obtained by the method of the present application, a substantial amount (up to 100%) of which exists in the form of colloidal size particles, i.e. 1-1000 μm, thus resulting in a solubility of the almost insoluble oleaginous powdered Olea europaea or wild olive and ensuring uniform distribution of the powdered Olea europaea or wild olive.

[0070] Other particularly preferred embodiments relate to Olea europaea or wild olive suspensions according to the present application, wherein the above Olea europaea or wild olive suspension is an aqueous suspension. The term as used herein includes in particular Olea europaea or wild olive suspensions which are water soluble via the use of surfactants, as well as each aqueous solution which is advantageously obtainable via the above use.

[0071] It is further preferred that the Olea europaea or wild olive composition according to the present application, wherein the above composition is a suspension, wherein the amount of non-toxic solubilizing agents, in particular surfactants, is less than 10%, preferably less than 5%, more preferably less than 3% of the total amount of Olea europaea or wild olive suspension. As mentioned above, the surfactants are preferably selected from the group consisting of Most preferred are RH40.

[0072] In another preferred embodiment of the present application, the composition described herein is in the form of an ointment, emulsion, cream, spray, gel, liquid, drop, capsule, or suppository. The above composition can be administered systemically, i.e. by transmucosal or transdermal means. For transmucosal or transdermal administration, penetration agents suitable for the barrier to be penetrated are used in the formulation. Such penetration agents are known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. For transmucosal administration, the composition according to the present application is configured as a liquid, drop, capsule, or suppository. For epicutaneous (transdermal) administration, the above composition is configured as an ointment, emulsion, cream, spray, or gel, as known in the art.

[0073] In its fourth aspect, the present application addresses the above-mentioned problems by providing a sterilized powdered Olea europaea or Olea ferruginea as described herein, or a composition of Olea europaea or Olea ferruginea as described herein, for use in medicine. More preferably, a sterilized powdered Olea europaea or Olea ferruginea as described herein, or a composition of Olea europaea or Olea ferruginea as described herein, for use in the prevention or treatment of an infection by a microorganism, such as a bacterium or a fungus, preferably for use in the prevention or treatment of an infection by an antibiotic-resistant bacterium or even a multi-drug resistant bacterium, in a mammal. Another aspect relates to the use of a sterilized powdered Olea europaea or Olea ferruginea as described herein, or a composition of Olea europaea or Olea ferruginea as described herein, for the manufacture of a medicament for the prevention or treatment of an infection by a microorganism, such as a bacterium or a fungus, preferably for the prevention or treatment of an infection by an antibiotic-resistant bacterium or even a multi-drug resistant bacterium, in a mammal.

[0074] As used herein, "mammal" can be a farm animal (e.g., a horse, cow, sheep, or pig), a pet (e.g., a cat, dog, rabbit, or guinea pig), a rodent, or, particularly, a human. Thus, the compositions according to the present application can be used to treat any of these mammals.

[0075] The term "treatment" as used herein includes the administration of the above-mentioned compositions to the above-mentioned mammals, preferably in a therapeutically effective amount, to slow the progression of a disease or a condition. Thus, an effective amount is that amount of the above-mentioned compositions or pharmaceutical compositions herein that results in a normalizing of the infected state of the mammal. This amount slows the symptoms associated with the infection and / or condition, without being toxic to the subject. The dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages will depend on many factors, including the size, surface area, age, and sex of the mammal, the particular compound being administered, the time and route of administration, the health of the mammal, and other drugs that can be administered concomitantly.

[0076] As used herein, the term "prevention" includes the administration of the above- mentioned compositions to the above-mentioned mammals, preferably in a prophylactically effective amount, to reduce the propensity or risk, no matter how slight, of a subject to become infected with an antibiotic-resistant bacterium or a multi-drug resistant bacterium. For prevention, the mammal is preferably one at risk of, or susceptible to, infection with an antibiotic-resistant bacterium or a multi-drug resistant bacterium, wherein the administration of the composition is preferably by injection. In addition, enteral and transdermal administration can be included in the context of the present application, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intradural, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and sternal injection and infusion.

[0077] In another preferred embodiment of the present application, the sterilized powdered Olea europaea or wild Olea europaea, according to the present application, or the Olea europaea or wild Olea europaea composition, according to the present application, is used for the prevention or treatment of bacterial or fungal infections in a mammal, preferably infections caused by antibiotic-resistant bacteria or multi-drug resistant bacteria, wherein the aforementioned bacteria are selected from the group consisting of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus hirae, Enterococcus faecalis, Enterobacter, Mycobacterium tuberculosis, Serratia, Proteus, Providencia, Morganella, Enterococcus faecium, Heliocobacter pylori, Campylobacter, Salmonella, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenza, Shigella, Acinetobacter baumannii, and drug-resistant and multi-drug resistant strains thereof, and for the prevention or treatment of fungal infections in a mammal, wherein the aforementioned fungi are selected from the group consisting of Candida albicans and Aspergillus brasiliensis.

[0078] In its fifth aspect, the present application addresses the aforementioned problems by providing a functional food additive comprising the sterilized powdered Olea europaea or wild Olea europaea, as described herein, or the Olea europaea or wild Olea europaea composition, as described herein. Furthermore, the aforementioned problems are also addressed by using the sterilized powdered Olea europaea or wild Olea europaea, as described herein, or the Olea europaea or wild Olea europaea composition, as described herein, as a functional food additive, in particular as an animal feed additive. As used herein, the term "animal feed additive" refers to the addition of the composition to the feed of farm animals.

[0079] Furthermore, as used herein, the term "functional food additive" refers to the use of the composition to increase the supply of the active ingredient in the composition to a mammal by supplementing the normal diet. The composition can also be taken as a dietary or food supplement in addition to the basic nutrition, in the form of a concentrate, or in the form of a medicament, in particular in the form of capsules, lozenges, tablets, pills, effervescent tablets, and other similar forms, powder sachets, liquid ampoules, drop bottles, and other similar liquid and powder forms for taking in suitable small doses. In particular, the term "animal feed additive" refers to the addition of the composition to the feed of farm animals, without excluding the feed for other animal species.

[0080] In a sixth aspect, the present application solves the above-mentioned problems by providing a cosmetic product comprising the sterilized powdered Olea europaea or Olea ferrunginea of the present application, the Olea europaea or Olea ferrunginea gel or suspension according to the present application, or the pharmaceutical composition according to the present application. Another aspect relates to the use of the sterilized powdered Olea europaea or Olea ferrunginea as described herein, the Olea europaea or Olea ferrunginea composition as described herein in a cosmetic product or a cosmetic method.

[0081] As described herein, the term "cosmetic" encompasses all fields of body care, or all fields of action for maintaining, repairing, or improving the appearance of a mammal. In particular, the fields of application of cleaning, care and protection, and in particular dental and mouth care, are included within the scope of the present application.

[0082] In the context of the present application, the terms "about" and "approximately" refer to an interval of accuracy that the person skilled in the art understands to still ensure the technical effect of the feature in question. The terms generally indicate a deviation of ± 20 %, ± 15 %, ± 10 %, and for example ± 5 % of the indicated numerical value. The person skilled in the art will be aware that the specific deviation of a numerical value will depend on the nature of the technical feature for a given technical effect. For example, natural or biological technical effects can essentially have a greater deviation than artificial or engineering technical effects. The person of ordinary skill in the art will be aware that the specific deviation of a numerical value will depend on the nature of the technical feature for a given technical effect. For example, natural or biological technical effects can essentially have a greater deviation than artificial or engineering technical effects. When used with an indefinite or definite article, e.g. "a", "an", or "the", also a plurality of the noun is included, unless specifically indicated otherwise.

[0083] The present application will be further illustrated in the following examples, but the present application is not limited thereto. For the purposes of the present application, all documents, patents and publications cited herein are fully incorporated by reference herein.

[0084] Example

[0085] Example 1: Extraction of Olea europaea components with supercritical carbon dioxide

[0086] The dried pre-ground parts of the Olea europaea tree are placed in the extractor. The extraction is performed in batch mode, as the extractor can only be emptied and refilled at atmospheric pressure. During the extraction, supercritical carbon dioxide (CO2) penetrates the raw material at high pressure and extracts soluble substances (i.e. the extract) from the raw material. The dissolved substances can be separated into fractions with different compositions by gradually reducing the pressure. In the first fraction (separator 1), the more poorly soluble substances can be accumulated, whereby in the following separator 2, the more soluble substances can be collected.

[0087] For the experiments performed, a modified extraction method was chosen with only one separation stage, whereby all dissolved substances are collected in separator 1. The amount of substances depends on the chosen extraction conditions (pressure and temperature), as well as the solubility and the amount of substances contained in the raw material.

[0088] Process flow: In the experiments, a high-pressure laboratory system (HDL4) was used. This is a laboratory device with one extractor and two separators. The device is designed for extraction pressures of up to 1000 bar and temperatures of up to 95°C. The extraction pressure is not higher than 1000 bar. Liquid CO2 from the CO2 tank is brought to the extraction pressure by a pump and to the extraction temperature by heat exchange. In the extractor, supercritical CO2 flows through the raw material and enriches the soluble substances. After the pressure is reduced, the mixture separates into gaseous CO2 and the extract. The extract can be collected in the separator and removed from the device. The gaseous and uncharged CO2 can then be liquefied again in a condenser and reused in the extraction cycle.

[0089] Execution: The raw material is placed in the extractor of the HDL4 for extraction. Next, valuable ingredients (e.g. terpenes and terpenoids) are to be extracted from the natural substance. Only one separation stage is used for the following extract separation, all extract is collected in separator 1. Since the obtained extract is not flowable at -60°C, it is difficult or impossible to perform a conventional extract extraction via the separator 1 outlet valve. The extract must be removed after each test run after opening the separator and must be additionally scraped out, which can partially wash out the solvent.

[0090] Example 2: Processing of Olea europaea material using a Wurster dryer

[0091] The original Olea europaea tree material is first subjected to thermal-mechanical processing, in which the particle size and the water content of the raw material are specifically changed. The technical implementation results in a dehydrated fine-grained product, possibly also a powder product, which is directly further used in the process chain and can also be used for intermediate process storage thereof.

[0092] Example 3: Sterilization procedure

[0093] Due to the natural contamination of the starting product with aerobic spores, several sterilization processes were applied. Different sterilization temperatures were used to determine the optimum temperature for retaining the active pharmaceutical ingredients: 121 °C and 134 °C. Sterilization was performed at different temperatures.

[0094] Example 4: Comparison of active substances in cultivated and wild Olea europaea leaves and the method of disruption

[0095] In the Casa de porros in Tarifa, Spain, the parts of wild and cultivated Olea europaea plants were collected by hand. Each plant part was processed as follows: fresh leaves, shoots, barks, and root barks were dried in a Taprogge®-ventilator, and the rest was broken in a hot mixer. The sample material was stored in sealed containers at room temperature in the dark. To have comparability, both sets of samples were sieved to the same particle size range. Next, Soxhlet extraction and chromatography were performed to analyze the chemical composition of the active agents in cultivated and wild Olea europaea. Positive effects were found in the oxygen radical absorbance capacity (ORAC) test; sampling of the active substances after Soxhlet extraction revealed that a higher extraction yield was achieved for the material broken in the Taprogge®-ventilator. For the material broken by the Taprogge®-ventilator, a favorable influence on ORAC (oxygen radical absorbance capacity) was detected, which in turn translates into positive protection properties and a particularly long shelf life. This can be illustrated by determining the antioxidant activity of the leaves and shoots processed by the Taprogge®-ventilator method over a period of 30 days compared to the antioxidant activity of fresh samples. The results are shown in Table 2.

[0096] Table 2: Comparison of the antioxidant activity of fresh samples and samples processed by the Taprogge®-ventilator method over a period of 30 days

[0097]

[0098] By comparing the active substances in wild and cultivated Olea europaea, it can be seen that a higher concentration of active substances is present in wild-type Olea europaea trees. Olea europaea and the forest variety differ in many ways: cultivated Olea europaea has larger fruits (fixed shape), which are therefore more suitable for the production of Olea oil. However, the tendency to produce larger fruits is evident, which shows that the remaining parts of the Olea europaea plant contain fewer active substances. Based on published data for three common species cultivated in the Andalusian region: Alberquina, Acebuche, and Picual, the two plants were compared. The spectrum of active substances was detected in fresh wild Olea europaea leaves (Table 3).

[0099] Table 3: Concentration of individual substances (mg / g)

[0100]

[0101] A comparison of the active substances of different cultivars of Olea europaea is shown in Figures 3-6 wherein Figure 3 a comparison of iridoids is shown, Figure 4 a comparison of flavonoids is shown, Figure 5 a comparison of terpenes is shown, and Figure 6 a comparison of tyrosol and hydroxytyrosol is shown.

[0102] The following table 4 compares the percentage of active substances of wild and cultivated Olea europaea.

[0103] Table 4: Amount of active substances in wild and identified cultivated Olea europaea

[0104]

[0105] Based on the comparison of these 9 substances, it shows that wild Olea europaea tree leaves have the highest concentration of phenols and monophenols, in particular flavonoids. From a pharmaceutical point of view, it is clear that wild Olea europaea (wild) and cultivated Olea europaea are completely different, and thus wild Olea europaea is a better raw material for potential pharmaceutical or cosmetic products.

[0106] Example 5: Study of antimicrobial efficacy of Olea europaea or wild Olea europaea suspension

[0107] The aim of the study was to test the antimicrobial efficacy of Olea europaea or wild Olea europaea suspensions after suspension in Kolliphor RH40 and water for injection (WFI) against an extended test microorganism panel (Table 5).

[0108] Table 5: Test microorganism panel for the microbial study of the present application

[0109]

[0110] Execution: Preparation of test microorganism suspensions: The test microorganisms were incubated with a maximum of 5 passages. The suspensions of the test microorganisms were adjusted to approximately 1000 CFU / 0.1 ml sterile 0.9% NaCI solution.

[0111] Sample preparation: 3.0 g of Olea ferruginea extract in 1.01 g of Kolliphor RH40 was heated in a water bath at 45°C for about 5 min, followed by heating in a water bath at 80°C for about 15 min and hand shaking. The Olea ferruginea extract and Kolliphor RH40 were mixed together in 15 ml of WFI to give a very thick, dark green to dark brown, non-pipetteable suspension. The next day, the stock was heated again in a water bath at 80°C and another 5 ml of WFI heated to 80°C was added. The stock was heated again to 80°C for about 5 min. The water bath was heated and hand shaken for about 1 min. The result was a stock containing 125 mg of Olea ferruginea extract / ml and 4.2% Kolliphor RH40. After the above treatment, the Olea ferruginea extract was a homogenous and pipetteable suspension with a dark brown to dark green color.

[0112] Evaluation of the antimicrobial efficacy of Olea ferruginea extract after preparation of the stock: For each test bacteria, 1 ml of the stock was transferred to 1 ml of double concentrated CaSo broth and Sabouraud broth (C. albicans). This preparation was then inoculated with 0.1 ml of each test bacteria suspension adjusted to about 1000 CFU / 0.1 ml. The number of bacteria in the test bacteria suspension was determined by surface plating on blood agar and Sabouraud agar. All media preparations were incubated for a maximum of 72 h at 30-35°C. After 24 h, 48 h and 72 h of incubation, subculturing of all 0.1 ml batches was performed by streaking on blood agar plates and Sabouraud agar, respectively. The agar plates were incubated aerobically for 24 h-48 h (yeast) at 30-35°C. Positive control: 1 ml of WFI was added to 1 ml of double concentrated CaSo broth or Sabouraud broth (C. albicans) and then inoculated with 0.1 ml of each test bacteria suspension adjusted to about 1000 CFU / 0.1 ml in a single determination. The positive control was used as a reference for the growth of the test bacteria in the double concentrated medium after 1 :2 dilution. The results are shown in the following tables.

[0113] Table 6: Results of the antimicrobial efficacy of Olea ferruginea suspension on the test bacteria C. albicans ATCC 10231; Inoculum: Effective number of bacteria inoculated in each test batch: 1160 CFU = 580 CFU / ml of medium batch

[0114]

[0115] += test bacteria grew in the nutrient medium preparation with visible turbidity

[0116] P = product turbidity, no visible test bacteria growth

[0117] Subculturing by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0118] Table 7: Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Enterococcus faecium ATCC BAA2317; Inoculum: number of viable inoculated bacteria per test batch: 1810 CFU = 905 CFU / ml medium batch

[0119] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product haze, no visible test bacteria growth

[0120] Passage by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0121] Table 8: Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Enterococcus hirae ATCC 10541; Inoculum: number of viable inoculated bacteria per test batch: 760 CFU = 380 CFU / ml medium batch

[0122] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product haze, no visible test bacteria growth

[0123] Passage by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0124] Table 9: Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Klebsiella pneumoniae subsp. pneumoniae CCUG 56233; Inoculum: number of viable inoculated bacteria per test batch: 1200 CFU = 600 CFU / ml medium batch

[0125]

[0126] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product haze, no visible test bacteria growth

[0127] Passage by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0128] Table 10: Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Klebsiella pneumoniae subsp. pneumoniae ATCC 10031; Inoculum: number of viable inoculated bacteria per test batch: 740 CFU = 370 CFU / ml medium batch

[0129]

[0130] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product haze, no visible test bacteria growth

[0131] Subcultures by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0132] Table 11 : Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Pseudomonas aeruginosa ATCC 9027; Inoculum: number of bacteria effectively inoculated in each test batch: 640 CFU = 320 CFU / ml of medium batch

[0133] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product turbidity, no visible test bacteria growth

[0134] Subcultures by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0135] Table 12: Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Pseudomonas aeruginosa ESBL 24600; Inoculum: number of bacteria effectively inoculated in each test batch: 680 CFU = 340 CFU / ml of medium batch

[0136] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product turbidity, no visible test bacteria growth

[0137] Subcultures by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0138] Table 13: Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Escherichia coli ATCC 8739; Inoculum: number of bacteria effectively inoculated in each test batch: 860 CFU = 430 CFU / ml of medium batch

[0139] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product turbidity, no visible test bacteria growth

[0140] Subcultures by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0141] Table 14: Results of the antimicrobial efficacy of Olea europaea suspension against the test bacteria Escherichia coli DSM 22312; Inoculum: number of bacteria effectively inoculated in each test batch: 670 CFU = 435 CFU / ml of medium batch

[0142] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product turbidity, no visible test bacteria growth

[0143] Subcultivation by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0144] Table 15: Results of the antimicrobial efficacy of Olea europaea L. suspension against the test bacteria Staphylococcus aureus ATCC 6538; Inoculum: number of bacteria effectively inoculated per test batch: 680 CFU = 340 CFU / ml of medium batch

[0145] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product turbidity, no visible test bacteria growth

[0146] Subcultivation by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0147] Table 16: Results of the antimicrobial efficacy of Olea europaea L. suspension against the test bacteria Staphylococcus aureus subsp. aureus ATCC 29213; Inoculum: number of bacteria effectively inoculated per test batch: 2020 CFU = 1010 CFU / ml of medium batch

[0148]

[0149] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product turbidity, no visible test bacteria growth

[0150] Subcultivation by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0151] For the two other test bacteria, Acinetobacter baumannii and Aspergillus brasiliensis, the Kolliphor RH40 content of the stock solution was reduced by 50%, i.e. from 4.2% to 2.1%. In addition to the original stock solution Al with Olea europaea extract at a concentration of 127.5 mg / ml, a 1 :2 dilution was prepared with water for injection, so that the Olea europaea extract had a concentration of 63.8 mg / ml (stock solution A2). Table 17: Results of the antimicrobial efficacy of Olea europaea L. suspension against the test bacteria Acinetobacter baumannii ATCC 19606; Inoculum: number of bacteria effectively inoculated per test batch: 4000 CFU = 2000 CFU / ml of medium batch

[0152] += test bacteria grew in the nutrient medium preparation with visible turbidity P = product turbidity, no visible test bacteria growth

[0153] Sub-culturing by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0154] Table 18: Results of the antimicrobial efficacy of Olea europaea suspensions against the test strain Acinetobacter baumannii NCTC 13420; inoculum: number of viable bacteria inoculated in each test batch: 890 CFU = 445 CFU / ml of medium batch

[0155] += test strain grew in the nutrient medium preparation at a visibly turbid degree

[0156] P = product turbidity, no visibly test strain growth

[0157] Sub-culturing by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0158] Table 19: Results of the antimicrobial efficacy of Olea europaea suspensions against the test strain Aspergillus brasiliensis ATCC 16404; inoculum: number of viable bacteria inoculated in each test batch: 620 CFU = 310 CFU / ml of medium batch

[0159]

[0160]

[0161] += test strain grew in the nutrient medium preparation at a visibly turbid degree

[0162] P = product turbidity, no visibly test strain growth

[0163] Sub-culturing by streaking (SC) of 0.1 ml of each nutrient medium preparation

[0164] As shown in the tests, after the respective sub-culturing, no growth of the test strains was observed after 72 hours of incubation of the nutrient medium preparations, except for the test strain Aspergillus brasiliensis ATCC 16404. Thus, it can be indicated that, under the test conditions, a good antimicrobial efficacy of the Olea europaea extract at a concentration of 62.5 mg / ml or 63.8 mg / ml, respectively. For the test strain Aspergillus brasiliensis, some isolated surviving viable spores were still detectable during the test in the presence of the Olea europaea suspension in stock A1 and stock A2, but a clear reduction of the CFU was also evident.

[0165] Example 6: Study of antimicrobial efficacy of Olea europaea or wild Olea europaea gel

[0166] ​Execution: A colloidal gel containing Kelcogel CG-HA was prepared from Olea europaea L. leaf powder. For this purpose, 0.1 g of Kelcogel CG-HA was dissolved in 100 ml of WFI by heating to 85-90 °C, after which 15 g of Olea europaea L. leaf powder was added, stirred until a visually homogeneous suspension was formed. After cooling in an ice bath, an Olea europaea L. leaf powder colloidal gel was obtained. The adjusted test bacteria suspension (105-106CFU / g) was inoculated in aliquots of 10 g of gel, and the number of microorganisms was tested according to the test times listed in Table 20.

[0167] Table 20: CFU values according to test time

[0168]

[0169] After 12 minutes, very good efficacy (T0h-value) was shown for E. coli, S. aureus and P. aeruginosa were below the detection limit of 100 CFU per gram and showed overall very good antimicrobial efficacy during the shorter exposure period.

[0170] Example 7: Study of antimicrobial efficacy of Olea europaea or wild Olea europaea gel

[0171] Table 21: CFU values according to test time

[0172]

[0173] Execution: A colloidal gel containing Kelcogel CG-HA was prepared from Olea europaea L. leaf powder. For this purpose, 0.1 g of Kelcogel CG-HA was dissolved in 100 ml of WFI by heating to 85-90 °C, after which 15 g of Olea europaea L. leaf powder was added, stirred until a visually homogeneous suspension was formed. After cooling in an ice bath, an Olea europaea L. leaf powder colloidal gel was obtained. The adjusted test bacteria suspension (105-106CFU / g) was inoculated in aliquots of 10 g of gel, and the number of microorganisms was tested according to the test times listed in Table 21.

[0174] Thus, after 6 minutes, very good efficacy was shown for E. coli, after 12 minutes, S. aureus was below the detection limit of 100 CFU per gram. For P. aeruginosa, the bacterial amount was reduced below the detection line after 30 minutes and showed overall very good antimicrobial efficacy during the shorter exposure period.

[0175] Example 8: Study of antimicrobial efficacy of Olea europaea or wild Olea europaea composition bars

[0176] Procedure: For each test, a dough block was prepared by mixing 500 mg of sample with 1 ml of WFI. After homogenization, 0.5 ml of the adjusted bacterial suspension was added and evenly mixed into the dough block. Subsequently, for untreated Osmanthus fragrans leaf powder products, as well as those steam-sterilized at 121°C for 15 minutes and 130°C for 30 minutes, both test bacteria showed very good efficacy after a 6-hour reaction time at room temperature.

[0177] Table 22: CFU values ​​based on test time

[0178]

[0179] Untreated Osmanthus fragrans leaf patches show a size of 1.4 × 10⁻⁶. 3 CFU-2.1×10 3 CFU per 500 mg of dough contains natural contamination from various aerobic spore-forming bacteria, which could not be killed during the 6-hour and 24-hour testing periods, respectively. In the test sample sterilized at 121°C for 15 minutes, the dough showed 1.1 × 10⁻⁶ CFU / mL. 3 CFU-2.2×10 2 Natural contamination within the range of 500 mg CFU. Both sterilization test samples were sterilized at 130°C for 30 minutes, and no contaminating bacteria were detected.

[0180] Example 9: Study of antimicrobial efficacy of Olea europaea or wild Olea europaea composition bars

[0181] Procedure: For each test of each test bacterium, a dough block was prepared by mixing 500 mg of osmanthus extract with 1 ml of WFI. After homogenization, 0.5 ml of the adjusted bacterial suspension was added and evenly mixed into the dough block. Subsequently, for untreated osmanthus leaves, as well as those steam-sterilized at 121°C for 15 minutes and 130°C for 30 minutes, both test bacteria showed very good efficacy after a 24-hour reaction time at room temperature (Table 23).

[0182] Table 23: CFU values ​​based on test time

[0183]

[0184] Untreated Osmanthus fragrans leaf patches show a size of 2.9 × 10⁻⁶. 4 The CFU level per 500mg of dough contains natural contamination from various aerobic spore-forming bacteria that could not be killed during the 24-hour test. No contaminating bacteria were detected in the sterilized test samples.

[0185] Example 10: Study of antimicrobial efficacy of Olea europaea or wild Olea europaea composition bars

[0186] Procedure: For each test, a bolus was prepared by mixing 500 mg of the olive wood material with 1 ml of WFI. After homogenization, 0.5 ml of the adjusted bacterial suspension was added and mixed evenly into the bolus. After 24 hours of reaction time at room temperature, both test bacteria showed very good efficacy for the untreated, as well as for the 121 °C 15 min and 130 °C 30 min steam sterilized stem bark / clone powder preparations (Table 24).

[0187] Table 24: CFU values according to test time

[0188]

[0189]

[0190] The untreated powder form of stem bark / clone showed 4.4 x 10 2 CFU per 500 mg bolus order of natural contamination with various aerobic spore-forming bacteria, which could not be killed during the 24 hours test period. In the 121 °C sterilized test sample, 1.1 x 10 2 CFU / bolus of contaminating bacteria were still detectable. The test sample was also sterilized at 121 °C. In the 130 °C sterilized test sample, no contaminating bacteria were detected.

[0191] Example 11: Study of antimicrobial efficacy of Olea europaea or wild Olea europaea composition bars

[0192] Procedure: For each test, a bolus was prepared by mixing 500 mg of the olive wood material with 1 ml of WFI. After homogenization, 0.5 ml of the adjusted bacterial suspension was added and mixed evenly into the bolus. After 24 hours of reaction time at room temperature, both test bacteria showed very good efficacy for the untreated, as well as for the 121 °C 15 min and 130 °C 30 min steam sterilized stem bark / clone powder preparations (Table 24).

[0193] Table 25: CFU values according to test time

[0194]

[0195]

[0196] The untreated powder form of stem bark / clone showed 1.1 x 10 6 CFU per 500 mg bolus order of natural contamination with various aerobic spore-forming bacteria, which could not be killed during the 24 hours test period. In the 121 °C and 130 °C sterilized test sample, 1.8 x 10 3 CFU / bolus and 8.8 x 10 2Contaminating bacteria in CFU / patch.

[0197] Example 12: Analysis of antimicrobial efficacy of hydroxytyrosol (single substance) against Staphylococcus aureus ATCC 6538

[0198] Olive oil stimulating aldehydes were found in substrates and extracts from Olea europaea trees.

[0199] Stock 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 water for injection (WFI), resulting in an effective concentration of 2.0 mg / ml of hydroxytyrosol.

[0200] Using the stock solution, 5 geometric concentrations of hydroxytyrosol from 2 mg hydroxytyrosol / ml to 0.125 mg hydroxytyrosol / ml were tested in a quantitative suspension test.

[0201] 2 ml of the stock solution and dilutions (V1 to V4), respectively, and as positive control a 2% methanol solution were inoculated with 0.1 ml of a test suspension of S. aureus ATCC 6538, adjusted to approximately 2.0 x 10 5 CFU / 0.1 ml, approximately 100.000 CFU / ml in each test.

[0202] After inoculation and incubation at 20-25°C, samples (0.1 ml) were taken at tO, 12, 30, and 60 minutes and at t24, 48 and 72 hours and diluted with 0.9 ml of a 0.9% NaCI solution. The samples were plated on blood agar plates and incubated at 30-35°C for up to 5 days.

[0203] The results are shown in Table 26 below.

[0204] Table 26: CFU values; initial cell amount: 1.7 x 10 5 CFU / ml, bacteria: S. aureus ATCC 6538, Hyd = hydroxytyrosol

[0205]

[0206] As shown in Table 26, 2% methanol did not have a significant antimicrobial effect at the start of the test. The decrease at the later time points is due to the natural loss of bacterial activity. Hydroxytyrosol showed a significant effect only after 24 hours, however, and this effect was very strong, i.e. at the detection limit of 100 CFU / ml of the test. Additional dilution further delayed the effect.

[0207] Example 13: Analysis of antimicrobial efficacy of oleocanthal (single substance) against Staphylococcus aureus ATCC 6538 Analysis

[0208] Olive oil stimulating aldehydes were found in substrates and extracts from Olea europaea trees.

[0209] A stock solution of 2 mg / ml oleocanthal concentration was prepared. The compound was dissolved in ethanol (99.8%) and WFI was added.

[0210] Using this stock solution, oleocanthal was tested in a quantitative suspension test in geometric concentrations from 1 mg oleocanthal / ml to 0.125 mg oleocanthal / ml.

[0211] 2 ml of the stock solution and dilutions (V1 to V4) respectively, and as positive control a 2.85% ethanol solution, were inoculated with 0.055 ml of the test suspension of S. aureus ATCC 6538 adjusted to about 3.6 x 10 5 CFU / 0.1 ml, about 99.000 CFU / ml in each test.

[0212] After inoculation and incubation at 20-25 °C, samples (0.1 ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48 and 72 hours and diluted with 0.9 ml of a 0.9% NaCI solution. The samples were plated on blood agar plates and incubated at 30-35 °C for up to 5 days.

[0213] The results are shown in Table 27 below.

[0214] Table 27: CFU values; initial cell amount: 9.9 x 10 4 CFU / ml, bacteria: S. aureus ATCC 6538, Ole = oleocanthal

[0215]

[0216] As can be seen from Table 27, 3.84% ethanol did not have a significant antimicrobial effect at the start of the test. The decrease at the later time points is due to the natural loss of bacterial activity. Oleocanthal showed a fast-acting effect at the higher concentrations, at the detection limit of 100 CFU / ml of the test. Dilution delayed these effects, however, the effect could still be seen.

[0217] Example 14: Analysis of antimicrobial efficacy of oleuropein (single substance) against Staphylococcus aureus ATCC 6538 Example 15: Analysis of antimicrobial efficacy of uvaol (single substance) against Staphylococcus aureus ATCC 6538

[0218] Oleocanthal was found in the base material and extracts from Olea europaea L. trees.

[0219] A stock solution of 1 mg / ml oleuropein concentration was prepared. The compound was dissolved in ethanol (99.8%) and WFI was added.

[0220] Using this stock solution, olivetolic acid was tested in a quantitative suspension test at concentrations of 0.25 mg olivetolic acid / ml and 0.125 mg olivetolic acid / ml.

[0221] 2 ml of the stock solution and the dilutions (V1 and V2), respectively, and as positive control a 0.44% ethanol solution, were inoculated with 0.055 ml of the test suspension of S. aureus ATCC 6538, adjusted to about 3.5 x 10 5 CFU / 0.1 ml, about 99.000 CFU / ml in each test.

[0222] After inoculation and incubation at 20-25°C, samples (0.1 ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48 and 72 hours and diluted with 0.9 ml of a 0.9% NaCI solution. The samples were plated on blood agar plates and incubated at 30-35°C for up to 5 days.

[0223] The results are shown in Table 28 below.

[0224] Table 28: CFU values; initial cell amount: 9.9 x 10 4 CFU / ml, bacteria: S. aureus ATCC 6538, Oci = olivetolic acid

[0225]

[0226] As can be seen from Table 28, 0.44% ethanol did not show a significant antimicrobial effect at the start of the test. The decrease at the later time points is due to the natural loss of bacterial activity. Olivetolic acid showed a faster effect at the concentrations tested, at the limit of detection of 100 CFU / ml of the test.

[0227] Example 16: Analysis of antimicrobial efficacy of homoeriodicyol (single substance) against Staphylococcus aureus ATCC 6538

[0228] Uvaol was found in the base material and extract from the Olea europaea tree.

[0229] A stock solution was prepared by adding 0.1 ml chloroform to 25 mg uvaol. After dissolving the compound, the solution was quantitatively transferred to 12.4 ml water for injection (WFI), effective concentration of uvaol: 2.0 mg / ml.

[0230] Using this stock solution, uvaol was tested in a quantitative suspension test at five (5) geometric concentrations of uvaol from 2 mg uvaol / ml to 0.125 mg uvaol / ml.

[0231] Two ml of stock and dilutions (V1-V4) respectively, and as positive control a 0.8% chloroform solution, were inoculated with 0.1 ml of test suspension of S. aureus ATCC 6538 adjusted to about 2.0 x 10 5 CFU / 0.1 ml, about 100.000 CFU / ml in each test.

[0232] After inoculation and incubation at 20-25°C, samples (0.1 ml) were taken at tO, 12, 30, and 60 minutes and at t24, 48 and 72 hours and diluted with 0.9 ml of a 0.9% NaCI solution. The samples were plated on blood agar plates and incubated at 30-35°C for up to 5 days.

[0233] The results are shown in Table 29 below.

[0234] Table 29: CFU values; initial cell amount: 2.0 x 10 5 CFU / ml, bacteria: S. aureus ATCC 6538, Uva = uvaol

[0235]

[0236] As can be seen from Table 29, 0.8% chloroform did not show a significant antimicrobial effect during the tests. Uvaol showed an effect after incubation at 0.25 Uva / ml or higher for more than 24 hours, at the detection limit of 100 CFU / ml of the test.

[0237] Cited Literature

[0238] Uvaol was found in the base material and extracts from Olea europaea L. trees.

[0239] A stock solution was prepared by adding 0.1 ml chloroform to 10.45 mg uvaol. After dissolving the compound, the solution was quantitatively transferred to 4.9 ml water for injection (WFI), the effective concentration of uvaol: 2.0 mg / ml.

[0240] Using the stock solution, five (5) geometric concentrations of uvaol from 2 mg uvaol / ml to 0.125 mg uvaol / ml were tested in quantitative suspension tests.

[0241] Two ml of stock and dilutions (V1-V4) respectively, and as positive control a 2% chloroform solution, were inoculated with 0.1 ml of test suspension of S. aureus ATCC 6538 adjusted to about 3.6 x 10 5 CFU / 0.1 ml, about 100.000 CFU / ml in each test.

[0242] After inoculation and incubation at 20-25°C, samples (0.1 ml) were taken at t0, 12, 30, and 60 minutes and at t24, 48 and 72 hours and diluted with 0.9 ml of a 0.9% NaCl solution. The samples were plated on blood agar plates and incubated at 30-35°C for up to 5 days.

[0243] The results are shown in the following table 30.

[0244] Table 30: CFU values; initial cell amount: 9.5 x 10 4 CFU / ml, bacteria: Staphylococcus aureus ATCC 6538, Ery = homoeriodicyol

[0245]

[0246] As can be seen from table 30, 2% chloroform did not show a significant antimicrobial effect at the beginning of the test. The decrease at the later time points is due to the natural loss of bacterial activity. Homoeriodicyol showed a stronger effect after incubation at a concentration of 2.0 Ery / ml, lower concentrations showed either a reappearance of bacteria at later times or no effect. It is likely that the inferior effect compared to the olivetolic acid or the olive oil stimulated aldehyde is at least partially caused by the lower solubility of homoeriodicyol, as encountered during the test.

[0247] ​

[0248] 1. AG, B. M. (2018). Multiresistente Erreger. 2018, from

[0249] https: / / www.bbraun.de / de / produkte-undtherapien / hyiene / multiresistente-erreger.html

[0250] 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.

[0251] 3. Effect, B. N. (2017). "Olivenblattextrakt Forschung." Retrieved 10.10.2017, from http: / / www.best-natural-effect.com / info / 5 / olivenblattextrakt-forschung.html.

[0252] 4. Fleming, H. P., et al. (1973). "Antimicrobial properties of oleuropein and products of its hydrolysis from green olives." Appl Microbiol 26(5): 777-782.

[0253] 5. Guinda, A., et al. (2010). "Pentacyclic triterpenoids from olive fruit and leaf." J Agric Food Chem 58(17): 9685-9691.

[0254] 6. Iming, S. (2005). Olivenblattextrakte - Heilmittel in der Praxis. Wien, Double-U GmbH.

[0255] 7. Khan, M. Y., et al. (2007). "Olea europaea: A phyto-pharmacological review." Pharmacognosy Reviews 1(1): 114-118.

[0256] 8. J. (2007). Lebensmittel-Mikrobiologie, Ulmer.

[0257] 9. Lieberei, R. R., C. (2007). Nutzpflanzenkunde. Stuttgart, Thieme Verlag.

[0258] 10. Liu, Y., et al. (2017). "Assessment of the Antimicrobial Activity of Olive Leaf Extract Against Foodborne Bacterial Patholgens." Front Microbiol 8: 113.

[0259] 11. Stiti, N., et al. (2007). "Formation of triterpenoids throughout Olea europaea fruit ontogeny." Lipids 42(1): 55-67.

[0260] 12. WHO, W.H.O. (2018). "Antibiotikaresistenz." from

[0261] http: / / www.euro.who.int / de / health-topics / disease-prevention / antimicrobial-resistance / antibiotic-resistance.

[0262] 13. Ahmed et al. (2014); "Antibacterial effect of olive (Olea europaea L.) leaves extract in raw peeled undeveined shrimp (Penaeus semisulcatus)

[0263] 14. US 7,429,008 B2; SYSTEM AND METHOD FOR PULVERIZING AND EXTRACTING MOISTURE; Graham et al.; 2008

[0264] 15. US 7,500,830 B2 SYSTEM AND METHOD FOR PULVERIZING AND EXTRACTING MOISTURE; Graham et al.; 2009

[0265] 16. US 7,909,577 B2 SYSTEM AND METHOD FOR PULVERIZING AND EXTRACTING MOISTURE; Graham et al.; 2011

[0266] 17. WO 2013 / 052583 A2 SYSTEMS AND METHODS FOR CONVERTING SEWAGE SLUDGE TO A COMBUSTIBLE FUEL; New et al.; 2013

[0267] 18. WO 2013 / 075003 Al; EGG SHELL POWDER COMPOSITIONS AND METHODS OF PRODUCING EGG SHELL POWDER COMPOSITIONS; Liu et al., 2013.

Claims

1. A method for preparing a composition of Olea europaea, preferably Olea europaea sylvestris, comprising the steps of: a) providing a part of an Olea europaea or Olea europaea sylvestris plant; and b) removing water from the part of Olea europaea or Olea europaea sylvestris by dehydrating or drying the part of Olea europaea or Olea europaea sylvestris; and c) breaking the part of Olea europaea or Olea europaea sylvestris of step b) by chopping, grinding, milling, pulverizing, or other method of breaking the part of Olea europaea or Olea europaea sylvestris to obtain a pulverized Olea europaea or Olea europaea sylvestris; d) sterilizing the pulverized Olea europaea or Olea europaea sylvestris, as appropriate, preferably at a temperature of 121 °C or 134 °C, to obtain a sterilized pulverized Olea europaea or Olea europaea sylvestris; and, optionally, e) mixing the sterilized pulverized Olea europaea or Olea europaea sylvestris with water and a thickening agent to obtain an Olea europaea or Olea europaea sylvestris gel having antibiotic or antifungal activity; or d') extracting, preferably CO2 extraction, an active agent having antibiotic or antifungal activity from the pulverized Olea europaea or Olea europaea sylvestris of step c); suspending the active agent in a non-toxic solubilizing and emulsifying agent to obtain an Olea europaea or Olea europaea sylvestris suspension having antibiotic or antifungal activity.

2. The method according to claim 1, wherein steps b) and c) are performed simultaneously by subjecting the Olea europaea or Olea europaea sylvestris to forces generated by a dynamic air flow, preferably by passing the Olea europaea or Olea europaea sylvestris through a venturi nozzle together with air, wherein the air is preferably pre-heated air.

3. The method according to any one of claims 1-2, wherein the thickening agent is selected from a gelling agent, such as gellan gum, and / or wherein the non-toxic solubilizing and / or emulsifying agent is selected from a surfactant.

4. The method according to any one of claims 1-3, wherein the part of Olea europaea or Olea europaea sylvestris is selected from a leaf, a branchlet, a twig, a root bark, a stem bark, a fruit, or an oil preparation residue, or a combination thereof.

5. The method according to any one of claims 1-4, wherein the active agent comprises an iridoid or a phenolic, and at least one organic compound selected from oleuropein, oleuropein aglycone, oleocanthal, tyrosol, hydroxytyrosol, uvaol, and homoeriodictyol.

6. An antibacterial or antifungal composition comprising at least one organic compound selected from oleuropein aglycone, hydroxytyrosol, oleocanthal, uvaol, and homoeriodictyol.

7. The composition according to claim 6 for use in the prevention or treatment of a bacterial or fungal infection in a mammal, preferably for use in the prevention or treatment of an infection caused by an antibiotic-resistant bacterium or a multi-drug resistant bacterium.

8. A sterilized pulverized Olea europaea or Olea europaea sylvestris having antibiotic or antifungal activity, obtained from steps a), b), c), and d) of the method according to claim 1.

9. The sterilized pulverized Olea europaea sylvestris having antibiotic or antifungal activity according to claim 8, wherein the pulverized Olea europaea or Olea europaea sylvestris is a powder and consists of more than 95% by weight of dry biomass. ​ ​ ​ ​ ​ ​ 10. Olea europaea or wild olive composition prepared according to any one of claims 1-5, optionally comprising a pharmaceutically or cosmetically acceptable carrier, diluent or excipient, wherein preferably the composition is a jelly, preferably a homogeneous colloidal gel, wherein preferably the Olea europaea or wild olive suspension is an aqueous suspension.

11. Olea europaea or wild olive composition according to claim 10, wherein the composition is a suspension and wherein the amount of non-toxic solubilizing agent is lower than 10%, preferably lower than 5%, more preferably lower than 3% of the total volume of the Olea europaea or wild olive suspension.

12. Composition according to any one of claims 10-11, in the form of an ointment, emulsion, cream, spray, gel, liquid, drops, capsule, or suppository.

13. Sterilized powdered Olea europaea or wild olive according to claim 8 or 9, Olea europaea or wild olive composition according to any one of claims 10-12, for use in the prevention or treatment of a bacterial or fungal infection in a mammal, preferably an infection caused by an antibiotic-resistant or multi-drug resistant bacterium, wherein preferably a) the bacterium is selected from the group consisting of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Mycobacterium tuberculosis, Enterococcus hirae, Enterococcus faecalis, Enterobacter, Serratia, Proteus, Providencia, Morganella, Enterococcus faecium, Helicobacter pylori, Campylobacter, Salmonella, Neisseria gonorrhoeae, Pneumococcus, Haemophilus influenzae, Shigella, Acinetobacter baumannii, and drug-resistant and multi-drug resistant strains thereof, and wherein b) the fungus is selected from the group consisting of Candida albicans and Aspergillus brasiliensis.

14. Use of sterilized powdered Olea europaea or wild olive according to claim 8 or 9, or Olea europaea or wild olive composition according to any one of claims 10-12, as a functional food additive, in particular an animal feed additive.

15. Use of sterilized powdered Olea europaea or wild olive according to claim 8 or 9, or Olea europaea or wild olive composition according to any one of claims 10-12, in a cosmetic product.

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