阅读说明：本技术 微生物混合物、从其衍生的分子、及其使用方法 (Microbial mixtures, molecules derived therefrom, and methods of use thereof ) 是由 R·杰利内克 O·马尔卡 D·凯森 M·梅杰勒 A·库什马洛 于 2019-08-08 设计创作，主要内容包括：本发明涉及组合物,其包含色醇衍生物和4-乙基-苯酚衍生物,以及至少一种可接受的载体。进一步提供了用于减少基于有机物的污染物的负载形成的方法。(The present invention relates to compositions comprising a tryptophol derivative and a 4-ethyl-phenol derivative, and at least one acceptable carrier. Further provided are methods for reducing load formation of organic-based contaminants.)

1. A composition comprising:

a. a tryptophol derivative; and

4-ethyl-phenol derivatives;

and at least one pharmaceutically acceptable carrier, wherein the ratio of the tryptophol derivative and the 4-ethyl-phenol derivative is 10: 1 to 1: a ratio of 10 w/w.

2. The composition of claim 1, wherein the tryptophol derivative and 4-ethyl-phenol derivative are each present within the composition at a concentration of at least 1 μ Μ.

3. The composition of any one of claims 1 or 2, wherein the concentration of the chromanol derivative within the composition is at least 0.1 μ Μ.

4. The composition of any one of claims 1 to 3, wherein the w/w ratio of the tryptophol derivative and the 4-ethyl-phenol derivative ranges from 2: 1(w/w) to 1: 2 (w/w).

5. The composition of any one of claims 1 to 4, wherein the tryptophol derivative is tryptophol acetate.

6. The composition of any one of claims 1 to 4, wherein the 4-ethyl-phenol derivative is selected from the group consisting of: tyrosol acetate, dopamine HCl and caffeic acid.

7. The composition of any one of claims 1 to 6, further comprising Kluyveromyces marxianus; and at least one probiotic microorganism.

8. The composition of any one of claims 1 to 7, for use in any one of reducing microbial activity, treating inflammatory diseases and amyloid aggregate-related diseases.

9. A microbial mixture comprising:

a. kluyveromyces marxianus; and

b. at least one probiotic micro-organism, wherein,

wherein the microbial mixture comprises at least 3% kluyveromyces marxianus.

10. The microbial blend of claim 9, wherein the probiotic microorganisms are probiotic bacteria.

11. The microbial blend of claim 10, wherein the probiotic bacteria are selected from the group consisting of lactobacillus, propionibacterium, lactococcus, and leuconostoc.

12. The microbial mixture of any one of claims 9 to 11, wherein the mixture is suspended in a culture medium.

13. The microbial mixture of claim 12, wherein the culture medium is milk.

14. The microbial mixture of claim 13, wherein the mixture is kefir.

15. The microbial mixture of any one of claims 9 to 14, wherein the mixture further comprises a tryptophol derivative, a 4-ethyl-phenol derivative, or a combination thereof.

16. The microbial mixture of claim 15, wherein the tryptophol derivative and the 4-ethyl-phenol derivative are produced by kluyveromyces marxianus.

17. A microbial mixture according to any one of claims 9 to 16 for use in a food product.

18. A microbial mixture according to any one of claims 9 to 17 for use in any one of reducing microbial activity, treating inflammatory diseases and amyloid aggregate-related diseases.

19. A method for treating a disease selected from the group consisting of: an inflammatory disease, an infectious disease, and a disease associated with amyloid aggregates, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any one of: at least one molecule selected from the group consisting of a tryptophol derivative and a 4-ethyl-phenol derivative, and a microbial mixture according to any one of claims 9 to 18.

20. The method of claim 19, wherein the infectious disease comprises a load of a microorganism, a biofilm derived therefrom, or both.

21. The method of claim 20, wherein the microorganism is selected from the group consisting of: viruses, fungi, parasites, yeasts, bacteria and protozoa.

22. The method of claim 21, wherein the fungus belongs to a genus selected from the group consisting of: botrytis, Penicillium, and Sclerotinia.

23. The method of claim 21, wherein the bacteria belong to a genus selected from the group consisting of: vibrio, salmonella, staphylococcus and pseudomonas.

24. The method of any one of claims 19 to 23, wherein the composition has a maximum half Inhibitory Concentration (IC) of 0.1-500 μ Μ₅₀)。

25. The method of any one of claims 19 to 24, wherein the subject has at least one disease selected from: inflammatory diseases, infectious diseases and amyloid-related diseases.

26. The method of any one of claims 19 to 25, wherein the inflammatory disease is inflammatory bowel disease.

27. The method of claim 26, wherein the inflammatory bowel disease is ulcerative colitis or crohn's disease.

28. The method of any one of claims 19 to 27, wherein the amyloid aggregate-associated disease is a neurodegenerative disease.

Technical Field

The present invention belongs to the field of microbiology.

Background

In view of the spread of bacterial resistance to existing antibiotics, there is a continuing and urgent need for new antibacterial agents that act through alternative bacterial killing or inhibition mechanisms. Promising strategies have recently focused on interfering with the bacterial communication pathway, also known as "quorum sensing" (QS). QS bacteria release a chemical molecule called "autoinducer" (auto-inducer), which in many cases affects gene expression in neighboring bacterial cells, thereby facilitating population regulation through bacterial-bacterial signaling. Both gram-positive and gram-negative bacteria use quorum sensing communication circuits to regulate a variety of physiological activities. Processes regulated by quorum sensing include virulence, competence, binding, antibiotic production, motility, and biofilm formation. Generally, gram-negative bacteria use Acylated Homoserine Lactones (AHLs) as autoinducers, and gram-positive bacteria use processed oligopeptides (DPDs). In addition, data are increasing which show that bacterial autoinducers also activate specific biological responses of the host organism.

It is important to identify and use substances that interfere with and/or disrupt the quorum sensing pathway of pathogenic bacteria and thus block their virulence. Related methods aim at developing compounds that activate the communication pathway of bacteria exhibiting beneficial health properties. Inhibition of QS by similar molecules may provide an alternative to conventional antibiotics, with the significant advantage of being less prone to develop resistance.

Disclosure of Invention

The present invention relates to compositions comprising a derivative of tryptophol and/or 4-Ethyl-Phenol (4-Ethyl-Phenol), such as for reducing biofilm production. The invention further relates to a microbial mixture comprising the yeast Kluyveromyces marxianus (Kluyveromyces marxianus) and at least one probiotic microorganism and uses thereof.

According to one aspect, there is provided a composition comprising: (1) a tryptophol derivative; and (2) 4-ethyl-phenol derivatives; and at least one pharmaceutically acceptable carrier, wherein the ratio of the tryptophol derivative and the 4-ethyl-phenol derivative is 10: 1 to 1: a ratio of 10 w/w.

According to another aspect, there is provided a microbial mixture comprising: (1) kluyveromyces marxianus; and (2) at least one probiotic microorganism, wherein the microbial mixture comprises at least 3% kluyveromyces marxianus.

According to another aspect, there is provided a method of treating a disease selected from the group consisting of: inflammatory diseases, infectious diseases, and amyloid-aggregate-related diseases (amyloid aggregatates-related diseases), the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any one of: at least one molecule selected from the group consisting of a tryptophol derivative and a 4-ethyl-phenol derivative, and a mixture of microorganisms disclosed herein.

In some embodiments, the tryptophol derivative and the 4-ethyl-phenol derivative are each present in the composition at a concentration of at least 1 μ Μ.

In some embodiments, the concentration of the chromanol derivative within the composition is at least 0.1 μ M.

In some embodiments, the w/w ratio of the tryptophol derivative and the 4-ethyl-phenol derivative ranges from 2: 1(w/w) to 1: 2 (w/w).

In some embodiments, the Tryptophol derivative is Tryptophol acetate (Tryptophol acetate).

In some embodiments, the 4-ethyl-phenol derivative is selected from: tyrosol acetate (Tyrosol acetate), dopamine HCl and caffeic acid.

In some embodiments, the composition further comprises kluyveromyces marxianus; and at least one probiotic microorganism.

In some embodiments, the composition is used for any one of reducing microbial activity, treating inflammatory diseases and amyloid aggregate-related diseases.

In some embodiments, the probiotic microorganism is a probiotic bacterium.

In some embodiments, the probiotic bacteria are selected from the group consisting of Lactobacillus (Lactobacillus), Propionibacterium (Propionibacterium), Lactococcus (Lactococcus), and Leuconostoc (Leuconostoc).

In some embodiments, the mixture is suspended in a culture medium.

In some embodiments, the medium is milk.

In some embodiments, the mixture is kefir.

In some embodiments, the mixture further comprises a tryptophol derivative, a 4-ethyl-phenol derivative, or a combination thereof.

In some embodiments, the tryptophol derivative and the 4-ethyl-phenol derivative are produced by kluyveromyces marxianus.

In some embodiments, the microbial mixture is used in a food product.

In some embodiments, the microbial mixture is used to reduce microbial activity, treat inflammatory diseases and amyloid aggregate associated diseases in any one of.

In some embodiments, the infectious disease comprises a load of a microorganism, a biofilm derived therefrom, or both.

In some embodiments, the microorganism is selected from: viruses, fungi, parasites, yeasts, bacteria and protozoa.

In some embodiments, the fungus belongs to a genus selected from the group consisting of: botrytis, Penicillium, and Sclerotinia.

In some embodiments, the bacterium belongs to a genus selected from: vibrio (Vibrio), Salmonella (Salmonella), Staphylococcus (Staphylococcus), and Pseudomonas (Pseudomonas).

In some embodiments, the composition has a maximum half Inhibitory Concentration (IC) of 0.1-500 μ M₅₀)。

In some embodiments, the subject has at least one disease selected from: inflammatory diseases, infectious diseases and amyloid-related diseases.

In some embodiments, the inflammatory disease is inflammatory bowel disease.

In some embodiments, the inflammatory bowel disease is ulcerative colitis or crohn's disease.

In some embodiments, the amyloid aggregate-associated disease is a neurodegenerative disease.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the following description of exemplary methods and/or materials is provided. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Further embodiments and areas of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

Fig. 1A-1C are identifications of a mixture of microorganisms in a probiotic yogurt. (1A) Is a distribution pie chart of microorganisms in probiotic yogurt obtained based on BLAST comparison of applied microbiology in an One Codex data platform. (1B) And (1C) light microscopy images of monoculture and kluyveromyces marxianus from probiotic yogurt, respectively.

Fig. 2A-2E depict portions of a microbial subpopulation in a probiotic yogurt. (2A) Bf detail intensity (19,539 events) is the autofluorescence intensity of population R5. Different sub-populations R3, R6 and R7 (including other complex sub-populations R8) can be seen. (2B-2E) are micrographs showing cells of populations R3, R6, R7, and R8, respectively.

Fig. 3A-3B are liquid chromatography-mass spectrometry (LC-MS) chromatograms of (3A) milk extract and (3B) probiotic yogurt. The two molecules corresponding to masses 180 and 203 (hereinafter referred to as "180" and "203" molecules) are only present in the probiotic yogurt and are enclosed.

FIGS. 4A-4B are LC-MS chromatograms of (4A) Kluyveromyces marxianus culture medium and (4B) Kluyveromyces marxianus crude product. The 180 and 203 molecules are only present in the crude kluyveromyces marxianus and are enclosed.

FIGS. 5A-5B are LC-MS chromatograms of crude Kluyveromyces marxianus extract fractions after separation of (5A)180 molecules and (5B)203 molecules.

Figures 6A-6C show weight and structure analysis of synthetic tyrosol acetate. (6A and 6B) are LC-MS (orbitrap) chromatograms of molecules with a mass of 180. (6A) Is retention time spectrum, and (6B) is m/z spectrum. (6C) Is the 1H NMR spectrum of tyrosol acetate with a peak distribution: δ 2.04(3H, s),2.87(2H, t, J ═ 7.0Hz), 4.28(2H, t, J ═ 7.0Hz),6.70(2H, ddd, J ═ 8.2, 2.4, 0.5Hz),6.95(2H, ddd, J ═ 8.2, 1.0, 0.5 Hz).

Fig. 7A-7C show weight and structural analysis of the synthesized tryptophol acetate. (7A and 7B) are LC-MS (orbitrap) chromatograms of molecules with mass 203. (7A) Is retention time spectrum, and (7B) is m/z spectrum. (7C) Is the 1H NMR spectrum of the tryptophol acetate with a peak distribution: δ 2.05(3H, s),3.08(2H, t, J ═ 5.2Hz),4.40(2H, t, J ═ 5.2Hz),6.93 to 7.12(2H, 6.98(ddd, J ═ 8.0, 7.8, 1.2Hz),7.07(ddd, J ═ 8.0, 7.8, 1.6Hz)),7.30 to 7.36(2H, 7.33(dddd, J ═ 8.0, 1.2, 0.5, 0.5Hz),7.32(t, J ═ 0.5Hz)),7.62(1H, dddd, J ═ 8.0, 1.6, 0.5, 0.5 Hz)).

FIGS. 8A-8E are vertical histograms showing a bioluminescence screen for the inhibition or activation of Quorum Sensing (QS) in the presence of synthetic tryptophol acetate. The assay was performed using mutant bacteria lacking the gene encoding the autoinducer. Bacteria were also cloned with bioluminescent reporter plasmids. N-Acyl Homoserine Lactones (AHLs) are useful for activation (agonists) or inhibition (antagonists) of the following bacteria: agrobacterium tumefaciens (Agrobacterium tumefaciens) which responds to C8 Autoinducer (AI), Vibrio cholerae (Vibrio cholerae) responds to CAI1 autoinducer, Pseudomonas aeruginosa (Pseudomonas aeruginosa) RhlA responds to C4 AI and Pseudomonas aeruginosa LasR responds to C12 AI. (8A) Tryptophol acetate was shown to inhibit QS in agrobacterium tumefaciens. (8B) Tryptophol acetate was shown to have low inhibitory effect on QS in pseudomonas aeruginosa (LasR). (8C-8D) shows that in Vibrio cholerae, low capacity tryptophol acetate activates QS and high capacity tryptophol acetate inhibits QS, where IC₅₀The concentration was 12.7. + -. 2.0. mu.M. (8E) It was shown that high capacity tryptophanol acetate has low inhibitory effect on QS in pseudomonas aeruginosa (RhlA).

FIG. 9 is a vertical bar graph showing that in Vibrio cholerae, low capacity tyrosol acetate activates QS and high capacity tyrosol acetate completely inhibits QS with IC₅₀24.4. + -. 2.4. mu.M.

Fig. 10 is a calibration curve of synthetic tryptophol acetate for quantification of secreted substances in crude probiotic yogurt extract, showing a concentration of 213 μ M in probiotic yogurt (measured in 800ml batches).

FIGS. 11A-11B are Confocal Laser Scanning Microscope (CLSM) images depicting Pseudomonas aeruginosa in the absence (11A, with 1% DMSO as a control) or in the presence (11B, 20 μ M) of tryptophol acetate. No effect on biofilm formation was observed.

Fig. 12A-12B are CLSM images depicting salmonella in the absence (12A, with 1% DMSO as control) or presence (12B, 50 μ M) of tryptophol acetate. A significant effect on biofilm formation was observed.

FIGS. 13A-13B are CLSM images depicting Staphylococcus aureus (Staphylococcus aureus) in the absence (13A, with 1% DMSO as a control) or presence (13B, 50 μ M) of tryptophol acetate. A significant effect on biofilm formation was observed.

Fig. 14A-14D are images depicting the biological activity of probiotic yogurt on fungal growth. The probiotic yogurt inhibits the growth of Sclerotinia sclerotiorum (14A-14B) for 12 days or (14C-14D) for 19 days on potato dextrose agar plates. Inhibition plates containing probiotic yogurt extracts (14B and 14D) and control plates showing sclerotinia growth (14A and 14C) are shown.

Fig. 15A-15B are images depicting the biological activity of probiotic yogurt extracts on fungal growth. (15A) Botrytis fungi grown on control plates; (15B) inhibited botrytis fungi inoculated on plates supplemented with probiotic yogurt extracts, recorded 22 days after inoculation.

Fig. 16A-16B are images depicting the biological activity of probiotic yogurt extracts on fungal growth. (16A) Penicillium fungi grown on control plates; (16B) inhibited penicillium fungi inoculated on plates supplemented with probiotic yogurt extracts recorded 22 days after inoculation.

FIGS. 17A-17B are graphs showing the synergistic effect of tryptophol acetate and tyrosol acetate on the activation of QS in Vibrio cholerae. (17A) Is shown as 1: ratio of 1Example Effect of a combination of tryptophol acetate and tyrosol acetate applied together, where IC₅₀11.6. + -. 0.9. mu.M. (17B) Is a vertical bar graph showing the comparative effect of each of tryptophol acetate and tyrosol acetate and combinations of both at the same concentration ranges described in (17A).

FIGS. 18A-18H are CLSM images depicting biofilm modulating activity of combinations of tryptophol acetate and tyrosol acetate detected in a mutant MM920 Vibrio cholerae strain (. DELTA.CqsA. DELTA.luxQ). The Vibrio cholerae CqsA mutant biofilms (18A and 18E) were much thicker and denser than the corresponding biofilms (18B and 18F) produced by the addition of autoinducers alone, which promote quorum sensing and thus disrupt biofilm formation. Tryptophol acetate and tyrosol acetate (18D and 18H), in particular, together these three compounds (18C and 18G) showed a significant synergistic effect in disrupting normal biofilm growth-leading to different biofilm morphologies. The upper row includes the top image and the lower row includes its corresponding 3D image.

FIG. 19 is a vertical bar graph showing the quantification of Vibrio cholerae toxin in the absence (0, with 1% (v/v) DMSO as a control) or in the presence of tryptophol acetate (25, 50, 100 and 200. mu.M) at different concentrations.

Fig. 20 is a vertical bar graph showing cell viability assays in the absence or presence of crude kefir extract. The results demonstrate that the crude kefir extract does not adversely affect the growth of bacterial cells.

FIGS. 21A-21D are graphs and vertical histograms showing the effect of tyrosol acetate at a concentration of 100. mu.M on Vibrio cholerae. (21A) Representative biofilm formation was obtained from confocal z-stacking using IMARIS software. Scale bar, 50 μm. (21B) Quantitative biofilm capacity was obtained by crystal violet assay using strain VC1 (wild type) and bioassay strain MM 920. (21C) The effect of tyrosol acetate on QS gene expression in wild type strains was assessed by RT-qPCR. (21D) Quantification of cholera toxin by GM 1-ELISA.

Figures 22A-22D are vertical histograms showing quorum sensing of crude probiotic yogurt biomass extracts. (22A-22C) QS inhibition/activation of Vibrio cholerae (22A), Agrobacterium tumefaciens (22B) and Vibrio harveyi (22C) reporter strains bioluminescence by crude yogurt biomass extract. (22D) Static anti-biofilm activity. The graph shows the biofilm capacity produced by three different wild-type bacteria (staphylococcus aureus, Salmonella enteritidis, and pseudomonas aeruginosa) in the absence or presence of a crude probiotic yogurt (e.g., kefir) extract. Data are mean ± SD. n is 3. P value: p <0.12, P <0.0l, P < 0.002.

FIGS. 23A-23C are micrographs and vertical histograms showing the effect of tryptophol acetate on Vibrio cholerae. (23Ai-23Av) is a representative image of biofilm formation obtained from confocal z-stack using IMARIS software. Scale bar, 50 μm. (23Ai) Vibrio cholerae VC1 (WT); (23Aii) Vibrio cholerae VC1 in the presence of 100. mu.M tryptophol acetate; (23Aiii) vibrio cholerae alone MM 920; (23Aiv) Vibrio cholerae MM920 with 900nM CAI-1; and (23Av) Vibrio cholerae MM920 with 900nM CAI-1 and in the presence of 100. mu.M tryptophol acetate. (23Avi) histogram showing the amount of biofilm capacity per unit area corresponding to the biofilm shown in 23Ai-23 Av. (23B) Vertical bar graph showing quantitative biofilm capacity of vibrio cholerae obtained by crystal violet staining in microtiter plates at different concentrations of tryptophanol acetate (0, 12.5, 25, 50, 100 and 200 μ M). Error bars represent standard deviations of 4 measurements. P <0.001, P <0.0001, P <0.000001vs. (23C) Vertical bar graphs assessed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) showing the effect of tryptophol acetate on QS gene expression in wild type strains. A relative measure of gene expression level is defined as the copy number of the cDNA of the genes in the QS pathway (hapR, vpsT, hapA, aphA and ctxA, normalized to the reference housekeeping gene that is not affected by treatment). Error bars represent standard deviations of 4 measurements. P <0.001, P <0.0001vs. untreated bacteria.

Fig. 24A-24B are vertical histograms showing that probiotic yogurt disclosed herein and the molecules identified therein reduce weight loss in a murine model of Inflammatory Bowel Disease (IBD). (24A) Experiment repetition No. 2 is shown, and (24B) experiment repetition No. 3 is shown. Weight loss was calculated as the percent change in body weight compared to body weight before the start of the experiment. At Y + DSS + Y compared to DSS + W; significant results of weight loss were observed between DSS + Y and DSS + Mole (at 25 μ M or 50 μ M). In addition, there was no significant weight loss effect in the group receiving the commercial yogurt (Yc + DSS + Yc or DSS + Yc). Group (2): control + W-regular food-no Dextran Sodium Sulfate (DSS); DDS + W-regular food; control + Y-test food (yogurt) -no DSS; DSS + Y — test food (yogurt); control + mole-molecular test items (oral, tyrosol acetate and tryptophol acetate), final concentration 25 μ M or 50 μ M) -no DSS; DSS + mole-molecular test items (oral, tyrosol acetate and tryptophol acetate) at a final concentration of 25 μ M or 50 μ M; control + Yc-test food (commercial yogurt) -no DSS; DSS + Yc — test food (commercial yogurt); y + DSS + Y — yogurt was applied early before and after DSS; and Yc + DSS + Yc — the commercial yogurt was applied early before and after DSS. P <0.000003 in (24A), P < 0.001. P <0.00005 in (24B), P < 0.05.

Figures 25A-25D are examples, images, and vertical histograms showing that the probiotic yogurt disclosed herein and the molecules identified therein reduce colon shortening in a murine IBD model. (25A) Examples of murine large intestinal tracts include: cecum, proximal colon, middle colon, distal colon, rectum, and anus. (25B) Colon length 7 days after DSS treatment. One of the 6 experiments performed was selected as a representative image. (25C) The measurement values on day 10 of experiment repetition No. 2 are shown, and (25D) the measurement values on day 10 of experiment repetition No. 3 are shown. The length of the colon from rectum to cecum was measured at the end of the experiment. A shortening of the colon was observed in mice treated with DSS (DSS + W) compared to the colon of mice treated with DSS and fed with the probiotic yogurt disclosed herein (Y + DSS + Y) before DSS. Furthermore, a statistically significant reduction in colon length was observed in mice treated with the molecules identified in the probiotic yogurt disclosed herein (50 μ M). P < 0.05. The experimental groups are shown in figure 24.

Fig. 26A-26B are vertical histograms showing the effect of the probiotic yogurt disclosed herein and the molecules identified therein on the Disease Activity Index (DAI) determined in experimental replicate 2(26A) and experimental replicate 3 (26B). DAI was determined based on the measured stool consistency and blood in the stool. Clinical signs were recorded every other day. The results show a significant reduction in clinical score in mice treated with yogurt before DSS (Y + DSS + Y) in replicate 2(26A), and a molecular concentration of 50 μ M (DSS + molecular) was identified in the probiotic yogurt disclosed herein in replicate 3 (26B). The experimental groups are shown in FIGS. 24-25.

FIGS. 27A-27D are photomicrographs of histological sections of the colon. (27A) Severe tissue damage was shown in all DSS-treated mice, with loss of crypt cells in the intestinal wall and infiltration of inflammatory cells into the colon. (27B) In the colon of mice fed yogurt (Y + DSS + Y) prior to DSS, tissue architecture was preserved with minimal initial signs of inflammation and colon tissue repair (circled). (27C) Sections of mouse colon fed with probiotic yogurt disclosed herein after DSS (DSS + Y). (27D) Is a control colon (e.g., a healthy colon).

Fig. 28A-28K are images depicting leishmaniasis ulcer healing after application of the probiotic yogurt disclosed herein. After conventional treatment failed to improve healing of the ulcer (including PENTOSTAM injection after local anesthesia with ESRACAIN and topical application of SALIKAREN), probiotic yogurt was applied topically (twice a day, e.g., in the morning and evening). After the start of the administration of the probiotic yogurt, (28A, 28G) day 0, (28B, 28H) day 1, (28C, 28I) day 2, (28D) day 4, (28J) day 5, (28E) day 6, (28F) day 11 and (28K) day 14. Accelerated healing of cutaneous leishmaniasis was evident from day 11 and day 14 (fig. 28F and 28K, respectively).

Fig. 29A-29E are images showing wound healing after administration of probiotic yogurt disclosed herein. Infected wounds were evident on day 0 (29A and 29C). In (29A) sutures are required for fusion of the incision, but since more than 24 hours since injury, fusion of the incision has not occurred, the probiotic yogurt disclosed herein is topically applied and the wound heals after four days (29B). Two days after the administration of the probiotic yoghurt had started, the growth of new tissue was evident (29D). Day 0 (29E), day 4 (29B), day 2 (29D) and day 7 (29E) after the start of the probiotic yogurt administration.

Fig. 30A-30B are vertical histograms showing the anti-inflammatory effect induced by the molecules identified in the probiotic yogurt disclosed herein. In the presence of tryptophol acetate and 4-ethyl-phenol derivatives (e.g., tyrosol acetate, dopamine HCl and caffeic acid), the production of IL-6(30A) and IL-1 α (30B) cytokines by macrophages is significantly reduced. Lipopolysaccharide (LPS).

Fig. 31 is a graph showing the results of a thioflavin t (tht) assay examining the effect of a mixture of molecules extracted from probiotic yogurt (e.g., kefir) disclosed herein and tryptophol acetate on amyloid β (α β 1-42) fibril formation. The results show that molecules extracted from the probiotic yogurt disclosed herein (e.g., kefir) and different concentrations of tryptophanol acetate both reduced α β 1-42 fibrillation. Mix-a mixture of molecules extracted from yoghurt; OM-tryptophol acetate.

Figures 32A-32B are graphs showing α β 1-42 fibrillation in the absence or presence of tryptophol acetate. (32A) Surface Plasmon Resonance (SPR) plots showing the degree of fibrillation as follows: only 30. mu.M α β 1-42(L1A 1-2-amyloid B protein), 30. mu.M α β 1-42 were suspended in PBS (L1A 3-4-bf); or 30. mu.M α β 1-42 in the presence of 50. mu.M tryptophol acetate (L1A 5-6-amyloid B protein + 203). (32B) Graph of SPR showing the degree of fibrillation of 30 μ M α β 1-42 in the presence of several concentrations of tryptophol acetate (6 μ M, 12 μ M, 25 μ M, 100 μ M and 200 μ M).

FIGS. 33A-33D are micrographs of a Transmission Electron Microscope (TEM). α β 1-42 was incubated at 37 ℃ for 24hr (33D), either alone (33A), or in the presence of 5 μ l of the crude probiotic yogurt extract disclosed herein (33B), 25 μ M tryptophol acetate (33C), or 50 μ M tryptophol acetate.

Detailed Description

In some embodiments, the present invention relates to antimicrobial molecules selected from the group consisting of a derivative of tryptophol and/or 4-ethyl-phenol, compositions and devices comprising the same, and methods of use thereof (including but not limited to inhibiting biofilm formation).

In some embodiments, the present invention relates to a method of treating a disease selected from the group consisting of: inflammatory diseases, infectious diseases, and amyloid aggregate-related diseases, the pharmaceutical composition comprising any one of: at least one of a chromanol derivative, a 4-ethyl-phenol derivative, and a microbial mixture comprising: kluyveromyces marxianus; and at least one probiotic microorganism, wherein at least 3% of the cells in the microbial mixture are kluyveromyces marxianus.

In some embodiments, the present invention relates to a method of inhibiting or reducing amyloid (e.g., amyloid beta protein) aggregates, the method comprising contacting a surface or cell with an effective amount of any one of: a tryptophol derivative, a 4-ethyl-phenol derivative, and a mixture of microorganisms as described herein. In some embodiments, amyloid aggregation comprises amyloid fibril formation, amyloid oligomerization, or a combination thereof.

In some embodiments, reducing the loading of organic-based contaminants comprises inhibiting or reducing amyloid.

As used herein, the terms "amyloid aggregates" and "amyloid aggregates" refer to aggregates or clumps of amyloid protein that refold into a conformation that allows multiple copies to aggregate, thereby producing aggregates, fibrils, oligomers, or any combination thereof. The type of protein characterized in amyloid will be apparent to one of ordinary skill in the art. In some embodiments, the amyloid protein comprises beta amyloid.

In some embodiments, any one of the molecules of the invention or the mixture of microorganisms of the invention (e.g., kefir) has any one of the following: antimicrobial activity, anti-inflammatory activity and anti-amyloid aggregation activity.

As used herein, the term "anti-amyloid aggregation activity" refers to the ability to prevent or reduce amyloid dimerization, amyloid oligomerization, amyloid aggregation, amyloid sedimentation, amyloid unfolding, amyloid non-native folding, or any combination thereof.

As used herein, the term "antimicrobial activity" refers to the ability to inhibit, prevent, reduce or delay bacterial growth, fungal growth, biofilm formation, amyloid aggregation (e.g., amyloid beta protein), on a surface or in a humid environment, or to eradicate viable bacterial cells or spores thereof, fungal cells or viruses in suspension. In some embodiments, inhibiting or reducing or delaying the formation of a microbial load refers to inhibiting or reducing the growth of a microorganism, the production of a biofilm by a microorganism, the production or aggregation of amyloid (e.g., amyloid beta protein), and/or the eradication of part or all of an existing population of microorganisms.

As used herein, the term "anti-inflammatory" refers to the ability to inhibit, prevent, reduce, or delay an inflammatory response of the body, including the production, secretion, or both of cytokines, immune cells priming, homing, activation, recruitment, or any combination thereof. In some embodiments, the inflammatory response comprises an autoinflammatory response.

The present invention is based in part on the following findings: derivatives of tryptophol and/or 4-ethylphenol (e.g., tryptophol acetate and tyrosol acetate, respectively) alter quorum sensing (bacterial and fungal) of microorganisms as well as reduce biofilm production and fungal growth. Anti-biofilm effects as well as anti-inflammatory and anti-amyloid aggregation effects were demonstrated using tryptophol and/or tyrosol derivatives obtained from the microbial cultures described herein and synthetic tryptophol and/or tyrosol derivatives.

As used herein, the term "compound or molecule of the present invention" refers to any of the tryptophol derivatives or 4-ethyl-phenol derivatives as described below having any of antimicrobial activity, anti-inflammatory activity, and anti-amyloid aggregation activity.

Tryptophol derivatives

In some embodiments, the tryptophol derivatives of the present invention have the following structure:

wherein "R₁”、“R₂”、“R₃"and" R₄"is selected from: methyl (CH)₃) Ethyl (CH)₃CH₂) Propyl group (CH)₃CH₂CH₂) And butyl (CH)₃CH₂CH₂CH₂)；

"n" is a carbon chain comprising: one, two, three or four carbons; and

"m" is selected from: methyl (CH)₃) Ethyl (CH)₃CH₂) Propyl group (CH)₃CH₂CH₂) And butyl (CH)₃CH₂CH₂CH₂)。

In some embodiments, the tryptophol derivative is tryptophol acetate.

Tryptophol acetate is known in the art to have the following structure:

4-ethylphenol derivative

In some embodiments, the 4-ethyl-phenol derivatives of the present invention have the following structure:

wherein each R is independently selected from: hydroxy, hydrogen, methyl (CH)₃) Ethyl (CH)₃CH₂) Propyl group (CH)₃CH₂CH₂) And butyl (CH)₃CH₂CH₂CH₂)；

"n" and "m" are 1 to 4,

R₁containing a heteroatom or absent;

represents a bond selected from: sp3 single C-C bond, sp2 double C-C bond, sp tri C-C bond; and

x is selected from: carboxylic acid derivatives, alkyl groups and hydrogen.

In some embodiments, the 4-ethyl-phenol derivatives of the present invention have the following structure:

r, R therein₁And X is as described above.

In some embodiments, each R is independently selected from: hydroxyl and hydrogen.

In some embodiments, R₁Selected from O, NH and NH₂。

In some embodiments, the 4-ethyl-phenol derivative is a dopamine derivative represented by the formula:

or a dopamine derivative represented by the formula:

wherein R and X are as described above.

In some embodiments, the dopamine derivative is represented by the formula:

or by the formula:

wherein R and X are as described above.

In some embodiments, X is hydrogen.

In some embodiments, the 4-ethyl-phenol derivative is dopamine or a salt thereof.

In some embodiments, the 4-ethyl-phenol derivatives of the present invention have the following structure:

wherein: each R is independently selected from: hydroxyl and hydrogen; and X is selected from the group consisting of carboxylic acid derivatives, alkyl groups and hydrogen.

In some embodiments, X isWherein R is₂Selected from: -OH, -SH, -NH₂Thioalkyl, alkoxy, aminoalkyl, hydrogen, alkyl, substituted alkyl.

In some embodiments, R₂Is hydrogen or alkyl.

In some embodiments, R₂Is C₁-C₅An alkyl group.

In some embodiments, R₂Is hydrogen.

In some embodiments, the 4-ethyl-phenol is a derivative of tyrosol acetate.

Tyrosol acetate is known in the art to have the following structure:

in some embodiments, the 4-ethyl-phenol derivative is a derivative of caffeic acid having the structure:

wherein each R is independently selected from: hydroxy, hydrogen, methyl (CH)₃) Ethyl (CH)₃CH₂) Propyl group (CH)₃CH₂CH₂) And butyl (CH)₃CH₂CH₂CH₂)；

"n" and "m" are 1 to 4,

represents a bond selected from: sp3 single C-C bond, sp2 double C-C bond, sp tri C-C bond; and

x is selected from: carboxylic acid derivatives, alkyl groups and hydrogen.

In some embodiments, each R is independently selected from: hydroxyl and hydrogen.

In some embodiments of the present invention, the substrate is,represents an unsaturated C-C bond. In some embodiments of the present invention, the substrate is,represents a double C-C bond.

In some embodiments, X is selected from: a carboxylic acid derivative and hydrogen.

In some embodiments, the derivative of caffeic acid has the following structure:

wherein R and X are as defined above.

In some embodiments, the derivative of caffeic acid has the following structure:

wherein R is₁As defined above, and R₃Selected from: hydrogen, -OH, -SH, -NH₂Thioalkyl, alkoxy, aminoalkyl, hydrogen, alkyl, substituted alkyl.

In some embodiments, R is selected from: hydrogen, -OH and alkyl.

In some embodiments, the derivative of caffeic acid has the following structure:

wherein R is₃As defined above.

As used herein, the term "carboxylic acid derivative" encompasses carboxyl, amide, carbonyl, anhydride, carbonate, and carbamate.

As used herein, the term "derivative" encompasses any compound having antimicrobial activity that is produced by a chemical reaction from a similar compound, or that is produced by substitution of one or more atoms from another compound. In some embodiments, the derivative comprises a structural analog.

In some embodiments, the compounds of the present invention are obtained by any chemical modification of tryptophol or 4-ethyl-phenol, as long as they have antimicrobial activity. In some embodiments, the tryptophol or 4-ethyl-phenol is chemically modified by the addition of at least one chemical group selected from acetylation, methylation, phosphorylation, amidation, or others. In some embodiments, the chemical modification comprises substitution. In some embodiments, the modification comprises adding an acetate group to the tryptophol or 4-ethyl-phenol. In some embodiments, the tryptophol acetate or tyrosol acetate further comprises at least one chemical group as described above.

As used herein, the compounds of the present invention do not comprise tryptophol or tyrosol.

In some embodiments, the disclosed invention relates to compositions comprising at least one molecule selected from the group consisting of: a tryptophol derivative and a 4-ethyl-phenol derivative, and at least one pharmaceutically acceptable carrier or diluent.

In some embodiments, the composition comprises tryptophol acetate, or tyrosol acetate, or any combination thereof, and at least one pharmaceutically acceptable carrier or diluent.

In some embodiments, the compounds of the invention are chemically synthesized or biosynthesized. Methods of biosynthesis are well known in the art and may include, but are not limited to: production in cell culture or production enzymatically without cells. In some embodiments, the compounds of the invention are biosynthesized using a cell culture comprising kluyveromyces marxianus. In some embodiments, the kluyveromyces marxianus-containing culture is a single culture or a multi-culture. In some embodiments, the compounds of the invention are biosynthesized from kluyveromyces marxianus. In some embodiments, according to the methods of the invention, tryptophol acetate or tyrosol acetate is biosynthesized from kluyveromyces marxianus.

Microorganism mixture

In some embodiments, the present invention relates to compositions comprising a mixture of microorganisms. In some embodiments, the composition comprises probiotic microorganisms. In some embodiments, the probiotic microorganism comprises a yeast. In some embodiments, the majority of the microorganisms within the disclosed inventive mixtures are probiotic yeast. In some embodiments, the probiotic yeast of the disclosed invention is kluyveromyces marxianus.

In one embodiment, Kluyveromyces marxianus is Kluyveromyces marxianus strain HA 63[ NRRL Y-8281, CBS 712 ].

As used herein, the term "probiotic" refers to any substance and/or microorganism that promotes growth, particularly the growth of microorganisms having beneficial properties (e.g., gut flora).

In some embodiments, the microorganism content (%) within the mixture of the invention is quantified based on the fraction of microorganism DNA in the total DNA of the mixture. In some embodiments, DNA quantification is directly based on the amount of extracted DNA. In some embodiments, DNA quantification further comprises enzymatic reactions including, but not limited to, restriction, ligation, amplification, sequencing, or any combination thereof. In some embodiments, DNA quantification is based on next generation sequencing. In some embodiments, DNA quantification is based on the ratio of the amount of microorganism-specific DNA reads compared to the total number of DNA reads for a mixture of microorganisms.

In some embodiments, the microbial content within a mixture of the invention includes the number of microbial cells (e.g., colony forming units [ CFU ]) per volume of mixture culture. In some embodiments, the amount of microorganisms within a mixture of the invention includes the number of cells of the microorganism compared to the total number of cells in the mixture. In some embodiments, the microbial content within the inventive mixture comprises the CFU of the microorganism.

In some embodiments, at least 1%, at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the cells in the microbial mixture are kluyveromyces marxianus cells. In some embodiments, 1-4%, 2-5%, 4-7%, 6-11%, 10-16%, 15-22%, 20-32%, 30-35%, 32-40%, 38-48%, 45-55%, 50-60%, or 55-75%, 60-80%, 65-90%, or 80-100% of the cells in the microbial mixture are Kluyveromyces marxianus cells. Each possibility represents a separate embodiment of the invention.

In some embodiments, the compositions of the disclosed invention comprise at least one probiotic bacterium. In one embodiment, the probiotic bacteria are selected from the genus lactobacillus. In one embodiment, the probiotic bacteria are selected from the genus propionibacterium. In one embodiment, the probiotic bacteria are selected from the genus. In one embodiment, the probiotic bacteria are selected from the genus lactococcus. In one embodiment, the probiotic bacteria are selected from the genus leuconostoc. In some embodiments, the at least one probiotic bacterium is at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten probiotic bacteria. In some embodiments, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, or at most 1% of the cells in the microbial mixture are probiotic bacteria. In some embodiments, 1-5%, 4-10%, 8-18%, 12-20%, 17-25%, or 22-30% of the cells in the microbial mixture are probiotic bacteria. Each possibility represents a separate embodiment of the invention.

In some embodiments, the microbial mixture of the disclosed invention further comprises other types of microorganisms. In one embodiment, the other microorganism is not a probiotic microorganism, such as a yeast or bacteria. In some embodiments, at most 15%, at most 13%, at most 11%, at most 10%, at most 9%, at most 7%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1% of the cells in the microbial mixture are of a microbial type other than the probiotic yeast and the at least one probiotic bacterium. In some embodiments, the microbial mixture does not comprise other types of microorganisms other than the probiotic yeast and the at least one probiotic bacterium.

In some embodiments, the microbial mixture is suspended in a culture medium. In some embodiments, the mixture of microorganisms is grown in a culture medium. In some embodiments, the culture medium is a cell culture medium suitable for the growth and maintenance of a mixture of microorganisms. In one embodiment, the cell culture medium (e.g., milk) is optimized for microbial growth.

As used herein, "cell culture medium" refers to any medium, liquid or solid that is capable of proliferating cells. Cell culture media are known in the art and can be selected according to the type of cell to be grown. For example, the cell culture medium used for growing the cells is Luria-Bertani (Luria-Bertani) liquid medium (broth) (LB; Miller liquid medium). In some embodiments, the microbial mixture is cultured under effective conditions that allow for increased production from the cultured microbial mixture. Non-limiting examples of increased yield include, but are not limited to, increased gene expression, protein production and secretion, biosynthesis of molecules, proliferation, and others. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactors, temperature, pH, and oxygen conditions that allow for increased production. In one embodiment, an effective medium refers to any medium in which a mixture of microorganisms is cultured to produce a compound of the invention. In some embodiments, the cell culture medium generally comprises an aqueous solution having assimilable sources of carbon, nitrogen, and phosphate, as well as appropriate salts, minerals, metals, and other nutrients (e.g., vitamins). In some embodiments, the microbial mixture of the invention may be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and culture dishes. In some embodiments, the culturing is performed at a temperature, pH, and oxygen content suitable for probiotic microorganisms (such as yeast or bacteria). In some embodiments, the culture conditions are within the expertise of one of ordinary skill in the art. A non-limiting example of a process for culturing the microbial mixture of the invention includes culturing the microbial mixture in milk at 28 ℃ for about 24 hours.

In one embodiment, the culturing process comprises culturing the mixture of microorganisms for a period of 12-16 hours, 14-18 hours, 12-24 hours, 16-24 hours, 18-28 hours, 10-20 hours, 22-36 hours. Each possibility represents a separate embodiment of the invention.

In one embodiment, the culturing process comprises culturing the mixture of microorganisms at a temperature of 20-26 deg.C, 24-28 deg.C, 22-34 deg.C, 26-34 deg.C, 28-38 deg.C, 20-30 deg.C, 32-46 deg.C. Each possibility represents a separate embodiment of the invention.

In some embodiments, the microbial mixture of the invention is cultured in milk. In some embodiments, the mixture of microorganisms cultured in milk produces a fermented milk product. In some embodiments, the fermented milk product is selected from: yoghurt, probiotic yoghurt or kefir.

In some embodiments, the fermented dairy product of the invention comprises a mixture of microorganisms, a tryptophol derivative or a tyrosol derivative or any combination thereof.

In some embodiments, the fermented milk product of the present invention is for human food consumption. In some embodiments, the fermented milk product of the present invention is consumed by the subject as part of a daily consumption. In one embodiment, the fermented milk product is consumed by the subject as part of a dietary consumption. In some embodiments, the fermented milk product may be used to treat skin lesions. In some embodiments, the fermented milk product is administered topically to the subject.

In some embodiments, the present invention provides methods for producing or obtaining a mixture of microorganisms disclosed herein. In some embodiments, the method comprises culturing kluyveromyces marxianus as described herein. In some embodiments, the method further comprises the step of determining to obtain or produce a mixture of microorganisms disclosed herein. In some embodiments, determining comprises determining that at least 3% of the cells in the mixture are kluyveromyces marxianus. In some embodiments, determining comprises determining that a tryptophol derivative or a 4-ethyl-phenol derivative is produced. In some embodiments, determining comprises determining that a tryptophol derivative and a 4-ethyl-phenol derivative are produced. In some embodiments, determining comprises determining that at least 3% of the cells in the mixture are kluyveromyces marxianus and producing the tryptophol derivative or the 4-ethyl-phenol derivative in a sufficient amount. In some embodiments, determining comprises determining that at least 3% of the cells in the mixture are kluyveromyces marxianus and producing the tryptophol derivative and the 4-ethyl-phenol derivative in sufficient amounts. As used herein, a sufficient amount includes a therapeutically effective amount as described herein (e.g., having or causing an anti-inflammatory response, an antimicrobial activity, an anti-amyloid aggregation activity, or a combination thereof).

Methods of use and compositions

According to some embodiments, there is provided a method of treating a disease selected from the group consisting of: inflammatory diseases, infectious diseases, and amyloid aggregate-associated diseases, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any one of: at least one molecule selected from the group consisting of a tryptophol derivative and a 4-ethyl-phenol derivative, and a mixture of microorganisms disclosed herein.

In some embodiments, the infectious disease comprises an organic-based contaminant. In some embodiments, the organic-based contaminant comprises a biofilm.

In some embodiments, the organic-based contaminant comprises amyloid aggregates (e.g., amyloid beta protein).

As used herein, the term "organic" refers to any of the following: carbon-containing compounds, substances produced or derived from organisms, organs and organisms.

In some embodiments, the contacting is administering. In some embodiments, the contacting is doping. In some embodiments, the contacting is administration to a subject. In some embodiments, the contact is incorporated into the surface. In some embodiments, the contacting is contacting a cell. In some embodiments, the cell is a unicellular microorganism. In some embodiments, the cell is a cell of a subject. In some embodiments, the cell is a cell of the nervous system. In some embodiments, the cell of the nervous system is a neuronal cell.

In some embodiments, the organic-based contaminant is on or within a surface, article, cell, or subject. In some embodiments, the subject comprises a cell. In some embodiments, the cell is an endogenous cell or an exogenous cell.

In some embodiments, there is provided a method of treating a biofilm-associated infectious disease or a symptom thereof in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one molecule selected from a tryptophol derivative or a 4-ethylphenol derivative, or a mixture of microorganisms disclosed herein, and at least one pharmaceutically acceptable carrier.

As used herein, "infectious disease associated with organic-based contaminants" refers to any disease or condition in a subject that results from increased loading of microorganisms and/or formation of a biofilm or biofouling. In some embodiments, the organism that induces the infectious disease associated with the organic-based contaminant is selected from the group consisting of: bacteria, viruses, fungi or parasites.

Non-limiting examples of infectious disease symptoms include, but are not limited to, fever, diarrhea, fatigue, muscle soreness, and cough.

Non-limiting examples of infectious diseases include urinary tract infection, gastrointestinal tract infection, enteritis, salmonellosis, diarrhea, non-tuberculous mycobacterial infection, legionnaires 'disease, nosocomial pneumonia, skin infection, cholera, septic shock, periodontitis, infection, inflammatory bowel disease, Ulcerative Colitis (UC), crohn's disease, and sinusitis. In some embodiments, the infection causes a condition selected from the group consisting of: bacteremia, skin infection, neonatal infection, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, skin scald syndrome, and food poisoning.

As used herein, the term "subject" refers to an animal, more particularly to non-human mammals and human organisms. Non-human animal subjects may also include prenatal forms of the animal (such as, for example, embryos or fetuses). Non-limiting examples of non-human animals include: horses, cattle, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, and pigs. In one embodiment, the subject is a human. The human subject may also include a fetus.

As used herein, the term "treatment" or "treating" of a disease, disorder or condition encompasses the reduction of at least one symptom thereof, the reduction of the severity thereof, or the inhibition of the progression thereof. Treatment does not necessarily mean a complete cure for the disease, disorder, or condition. To be an effective treatment, a composition useful herein need only reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of a patient or subject.

As used herein, the term "prevention" of a disease, disorder or condition encompasses the delay, prevention, suppression or inhibition of the onset of the disease, disorder or condition. As used in accordance with the presently described subject matter, the term "prevention" relates to a process of prevention (prophyxiases) in which a subject is exposed to a presently described peptide prior to induction or onset of a disease/disorder process. This may be done when the individual has a genetic lineage indicative of a predisposition to the development of the disease/disorder to be prevented. This may be true, for example, for individuals whose ancestors exhibit predisposition to certain types of disorders, such as inflammatory disorders. The term "suppression" is used to describe a situation in which the disease/disorder process has begun but no apparent symptoms of the disorder have been achieved. Thus, the cells of an individual may be afflicted with a disease/disorder, but the external signs of the disease/disorder are not yet clinically recognized. In either case, the term "prevention (prophyxias)" can be applied to encompass both prevention (prevention) and suppression. Conversely, the term "treatment" refers to the clinical application of an active agent to combat an already existing condition in which a clinical manifestation has been achieved in a patient.

As used herein, the term "condition" includes deviations from normal anatomy and physiology, which constitute a impairment of the normal state of a living animal or one of its parts, which interrupts or alters the performance of a bodily function.

In some embodiments, the compositions disclosed herein relate to killing or reducing the formation of microorganisms on or in living tissue or on or in an article.

In some embodiments, the present invention relates to a method of inhibiting or reducing the formation of a load of organic-based contaminants on or within an article, comprising incorporating or coating a composition comprising at least one molecule selected from a chromanol derivative and a 4-ethylphenol derivative, or a mixture of microorganisms disclosed herein, and an acceptable carrier on and/or within the article.

In some embodiments, the loading of the organic-based contaminant is the loading of microorganisms, and/or the formation of a biofilm or biofouling, in and/or on the article of manufacture. In some embodiments, the microorganism is selected from: viruses, fungi, parasites, yeasts, bacteria and protozoa.

According to some embodiments, the method comprises treating or ameliorating an infectious disease associated with an organic-based contaminant or a symptom thereof in a subject in need thereof, comprising administering to the subject any one of: a compound of the invention or a pharmaceutical composition comprising the same; or a microbial mixture as disclosed herein, or a fermented milk product comprising the same.

In some embodiments, there is provided a use of a composition comprising an effective amount of a compound of the invention, or a mixture of microorganisms of the invention, in the manufacture of a medicament for treating, reducing or preventing an infectious disease associated with an organic-based contaminant, or a symptom thereof, in a subject in need thereof. In some embodiments, the invention provides the use of a composition comprising an effective amount of one or more molecules or any derivative thereof, or a mixture of microorganisms disclosed herein, in the manufacture of a medicament for treating an infectious disease or a symptom thereof in a subject in need thereof.

In one embodiment, the compound of the invention or the microbial mixture of the invention is provided to the subject itself. In one embodiment, one or more of the compounds of the invention is provided to the subject itself. In one embodiment, a compound of the invention or a mixture of microorganisms of the invention is provided to a subject as part of a pharmaceutical composition, wherein it is admixed with a pharmaceutically acceptable carrier. In one embodiment, one or more of the compounds of the present invention are provided to a subject as part of a pharmaceutical composition, wherein they are admixed with a pharmaceutically acceptable carrier.

As used herein, the term "Quorum Sensing (QS)" refers to a system that modifies gene expression pathways in response to cell population density dynamics. In some embodiments, the molecule of the invention, or any derivative thereof, is used in a method of inhibiting QS. Any method known in the art can be used to assess the effect of a molecule on QS. Non-limiting examples for QS checks include the use of bioluminescent assays based on engineered bacteria lacking an active auto-inducible gene and further cloned with a DNA vector containing a fluorescent reporter gene.

As used herein, the term "biofilm" refers to a group of microorganisms that adhere to each other, embedded within a self-produced and autocrine extracellular polymer comprising DNA, proteins and polysaccharides. In some embodiments, the biofilm is adhered to a surface of a living host. In one embodiment, the biofilm is adhered to a non-living surface. In some embodiments, the QS activity is associated with a level of biofilm formation.

In some embodiments, any of the compositions, fermented milk products, or methods of the present invention alters QS activity by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, or any value or range therebetween, as compared to a control. Each possibility represents a separate embodiment of the invention. In some embodiments, altering QS activity is by 1-5%, 3-6%, 4-10%, 8-20%, 15-30%, 28-40%, 35-50%, 45-60%, 50-70%, 65-80%, 70-90%, or 90-100% as compared to a control. In some embodiments, the QS activity is altered at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold as compared to a control. Each possibility represents a separate embodiment of the invention.

In some embodiments, any of the compositions, fermented milk products, or methods of the invention reduces biofilm production by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, or any value and range therebetween, as compared to a control. Each possibility represents a separate embodiment of the invention. In some embodiments, biofilm production is reduced by 1-5%, 3-6%, 4-10%, 8-20%, 15-30%, 28-40%, 35-50%, 45-60%, 50-70%, 65-80%, 70-90%, or 90-100% as compared to a control. In some embodiments, biofilm production is reduced by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, or any value and range therebetween, as compared to a control. Each possibility represents a separate embodiment of the invention.

In one embodiment, the control is a non-fermented dairy product. In one embodiment, the control is a fermented milk product with a different microbial mixture compared to the microbial mixture of the disclosed invention. In one embodiment, the control is a fermented or non-fermented dairy product without kluyveromyces marxianus. In one embodiment, the control is a fermented or unfermented milk product comprising a microbial mixture comprising 2% or less kluyveromyces marxianus. In one embodiment, the control is a fermented or unfermented dairy product comprising a microbial mixture comprising inactivated kluyveromyces marxianus. A non-limiting example of inactivation is heat inactivation. In some embodiments, the control comprises tyrosol, or tryptophol, or any combination thereof, as disclosed above. In some embodiments, the control does not comprise a tyrosinol derivative, or a tryptophol derivative, or a combination thereof.

In some embodiments, the present invention relates to compositions comprising an effective amount of a compound of the present invention as an active ingredient, together with acceptable carriers and/or diluents. In some embodiments, the present invention relates to compositions comprising an effective amount of tryptophol acetate, tyrosol acetate, dopamine HCl, caffeic acid, or any combination thereof as an active ingredient. In some embodiments, an acceptable carrier facilitates incorporation or coating of the active ingredient into a substrate.

In some embodiments, the composition further comprises a substrate. In some embodiments, will comprise at least one of: a chromanol derivative or a 4-ethylphenol derivative, incorporated into and/or on at least a portion of a substrate.

In some embodiments, the present invention relates to a composition comprising a substrate having incorporated in and/or on at least a portion thereof at least one of: a tryptophol derivative or a 4-ethylphenol derivative.

As used herein, the term "portion thereof" refers to, for example, a surface of a solid or semi-solid substrate, or a portion thereof, and/or a body thereof, or a portion thereof; or the volume of liquids, gels, foams and other non-solid substrates or portions thereof.

As described herein, substrates having widely different chemistries can be successfully utilized to incorporate thereon (e.g., deposited on a surface thereof) at least one of: a tryptophol derivative or 4-ethylphenol, or a composition comprising the same. The term "successfully utilized" refers to the result, which means: (i) at least one of the following: the chromanol derivative or tyrosol derivative, or a composition comprising the same, successfully forms a uniform and homogeneous coating on the substrate surface; and (ii) the resulting coating imparts durable desirable properties (e.g., antimicrobial properties) to the substrate surface.

Thus, substrates useful according to some embodiments of the present invention may be hard (rigid) or soft, solid, semi-solid, or liquid substrates, and may take the form of foams, solutions, emulsions, gels, creams, or any mixture thereof.

Substrates useful according to some embodiments of the present invention may have, for example, inorganic or organic surfaces, including, but not limited to, glass surfaces; porcelain watchKneading; a ceramic surface; silicon or silicone surfaces, metal surfaces (e.g., stainless steel); mica, polymeric surfaces (such as, for example, plastic surfaces), rubber surfaces, paper, wood, polymers, metals, carbon, biopolymers, silicon minerals (rock or glass), surfaces, wool, silk, cotton, hemp, leather, fur, feathers, skin (rawhide, fur, or hair) surfaces, plastic surfaces, and surfaces comprising or made of polymers (polymers such as, but not limited to, polypropylene (PP), Polycarbonate (PC), Polyethylene (PET), High Density Polyethylene (HDPE), Low Density Polyethylene (LDPE), Polyester (PE), unplasticized polyvinyl chloride (PVC), and fluoropolymers (including, but not limited to, polytetrafluoroethylene (PTFE,) ); or may comprise or be made from any of the foregoing, or any mixture thereof.

Alternatively, other portions or the entire substrate are made of the above-described materials.

In some embodiments, at least one of the following is incorporated, as described herein: the substrate of the chromanol derivative or the 4-ethylphenol derivative, or a composition comprising the same, is or forms part of an article.

According to some embodiments, an article (e.g., an article of manufacture) comprises a substrate having incorporated into and/or on at least a portion of at least one of a chromanol derivative or a 4-ethylphenol derivative or a composition comprising the same.

The article may be any article that can benefit from the antimicrobial and/or antibiofilm forming activity of the tryptophol derivative or the 4-ethylphenol derivative.

Non-limiting examples of articles include, but are not limited to, medical equipment, organic waste treatment equipment, fluidic equipment, agricultural equipment, packaging, sealed articles, fuel containers, water and cooling system equipment, and construction elements.

As described herein, at least one of the following may be incorporated: non-limiting examples of devices of the chromanol derivative or the 4-ethylphenol derivative or a composition comprising the same advantageously include pipes, pumps, drains or waste water pipes, screw plates (screw plates) and the like.

Non-limiting examples of articles include, but are not limited to, components used in water treatment systems (e.g., for containing and/or transporting and/or treating aqueous media or water), equipment, containers, filters, pipes, solutions and gases, and the like.

Non-limiting examples of articles include, but are not limited to, elements in organic waste treatment systems (e.g., for containing and/or disposing of and/or transporting and/or processing organic waste), equipment, containers, filters, pipes, solutions, and gases and the like.

In some embodiments, the present invention relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present invention or a mixture of microorganisms of the present invention as an active ingredient, together with a pharmaceutically acceptable carrier and/or diluent. In some embodiments, the present invention relates to a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of tryptophol acetate, or tyrosol acetate, or any combination thereof. In some embodiments, a pharmaceutically acceptable carrier facilitates administration of a compound of the invention to an organism. For example, the term "pharmaceutically acceptable" may mean approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

In some embodiments, the disclosed invention relates to compositions for altering pathogenic activity, microbial growth, microbial activity, altering inflammatory responses, altering amyloid aggregation, or any combination thereof. As defined herein, "change" encompasses an increase or decrease depending on the desired result. In some embodiments, the pathogenic activity comprises a microbial activity. In some embodiments, the microbial activity comprises any activity selected from the group consisting of: proliferation, antibiotic resistance, cellular communication or quorum sensing, biofilm production, amyloid aggregation, fibril formation or oligomerization, or any combination thereof, toxin production or secretion, or a combination thereof. Microbial activity can be determined using any known method, non-limiting examples include, but are not limited to, spectrophotometry, resistance assays using selective substrates, bioluminescence assays, liquid chromatography and mass spectrometry, or others, some of which are exemplified herein below, and all of which are well known to those of ordinary skill in the art.

In some embodiments, the disclosed invention relates to methods for treating an inflammatory disease, an infectious disease, an amyloid aggregation-related disease, or a combination thereof.

As used herein, the term "amyloid aggregation-related disease" encompasses any pathogenic condition involving amyloid as part of the disease pathogenesis or pathophysiology. In some embodiments, the amyloid-related disease comprises any one of the following: increased amyloid aggregation, increased rate of amyloidogenesis, increased rate of amyloid fibril formation, increased rate of amyloid oligomerization, amyloid-induced toxicity, amyloid-induced apoptosis, amyloid-induced cell death, and any combination thereof.

In some embodiments, the disease associated with amyloid aggregation is a neurodegenerative disease. As used herein, a neurodegenerative disease may also include a neuromuscular degenerative disease. Non-limiting examples include autonomic neuropathy, Hohner syndrome, multiple system atrophy, simple autonomic failure, delirium, dementia, Alzheimer's disease, chronic traumatic encephalopathy, frontotemporal dementia, Lewy body dementia, Parkinson's disease, multiple sclerosis, neuromyelitis optica, Huntington's chorea, progressive supranuclear palsy, neuroophthalmic and cranial nerve disorders, neuromuscular stiffness, stiff person syndrome, Guilin-Barre syndrome (GBS), Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), hereditary neuropathy, hereditary pressure-susceptibility peripheral neuropathy (HNPP), Amyotrophic Lateral Sclerosis (ALS) and other Motor Neuron Diseases (MND), myasthenia gravis, radiculopathy, nucleus pulposus herniation, peripheral neuropathy, mononeuropathy, polyneuropathy, brachial plexus and lumbosacral plexus disorders, Spinal Muscular Atrophy (SMAs), thoracic outlet pressure syndrome (TOS), creutzfeldt-jakob disease (CJD), gerstmann-schwerner-schneike disease (GSS), seizures, spinal cord disorders, and stroke.

In some embodiments, the change is at least 5%, at least 15%, at least 25%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99%, or any value or range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the alteration is by 1-5%, 7-15%, 10-25%, 20-40%, 35-50%, 45-65%, 55-75%, 70-85%, 80-90%, 87-95%, or 92-100%. Each possibility represents a separate embodiment of the invention.

In some embodiments, the reduction is a reduction of at least 5%, at least 15%, at least 25%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99%, or any value or range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the reduction is a reduction of 1-5%, 7-15%, 10-25%, 20-40%, 35-50%, 45-65%, 55-75%, 70-85%, 80-90%, 87-95%, or 92-100%. Each possibility represents a separate embodiment of the invention.

The terms "reduce" and "inhibit" are used interchangeably herein.

In some embodiments, the enhancement is an enhancement of at least 5%, at least 15%, at least 25%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99%, or any value or range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the enhancement is an enhancement of 1-5%, 7-15%, 10-25%, 20-40%, 35-50%, 45-65%, 55-75%, 70-85%, 80-90%, 87-95%, or 92-100%. Each possibility represents a separate embodiment of the invention.

In some embodiments, the compositions described herein comprise a ratio of 1: 15 to 15: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 14 to 14: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 13 to 13: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 12 to 12: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 11 to 11: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 10 to 10: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 9 to 9: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 8 to 8: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 7 to 7: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 6 to 6: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 5 to 5: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 4 to 4: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 3 to 3: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 2 to 2: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. In some embodiments, the compositions described herein comprise a ratio of 1: 1 to 1: 1w/w of a tryptophol derivative or a 4-ethylphenol derivative. Each possibility represents a separate embodiment of the invention.

In some embodiments, within the composition, the tryptophol derivative or the 4-ethylphenol derivative is present at a concentration of at least 1 μ M, at least 2 μ M, at least 5 μ M, at least 10 μ M, at least 15 μ M, at least 20 μ M, at least 30 μ M, at least 40 μ M, at least 50 μ M, at least 75 μ M, at least 100 μ M, at least 150 μ M, at least 200 μ M, at least 225 μ M, or at least 350 μ M, or any value or range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the tryptophol derivative or the 4-ethylphenol derivative is present in the composition at a concentration of 1-10. mu.M, 5-20. mu.M, 10-30. mu.M, 20-40. mu.M, 25-50. mu.M, 30-60. mu.M, 40-70. mu.M, 50-80. mu.M, 65-90. mu.M, 70-100. mu.M, 80-110. mu.M, 90-120. mu.M, 110-160. mu.M, 150-275. mu.M, or 250-500. mu.M. Each possibility represents a separate embodiment of the invention.

In some embodiments, the composition or microbial mixture comprises each of a tryptophol derivative and a 4-ethylphenol derivative, and the concentration of each of the tryptophol derivative and the 4-ethylphenol derivative is at least 50nM, at least 100nM, at least 1 μ Μ, at least 2 μ Μ, at least 5 μ Μ, at least 10 μ Μ, at least 15 μ Μ, at least 20 μ Μ, at least 30 μ Μ, at least 40 μ Μ, at least 50 μ Μ, at least 75 μ Μ, at least 100 μ Μ, at least 150 μ Μ, at least 200 μ Μ, at least 225 μ Μ, or at least 350 μ Μ, or any value and range therebetween, within the composition. Each possibility represents a separate embodiment of the invention. In some embodiments, the composition or mixture of microorganisms comprises each of a tryptamine alcohol derivative and a 4-ethylphenol derivative, and the concentration of each of the tryptamine alcohol derivative and the 4-ethylphenol derivative within the composition is 10-50nM, 40-100nM, 75-500nM, 450-900nM, 0.75-1.5. mu.M, 1-10. mu.M, 5-20. mu.M, 10-30. mu.M, 20-40. mu.M, 25-50. mu.M, 30-60. mu.M, 40-70. mu.M, 50-80. mu.M, 65-90. mu.M, 70-100. mu.M, 80-110. mu.M, 90-120. mu.M, 110-160. mu.M, 150-275. mu.M, or 250-500. mu.M. Each possibility represents a separate embodiment of the invention.

As defined herein, the term "maximum half Inhibitory Concentration (IC)₅₀) "refers to a measure of the effectiveness of a substance to inhibit a particular biological or biochemical function. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a tryptophol derivative, a 4-ethylphenol derivative, or any combination thereof has an IC at a micromolar level₅₀. In some embodiments, a composition comprising a mixture of microorganisms disclosed herein has an IC at a micromolar level₅₀. In some embodiments, the micromolar level comprises at most 1,000 μM, at most 900. mu.M, at most 800. mu.M, at most 700. mu.M, at most 600. mu.M, at most 500. mu.M, at most 400. mu.M, at most 300. mu.M, at most 200. mu.M, at most 100. mu.M, at most 75. mu.M, at most 50. mu.M, at most 35. mu.M, at most 20. mu.M, at most 15. mu.M, at most 10. mu.M, at most 5. mu.M, or at most 1. mu.M, or any value or range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the micromolar levels comprise 1-10 μ M, 5-20 μ M, 15-30 μ M, 25-500 μ M, 40-75 μ M, 70-120 μ M, 100-. Each possibility represents a separate embodiment of the invention.

The term "carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such carriers can be sterile liquids (e.g., aqueous and oily, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like), polyethylene glycols, glycerol, propylene glycol or other synthetic solvents.

Water may be used as a carrier, such as when the active compound is contained in a pharmaceutical composition for intravenous administration. Saline solutions as well as aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents (e.g., acetates, citrates or phosphates). Antibacterial agents (such as benzyl alcohol or methyl paraben) are also envisioned; antioxidants (such as ascorbic acid or sodium bisulfite); and agents for regulating tonicity (such as sodium chloride or dextrose). The carriers may collectively comprise from about 0.1% to about 99.99999% by weight of the compositions set forth herein.

Embodiments of the present invention relate to molecules of the present invention or derivatives thereof in unit dosage form and prepared by any method known in the pharmaceutical art. In one embodiment, the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial, or pre-filled syringe.

In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the nature of the disease or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose response curves derived from in vitro or in vivo animal model test bioassays or systems.

In one embodiment, the composition of the invention is administered in the form of a pharmaceutical composition comprising at least one of the active ingredients of the invention (e.g., a tryptophol derivative or a 4-ethylphenol derivative) and a pharmaceutically acceptable carrier or diluent. In another embodiment, the compositions of the present invention may be administered separately or together in any conventional oral, parenteral or transdermal dosage form.

As used herein, the term "administering" and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in sound medical practice.

In some embodiments, the pharmaceutical composition comprising a compound of the invention, or any derivative or combination thereof, or a mixture of microorganisms of the invention is administered via an oral (i.e., enteral), rectal, vaginal, topical, nasal, ocular, transdermal, subcutaneous, intramuscular, intraperitoneal, or intravenous route of administration. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injection, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal and any other injection means known in the art. In addition, it may be desirable to introduce the pharmaceutical compositions of the present invention by any suitable route (including intraventricular and intrathecal injections); intraventricular injection may be facilitated by, for example, an intraventricular catheter attached to a reservoir. Pulmonary administration can also be performed, for example, by using an inhaler or nebulizer.

For topical application, the compounds of the invention, or any derivative or combination thereof, or the microbial mixture of the invention, may be combined with a pharmaceutically acceptable carrier to deliver an effective dose based on the desired activity. The carrier may be in the form of, for example, but not limited to, an ointment, cream, gel, paste, foam, aerosol, suppository, pad, or gel stick.

For oral use, the pharmaceutical composition may be in the form of a tablet or capsule, which may contain any of the following ingredients or compounds of similar nature: binders (such as microcrystalline cellulose, tragacanth or gelatin; excipients (such as starch or lactose), disintegrants (such as alginic acid, Primogel or corn starch), lubricants (such as magnesium stearate), or glidants (such as colloidal silicon dioxide). when the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier (such as a fatty oil). additionally, the dosage unit form may contain coatings of various other materials that modify the physical form of the dosage unit, such as sugar, shellac or other enteric agents Probiotic yogurt, kefir, fermented milk or others.

For the purpose of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol may be used, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered if necessary, and the liquid diluent rendered isotonic first with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.

Compositions also include incorporation of the active substance into or onto a particulate preparation of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels, etc.), or into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroids. Such compositions will affect physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

In one embodiment, the present invention provides a combination formulation. In one embodiment, a "combined preparation" specifically defines a "kit of parts" in the sense that the combination partners defined above can be administered independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e. simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the set of parts may then be administered, e.g., simultaneously or chronologically staggered, i.e., at different time points and with equal or different time intervals for any part of the set of parts. In some embodiments, the proportion of the total amount of the combination partner may be administered in a combined preparation. In one embodiment, the combined preparation may be varied, for example, in order to cope with the needs of a patient sub-population to be treated or the needs of an individual patient, the different needs of which may be caused by a specific disease, the severity of the disease, the age, sex or body weight, which can be easily determined by a person skilled in the art.

In one embodiment, it will be appreciated that the molecules of the invention, or any derivative or combination thereof, or the microbial mixture of the invention, may be provided to an individual along with other active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures are taken (e.g., dosage and selection of supplements) against adverse side effects associated with combination therapy.

In one embodiment, the dose may be administered in a single or multiple administrations, depending on the severity and responsiveness of the condition to be treated, with the course of treatment lasting from days to weeks or until a cure is effected or a diminution of the disease state is achieved.

In some embodiments, the compositions of the present invention are administered in a therapeutically safe and effective amount. As used herein, the term "safe and effective amount" refers to an amount of a component sufficient to produce a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner presently described. In another embodiment, a therapeutically effective amount of a molecule, or any derivative or combination thereof, is the amount of the molecule mentioned herein necessary for a measurable expected biological effect in vivo. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g., determining dosage, timing, etc., is within the responsibility of a general practitioner or specialist, and generally takes into account the condition to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of techniques and protocols can be found in Remington, The Science and Practice of Pharmacy,21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005). In some embodiments, the preparation of an effective amount or dose can be initially estimated from in vitro assays. In one embodiment, the dose can be formulated in animal models, and this information can be used to more accurately determine a dose useful for humans.

In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell culture, or in experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used to develop a series of doses for use in humans. In one embodiment, the dosage varies depending on the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration, and dosage may be selected by the individual physician according to the condition of the patient. [ see, for example, Fingl, et al, (1975) "The pharmaceutical Basis of Therapeutics", Ch.1p.1 ].

Pharmaceutical compositions containing as active ingredient a molecule of the invention, or any derivative or combination thereof, or a mixture of microorganisms of the invention, may be prepared according to conventional pharmaceutical compounding techniques. See, e.g., Remington's Pharmaceutical Sciences,18^thEd., Mack Publishing co., Easton, Pa. (1990). In addition, Remington, The Science and Practice of Pharmacy,21st Ed., Lippincott Williams&Wilkins,Philadelphia,Pa.(2005)。

In one embodiment, a composition comprising a formulation of the present invention formulated in a compatible pharmaceutical carrier is prepared, placed in an appropriate container, and labeled for treatment of the indicated condition.

In one embodiment, the compositions of the present invention are presented in a pack or dispenser device (e.g., an FDA approved kit containing one or more unit dosage forms containing an active ingredient). In one embodiment, the package comprises, for example, a metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the package or dispenser accommodates a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice reflects approval by the agency of the composition or form of human or veterinary administration. In one embodiment, such notification is a label approved by the U.S. food and Drug Administration for prescription drugs or a label for an approved product insert.

Unless otherwise indicated, any concentration range, percentage range, or ratio range recited herein is to be understood as including the concentration, percentage, or ratio of any integer and fraction thereof within that range (e.g., one tenth and one hundredth of an integer).

Unless otherwise indicated, any numerical range recited herein that relates to any physical characteristic (e.g., polymer subunit, size, or thickness) should be understood to include any integer within the recited range.

In the discussion, unless otherwise specified, adjectives such as "substantially" and "about" that modify a condition or relational characteristic of one or more features of an embodiment of the invention are understood to mean that the condition or characteristic is defined to be within an acceptable tolerance (which is acceptable for operation of the embodiment for which it is intended). Unless otherwise indicated, the word "or" in the specification and claims is considered to be an inclusive "or" rather than an exclusive "or" and indicates at least one or any combination of the items it incorporates.

It should be understood that the terms "a" and "an," as used above and elsewhere herein, refer to "one or more" of the enumerated components. It will be clear to those of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Thus, in the present application, the terms "a" and "at least one" are used interchangeably.

For a better understanding of the present teachings, and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In the description and claims of this application, the verbs "comprise," "include," "have," and "have" are each used to indicate that the object or objects of the verb are not necessarily a complete list of parts, elements, or portions of the subject or subjects of the verb.

Other terms as used herein are intended to be defined by their well-known meanings in the art.

Other objects, advantages and novel features of the present invention will become apparent to one of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. Furthermore, each of the various embodiments and aspects of the present invention as described hereinabove and as claimed in the claims section below finds experimental support in the following examples.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiments are inoperable without these elements.

Examples

Generally, nomenclature used herein and laboratory procedures employed in the invention include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained extensively in the literature. See, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); ausubel et al, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); perbal, "A Practical Guide to Molecular Cloning," John Wiley & Sons, New York (1988); watson et al, "Recombinant DNA", Scientific American Books, New York; birren et al (eds.) "Genome Analysis: A Laboratory Manual Series", Vols.1-4, Cold Spring Harbor Laboratory Press, New York (1998); U.S. patent nos. 4,666,828; 4,683,202; 4,801,531, respectively; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freesney, Wiley-Liss, N.Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J.E., ed. (1994); stits et al (eds), "Basic and Clinical Immunology" (8th Edition), apple & Lange, Norwalk, CT (1994); mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference. Other general references are provided throughout this document.

Materials and methods

Samples and cultures of probiotic yogurt (e.g., kefir)

Fifty (50) gr kefir particles were inoculated into a 1000ml flask containing 800ml pasteurized cow milk (TARA, israel) and covered with sterile gauze. The mixture was then incubated at 28 ℃ for 24 h. The probiotic yogurt is then filtered to separate the particles from the fermented milk. The microorganisms cultured in the fermented milk were subjected to sequence analysis.

According to manufacturer's instructions, useIsolation kits (MoBio Laboratories, Loker Avenue West Carlsbad; CA) extract DNA from probiotic yogurt (e.g., kefir) cultures. The total DNA was sent to the DNA services (DNAS) facility at the research resource center at Chicago University (UIC) at Illinois for shotgun sequencing. An illumine Nex-Keg 500 sequencer with a sequencing method of Nextera XT and paired-end 2 x 150 reads was performed. The readings are uploaded as FASTQ to an One Codex website and the metagenomic taxonomy annotation results are analyzed.

Imaging flow cytometer

Probiotic yogurt (e.g., kefir) was mixed with water 1: 10 dilutions were made and analyzed by ImageStreamX Mk II (Amnis Corporation, Seattle, WA, USA). A default mask of (bright field) BF is used to identify the microorganisms in focus and to plot a Root Mean Square (RMS) gradient. Population analysis was performed based on the intensity of Infrared (IR) absorbance detected from bacterial cells, but not from fungal cells. For the purpose of calculating the count value, a custom mask was applied to the fungal cells on the autofluorescence channel. The microorganisms are then divided into subpopulations based on the intensity values calculated by the instrument.

Extraction of probiotic yoghurt-derived organic molecules

The resulting probiotic yogurt (e.g., kefir) was centrifuged at 4,000rpm for 10min to separate the precipitate and supernatant. Five hundred (500) ml of the supernatant was then transferred to a separatory funnel and mixed with 500ml of ethyl acetate. The organic phase was separated from the supernatant and transferred to a round bottom flask to evaporate all the solvent until a solid residue was obtained containing organic molecules from the probiotic yogurt.

Biological activity of extracted organic molecules

Bioluminescence assays were performed using mutant bacteria lacking nucleotide sequences encoding the relevant Autoinducer (AI). In addition, bacteria were cloned with bioluminescent reporter plasmids to measure their luminescence intensity as quorum sensing gene expression occurs. Ninety-six (96) well microtiter plate assays were used to assess AI activation (agonists) or inhibition (antagonists) of the following bacteria, agrobacterium tumefaciens, which responds to C8 autoinducer, and vibrio cholerae and vibrio harveyi, both of which respond to (S) -4, 5-dihydroxy-2, 3-pentanedione (DPD) autoinducer for communication.

A.tumefaciens A136 pCF218 pMV26 culture was grown for 24h at 28-30 ℃ in Luria-Bertani broth (LB; Miller's broth) supplemented with 25. mu.g/ml kanamycin and 4.5. mu.g/ml tetracycline. Vibrio harveyi and Vibrio cholerae were both grown in LB broth (broth) (Lennox) supplemented with tetracycline for 24h at 30 ℃. The overnight culture was diluted to an absorbance density (OD) of 0.05 with fresh LB medium₆₀₀). Probiotic yogurt (e.g. kefir) extracted molecules were tested in gradient concentrations in two types of experiments: OD in the absence (activation/agonist) or presence (inhibition/antagonist) of 400pM 3-oxo-C8-HSL (Agrobacterium tumefaciens A136 pCF 2118 pMV26) CAI1 (Vibrio cholerae MM920)200nM DPD (Vibrio harveyi MM30)₆₀₀Competition assay was performed at 0.05 (by fresh LB medium). Clear bottom 96-well microliter plates were prepared, where wells contained test compounds (in triplicate) serially diluted into LB medium. A total of 100 μ l of diluted cells was added to each well. The control sample contained bacteria and specific AI molecules, but no yogurt extracted molecules. Luminescence was measured every 20min for 19h using a Microtiter Plate Reader (Microtiter Plate Reader) (Varioskan Flash, Thermo) with continuous shaking at 30 ℃. Average luminance value divided by OD₆₀₀Values are plotted against the concentration of compound added.

Extraction of organic molecules from Kluyveromyces marxianus

Kluyveromyces marxianus strain HA 63[ NRRL Y-8281, CBS 712] was cultured in 300ml of yeast and malt LB, and grown at 28 ℃ for 24h with stirring at 100 rpm. The culture was centrifuged at 4,000rpm for 10min, and the supernatant was collected. The supernatant was then transferred to a separatory funnel and mixed with an equal volume of ethyl acetate for extraction. The mixture was shaken for 10min, the organic phase was transferred to a new tube, and the extraction was repeated 3 times. Next, the extract is evaporated to remove all liquid and finally dried on a lyophilizer to avoid the presence of water.

Liquid chromatography-Mass Spectrometry (LC-MS)

Crude kluyveromyces marxianus extract was injected into LC-MS to identify yeast-derived metabolites. The chromatogram of the crude Kluyveromyces marxianus extract is compared to the chromatogram of the medium crude extract to identify molecules specifically associated with Kluyveromyces marxianus metabolism. Molecular weight was determined by MS using LTQ XL Orbitrap with static nanospray of positive ion mode (Waters Acquity QDA with PDA and QDA detector).

Anti-biofilm Activity of the synthetic molecules (203)

For static biofilm assays, overnight cultures of pseudomonas aeruginosa (PA01), salmonella and staphylococcus aureus strains were grown in fresh LB medium at a 1: 10 dilutions, LB medium contained synthetic molecules (203) at a final concentration of 20 or 50. mu.M, or DMSO (up to 1%) as controls. Biofilms were grown in 96-well plates (Thermo Scientific, Rochester, NY, usa) at 37 ℃ under non-shaking conditions. By usingThe Dead cells stained green and Dead cells stained red were visualized by the Dead Dead/Live kit (Invitrogen, Eugene, OR, USA), and the use of the synthetic molecule (203) did not affect cell growth. No increase in dead cells was observed in the presence of the synthetic molecule (203) compared to the control biofilm. Stained cells were washed twice with PBS. The biofilm images were taken by CLSM (Plan-Apochromat 20X/0.8M27, Zeiss LSM880, Germany).

Biofilm modulating activity of synthetic tryptophol acetate and tyrosol acetate

The various pathogenicity indicator strains used in this study were the Vibrio cholerae strain MM920(ACqsA AluxQ), the Vibrio harveyi strain MM30(LuxS, Tn 5). To initiate biofilm formation, bacteria were grown in the absence of vibrio cholerae autoinducer 1(CAI 1). Furthermore, the bacteria grow in the presence of: 900nM CAI1 (Vibrio cholerae MM920), 900nM CAI1, 200. mu.M tryptophol acetate and 200. mu.M tyrosol acetate, and 200. mu.M tryptophol acetate and 200. mu.M tyrosol acetate.

Salmonella enteritidis is provided. Staphylococcus aureus group IV strain RN 8242. Pseudomonas aeruginosa PA01, Salmonella and Staphylococcus aureus strains were incubated in LB liquid medium at 37 ℃ for 24 h.

Biofilms were grown in 96-well plates (Thermo Scientific, Rochester, NY, usa) under shaking conditions at 30 ℃. As can be seen from the above, the use of synthetic molecules does not affect cell growth. Stained cells were washed twice with PBS. The biofilm images were taken by CLSM (Plan-Apochromat 20X/0.8M27, Zeiss LSM880, Germany).

Antifungal Activity

Three types of fungi (Sclerotinia (sclerotina), Botrytis (Botrytis) and Penicillium (Penicillium)) were used to assess antifungal activity. Fungi were grown in Potato Dextrose Broth (PDB) and a piece of agar from the plate (on which the fungus was found) was placed in Falcon containing 30ml of PDB and incubated at 22 ℃ until it produced mycelia. Fungal suspensions were purified with fresh PDB 1: 1 dilution and incubation with 75 μ l crude probiotic yoghurt (e.g. kefir) extract in DMSO or with DMSO as control (refreshed sample) at 22 ℃ for about 48 hours. After 48 hours, the fungal suspension was filtered and 10 μ l of the filtrate was transferred to the center of a Potato Dextrose Agar (PDA) plate containing 1.5% (v/v) crude probiotic yogurt extract or DMSO (as control).

Chemical synthesis of tryptophol acetate and tyrosol acetate

Homovanillyl alcohol (1mmol) was dissolved in 3ml Dichloromethane (DCM) and then 1.2mmol acetyl chloride was added to the reaction mixture and allowed to stir at room temperature for 24h until the substrate disappeared. After completion of the reaction, DCM was evaporated under vacuum and the mixture was dissolved with ethyl acetate and washed with a saturated solution of NaCl. The aqueous phase was extracted with ethyl acetate (3X 50mL) and washed with 50mL saturated NaCl and then dried over magnesium sulfate. Purification by flash chromatography on silica gel eluting with EtOAc-hexanes (2: 1) gave tyrosol acetate (2%, 18 mg). Rf 0.43 (30% EtOAc-70% hexanes); verified by LC-MS and 1H NMR.

3- (2-Hydroxyethyl) indole (3- (2-Hydroxyethyl) indole) (3.224gr, 20mmol) was dissolved in 40mL pyridine and 2,648. mu.L (28mmol) acetic anhydride was added dropwise and allowed to stir at room temperature for 15 h. The solution was poured into 160mL of H₂O solution, and stirring for 20 min. Separating the heterogeneous mixture with CH₂Cl₂The aqueous layer was extracted (4X 40 mL). The combined organic layers were washed with brine and dried (MgSO)₄) Filtered through celite and concentrated under reduced pressure. Purification by flash chromatography on silica gel eluting with EtOAc/hexanes provided the alcohol acetate (5.2%, 0.212gr) as a colorless oil. Rf 0.46 (50% EtOAc/hexanes); verified by LC-MS and 1H NMR.

Determination of quorum sensing Activity

The effect of kefir crude biomass extract molecules on the following bacteria, Agrobacterium tumefaciens A136, Vibrio cholerae MM920 and Vibrio harveyi MM30, was measured as described by Brenier et al (2008). The overnight culture was diluted to an absorbance density (OD) of 0.05 with fresh LB medium₆₀₀). Clear bottom 96-well microliter plates were prepared, where the wells contained test compounds (in triplicate) serially diluted into LB medium. A total of 100 μ L of diluted cells was added to each well. The control sample contained bacteria and specific autoinducer molecules, but no crude extracted molecules. Luminescence was measured every 20min for 19h using a microtiter plate reader (Varioskan Flash, Thermo) with continuous shaking at 30 ℃. Two types of experiments were performed: the competition assay was performed in the presence of 400pM 3-oxo-C8-HSL (Agrobacterium tumefaciens A136), CAI-1 (Vibrio cholerae MM920), 200nM DPD (Vibrio harveyi MM30), and another in the absence of these AI's to measure the agonistic activity of the crude extracted molecules. Will be divided by OD₆₀₀The mean luminescence value of the values is plotted against the concentration of the added compound.

Biofilm modulating activity of kefir crude biomass extract molecules

Pseudomonas aeruginosa PA01, Salmonella and Staphylococcus aureus strains were incubated at 37 ℃ for 24 h. Suspending bacteriaThe solution was diluted with fresh LB at 1: 10 dilutions and incubation with kefir crude biomass extract molecules or with DMSO as control (refreshed samples) for 3 h. DMSO concentrations were up to 1% (v/v) in all cultures. 200 μ l of liquid medium mixture with yoghurt extract or with DMSO as control was placed in each well of a 96-well plate (Thermo Scientific, Rochester, NY, USA). Add 2. mu.l of refreshed sample to each appropriate well. The plates were incubated at 37 ℃ for 24 hr. After incubation, the samples were washed 3 times with PBS. For visualizing living cells, use is made ofThe Dead cells were stained red and the bacteria were stained green using the Dead/Live kit (Invitrogen, Eugene, OR, USA). Stained cells were washed twice with PBS. Biofilm images were taken by CLSM (Olympus, tokyo, japan). Image processing was done using IMARIS software (Bitplane, zurich, switzerland).

Biofilm modulating activity of synthetic tryptophol acetate and tyrosol acetate

For static biofilm assays, overnight cultures of vibrio cholerae strain MM920 were grown in fresh LB medium at a 1: 10 dilutions, the medium contained 100 μ M or DMSO (up to 1%) final concentration of each of tryptophol acetate and tyrosol acetate as controls. The inventors analyzed two types of samples. One without the presence of Vibrio cholerae autoinducer (CAI-1) and the other with 900nM CAI-1. Biofilms were grown in 96-well plates (Thermo Scientific, Rochester, NY, usa) at 37 ℃ under non-shaking conditions. By usingThe Dead cells stained green and Dead cells stained red were visualized by the Dead Dead/Live kit (Invitrogen, Eugene, OR, USA), and the use of synthetic molecules did not affect cell growth. No increase in dead cells was observed in the presence of both synthetic molecules compared to the control biofilm. Stained cells were washed twice with PBS. The biofilm images were taken by CLSM (Plan-Apochromat 20X/0.8M27, Zeiss LSM880, Germany).

Nuclear Magnetic Resonance (NMR)

Recording at Room Temperature (RT) using a Bruker Avance DPX500 instrument (500MHz)¹H and¹³spectrum of C Nuclear Magnetic Resonance (NMR).

Quantitative real-time PCR (RT-PCR) analysis

Following the manufacturer's instructions (including the manufacturer's description of on-column DNase I digestion), using RNA Protect Bacteria reagent andthe kit (Qiagen, Valencia, CA) was grown to approximately 1.0 from culture in LB lennox medium (with or without 100. mu.M tryptophol acetate or tyrosol acetate)_ODRNA was extracted from the wild type strain VC1 of A600 (see above). The purified RNA was quantified using a Banalyzer (Eppendorf, Hamburg, Germany). cDNA was synthesized from 1. mu.g of RNA using PrimeScriptTM RT kit (Takara, Ohtsu, Japan). The reaction was incubated at 37 ℃ for 30min and 2. mu.l of DNA was analyzed by RT-PCR on an AB Step One Plus PCR system (Applied Biosystems, Carlsbad, Calif.) using qPCRBIO SyGreen Blue mix Hi-ROX (PCR Biosystems, London, UK). RT-PCR was performed in triplicate in 96-well plates (Bio-Rad) at a volume of 20- μ l. The mdh gene was used as an endogenous loading control for the reaction. The amount of transcripts was analyzed with StepOneNus software V2.3(Applied Biosystems Carlsbad, Calif.). Mdh forward primer 5'-CTGGCGGCATTGGTCAAGCCC-3' (SEQ ID NO: 1); mdh reverse primer 5'-ACCCGGTGTGACAGGCGCAA-3' (SEQ ID NO: 2); vpsT forward primer 5'-CGCAGTATTCAGATGCTGGTG-3' (SEQ ID NO: 3); vpsT reverse primer 5'-GACCTCTTTCGCATCAGGACA-3' (SEQ ID NO: 4); ctxA forward primer 5'-AGCAGTCAGGTGGTCTTATGC-3' (SEQ ID NO: 5); ctxA reverse primer 5'-CCCGTCTGAGTTCCTCTTGC-3' (SEQ ID NO: 6); aphA forward primer 5'-ACCGGGTACGATATAACCAAAGAG-3' (SEQ ID NO: 7); aphA reverse primer 5'-GATGGCTGGCTTTCCAGAAG-3' (SEQ ID NO: 8). For construction of transcriptome libraries we used purified RNA as described in the quantitative real-time PCR (RT-PCR) analysis section. Ribosomal RNA was removed using the MICROBExpress kit (Invitrogen by Thermo Fisher Scientific, Lithounia). Library preparation was performed using SMARTer Standard RNA-Seq kit (Takara Bio, USA) according to the manufacturing instructions. High throughput sequencing analysis was performed by Illumina HiSeq 2500, SR 50bp and barcode Raw data quality value phred + 33. The number and percentage of reads through the sequencer's automatic quality filter, control mapping (mapping) values, error rates, and quality scores per base indicate that sequencing is of high quality. Due to trimming, only a small fraction (a small percentage of) reads are discarded and the peak of the length distribution is at the original length 51. The software and application used for comparative analysis was Library quality control (Library quality control): FASTQC version 0.11.5; trim _ galore (using cutatapt version 1.10); mapping (Mapping): tophat2 version 2.1.0 (using Bowtie2 version 2.2.6); and (3) gene counting: HTseq-count version 0.6.1. For normalization and differential expression analysis, DESeq 2R software package version 1.18.1 input file. The reference genome used in the analysis was vibrio cholerae O1 biovar El Tor str.n 16961 from the following links: ftp:// ftp. ncbi. nlm. nih. gov/genes/all/GCF/000/006/745/GCF _000006745.1_ ASM674v 1. The counting is performed using the annotation files downloaded from the following links: ftp/ftp. ncbi. nlm. nih. gov/genes/all/GCF/000/006/745/GCF _000006745.1_ ASM674v1/GCF _000006745.1_ ASM674v1_ genetic. gtf. gz. All these files can be found in our FTP server, see section 4-analysis results file.

GM1 enzyme-linked immunosorbent assay (ELISA) -cholera toxin detection assay

GM1 (monosialotetrahexosylganglioside) was inoculated by the following procedure and immobilized on 96-well white/clear bottom microtiter plates (Greiner): stock GM1 (2mg/mL in PBS) was diluted with PBS (final concentration 10. mu.g/mL). mu.L of GM1 solution was added to each well and incubated at 37 ℃ for 4-16 hours without shaking. The plate was washed with PBS (× 3). Bovine Serum Albumin (BSA) was dissolved in PBS (final concentration 4mg/mL) and 200. mu.L of BSA-PBS was added to each well and incubated at 37 ℃ for at least 4 hours without shaking. Plates were washed with PBS (× 3) and kept in a refrigerator until use. VC1 wild-type strain with test compounds (tryptophol acetate and tyrosol acetate both at a concentration of 100. mu.M) and a control with bacteria only were grown and incubated with aeration and shaken overnight at 30 ℃ in LB medium. The culture was centrifuged (spun-down) at 5,000rcf for 5min and the supernatant was taken and washed with BSA-PBS 4mg/mL solution at 1: and 2, diluting. 200 μ L of diluted supernatant was added in six replicates to each GM1 coated well and the plates were incubated at 37 ℃ for at least 30min with gentle shaking. Meanwhile, rabbit antitoxin serum (RSA) stock was diluted with BSA-PBS at 1: 999 dilution. The plates were washed with PBS (× 3), 200 μ L of RSA solution was added to each well, and the plates were incubated at 37 ℃ for at least 30min with gentle shaking. Meanwhile, goat anti-rabbit immunoglobulin g (igg) H & L alkaline phosphatase stock (1mg/mL in DDW) was mixed with BSA-PBS at 1: 1,499 dilution. The plates were washed with PBS (× 3), 200 μ L of IgG solution was added to each well, and the plates were incubated at 37 ℃ for at least 30min with gentle shaking. At the same time, a luminol working solution was prepared by preparing two stock solutions. Stock a was prepared by adding 0.1mL luminol 250mM in DMSO, 44 μ L coumaric acid 90mM in DMSO, 1mL1M tris-HCl at pH 8.5 and DDW to a final volume of 10 mL. Stock B was prepared by adding 6.4 μ l of 30% hydrogen peroxide, 1ml of 1M tris-HCl at pH 8.5 and DDW to a final volume of 10 ml. The plate was washed with PBS (x 3) and equal volumes of stock solutions A and B were mixed rapidly to make luminol working solutions. 100 μ L of luminol working solution was added to each well, the plate was shaken for 1.5-2min, and luminescence was measured using a microtiter plate reader (Varioskan Flash, Thermo). Luminescence values were normalized to the control.

Macrophage collection

Peritoneal macrophages were harvested using the protocol previously described (Zhang et al, 2008). Induced macrophages (induced macrophages) were harvested from WT mice that had received intraperitoneal administration of 2.5ml of 3% Brewer mercaptoacetate liquid medium (Sigma, UK) 72h prior to harvest.

In vitro LPS stimulation

Peritoneal macrophages (5X 10)⁵Per ml) and 0.1 μ g/ml lipopolysaccharide (LPS-Escherichia coli) serotype 0127: b8, Sigma-Aldrich) and incubated in the presence of 100. mu.M-tryptophol acetate, tyrosol acetate, dopamine HCl, caffeic acid, or a combination of tryptophol acetate and tyrosol acetate-for 24hr. After incubation, supernatants were collected and stored at-20 ℃ for detection of IL-6 cytokines. For the IL-1 α assay, the inventors induced macrophage lysis by adding fresh RPMI medium and thawing 3 times.

Cytokine ELISA

IL-1 α and IL-6 levels in peritoneal exudate and LPS stimulated culture supernatants were measured by ELISA. For the detection of cytokines, R & D ELISA kits (catalog numbers MLA00 and DY406, respectively) were used according to the manufacturer's instructions. A standard curve was constructed starting at a concentration of 15ng/ml using serial dilutions of purified IL-1. alpha. or IL-6.

Thioflavin T (ThT) assay

ThT fluorescence measurements were performed on a Biotek Synergy H1 plate reader using 96-well black plates at 37 ℃. Samples containing 30 μ M amyloid β [ α β (1-42) ] protein were measured in the absence or presence of different concentrations of tryptophol acetate. 120- μ L aliquots of the aggregation reaction were mixed with 10 μ M ThT in phosphate buffer (pH 7.4). Fluorescence intensity was measured every 30min for 24hr at λ excitation (λ ex) 440 and λ emission (λ em) 485 nm.

Surface Plasmin Resonance (SPR)

SPR sensor chips with oligomer a11 antibody (anti-amyloid oligomer conformation specific antibody) were tested using samples containing 30 μ M α β (1-42) in the absence or presence of varying concentrations of tryptophol acetate.

Transmission Electron Microscope (TEM)

TEM grids of samples containing 30. mu.M. alpha.beta. (1-42) were prepared after 24hr incubation at 37 ℃ in the absence or presence of different concentrations of tryptophol acetate.

Example 1

Identification of microorganisms in probiotic yogurt (e.g., kefir)

The inventors found that the main bacterial species in probiotic yoghurt were Lactobacillus equi (Lactobacillus kefiranofaciens), Lactobacillus, Lactococcus lactis (Lactococcus lactis), propionibacterium and Leuconostoc mesenteroides (Leuconostoc mesenteroides). There are many other genera that exist in probiotic yogurts; however, they generally represent less than 0.5% of the microflora. Significant findings in sequencing analysis and in contrast to other reported probiotic yoghurts (e.g., kefir), the probiotic yeast kluyveromyces marxianus was identified, which was found to be the dominant species (59.04%) in the disclosed probiotic yoghurt (fig. 1A). The morphology of this yeast in probiotic yogurt was found to be similar to that of its single culture (fig. 1B and 1C).

Example 2

Composition of microbial subpopulations in probiotic yogurt (e.g., kefir)

As summarized in the following table, the inventors obtained a scatter plot of autofluorescence intensity versus BF detail intensity for population R5 (19539 events; FIG. 2A). Different sub-populations R3, R6 and R7, as well as other complex sub-populations (R8) can be seen on the graph (fig. 2A). The different colors selected for the different gates correspond to the set of images displayed at the periphery of the graph. R3 (orange dots; FIG. 2A) is defined as the population of bacterial cells (86.1%; FIG. 2B), R6 (light blue dots; FIG. 2A) is defined as the population of fungal cells (4.94%; FIG. 2C) and R7 (light yellow dots; FIG. 2A) is defined as the population of fungi and bacterial aggregates (7.58%; FIG. 2D). The R8 (brownish red dots; fig. 2A) population is a larger aggregate (fig. 2E) and is therefore defined as a sub-population of R7.

Example 3

Characterization and Mass Spectrometry (MS) analysis of crude extracts

Crude kluyveromyces marxianus monoculture extracts were injected into LC-MS to identify molecules derived from yeast metabolism. The chromatograms of the crude extracts were compared with the medium crude extracts to check which molecules specifically bind to the metabolism of kluyveromyces marxianus. Two molecules were identified only in the kluyveromyces marxianus crude extract chromatogram (fig. 4B) but not in the medium extract chromatogram (fig. 4A), with Retention Times (RT) of 11.68 and 13.84 min. The molecular weights were determined by MS and the m/z peaks of 180 and 203, respectively, predominate. Furthermore, two peaks were observed in the LC-MS chromatogram of a crude extract of a probiotic yogurt (e.g. kefir) comprising kluyveromyces marxianus (fig. 3B). These peaks were not observed in the extract of milk used to ferment probiotic yogurt (fig. 3A).

Example 4

Synthesis and characterization of tryptophol acetate and tyrosol acetate

The inventors have isolated two molecules with masses of 180 and 203, respectively, from crude kluyveromyces marxianus extract, which was verified by LC-MS (fig. 5). After that, the inventors chemically synthesized two molecules, and verified the weight and structure analysis using LC-MS and 1H NMR, respectively (fig. 6 and 7).

Example 5

Effect of crude yogurt molecules on the QS System

QS activation activity was calculated by normalization to the inducer bioluminescence signal alone. Crude extract containing 203 molecules activated QS in agrobacterium tumefaciens (fig. 8A) and pseudomonas aeruginosa (fig. 8B). In vibrio cholerae, low-volume crude 203 molecules activated QS, while high-volume crude 203 molecules inhibited QS (fig. 8C and 8D). In Vibrio cholerae, 180 molecules with low capacity activate QS, while 180 molecules with high capacity completely inhibit QS (FIG. 9), where IC is₅₀24.4. + -. 2.4. mu.M.

Example 6

Effect of probiotic yogurt (e.g. kefir) on fungal growth

The effect of probiotic yoghurt on fungal survival was further examined. Probiotic yogurt was supplemented onto potato dextrose agar plates that were cultured with the fungus sclerotinia sclerotiorum. Sclerotinia cultured on agar plates supplemented with probiotic yogurt was shown to be inhibited for up to 12 days (fig. 14A) or 19 days (fig. 14B). Similar inhibition was observed up to 22 days after inoculation in Botrytis (FIG. 15) and Penicillium (FIG. 16), but to a lesser extent.

Example 7

Synthesis of tryptophol acetate molecules affecting biofilm production

The inventors examined the effect of synthetic tryptophol acetate on biofilm formation by several types of bacteria. When provided at 20 μ M, tryptophol acetate did not appear to affect biofilm formation in P.aeruginosa (FIG. 11). In contrast, 50 μ M tryptophol acetate had a significant effect on biofilm formation in salmonella (fig. 12) and staphylococcus aureus (fig. 13).

Example 8

Synergistic effect of tyrosol acetate and tryptophol acetate on QS (Vibrio cholerae)

A synergistic effect of tryptophol acetate and tyrosol acetate on QS activation in vibrio cholerae was observed (fig. 17). Mixing the raw materials in a ratio of 1: 1 has an IC of 11.6 + -0.9 μ M₅₀。

In addition, various combinations of tryptophol acetate and tyrosol acetate were examined in the mutant MM920 vibrio cholerae strain (Δ CqsA Δ luxQ). The mutant Vibrio cholerae produced biofilms (FIGS. 18A and 18E) that were much thicker and denser than the corresponding biofilms (FIGS. 18B and 18F) produced when the autoinducer alone was added, which promoted quorum sensing and thus disrupted biofilm formation. Tryptophol acetate and tyrosol acetate (fig. 18D and 18H), and in particular these three compounds together (fig. 18C and 18G), showed significant synergy in the disruption of normal biofilm growth-leading to different biofilm morphology.

Example 9

Synthetic tryptophol acetate reduces the load of Vibrio cholerae toxin

The inventors then examined whether the synthesized tryptophol acetate affects the level of production of the vibrio cholerae toxin. Indeed, synthetic tryptophol acetate was found to reduce vibrio cholerae toxin levels in a dose-dependent manner (fig. 19).

Example 10

Synthesis of tyrosol acetate molecules affecting biofilm production

The inventors examined the effect of synthetic tyrosol acetate on Vibrio cholerae. Tyrosol acetate was found to inhibit biofilm and toxin production of vibrio cholerae. Further, tyrosol acetate has been shown to affect the expression of QS related genes (fig. 21).

Example 11

Effect of probiotic yogurt (e.g., kefir) on bacterial growth and biofilm production

The effect of probiotic yogurt on bacterial growth was further examined. Crude kefir extracts interfered with the QS pathway of all three bacterial strains examined. Crude kefir extract had a significant Quorum Sensing Inhibition (QSI) effect if the vibrio cholerae MM920 mutant lacked the ability to synthesize its CAI-1 autoinducer, as a direct correlation between kefir crude extract dilution and bioluminescence decay reflecting QS inhibition in this strain was evident (fig. 22A). Further, kefir extract has been shown to have a concentration-dependent QSI effect on agrobacterium tumefaciens a136, wherein the QS pathway is induced by a 3-oxo-octyl (3-oxo-octanyl) autoinducer (fig. 22B). Interestingly, MM30 bioluminescence assay of vibrio harveyi using DPD autoinducer appeared to show that the crude kefir extract induced a Quorum Sensing Activation (QSA) effect at all dilutions (fig. 22C). With the bioluminescence assay demonstrating that substances in kefir extracts affect QS pathways (inhibition or activation) of different bacteria (fig. 22A-22C), the inventors further investigated whether kefir extracts affect the formation of biofilm matrix assembled by pathogenic bacteria. Indeed, crude kefir extracts have been shown to significantly inhibit biofilm production by the important pathogenic bacteria pseudomonas aeruginosa, salmonella enteritidis and staphylococcus aureus (fig. 22D). Specifically, the results show that when the bacteria were co-incubated with kefir extract, the calculated biofilm capacity of each bacteria was reduced by about 30% to 40% compared to untreated bacteria by image analysis of three-dimensional confocal microscope images of the biofilm layer (fig. 22D). Importantly, cell viability assays demonstrated that crude kefir extract did not adversely affect proliferation and viability of bacterial cells (fig. 20), thus indicating that disruption of cell-cell communication is a possible factor leading to kefir reduction in biofilm formation.

Example 12

Effect of tryptophol acetate on Vibrio cholerae biofilm

The inventors examined the effect of tryptophol acetate on the Vibrio cholerae biofilm, a key component of Vibrio cholerae proliferation and pathogenicity, using the Vibrio cholerae reporter strain MM920 lacking the oligonucleotide sequence encoding QS autoinducer CAI-1.

The biofilm matrix of Vibrio cholerae VC1 WT (FIGS. 23Ai and 23Aii), and the biofilm matrix of the Vibrio cholerae MM920 mutant (FIGS. 23Aiii and 23Aiv) were grown for 24 hours in growth medium with or without tryptophol acetate and usedThe kit was stained to distinguish viable cells. Fluorescence microscopy analysis demonstrated that tryptophol acetate had a significant effect on the morphology and tissue of the biofilm, consistent with the anti-QS effect of this compound (e.g., fig. 17B). Specifically, in the case of vibrio cholerae VC1 WT, the control biofilm matrix (without addition of tryptophol acetate) appeared thin and uneven (fig. 23 Ai); this biofilm appearance was attributed to recent reports on the correlation between the functional QS pathway in the vibrio cholerae VC1 WT strain and the assembly of the biofilm matrix by this bacterium. It was further demonstrated that proliferating bacteria form significantly denser biofilms after incubation with tryptophol acetate (100 μ M, FIG. 23Aii), which inhibits the important QS CAI-1 cascade. Similar significant effects of tryptophol acetate on biofilm assembly were evident in the case of the vibrio cholerae MM920 mutant (fig. 23Aiii and 23 Av). Due to the lack of CAI-1 QS cascade, biofilms of mutant bacteria alone appeared thick and dense (fig. 23 Aiii). Addition of CAI-1 autoinducer to bacterial growth media reintroduced QS while destroying the integrity of the biofilm (figure 23 Aiv). However, co-addition of tryptophanol acetate and CAI-1 caused the reformation of a dense and uniform biofilm layer (fig. 23Av), reflecting the inhibition of the CAI QS pathway (fig. 23 Ai).

The results of fluorescence microscopy in fig. 23A, confirmed by biofilm volume analysis using a quantitative Crystal Violet (CV) assay (depicted in fig. 23B), provides additional evidence of the inhibition of QS by tryptophol acetate. Indeed, considering the fact that activation of QS leads to a decrease in biofilm formation at high cell densities, the increased biofilm capacity recorded after addition of tryptophol acetate supports direct inhibition of the QS pathway in vibrio cholerae.

To identify genes that could potentially be regulated by tryptophol acetate, the inventors performed a real-time quantitative PCR (RT-qPCR) analysis to assess gene expression of vibrio cholerae VC1 WT under high cell density conditions (fig. 23C). The RT-qPCR results provided evidence that tryptophol acetate at a concentration of 100 μ M had a significant effect on gene expression pathways, particularly genes associated with QS, biofilm, and regulation of virulence, suggesting phenotypic changes induced by this compound (fig. 23A-23B). Specifically, RT-qPCR data revealed significant downregulation due to tryptol acetate hapR (fig. 23C). This result is significant since hapR inhibition occurs at low cell density conditions; indeed, mimicking low cell density by hapR down-regulation probably explains the enhanced biofilm formation observed when vibrio cholerae was incubated with tryptophol acetate (fig. 23A-23B). This explanation was confirmed by the significant upregulation of vpsT by tryptophol acetate (fig. 23C), and the expression of this gene, which is required for biofilm formation, was enhanced under low cell density conditions. In addition, tryptophol acetate also resulted in reduced expression of hapA (downstream gene regulated by hapR) (fig. 23C). Inhibition of hapA may actually indicate a potential therapeutic benefit of the probiotic yogurt disclosed herein (e.g., kefir), as hapA is associated with a variety of pathogenic effects induced by vibrio cholerae (e.g., fluid production and diarrhea). Importantly, it is expected that mimicking the low cell density for vibrio cholerae by tryptophol acetate facilitates the genetic cascade, leading to enhanced virulence. Indeed, RT-qPCR experiments demonstrated upregulation of aphA associated with Vibrio cholerae toxin production (FIG. 23C). However, surprisingly, RT-qPCR analysis revealed that ctxA, a virulence inducing gene downstream of the aphA-induced virulence cascade, was in fact significantly inhibited by tryptophol acetate (fig. 23C). To assess the phenotypic aspects of this result, the inventors performed measurements and showed that tyrosol acetate reduced toxin production of vibrio cholerae in a dose-dependent manner (fig. 19).

Example 13

Probiotic yoghurt (e.g. kefir) for use in the treatment of inflammatory bowel disease

Summary of the experiment:

study duration (per cycle): 10 days; group size: n is 4 or 6; the group number is 10; animals: male black mice 6-8 weeks old at the beginning of the study; model: colitis/Crohn's disease was induced in mice by supplementation of 2.5% (w/v) Dextran Sodium Sulfate (DSS) in drinking water. DSS-water was changed daily for 7 days.

Treatment:

and (3) testing items: probiotic yoghurt, or a mixture of two molecules (tryptophol acetate and tyrosol acetate) at a final concentration of 25 μ M (repeat 2) or 50 μ M (repeat 3)), was delivered by the oral route (gavage) as described below, once daily from day 1 to day 7.

TABLE 1 summary of examination of test items in murine IBD models

Number of groups	Group name	Description of the invention	DSS
				1	Control + W	Conventional food-no DSS	-
2	DSS+W	Conventional food	+
				3	Control + Y	Test food (yogurt) -No DSS	-
4	DSS+Y	Test food (yoghourt)	+
				5	Control + mole	Molecular test items (oral)	-
6	DSS+mole	Molecular test items (oral)	+
				7	Control + Yc	Test food (commercial yogurt) -No DSS	-
8	DSS+Yc	Test food (commercial yoghourt)	+
				9	Y+DSS+Y	Early application of yogurt before and after DSS	+
10	Yc+DSS+Yc	Early application of commercial yogurt before and after DSS	+

Examination throughout the experiment:

morbidity and mortality-once a day; clinical signs-every other day; body weight-every other day on the first day and thereafter; rectal bleeding and stool consistency scores (as indicated in table 2 below) -on day one (day 0 or baseline) and day 7.

And (4) terminating:

bleeding for cytokines; clinical score (according to table 2); animals were sacrificed on day 10 and the colon was excised, measured and weighed; the colon was fixed in 10% buffered formalin and the distal dissected area of the colon was paraffin embedded for histological examination. Histological samples were stained with hematoxylin and eosin for histopathological analysis and scored for loss of crypt cells in the intestinal wall and infiltration of inflammatory cells into the colon.

TABLE 2 clinical scores

Scoring (score)	Stool consistency	Bleeding of rectum
			0	Is normal	Negative of
1	Loose stool	Negative of
			2	Loose stool	Latent
3	Diarrhea (diarrhea)	Latent
			4	Diarrhea (diarrhea)	Positive for

The inventors show that the probiotic yogurt disclosed herein and the molecules identified therein reduce weight loss in a murine model of Inflammatory Bowel Disease (IBD). Compared with DSS + W, the method is carried out by using Y + DSS + Y; in mice treated with DSS + Y and DSS + Mole (at 25 μ M or 50 μ M), significantly reduced weight loss was observed (figure 24). In addition, there was no significant weight loss effect in the group receiving the commercial yogurt (Yc + DSS + Yc or DSS + Yc).

The inventors showed that the probiotic yoghurt disclosed herein and the molecules identified therein reduced colon shortening in murine models of IBD (figure 25). Colon length was measured from rectum to cecum at the end of the experiment. A shortening of the colon was observed in mice treated with DSS (DSS + W) compared to the colon of mice treated with DSS and fed with the probiotic yogurt disclosed herein before DSS (Y + DSS + Y). Furthermore, a statistically significant reduction in colon length was observed in mice treated with the molecules identified in the probiotic yogurt disclosed herein (50 μ M).

The inventors further examined the effect of the probiotic yoghurts disclosed herein and the molecules identified therein on the Disease Activity Index (DAI). The results show a significant reduction in clinical score in mice treated with yogurt (Y + DSS + Y; fig. 26A) before DSS, or with the molecules identified in the probiotic yogurt disclosed herein at a concentration of 50 μ M (DSS + molecular; fig. 26B).

Colon histopathological sections revealed that severe tissue damage was accompanied by loss of crypt cells in the intestinal wall and infiltration of inflammatory cells into the colon in all DSS treated mice (fig. 27A). In the colon of mice fed yoghurt before DSS (Y + DSS + Y), tissue architecture was preserved with only minimal initial signs of inflammation and colon tissue repair (circled; fig. 27B).

Example 14

Administration of probiotic yogurt (e.g. kefir) in skin ulcer treatment

The inventors show that leishmaniasis ulcers can be treated using the probiotic yoghurt disclosed herein. In short, healing was observed after application of the yoghurt mix, with application after three months of conventional treatment (which failed to help patients). Conventional treatment includes injection of PENTOSTATAM after local anesthesia with ESRACAIN injection. In addition, the patient was coated SALIKAREN due to the thick coating. In yogurt treatment, patients applied probiotic yogurt to all of their ulcers and bandaged the ulcers twice a day (morning and evening). The results show accelerated healing of cutaneous leishmaniasis after 11-14 days of treatment (fig. 28).

Furthermore, in the case where sutures were required for fusion of the incision, since no suture was made since the injury had been over 24 hours, probiotic yogurt was applied and wound healing was evident as early as four days after the initial application (fig. 29).

Example 15

Anti-inflammatory activity of tryptophol acetate and tyrosol acetate

The inventors showed that the production of both pro-inflammatory cytokines IL-1 α and IL-6 was significantly reduced in macrophages cultured in the presence of tryptophol acetate, tyrosol acetate, or both (figure 30). Other 4-ethylphenol derivatives, dopamine HCl and caffeic acid showed similar results.

Example 16

Anti-neurodegenerative activity of tryptophol acetate

The inventors examined the activity of the composition comprising tryptophol acetate in a common in vitro neurodegenerative model. Both the molecular mixture extracted from the probiotic yogurt disclosed herein and the tryptophanol acetate were shown to be effective in reducing amyloid β fibril formation after 12 hours of incubation (fig. 31). Further, tryptophol acetate has been shown to be dose-dependent in its inhibitory effect on amyloid beta (fig. 32).

The inventors further examined the effect of the crude extract of probiotic yogurt or tryptophanol acetate disclosed herein on amyloid β fibril formation after an incubation period of 24 hr. In the presence of crude extracts of probiotic yoghurts disclosed herein, fibril formation was completely inhibited (fig. 33B). Fibrils were also significantly inhibited in a dose-dependent manner in the presence of tryptophol acetate (fig. 33C-33D).

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Sequence listing

<110> Ben-Gulian university B.G. Navy technology and applications

<120> microbial mixtures, molecules derived therefrom, and methods of use thereof

<130> BGU-P-071-PCT

<150> 62/715,875

<151> 2018-08-08

<160> 8

<170> PatentIn version 3.5

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