COMPOSITIONS OF THE OLEA EUROPAEA V. SYLVESTRIS WITH ANTIMICROBIAL EFFICACY

Abstract

The invention relates to methods for preparing an olive (Olea europaea subsp. europaea), preferably acebuche (Olea europaea subsp. europaea var. sylvestris) composition, the olive or acebuche composition as prepared, and uses thereof in the prevention or treatment of a bacterial or fungal infection in mammals, preferably infection by antibiotic resistant or multi-resistant bacteria. Other uses relate to functional food additives, in particular an animal feed additive, or to uses in cosmetics.

Claims

1. A method for preparing an olive (Olea europaea subsp. europaea), composition, comprising the steps of: a) providing parts of an olive or acebuche plant; and b) removing moisture from said olive or acebuche parts by dewatering or drying of said olive or acebuche parts; and c) comminution of said olive or acebuche parts of step b) to obtain a pulverized olive or acebuche; d) sterilizing said pulverized olive or acebuche to obtain a sterilized pulverized olive or acebuche; and, optionally, e) blending said sterilized pulverized olive or acebuche with water and a thickening agent to obtain an antibiotically or antifungally active olive or acebuche gel; or; d′) extracting an antibiotically or antifungally active agent from the pulverized olive or acebuche from step c) by extraction; suspending said active agent in a non-toxic solubilizer and emulsifying agent to obtain an antibiotically or antifungally active olive or acebuche suspension.

2. The method according to claim 1, wherein steps b) and c) are performed simultaneously by subjecting said olive or acebuche to forces generated by dynamic airflow.

3. The method according to claim 1, wherein said thickening agent is a gelling agent and/or wherein said non-toxic solubilizer and/or emulsifying agent is a tenside.

4. The method according to claim 1, wherein said olive or acebuche parts are selected from leaves, branches, twigs, root bark, stem bark, fruit or oil production residues, and combinations thereof.

5. The method according to claim 1, wherein said active agent comprises iridoids or phenols, and at least one organic compound selected from the group of oleuropein, oleacein, oleocanthal, tyrosol, hydroxytyrosol, uvaol and erythrodiol.

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

7. (canceled)

8. An antibiotically or antifungally active sterilized pulverized olive or acebuche, obtained by steps a, b), c), and d) of the method according to claim 1.

9. The antibiotically or antifungally active sterilized pulverized acebuche according to claim 8, wherein said pulverized olive or acebuche is a powder and consists of more than 95% by weight of dry biomass.

10. An olive or acebuche composition, produced according to claim 1, optionally comprising a pharmaceutically or cosmetically acceptable carrier, diluent, or excipient.

11. The olive or acebuche composition according to claim 10, wherein said composition is a suspension, comprising a non-toxic solubilizer, wherein the non-toxic solubilizer is less than 10% of the overall volume of the olive or acebuche suspension.

12. The composition according to claim 10 in the form of a salve, a lotion, a cream, spray, a gel, a liquid, a drop, a capsule or a suppository.

13. A method for prevention or treatment of a bacterial or fungal infection in a mammal, wherein said method comprises administering, to a mammal in need of such prevention or treatment, the sterilized pulverized olive or acebuche composition according to claim 10.

14. The olive or acebuche composition according to claim 10 formulated as a functional food additive.

15. The olive or acebuche composition according to claim 10, comprising a cosmetically acceptable carrier.

16. The method according to claim 1, wherein said olive is Olea europaea subsp. europaea var. sylvestris.

17. The composition according to claim 10, wherein said composition is a homogenous colloidal gel, and wherein said olive or acebuche suspension is an aqueous suspension.

18. The method according to claim 13, wherein the infection is by antibiotic resistant or multi-resistant bacteria.

19. The method according to claim 13, wherein the infection is by bacteria selected from 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, Streptococcus pneumoniae, Haemophilus influenza, Shigella, Acinetobacter baumannii, and resistant and multi-resistant strains thereof, or the infection is by a fungus selected from Candida albicans and Aspergillus brasiliensis.

20. The composition according to claim 14, which is formulated as an animal feed additive.

Description

[0076] In the Figures,

[0077] FIG. 1 shows the schematic method steps for the preparation of an antibiotic/antifungal gel according to the invention or for the preparation of an antibiotic/antifungal suspension according to the invention, including respective intermediate products.

[0078] FIG. 2 shows the principle of the device used for the method steps of removing moisture from olive or acebuche parts through dewatering or drying; and the comminution of olive or acebuche parts by chopping, grinding, milling, pulverizing in order to obtain a pulverized olive or acebuche. The steps are performed simultaneously by subjecting the olive or acebuche parts to forces generated by dynamic airflow, preferably by passing said olive or acebuche together with air, preferably pre-heated air, through a Venturi nozzle.

[0079] FIG. 3 shows a comparison of active substances (iridoids) of different olive cultivars.

[0080] FIG. 4 also shows a comparison of active substances (flavinoids) of different olive cultivars.

[0081] FIG. 5 shows another comparison of active substances (terpenes) of different olive cultivars.

[0082] FIG. 6 shows yet another comparison of active substances (tyrosol and hydroxytyrosol) of different olive cultivars.

EXAMPLES

Example 1: Extraction of Olive Tree Components with Supercritical Carbon Dioxide

[0083] The dried and pre-grinded olive tree components are presented to the extractor. The extractions are always carried out in batch mode, as the extractor can only be emptied and refilled at atmospheric pressure. During the extraction, the supercritical carbon dioxide (CO.sub.2) percolates at high pressure through the raw material and extracts the soluble substances from the raw material (i.e. extract). The dissolved substances can be divided into fractions with different compositions, by gradually reducing the pressure. In the first fraction (separator 1) the pressure can be the more difficult soluble substances accumulate, whereby in the following separator 2 the more easily soluble substances can be collected.

[0084] An extraction variant with only one separation stage was selected for the experiments carried out, whereby all dissolved substances were collected in separator 1. The amount of substances depends on the selected extraction conditions (pressure and temperature), as well as on the solubility and the amount of substances contained in the raw material.

[0085] Process flow: The high-pressure laboratory system (HDL 4) was used for the experiments. This is a laboratory plant with one extractor and two separators. The plant is designed for an extraction pressure of up to 1000 bar and temperatures of up to 95° C. The extraction pressure is not higher than 1000 bar. Liquid CO.sub.2 from the CO.sub.2 tank is brought to extraction pressure by means of a pump and to extraction temperature by means of a heat exchanger. In the extractor, the now supercritical CO.sub.2 flows through the raw material and is enriched with the soluble substances. After a pressure reduction, the mixture is separated into gaseous CO.sub.2 and extract. The extract can be collected in the separator and removed from the plant. The now gaseous and uncharged CO.sub.2 can then be liquefied again in the condenser and reused for the extraction cycle.

[0086] Performance: The raw material was presented to the extractor of the HDL 4 for extraction. Subsequently, an attempt was made to remove the valuable ingredients (such as terpenes and terpenoids) from the natural substance. Only one separation stage was used for the subsequent extract separation and the entire extract was collected in separator 1. Since the extract obtained is not flowable at −60° C., conventional extract extraction via the outlet valve of the separator 1 is difficult to not possible. The extract had to be removed after each test run and additionally scraped off after opening the separator and partly solvents can be washed out.

Example 2: Processing of Olive Tree Material Using the Venturi Dryer

[0087] The original olive tree material is first subjected to a thermal-mechanical process in which the particle size of the raw material and its moisture content are specifically altered. The technical application result is a dehydrated, fine-grained, possibly also powdery product for direct further use in the process chain, possibly also for its intermediate storage.

Example 3: Sterilisation Process

[0088] Due to the natural contamination of the starting product with aerobic spore formers, various sterilization procedures were applied. Different sterilization temperatures were used to determine the optimal temperature for preserving the pharmaceutically active ingredients: 121° C. and 134° C. The sterilization was carried out at different temperatures.

Example 4: Comparison of Active Substances in Cultivated and Wild Olive Leaves, and the Comminution Method

[0089] The sampling of the wild and cultivated olive plant parts was carried out by hand in the region of Casa de Porros, Valdevaqueros, Tarifa, Spain. The plant parts were each processed as follows: A portion of fresh leaves, twigs, stem bark and root bark were dried in a Taprogge Venturi device, the other portion pulverized in a Thermomix. The sample material was stored in tightly closed containers protected from light at ambient temperature. For comparability, both samples were sieved to an identical particle size spectrum. Subsequently, Soxleth extraction, and chromatographic methods were performed to analyse the chemical composition of active agents both in cultivated and wild olive. A positive effect was found in Oxygen Radical Absorption Capacity (ORAC) testing; sampling of active substances after soxleth extraction revealed that higher yields in extraction were achieved for materials that had been comminuted in the Taprogge venturi device. Favorable influence on ORAC (Oxygen, Radical Absorption Capacity) was detected for materials comminuted by the Taprogge Venturi device, which, in turn, translates into positive conservation properties, and a particularly long shelf life. This was demonstrated by measuring the antioxidative activity of leaves and twigs processed by the Taprogge Venturi device method compared to the antioxidative activity of fresh samples over the course of 30 days while the value of the samples processed by the Taprogge Venturi device method remains constant. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Comparison of the antioxidative activity of fresh samples and samples processed by the Taprogge Venturi device method over a 30-day period Leaves and twigs processed by VD Fresh leaves and (μmol Trolox twigs (μmol Trolox equivalent/g of equivalent/g of sample) sample) Day 1 521.98 502.14 (constant value) Day 5 678.10 Day 10 678.77 Day 20 657.23 Day 30 618.49

[0090] By comparing the active substances in the wild and cultivated olive, it can be seen that higher concentrations of active substances are present in the wild form of the olive tree. Olea europaea var. europaea and var. sylvestris differ in many aspects: The cultivated olive has larger fruits in a constant shape and is therefore more suitable for the production of olive oil. However, the tendency to produce larger fruits apparently means that the rest of the olive plant contains fewer active substances. The comparison of the two plants was made on the basis of already published data on the three most common species of cultivated olive in Andalusia: Arbequina, Hojiblanca and Picual. Fresh Acebuche olive leaves were examined for their profile of active substances (Table 3).

TABLE-US-00003 TABLE 3 Concentrations of single substances in mg/g var. var. europaea sylvestris Arbequina Hojiblanca Picual substances [mg/g] [mg/g] [mg/g] [mg/g] Oleuropein 82.30 29.84 33.48 25.37 Oleuroside 42.86 13.34 8.11 8.10 Ligstrosid 12.55 3.85 3.48 3.25 Verbacosid 9.66 4.07 1.16 1.13 Apigenin 4.00 0.28 0.23 0.39 Luteolin 4.80 0.39 0.37 0.50 Oleanolic acid 8.32 4.72 2.57 3.29

[0091] A comparison of active substances of different olive cultivars is shown in FIGS. 3 to 6, wherein FIG. 3 shows the comparison of iridoids, FIG. 4 shows the comparison flavinoids, FIG. 5 shows the comparison of terpenes, and FIG. 6 shows the comparison of tyrosol and hydroxytyrosol.

[0092] The following table 4 compares the percentage of active substances of acebuche vs. the cultivated olive.

TABLE-US-00004 TABLE 4 Amounts of active substances in acebuche vs. cultivated olive as identified var. var. % substances sylvestris europaea sylvestris/europaea Oleuropein 82.30 29.53 278.7% Oleuroside 42.86 9.85 435.1% Ligstroside 12.55 3.53 355.5% Verbacoside 9.66 2.12 455.7% Apigenine 4.00 0.30 1333.3% Luteolin 4.80 0.42 1142.8% Oleanolic acid 8.32 3.53 235.7% Maslinic acid 10.10 2.91 347.1% Tyrosol/Hydroxytyrosol 0.91 0.37 246.0%

[0093] The comparison based on these nine substances shows that acebuche olive leaves have the highest concentrations of phenols and single phenols, especially flavinoids. It is apparent that from a pharmaceutical view wild olive (acebuche) and cultivated olive are absolutely distinct, and the wild olive is therefore the better raw material for a potentially therapeutic or cosmetic product.

Example 5: Investigation of Antimicrobial Efficacy of Olive or Acebuche Suspension

[0094] The aim of the investigations was to test the antimicrobial efficacy of olive or acebuche suspension after suspension in Kolliphor RH40 and water for injection (WFI) against the extended test germ spectrum (Table 5).

TABLE-US-00005 TABLE 5 Test germ spectrum of the microbiological investigations of the present disclosure Escherichia Staphylococcus Pseudomonas Klebsiella Candida Acinetobacter Aspergillus coli aureus aeruginosa pneumoniae Enterococcus albicans baumannii brasiliensis ESBL MRSA ESBL subsp. Hirae Faecalis/ pneumoniae VRE ATCC DSM ATCC ATCC ATCC DSM ATCC CCUG ATCC ATCC ATCC ATCC NCTC ATCC 8739 22312 6538 29213 9027 24600 10031 56233 10541 BAA 10231 19606 13420 16404 2317 KPC Vanco- Carba- mycin penem resistant resistant

[0095] Performance: Preparation of the test germ suspension: The test bacteria were cultivated with a maximum passage number of 5. The test bacteria suspensions were adjusted to approximately 1000 CFU/0.1 ml in sterile 0.9% NaCl solution.

[0096] Sample preparation: 3.00 g olive extract in 1.01 g Kolliphor RH40 were heated for approximately 5 min. at 45° C. in a water bath, then for approximately 15 min. at 80° C. in a water bath and shaken by hand. The olive extract and Kolliphor RH40 were mixed together with 15 ml WFI, resulting in a very viscous, dark green to dark brown suspension that could not be pipetted. The next day the stock solution was heated again at 80° C. in a water bath and another 5 ml WFI heated to 80° C. was added. The stock solution was again heated to 80° C. for approximately 5 minutes. Water bath warmed up and shaken approx. 1 min. by hand. The result was a stock solution containing 125 mg olive extract per ml and 4.2% Kolliphor RH40. After this processing, the olive extract was available as a homogeneous and pipettable suspension with a dark brown to dark green color.

[0097] Evaluation of the antimicrobial efficacy of olive extract after preparation of stock solutions: For each test germ 1 ml of the stock solution was converted to 1 ml of double concentrated CaSo bouillon and Sab bouillon (C. albicans). The preparations were then inoculated with 0.1 ml each of the test germ suspensions adjusted to approx. 1000 CFU/0.1 ml. The bacterial count in the test germ suspensions was checked for blood agar and sab-agar by means of the spatula method. All culture media preparations were incubated for a maximum of 72 hours at (30-35° C.). After 24 h, 48 h, and 72 hours incubation subcultures of all 0.1 ml batches were performed by streaking on blood agar plates and Sab-agar respectively. The agar plates were tested at 30-35° C. for 24 h-48 h (yeast) aerobically incubated. Positive controls: 1 ml WFI were added to 1 ml double concentrated CaSo bouillon or Sab bouillon (C. albicans) and then inoculated with 0.1 ml each of the test germ suspensions adjusted to approx. 1000 KBE/0.1 ml in single determination. The positive controls were used as a reference for the growth of test bacteria in the double concentrated culture medium after 1:2 dilutions. The results are presented in the following tables.

TABLE-US-00006 TABLE 6 Results of the antimicrobial efficacy of olive suspensions with the test germ C. albicans ATCC10231; Inoculum: effectively inoculated bacterial count per test batch: 1160 CFU = 580 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00007 TABLE 7 Results of the antimicrobial efficacy of olive suspension with the test bacterium E. faecium ATCC BAA2317; Inoculum: effectively inoculated bacterial count per test batch: 1810 CFU = 905 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00008 TABLE 8 Results of the antimicrobial efficacy of olive suspension with the test bacterium E. hirae ATCC 10541; Inoculum: effectively inoculated bacterial count per test batch: 760 CFU = 380 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00009 TABLE 9 Results of the antimicrobial efficacy of olive suspension with the test bacterium Kleb. pneu. subsp. Pneumoniae CCUG 56233; Inoculum: effectively inoculated bacterial count per test batch: 1200 CFU = 600 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00010 TABLE 10 Results of the antimicrobial efficacy of olive suspension with the test bacterium Kleb. pneu. subsp. pneumoniae ATCC 10031; Inoculum: effectively inoculated bacterial count per test batch: 740 CFU = 370 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00011 TABLE 11 Results of the antimicrobial efficacy of olive suspension with the test bacterium P. aeruginosa ATCC 9027; Inoculum: effectively inoculated bacterial count per test batch: 640 CFU = 320 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00012 TABLE 12 Results of the antimicrobial efficacy of olive suspension with the test bacterium P. aeruginosa ESBL DSM 24600; Inoculum: effectively inoculated bacterial count per test batch: 680 CFU = 340 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00013 TABLE 13 Results of the antimicrobial efficacy of olive suspension with the test bacterium E. coli ATCC 8739; Inoculum: effectively inoculated bacterial count per test batch: 860 CFU = 430 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00014 TABLE 14 Results of the antimicrobial efficacy of olive suspension with the test bacterium E. coli DSM 22312; Inoculum: effectively inoculated bacterial count per test batch: 670 CFU = 435 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00015 TABLE 15 Results of the antimicrobial efficacy of olive suspension with the test bacterium S. aureus ATCC 6538; Inoculum: effectively inoculated bacterial count per test batch: 680 CFU = 340 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00016 TABLE 16 Results of the antimicrobial efficacy of olive suspension with the test bacterium S. aureus supsp. aureus ATCC 29213; Inoculum: effectively inoculated bacterial count per test batch: 2020 CFU = 1010 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually (125 mg/ml (62.5 mg/ml recognizable/ recognizable/ recognizable/ product) product) No growth No growth No growth after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

[0098] For investigation of two further test germs, the bacteria Acinetobacter baumannii and Aspergillus brasiliensis, the Kolliphor RH40 content of the stock solution was reduced by 50% from 4.2% to 2.1%. In addition to this origin stock solution A1 with the olive extract in a concentration of 127.5 mg/ml a 1:2 dilution with water for injection was prepared so that the olive extract had a concentration of 63.8 mg/ml (stock solution A2).

TABLE-US-00017 TABLE 17 Results of the antimicrobial efficacy of olive suspension with the test bacterium A. baumannii ATCC 19606; Inoculum: effectively inoculated bacterial count per test batch: 4000 CFU = 2000 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually A1 A1 recognizable/ recognizable/ recognizable/ (127.5 mg/ml (63.8 mg/ml No growth No growth No growth product) product) after after after subculture subculture subculture stock stock P P P solution solution not visually not visually not visually A2 A2 recognizable/ recognizable/ recognizable/ (63.8 mg/ml (31.9 mg/ml No growth growth growth product) product) after after after subculture subculture, subculture, comparable comparable with with control control =Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00018 TABLE 18 Results of the antimicrobial efficacy of olive suspension with the test bacterium A. baumannii NCTC 13420; Inoculum: effectively inoculated bacterial count per test batch: 890 CFU = 445 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (layer) (layer) (layer) stock stock P P P solution solution not visually not visually not visually A1 A1 recognizable/ recognizable/ recognizable/ (127.5 mg/ml (63.8 mg/ml No growth No growth No growth product) product) after after after subculture subculture subculture stock stock P P P solution solution not visually not visually not visually A2 A2 recognizable/ recognizable/ recognizable/ (63.8 mg/ml (31.9 mg/ml No growth No growth No growth product) product) after after after subculture subculture subculture + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

TABLE-US-00019 TABLE 19 Results of the antimicrobial efficacy of olive suspension with the test germ A. brasiliensis ATCC 16404; Inoculum: effectively inoculated bacterial count per test batch: 620 CFU = 310 CFU/ml culture medium batch Test concen- tration in the Results nutrient Growth Growth Growth sample ID medium after 24 h after 48 h after 72 h positive ./. + + + control After After After subculture subculture subculture growth growth growth (4 CFU) (layer) (layer) stock stock P P P solution solution not visually not visually not visually A1 A1 recognizable/ recognizable/ recognizable/ (127.5 mg/ml (63.8 mg/ml After After After product) product) subculture subculture subculture growth of growth of 6 growth of 2 approx. 28 CFU/0.1 CFU/0.1 CFU/0.1 ml, not ml, not ml, comparable comparable comparable with with with control control control stock stock P P P solution solution not visually not visually not visually A2 A2 recognizable/ recognizable/ recognizable/ (63.8 mg/ml (31.9 mg/ml After After After product) product) subculture subculture subculture growth of 9 growth of 2 growth of 1 CFU/0.1 CFU/0.1 CFU/0.1 ml, ml, ml, comparable comparable comparable with with with control control control + = Growth of the test germ in the form of visually visible turbidity in the nutrient media preparations P = Product turbidity, no visual assessment of the growth of the test germ possible Subcultures (SC) by streaking of 0.1 ml each of the nutrient media preparation

[0099] As the tests show, no growth of test germs, with the exception of the test germ A. brasiliensis ATCC 16404, can be detected after 72 hours of incubation of the nutrient media preparations after carrying out the corresponding subcultures. Thus, a good antimicrobial efficacy of the olive extract in a concentration of 62.5 mg/ml or 63.8 mg/ml respectively can be demonstrated under the test conditions. For the test germ A. brasiliensis in the presence of the olive suspension from stock solution A1 and stock solution A2, a few isolated surviving live spores are still detectable throughout the test, but the reduction of CFU is also significant here.

Example 6: Investigation of Antimicrobial Efficacy of Olive or Acebuche Gel

[0100] Performance: A colloidal gel with Kelcogel CG-HA was produced from olive leaf powder. For this purpose, 0.1 g Kelcogel CG-HA was dissolved in 100 ml WFI by heating to 85-90° C. and then 15 g olive leaf powder was added while stirring until a homogeneous suspension was visually formed. After cooling in the ice bath, a colloidal olive leaf powder gel was obtained. Aliquots of 10 g gel were inoculated with the adjusted test germ suspension (10.sup.5-10.sup.6 CFU/g) and tested for germ count according to the test times listed in the table 15.

TABLE-US-00020 TABLE 20 Number of CFU according to the test time. Initial t = 6 t = 12 t = 30 t = 60 load t.sub.0 min min min min Test germs CFU/g CFU/g CFU/g CFU/g CFU/g CFU/g Staphylococcus 4.0 × 10.sup.5 2.7 × 10.sup.3 1.0 × 10.sup.2 <100 <100 <100 aureus ATCC 6538 Pseudomonas 7.5 × 10.sup.5 5.6 × 10.sup.3 4.0 × 10.sup.2 <100 <100 <100 aeruginosa ATCC 15442 Escherichia 6.3 × 10.sup.5 <100 <100 <100 <100 <100 coli ATCC 8739

[0101] After 12 minutes, E. coli already showed a very good efficacy at the to h-value and S. aureus and P. aeruginosa below the detection limit of 100 CFU per gram, and proves the overall very good antimicrobial efficacy within a short exposure period.

Example 7: Investigation of Antimicrobial Efficacy of Olive or Acebuche Gel

[0102] Performance: A colloidal gel with Kelcogel CG-HA was produced from olive leaf powder. For this purpose, 0.1 g Kelcogel CG-HA was dissolved in 100 ml WFI by heating to 85-90° C. and then 15 g olive leaf powder was added while stirring until a homogeneous suspension was visually formed. After cooling in the ice bath, a colloidal olive leaf powder gel was obtained. Aliquots of 10 g gel were inoculated with the adjusted test germ suspension (10.sup.5-10.sup.6 CFU/g) and tested for germ count according to the test times listed in the table 21.

TABLE-US-00021 TABLE 21 Number of CFU according to the test time. Initial t = 6 t = 12 t = 30 t = 60 load t.sub.0 min min min min Test germs CFU/g CFU/g CFU/g CFU/g CFU/g CFU/g Staphylococcus 4.0 × 10.sup.5 6.1 × 10.sup.3 6.1 × 10.sup.2 <100 <100 <100 aureus ATCC 6538 Pseudomonas 7.5 × 10.sup.5 5.9 × 10.sup.3 6.1 × 10.sup.2 1.0 × 10.sup.2 <100 <100 aeruginosa ATCC 15442 Escherichia 6.3 × 10.sup.5 3.0 × 10.sup.2 <100 <100 <100 <100 coli ATCC 8739

[0103] Accordingly, E. coli shows very good efficacy after 6 minutes and S. aureus after 12 minutes up to below the detection limit of 100 CFU per gram. For P. aeruginosa the bacterial count is reduced to below the detection limit after 30 minutes and proves the overall very good antimicrobial efficacy within a short exposure period.

Example 8: Investigation of Antimicrobial Efficacy of Olive or Acebuche Composition with Dough Pieces

[0104] Performance: Dough pieces were prepared per test time by mixing 500 mg sample with 1 ml WFI. After homogenization, 0.5 ml of adjusted germ suspension was added and homogeneously mixed into the dough. Afterwards both test germs showed a very good efficacy already after 6 hours reaction time at room temperature with the untreated as well as with the 15 minutes at 121° C. and 30 minutes at 130° C. steam-sterilized product olive leaf in powder form. (Table 22)

TABLE-US-00022 TABLE 22 Number of CFU according to the test time. Initial load t.sub.0 6 h 24 h CFU/dough CFU/dough CFU/dough CFU/dough Test germ pieces pieces pieces pieces Staphylococcus 2.0 × 10.sup.5 1.8 × 10.sup.5 <100 <100 aureus MRSA-strain ATCC 29213 Untreated olive leaf powder Staphylococcus 2.0 × 10.sup.5 2.4 × 10.sup.5 <100 <100 aureus MRSA-strain ATCC 29213 olive leaf powder sterilized at 121° C. for 15 min. Staphylococcus 2.0 × 10.sup.5 3.4 × 10.sup.5 <100 <100 aureus MRSA-strain ATCC 29213 olive leaf powder sterilized at 130° C. for 30 min.

[0105] The untreated olive leaf dough pieces show a natural contamination of the order of 1.4×10.sup.3 CFU-2.1×10.sup.3 CFU per 500 mg dough piece with various aerobic spore formers, which was not killed within the test period after 6 hours and 24 hours respectively. In the sterilized test samples at 121° C. for 15 minutes, the dough pieces showed a natural contamination in the range of 1.1×103 CFU-2.2×102 CFU per 500 mg. Both sterilized test specimens were sterilized for 30 minutes at 130° C. and no contamination germs were detected.

Example 9: Investigation of Antimicrobial Efficacy of Olive or Acebuche Composition with Dough Pieces

[0106] Performance: Dough pieces were prepared by mixing 500 mg olive substrate with 1 ml WFI per test germ and test time. After homogenization, 0.5 ml of adjusted germ suspension was added and homogeneously mixed into the dough. Afterwards both test germs showed a very good efficacy already after 24 hours reaction time at room temperature both at untreated and at 15 minutes at 121° C. and 30 minutes at 130° C. steam sterilized olive leaf. (Table 23)

TABLE-US-00023 TABLE 23 Number of CFU according to the test time. Initial load t.sub.0 24 h CFU/dough CFU/dough CFU/dough Test germ pieces pieces pieces Escherichia coli 1.3 × 10.sup.5 1.3 × 10.sup.5 <100 ATCC 8739 Untreated olive leaf powder Pseudomonas 2.2 × 10.sup.5 2.1 × 10.sup.5 <100 aeruginosa ATCC 9027 Untreated olive leaf powder Staphylococcus 1.4 × 10.sup.5 1.8 × 10.sup.5 <100 aureus ATCC 6538 Untreated olive leaf powder Escherichia coli 1.3 × 10.sup.5 1.3 × 10.sup.5 <100 ATCC 8739 olive leaf powder sterilized at 121° C. for 15 min. Pseudomonas 2.2 × 10.sup.5 2.2 × 10.sup.5 <100 aeruginosa ATCC 9027 olive leaf powder sterilized at 121° C. for 15 min. Staphylococcus 1.4 × 10.sup.5 2.4 × 10.sup.5 <100 aureus ATCC 6538 olive leaf powder sterilized at 121° C. for 15 min. Escherichia coli 1.3 × 10.sup.5 2.3 × 10.sup.5 <100 ATCC 8739 olive leaf powder sterilized at 130° C. for 30 min. Pseudomonas 2.2 × 10.sup.5 1.6 × 10.sup.5 <100 aeruginosa ATCC 9027 olive leaf powder sterilized at 130° C. for 30 min. Staphylococcus 1.4 × 10.sup.5 1.9 × 10.sup.5 <100 aureus ATCC 6538 olive leaf powder sterilized at 130° C. for 30 min.

[0107] The untreated olive leaf dough pieces show a natural contamination of the order of 2.9×10.sup.4 CFU per 500 mg dough piece with various aerobic spore formers, which were not killed within the test period after 24 hours. No contamination germs are detectable in the sterilized test samples.

Example 10: Investigation of Antimicrobial Efficacy of Olive or Acebuche Composition with Dough Pieces

[0108] Performance: Dough pieces were prepared by mixing 500 mg olive substrate with 1 ml WFI per test germ and test time. After homogenization, 0.5 ml of adjusted germ suspension was added and homogeneously mixed into the dough. Afterwards, both test germs showed a very good efficacy after 24 hours reaction time at room temperature both at untreated and at 15 minutes at 121° C. and 30 minutes at 130° C. steam-sterilized product bark/strain in powder form. (Table 24)

TABLE-US-00024 TABLE 24 Number of CFU according to the test time. Initial load t.sub.0 24 h CFU/dough CFU/dough CFU/dough Test germ pieces pieces pieces Escherichia coli 1.4 × 10.sup.5 1.2 × 10.sup.5 <100 ATCC 8739 Untreated bark/strain powder Pseudomonas 1.3 × 10.sup.5 2.7 × 10.sup.5 <100 aeruginosa ATCC 9027 Untreated bark/strain powder Staphylococcus 2.1 × 10.sup.5 5.3 × 10.sup.5 <100 aureus ATCC 6538 Untreated bark/strain powder Escherichia coli 1.4 × 10.sup.5 1.3 × 10.sup.5 <100 ATCC 8739 bark/strain powder sterilized at 121° C. for 15 min. Pseudomonas 1.3 × 10.sup.5 2.0 × 10.sup.5 <100 aeruginosa ATCC 9027 bark/strain powder sterilized at 121° C. for 15 min. Staphylococcus 2.1 × 10.sup.5 1.8 × 10.sup.5 <100 aureus ATCC 6538 bark/strain powder sterilized at 121° C. for 15 min. Escherichia coli 1.4 × 10.sup.5 1.6 × 10.sup.5 <100 ATCC 8739 bark/strain powder sterilized at 130° C. for 30 min. Pseudomonas 1.3 × 10.sup.5 1.8 × 10.sup.5 <100 aeruginosa ATCC 9027 bark/strain powder sterilized at 130° C. for 30 min. Staphylococcus 2.1 × 10.sup.5 1.2 × 10.sup.5 <100 aureus ATCC 6538 bark/strain powder sterilized at 130° C. for 30 min.

[0109] The untreated bark/strain in powder form showed a natural contamination of the order of 4.4×10.sup.2 CFU per 500 mg dough with various aerobic spore formers, which was not killed within the test period after 24 hours. Contamination germs of 1.1×10.sup.2 CFU/dough piece are still detectable in the test specimen sterilized at 121° C. The test specimen is also sterilized at 121° C. In the test specimen sterilized at 130° C. no contamination germs are detectable.

Example 11: Investigation of Antimicrobial Efficacy of Olive or Acebuche Composition with Dough Pieces

[0110] Performance: Dough pieces were prepared by mixing 500 mg olive substrate with 1 ml WFI per test germ and test time. After homogenization, 0.5 ml of adjusted germ suspension was added and homogeneously mixed into the dough. Afterwards both test germs showed a very good efficacy already after 24 hours reaction time at room temperature both at untreated and at 15 minutes at 121° C. and 30 minutes at 130° C. steam-sterilized product bark/root in powder form. (Table 25)

TABLE-US-00025 TABLE 25 Number of CFU according to the test time Initial load t.sub.0 24 h CFU/dough CFU/dough CFU/dough Test germ pieces pieces pieces Escherichia coli 1.4 × 10.sup.5 1.8 × 10.sup.5 <100 ATCC 8739 Untreated bark/root powder Pseudomonas 1.3 × 10.sup.5 3.3 × 10.sup.5 <100 aeruginosa ATCC 9027 Untreated bark/root powder Staphylococcus 2.1 × 10.sup.5 1.1 × 10.sup.5 <100 aureus ATCC 6538 Untreated bark/root powder Escherichia coli 1.4 × 10.sup.5 1.4 × 10.sup.5 <100 ATCC 8739 Bark/root powder sterilized at 121° C. for 15 min. Pseudomonas 1.3 × 10.sup.5 2.4 × 10.sup.5 <100 aeruginosa ATCC 9027 bark/root powder sterilized at 121° C. for 15 min. Staphylococcus 2.1 × 10.sup.5 3.1 × 10.sup.5 <100 aureus ATCC 6538 bark/root powder sterilized at 121° C. for 15 min. Escherichia coli 1.4 × 10.sup.5 2.3 × 10.sup.5 <100 ATCC 8739 bark/root powder sterilized at 130° C. for 30 min. Pseudomonas 1.3 × 10.sup.5 2.3 × 10.sup.5 <100 aeruginosa ATCC 9027 bark/root powder sterilized at 130° C. for 30 min. Staphylococcus 2.1 × 10.sup.5 1.3 × 10.sup.5 <100 aureus ATCC 6538 bark/root powder sterilized at 130° C. for 30 min.

[0111] The untreated bark/root in powder form showed a natural contamination in the range of 1.1×10.sup.6 CFU per 500 mg dough with various aerobic spore formers, which was not killed within the test period after 24 hours. In the test samples sterilized at 121° C. and 130° C., contamination germs of 1.8×10.sup.3 CFU/dough piece and 8.8×10.sup.2 CFU/dough piece are still detectable.

Example 12: Analysis of Antimicrobial Efficacy of Hydroxytyrosol (Mono-Substance) Against Staphylococcus aureus ATCC 6538

[0112] Hydroxytyrosol is found in substrates and extracts derived from olive trees.

[0113] A stock solution was prepared by adding 0.1 ml methanol to 10.48 mg of hydroxytyrosol. After dissolving of the compound, the solution was quantitatively transferred into 4.9 ml water for injection (WFI), effective concentration of hydroxytyrosol: 2.0 mg/ml.

[0114] Using the stock solution, five (5) concentrations of hydroxytyrosol in a geometric series of 2 mg hydroxytyrosol/ml to 0.125 mg hydroxytyrosol/ml were tested in a quantitative suspension assay.

[0115] 2 ml of the stock solution and the dilutions (V1 to V4), respectively, and a methanol-solution of 2% as a positive control were inoculated with 0.1 ml of a test suspension of S. aureus ATCC 6538, adjusted to about 2.0×10.sup.5 CFU/0.1 ml, resulting in about 100,000 CFU/ml in the individual assays.

[0116] Samples (0.1 ml) were then taken at t 0, 12, 30, and 60 minutes as well as t 24 48, and 72 hours after inoculation and culturing at between 20-25° C., and were diluted with 0.9 ml of 0.9% NaCl solution. The samples were plated on blood agar plates, and incubated up to 5 days at between 30-35° C.

[0117] Results are shown in the following Table 26.

TABLE-US-00026 TABLE 26 Number of CFUs; Intial cell load: 1.7 × 10.sup.5 CFU/ml, bacterium: S. aureus ATCC 6538, Hyd = Hydroxytyrosol Results after 1:10 dilution CFU/ml of assay t 0 min t 12 min t 30 min t 60 min t 24 h t 48 h t 72 h Positive 7.0 × 10.sup.4 8.0 × 10.sup.4 6.6 × 10.sup.4 7.8 × 10.sup.4 1.2 × 10.sup.4 3.1 × 10.sup.3 7.1 × 10.sup.2 control Stock 7.0 × 10.sup.4 8.9 × 10.sup.4 5.9 × 10.sup.4 5.9 × 10.sup.4 <100 <100 <100 solution 2.0 Hyd/ml V1 1.0 6.8 × 10.sup.4 7.8 × 10.sup.4 7.4 × 10.sup.4 7.2 × 10.sup.4 <100 <100 <100 Hyd/ml V2 0.5 7.0 × 10.sup.4 6.6 × 10.sup.4 6.4 × 10.sup.4 6.1 × 10.sup.4 2.0 × 10.sup.2 <100 <100 Hyd/ml V3 0.25 5.7 × 10.sup.4 5.1 × 10.sup.4 5.9 × 10.sup.4 7.4 × 10.sup.4 1.4 × 10.sup.3 <100 <100 Hyd/ml V4 0.125 7.0 × 10.sup.4 7.4 × 10.sup.4 7.0 × 10.sup.4 6.8 × 10.sup.4 2.8 × 10.sup.3 <100 7.1 × 10.sup.2 Hyd/ml

[0118] As can be seen in table 26, methanol at 2% had no significant antimicrobial effect at the beginning of the test. The reduction at the later points in time is due to the natural loss of viability of the bacteria. Hydroxytyrosol only shows a significant effect only after 24 hours, nevertheless, this effect is strong, i.e. below the detection limit of the assay at 100 CFU/ml. An additional dilution further delays the effects.

Example 13: Analysis of Antimicrobial Efficacy of Oleocanthal (Mono-Substance) Against Staphylococcus aureus ATCC 6538

[0119] Oleocanthal is found in substrates and extracts derived from olive trees.

[0120] A stock solution was prepared at a concentration of 2 mg/ml of oleocanthal. The compound was dissolved by in ethanol (99.8%) and WFI was added.

[0121] Using the stock solution, concentrations of oleocanthal in a geometric series of 1 mg oleocanthal/ml to 0.125 mg oleocanthal/ml were tested in a quantitative suspension assay.

[0122] 2 ml of the stock solution and the dilutions (V1 to V4), respectively, and a ethanol-solution of 2.85% as a positive control were inoculated with 0.055 ml of a test suspension of S. aureus ATCC 6538, adjusted to about 3.6×10.sup.5 CFU/0.1 ml, resulting in about 99,000 CFU/ml in the individual assays.

[0123] Samples (0.1 ml) were then taken at t 0, 12, 30, and 60 minutes as well as t 24 48, and 72 hours after inoculation and culturing at between 20-25° C., and were diluted with 0.9 ml of 0.9% NaCl solution. The samples were plated on blood agar plates, and incubated up to 5 days at between 30-35° C.

[0124] Results are shown in the following Table 27.

TABLE-US-00027 TABLE 27 Number of CFUs; Intial cell load: 9.9 × 10.sup.4 CFU/ml, bacterium: S. aureus ATCC 6538, Ole = oleocanthal Results after 1:10 dilution CFU/ml of assay t 0 min t 12 min t 30 min t 60 min t 24 h t 48 h t 72 h Positive 5.7 × 10.sup.5 5.6 × 10.sup.5 .sup. 1 × 10.sup.5 6.1 × 10.sup.4 3.7 × 10.sup.4 4.1 × 10.sup.4 3.3 × 10.sup.3 control Stock <100 <100 <100 <100 <100 <100 <100 solution 2.0 Ole/ml V1 1.0 <100 <100 <100 <100 <100 <100 <100 Ole/ml V2 0.5 8.3 × 10.sup.3 6.1 × 10.sup.3 5.1 × 10.sup.3 2.4 × 10.sup.3 <100 <100 <100 Ole/ml V3 0.25 1.7 × 10.sup.4 1.8 × 10.sup.4 2.0 × 10.sup.4 1.6 × 10.sup.4 7.2 × 10.sup.3 8.3 × 10.sup.3 <100 Ole/ml V4 0.125 2.0 × 10.sup.4 1.6 × 10.sup.4 1.6 × 10.sup.4 1.1 × 10.sup.4 5.4 × 10.sup.3 <100 <100 Ole/ml

[0125] As can be seen in table 27, ethanol at 3.84% had no significant antimicrobial effect at the beginning of the test. The reduction at the later points in time is due to the natural loss of viability of the bacteria. Oleocanthal shows an immediate effect at the higher concentrations, below the detection limit of the assay at 100 CFU/ml. A dilution delays these effects, which can, nevertheless, still be identified.

Example 14: Analysis of Antimicrobial Efficacy of Oleacin (Mono-Substance) Against Staphylococcus aureus ATCC 6538

[0126] Oleacin is found in substrates and extracts derived from olive trees.

[0127] A stock solution was prepared at a concentration of 1 mg/ml of oleacin. The compound was dissolved by in ethanol (99.8%) and WFI was added.

[0128] Using the stock solution, concentrations of oleacin at 0.25 mg oleacin/ml and 0.125 mg oleacin/ml were tested in a quantitative suspension assay.

[0129] 2 ml of the stock solution and the dilutions (V1 and V2), respectively, and a ethanol-solution of 0.44% as a positive control were inoculated with 0.055 ml of a test suspension of S. aureus ATCC 6538, adjusted to about 3.5×10.sup.5 CFU/0.1 ml, resulting in about 99,000 CFU/ml in the individual assays.

[0130] Samples (0.1 ml) were then taken at t 0, 12, 30, and 60 minutes as well as t 24 48, and 72 hours after inoculation and culturing at between 20-25° C., and were diluted with 0.9 ml of 0.9% NaCl solution. The samples were plated on blood agar plates, and incubated up to 5 days at between 30-35° C.

[0131] Results are shown in the following Table 28.

TABLE-US-00028 TABLE 28 Number of CFUs; Intial cell load: 9.9 × 10.sup.4 CFU/ml, bacterium: S. aureus ATCC 6538, Oci = oleacin Results after 1:10 dilution CFU/ml of assay t 0 min t 12 min t 30 min t 60 min t 24 h t 48 h t 72 h Positive 1.2 × 10.sup.5 1.3 × 10.sup.5 1.0 × 10.sup.5 1.1 × 10.sup.5 3.7 × 10.sup.4 9.0 × 10.sup.3 3.0 × 10.sup.3 control V1 0.25 1.8 × 10.sup.3 <100 <100 <100 <100 <100 <100 Oci/ml V2 0.125 4.3 × 10.sup.3 <100 <100 <100 <100 <100 <100 Oci/ml

[0132] As can be seen in table 28, ethanol at 0.44% had no significant antimicrobial effect at the beginning of the test. The reduction at the later points in time is due to the natural loss of viability of the bacteria. Oleacin shows an immediate effect at the concentrations tested, below the detection limit of the assay at 100 CFU/ml.

Example 15: Analysis of Antimicrobial Efficacy of Uvaol (Mono-Substance) Against Staphylococcus aureus ATCC 6538

[0133] Uvaol is found in substrates and extracts derived from olive trees.

[0134] A stock solution was prepared by adding 0.1 ml chloroform to 25 mg of uvaol. After dissolving of the compound, the solution was quantitatively transferred into 12.4 ml water for injection (WFI), effective concentration of uvaol: 2.0 mg/ml.

[0135] Using the stock solution, five (5) concentrations of uvaol in a geometric series of 2 mg uvaol/ml to 0.125 mg uvaol/ml were tested in a quantitative suspension assay.

[0136] 2 ml of the stock solution and the dilutions (V1 to V4), respectively, and a chloroform-solution of 0.8% as a positive control were inoculated with 0.1 ml of a test suspension of S. aureus ATCC 6538, adjusted to about 2.0×10.sup.5 CFU/0.1 ml, resulting in about 100,000 CFU/ml in the individual assays.

[0137] Samples (0.1 ml) were then taken at t 0, 12, 30, and 60 minutes as well as t 24 48, and 72 hours after inoculation and culturing at between 20-25° C., and were diluted with 0.9 ml of 0.9% NaCl solution. The samples were plated on blood agar plates, and incubated up to 5 days at between 30-35° C.

[0138] Results are shown in the following Table 29.

TABLE-US-00029 TABLE 29 Number of CFUs; Intial cell load: 2.0 × 10.sup.5 CFU/ml, bacterium: S. aureus ATCC 6538, Uva = uvaol Results after 1:10 dilution CFU/ml of assay t 0 min t 12 min t 30 min t 60 min t 24 h t 48 h t 72 h Positive 5.9 × 10.sup.4 5.3 × 10.sup.4 6.4 × 10.sup.4 7.8 × 10.sup.4 1.2 × 10.sup.4 1.1 × 10.sup.4 9.2 × 10.sup.3 control Stock 7.2 × 10.sup.4 5.9 × 10.sup.4 7.0 × 10.sup.4 7.0 × 10.sup.4 <100 <100 <100 solution 2.0 Uva/ml V1 1.0 7.8 × 10.sup.4 7.8 × 10.sup.4 7.4 × 10.sup.4 5.9 × 10.sup.4 <100 <100 <100 Uva/ml V2 0.5 7.0 × 10.sup.4 7.8 × 10.sup.4 7.6 × 10.sup.4 5.7 × 10.sup.4 <100 <100 <100 Uva/ml V3 0.25 5.3 × 10.sup.4 8.1 × 10.sup.4 5.3 × 10.sup.4 5.7 × 10.sup.4 <100 <100 <100 Uva/ml V4 0.125 6.3 × 10.sup.4 5.7 × 10.sup.4 7.6 × 10.sup.4 6.6 × 10.sup.4 4.7 × 10.sup.3 4.2 × 10.sup.3 8.2 × 10.sup.2 Uva/ml

[0139] As can be seen in table 29, chloroform at 0.8% had no significant antimicrobial effect during the test. Uvaol shows an effect after an incubation of more than 24 hours at concentrations of 0.25 Uva/ml or more, but below the detection limit of the assay at 100 CFU/ml.

Example 16: Analysis of Antimicrobial Efficacy of Erythrodiol (Mono-Substance) Against Staphylococcus aureus ATCC 6538

[0140] Erythrodiol is found in substrates and extracts derived from olive trees.

[0141] A stock solution was prepared by adding 0.1 ml chloroform to 10.45 mg of erythrodiol. After dissolving of the compound, the solution was quantitatively transferred into 4.9 ml water for injection (WFI), effective concentration of erythrodiol: 2.0 mg/ml.

[0142] Using the stock solution, five (5) concentrations of uvaol in a geometric series of 2 mg uvaol/ml to 0.125 mg uvaol/ml were tested in a quantitative suspension assay.

[0143] 2 ml of the stock solution and the dilutions (V1 to V4), respectively, and a chloroform-solution of 2% as a positive control were inoculated with 0.1 ml of a test suspension of S. aureus ATCC 6538, adjusted to about 3.6×10.sup.5 CFU/0.1 ml, resulting in about 100.000 CFU/ml in the individual assays.

[0144] Samples (0.1 ml) were then taken at t 0, 12, 30, and 60 minutes as well as t 24 48, and 72 hours after inoculation and culturing at between 20-25° C., and were diluted with 0.9 ml of 0.9% NaCl solution. The samples were plated on blood agar plates, and incubated up to 5 days at between 30-35° C.

[0145] Results are shown in the following Table 30.

TABLE-US-00030 TABLE 30 Number of CFUs; Intial cell load: 9.5 × 10.sup.4 CFU/ml, bacterium: S. aureus ATCC 6538, Ery = erythrodiol Results after 1:10 dilution CFU/ml of assay t 0 min t 12 min t 30 min t 60 min t 24 h t 48 h t 72 h Positive 1.0 × 10.sup.5 1.1 × 10.sup.5 1.0 × 10.sup.5 7.8 × 10.sup.4 4.1 × 10.sup.4 1.2 × 10.sup.4 3.5 × 10.sup.3 control Stock <100 <100 <100 <100 8.2 × 10.sup.3 <100 <100 solution 2.0 Ery/ml V1 1.0 <100 <100 <100 <100 1.7 × 10.sup.4 1.0 × 10.sup.4 8.8 × 10.sup.3 Ery/ml V2 0.5 1.9 × 10.sup.4 2.0 × 10.sup.4 1.8 × 10.sup.4 1.0 × 10.sup.4 >10.sup.4 >10.sup.4 >10.sup.4 Ery/ml V3 0.25 2.8 × 10.sup.4 2.9 × 10.sup.4 2.0 × 10.sup.4 1.7 × 10.sup.4 >10.sup.4 >10.sup.4 >10.sup.4 Ery/ml V4 0.125 >10.sup.4 >10.sup.4 >10.sup.4 >10.sup.4 >10.sup.4 >10.sup.4 >10.sup.4 Ery/ml

[0146] As can be seen in table 30, chloroform at 2% had no significant antimicrobial effect at the beginning of the test. The reduction at the later points in time is due to the natural loss of viability of the bacteria. Erythrodiol shows a strong effect after incubation at concentrations of 2.0 Ery/ml, lower concentrations either show a later re-appearance of the bacteria or no effect. It seems likely that the lower effect compared with oleacin or oleocanthal is at least in part due to the poor solubility of erythrodiol, as was encountered during the assays.

REFERENCES AS CITED

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