[1] The present invention is directed to a method of propagating an antimicrobial culture. In particular, the present invention relates to a method of propagating antimicrobial culture comprising incubating a viable microorganism in the presence of a microbial lysate under suitable conditions allowing the accumulation of antimicrobial metabolites. The present invention also relates to a propagated antimicrobial culture and uses thereof as an antimicrobial agent.

INTRODUCTION

[2] Infectious diseases including bacterial infections continue to be a serious health problem worldwide. Probiotics are defined as“live microorganisms which when administered in adequate amounts confer a health benefit on the host”. In the last several years, the use of probiotics for human has received increasing attention as scientific evidence continues to accumulate on the properties, functionality and beneficial effects of probiotic bacteria. The search for more new probiotics is driven by the growing demand for probiotic functional food and beverages and dietary supplements due to rising levels of health consciousness and growing consumer awareness regarding gut health and the concept of preventive health care. We know that some of the infections and disorders, such as irritable bowel syndrome, inflammatory bowel disease and antibiotic- induced diarrhoea are associated with deficient or compromised intestinal microflora where certain probiotics are considered to provide benefits in controlling such disorders.

[3] Lactic acid bacteria in particular, especially Lactobacillus, are the most commonly used microorganisms as probiotics -“Generally Recognized As Safe” (GRAS). Acidity, presence of bile salts, and pancreatic enzymes in the gastrointestinal tract (GIT) are some of the major stresses that an orally taken probiotic experiences in the GIT. Apart from being able to survive, a probiotic strain also has to be able to adhere to and subsequently colonize (at least temporarily) the intestinal tract. Since the GIT is a dynamic environment, the flow of the gut digesta may wash out any bacterium not attached to the intestinal mucosa. Thus, probiotic strains with adherent ability are more likely to have an increased opportunity to colonize the GIT and provide beneficial effects.

[4] The interest in using probiotics in industry has increased dramatically. It is important that these probiotics and products containing probiotics can be produced on an industrial scale in a reproducible manner without compromising on the beneficial effects. [5] Different methods of producing probiotics have been described and are well documented. Some of the ese methods describe producing mixed microbial cultures in liquid growth medium. The production of such mixed cultures is greatly challenged by microbial competition among the diverse populations in the culture medium. One way of producing mixed microbial cultures is to separately propagate the different strains and to afterwards combine the propagated strains in the desired ratio. This is however undesirably laborious and costly. Therefore, there are still a need for developing efficient and cost-effective methods for producing antimicrobial cultures such as probiotics.

BRIEF SUMMARY OF THE INVENTION

[6] The inventors have developed a method of reproducibly producing an antimicrobial culture by propagating a microorganism in the presence of microbial lysate.

[7] According to one aspect, the method of the invention comprises the steps of: a. inoculating an aqueous growth medium with an inoculum comprising a microorganism to produce an inoculated medium containing about 10 ² - 5x10 ¹⁰ viable cells/ml;

b. mixing the inoculated medium with a microorganism lysate to produce a mixture;

c. incubating the mixture at a first incubation temperature (ITc);

d. cooling the mixture of step c. to a second incubation temperature (2Tc) that is at least about 0.5°C below the first incubation temperature (ITc) and incubating the cooled mixture at the 2Tc to produce a propagated antimicrobial culture; and optionally

e. collecting the propagated antimicrobial culture.

[8] The method according to the invention enables industrial scale or largescale production of propagated antimicrobial cultures.

[9] According to one aspect, the method of the invention comprising the steps of: f. adding to a largescale aqueous growth medium a propagated antimicrobial culture according to claim 1 as an inoculum; g. incubating the inoculated medium at a first incubation temperature (ITc) to produce a propagated antimicrobial culture; and optionally h. collecting the propagated antimicrobial culture. [10] The present method is perfectly suited for producing propagated antimicrobial cultures that contain a large number of different antimicrobial metabolites.

[11] In addition, the present method can be carried out using only food grade materials.

[12] Although the inventors do not wish to be bound by theory, it is believed that the method of the present invention benefits from the quorum sensing capabilities of microorganisms, particularly bacteria such as lactic acid bacteria.

[13] The present inventors surprisingly and unexpectedly observed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, particularly bacteria such as lactic acid bacteria, due to the metabolic stress they experience, increased the release of different antimicrobial metabolites. In particular, the present inventors surprisingly and unexpectedly observed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, particularly bacteria such as lactic acid bacteria, due to a combination of the metabolic stress and quorum sensing capabilities of the microorganisms, maximise the release of different antimicrobial metabolites.

[14] In some embodiments, the propagated antimicrobial culture comprises Lactobacilli and microorganism lysate. In some embodiments, the propagated antimicrobial Lactobacilli is Lactobacillus delbrueckii. In some embodiments, the propagated antimicrobial Lactobacilli is Lactobacillus delbrueckii subsp. bulgaricus. In some embodiments, the Lactobacilli is Lactobacillus delbrueckii subsp. bulgaricus strain GLB 44.

[15] The antimicrobial metabolites can be toxic to pathogenic microorganisms such as bacterial pathogens.

[16] In various embodiments, the propagated antimicrobial culture is useful for the treatment of a variety of human, animal and plant diseases or conditions such as microbial disease or conditions. In various embodiments, the propagated antimicrobial culture is useful for the treatment of a variety of human, animal and plant diseases or conditions such as bacterial disease or conditions. [17] In some embodiments, the propagated antimicrobial culture is useful for treating diseases or conditions associated with bacterial pathogens such as for example E.coli and Helicobacter pylori.

BRIEF DESCRIPTION OF THE DRAWINGS

[18] FIG. 1 illustrates an antibacterial activity of L. bulgaricus GLB44 against H. pylori - Inhibition Zones vs Fermentation Time.

DETAILED DESCRIPTION OF THE INVENTION

[19] Disclosed herein is a method of propagating an antimicrobial culture where incubating a microorganism in the presence of an optimised amount of microbial lysate under suitable conditions leads to enhanced levels of antimicrobial metabolites in the propagated culture.

[20] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this disclosure pertains.

[21] According to one aspect, there is provided a method for propagating an antimicrobial culture, the method comprising the steps of:

a. inoculating an aqueous growth medium with an inoculum comprising a microorganism to produce an inoculated medium containing between about 10 ² -5xl0 ¹⁰ viable cells/ml;

b. mixing the inoculated medium with a microorganism lysate to produce a mixture;

c. incubating the mixture at a first incubation temperature (ITc);

d. cooling the mixture of step c. to a second incubation temperature (2Tc) that is at least about 0.5°C below the first incubation temperature (ITc) and incubating the cooled mixture at the second incubation temperature to produce a propagated antimicrobial culture; and optionally

e. collecting the propagated antimicrobial culture.

[22] There is also provided a method for propagation of an antimicrobial culture, the method comprising the steps of:

f. adding to a largescale aqueous growth medium a propagated antimicrobial culture according to claim 1 as an inoculum; g. incubating the inoculated medium at a first incubation temperature (1TC) to produce a propagated antimicrobial culture; and optionally

h. collecting the propagated antimicrobial culture.

[23] As used herein, certain terms may have the following defined meanings.

[24] As used herein, the singular terms "a" and "the" are synonymous and can be used interchangeably with "one or more" and "at least one," unless the language and/or context clearly indicates otherwise. For example, the term“a cell” includes a single cell as well as a plurality of cells, including mixtures thereof.

[25] As used herein, the term "comprising" means including, made up of, composed of encompass, consist of, constitute and incorporate.

[26] All numbers or numerals as used herein that indicate amounts, ratios of materials, physical properties of materials, and/or use are to be understood as modified or qualified by the term "about," except as otherwise explicitly indicated.

[27] As used herein, the term "about" includes the recited number or number and +/- 10% from the recited numeral or number. By way of non-limiting example, the term "about ten (10)" would encompass nine (9) to eleven (11) or 9-11.

[28] As used herein the term“antimicrobial” refers to destroying or inhibiting the growth of microorganisms and especially pathogenic microorganisms such as pathogenic bacteria.

[29] As used herein, the term“propagating an antimicrobial culture” means a method of multiplying or increasing the numbers of microorganisms such as bacteria by letting them reproduce, grow or proliferate in predetermined culture medium such as aqueous medium, under controlled incubation conditions. The incubation conditions would vary between different microorganisms and these would be known to the skilled person and adapted as required in order to perform the methods of the present invention.

[30] In some embodiments the propagated antimicrobial culture obtained by the method of the present invention is lyophilised. In some embodiments the propagated antimicrobial culture obtained by the method of the present invention is freeze dried.

[31] In some embodiments the inoculum is lyophilised. [32] In some embodiments the inoculum is freeze dried.

[33] In some embodiments the propagated antimicrobial culture obtained by the method of the present invention is collected for further processing. In some embodiments the collected antimicrobial culture is lyophilised. In some embodiments the collected antimicrobial culture is freeze dried.

[34] In some embodiments the inoculum is collected for further processing. In some embodiments the collected inoculum is lyophilised. In some embodiments the collected inoculum is freeze dried.

[35] The term "aqueous medium" as used herein refers to made from, with or by water growth medium or propagation medium, that supports the growth of the microorganisms.

[36] In some embodiments the aqueous medium is inoculated with microorganisms. The term“inoculated” as used herein refers to introducing or adding microorganisms, cells, lysate or combinations thereof, into the aqueous medium.

[37] In some embodiments, the aqueous medium employed in the present method typically contains at least 70wt.% water. More preferably, the aqueous culture medium contains at least 80 wt.%, most preferably 90 wt.% water. Besides water, the aqueous culture medium contains a carbon and nitrogen source and optionally any other ingredients needed by the organisms to grow, such as salts providing essential elements such as magnesium, phosphorus and sulphur.

[38] In some embodiments, the aqueous medium is animal milk or dairy milk. In some embodiments the animal milk or dairy milk can be derived from a cow, a sheep, a goat, a camel, a reindeer, a water buffalo, a yak, a horse, a donkey etc.

[39] In some embodiments, the aqueous medium is non-animal milk or non-dairy milk. In some embodiments the non-animal milk or non-dairy milk can be derived from almonds, soya, rice, cashew, coconut, oats, flax, hemp etc.

[40] In some embodiments the aqueous medium is substantially free of animal or dairy products or by-products.

[41] As used herein the term“animal products” means any material derived from the body of an animal. Animal products include milk, eggs, fat, flesh, blood, fish, crustacean shellfish etc.. As used herein the term“animal by-product” is a product harvested or manufactured from livestock other than muscle meat.

[42] As used herein, the term "substantially free" means that the content is sufficiently low or negligent that no appreciable danger to humans will result from contact with the cultures described herein. In some embodiments, the aqueous medium is plant medium.

[43] In some embodiments, the plant medium can be juice or extract from an acai, an agave, an almond, an aloe, an apple, an apricot, an arugula, an avocado, a beet, a bell pepper, a blackberry, a blue green algae, a blueberry, a carrot, a cayenne, a celery, a chia, a cilantro, a clove, a coconut, a cucumber, a dandelion, a date, a fennel, a garlic, a ginger, a ginkgo, a grapefruit, a guayusa, a hemp, a jalapeno pepper, a kale, a kiwi, a lemon, a lemon grass, a lime, a maca, a mandarin, an onion, an orange, a parsley, a peach, a pear, a pineapple, a raspberry, a spearmint, a spinach, a spirulina, a strawberry, a sweet potato, a tomato, a turmeric root, a watermelon, or a wheatgrass or the plant medium is soya milk, rice milk, almond milk, coffee, flax oil, herbal tea, or maple syrup or honey.

[44] In some embodiments the plant medium comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the aqueous growth medium.

[45] In some embodiments the aqueous medium is a vegetable medium. In some embodiments the vegetable medium can be juice or extract from carrot, peas, tomato peals, artichoke, broccoli, broccoli blowers, caper, cauliflower, asparagus, beetroot, canna, cassava, ginger, parsnip, yam or turmeric.

[46] In some embodiments the vegetable medium comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the aqueous growth medium.

[47] In some embodiments the vegetable medium comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% carrot juice. [48] In some embodiment the aqueous growth medium comprises a combination of one or more plant based medium and one or more vegetable based medium at different ratios.

[49] In some embodiments the aqueous growth medium can be about 50-50 ratio of carrot juice and peas juice. In some embodiments, the aqueous medium can be 50-50 ratio of spirulina extract and carrot juice. In some embodiments the aqueous growth medium can be 50-50 ratio of tomato peal extract and carrot juice.

[50] In some embodiments the aqueous medium is a vegan medium. As used herein the term “vegan” means containing no components (such as meat, eggs, or dairy products) that come from animals or derived from animal products.

[51] As used herein, the term“inoculum” means a microbial material or cell culture which is added to some other material or substance, such as aqueous growth medium. In some embodiments the inoculum comprises live or viable microorganism cells. In some embodiment, the inoculum employed in the present method comprises at least one microorganism. The microorganism that can be employed can include prokaryote or eukaryote. The microorganism can be selected from viruses, bacteria or fungi.

[52] Preferably the microorganism is selected from bacteria. The bacteria kingdom consists of over 70,000 species and each carry very different characteristics. The microorganism that are propagated using the present method can be sampled from, for instance, complex cultures for food or feed fermentation, mixed cultures for bioprotection, complex probiotics, from microbiota.

[53] In some embodiments the inoculum contains between about 10 ² -5xl0 ¹⁰ viable cells/ml or more. According to some embodiments the inoculum can be produced using microfluidic systems.

[54] In some embodiments the inoculum employed in the present method comprises lactic acid bacteria.

[55] Probiotics usually involve one of three genuses of bacteria: Lactobacillus, Bifidobacteria, and Streptococcus. In each of these three genuses of bacteria, there are multiple species. [56] The genus Lactobacillus contains over 180 species. In some embodiments, the Lactobacilli are of the species L. acidophilus, L. brevis, L. buchneri, L. casei, L. curvatus, L. delbrueckii, L. fermentum, L. helveticus, L. plantarum, L. reuteri, L. sakei, or L. salivarius.

[57] In some embodiments, the Lactobacilli are from the species L. delbrueckii. In some embodiments, the Lactobacilli is Lactobacilli delbrueckii bulgaricus, Lactobacilli delbrueckii lactis, Lactobacilli delbrueckii delbrueckii, or Lactobacilli delbrueckii indicus. In some embodiments, the Lactobacilli is Lactobacilli delbrueckii bulgaricus.

[58] In some embodiments, the microorganism is Lactobacillus plantarum GLP3.

[59] In some embodiments, the microorganism is Lactobacillus delbrueckii bulgaricus strain GLB 44.

[60] In some embodiments, the Lactobacilli is Lactobacillus delbrueckii bulgaricus strain GLB 44. Lactobacillus delbrueckii subsp. bulgaricus GLB 44, has been deposited with the National Bank for Industrial Microorganisms and Cell Cultures in Sofia, Bulgaria on April 17, 2014, Accession Number NBIMCC No. 8814.

[61] Different strains of Lactobacillus delbrueckii subsp. bulgaricus have different morphologies. By way of example, some are straight, some are curved, some a single cell, some are in pairs or lines of three. The cell morphology of Lactobacillus delbrueckii subsp. bulgaricus GLB 44 is long with an average length of about 14 pm to about 16 pm. Lactobacillus delbrueckii subsp. bulgaricus GLB 44 also commonly occurs in short chains of two or three straight rods that are attached together.

[62] In some embodiments, Lactobacillus delbrueckii subsp. bulgaricus can be used as bacteria in the present invention. Lactobacillus delbrueckii subsp. bulgaricus is found naturally on four different plants in Bulgaria: Cornus mas (Cornelian cherry), Galanthus nivalis (snowdrop), Calendula officinalis (common marigold), and Prunus spinosa (black thorn).

[63] In some embodiments, the inoculated aqueous medium contains between about 10 ² - 5x10 ¹⁰ viable cells/ml. In some embodiments, the inoculated aqueous medium contains between about 10 ² -5xl0 ⁹ viable cells/ml. In some embodiments, the inoculated aqueous medium contains between about 10 ² -5xl0 ⁸ viable cells/ml. In some embodiments, the inoculated aqueous medium contains between about 10 ² -5xl0 ⁷ viable cells/ml. In some embodiments, the inoculated aqueous medium contains between about 10 ² -5xl0 ⁶ viable cells/ml. In some embodiments, the inoculated aqueous medium contains between about 10 ² -5xl0 ⁵ viable cells/ml.

[64] In some embodiments, the inoculated aqueous medium contains about 10 ¹⁰ viable cells/ml. In some embodiments, the inoculated aqueous medium contains about 10 ⁹ viable cells/ml. In some embodiments, the inoculated aqueous medium contains about 10 ⁸ viable cells/ml. In some embodiments, the inoculated aqueous medium contains about 10 ⁷ viable cells/ml. In some embodiments, the inoculated aqueous medium contains about 10 ⁶ viable cells/ml. In some embodiments, the inoculated aqueous medium contains about 10 ⁵ viable cells/ml.

[65] The skilled person would know that viability of microbial cells can be assessed or estimated using colony-forming units per millilitre (CFU/mL) in case of a liquid being tested or grams (CFU/g) if a solid material is tested.

[66] If the propagated antibacterial culture is a liquid, the concentration of the viable cells can be estimated via colony-forming units per millilitre (CFU/mL or growth medium). In some embodiments, the concentration of the viable cells in the composition is from 0.5 million to 1 billion CFU/mL, 0.5 million to 500 million CFU/mL, 0.5 million to 400 million CFU/mL, 0.5 million to 300 million CFU/mL, 0.5 million to 200 million CFU/mL, 0.5 million to 150 million CFU/mL, 0.5 million to 125 million CFU/mL, 0.5 million to 100 million CFU/mL, 0.5 million to 75 million CFU/mL, 0.5 million to 50 million CFU/mL, 0.5 million to 10 million CFU/mL, 0.5 million to 5 million CFU/mL, 0.5 million to 1 million CFU/mL, 1 million to 1 billion CFU/mL, 1 million to 500 million CFU/mL, 1 million to 400 million CFU/mL, 1 million to 300 million CFU/mL, 1 million to 200 million CFU/mL, 1 million to 150 million CFU/mL, 1 million to 125 million CFU/mL, 1 million to 100 million CFU/mL, 1 million to 75 million CFU/mL, 1 million to 50 million CFU/mL, 1 million to 10 million CFU/mL, 1 million to 5 million CFU/mL, 5 million to 1 billion CFU/mL, 5 million to 500 million CFU/mL, 5 million to 400 million CFU/mL, 5 million to 300 million CFU/mL, 5 million to 200 million CFU/mL, 5 million to 150 million CFU/mL, 5 million to 125 million CFU/mL, 5 million to 100 million CFU/mL, 5 million to 75 million CFU/mL, 5 million to 50 million CFU/mL, 5 million to 10 million CFU/mL, 10 million to 1 billion CFU/mL, 10 million to 500 million CFU/mL, 10 million to 400 million CFU/mL, 10 million to 300 million CFU/mL, 10 million to 200 million CFU/mL, 10 million to 150 million CFU/mL, 10 million to 125 million CFU/mL, 10 million to 100 million CFU/mL, 10 million to 75 million CFU/mL, 10 million to 50 million CFU/mL, 50 million to 1 billion CFU/mL, 50 million to 500 million CFU/mL, 50 million to 400 million CFU/mL, 50 million to 300 million CFU/mL, 50 million to 200 million CFU/mL, 50 million to 150 million CFU/mL, 50 million to 125 million CFU/mL, 50 million to 100 million CFU/mL, 50 million to 75 million CFU/mL, 100 million to 1 billion CFU/mL, 100 million to 500 million CFU/mL, 100 million to 400 million CFU/mL, 100 million to 300 million CFU/mL, 100 million to 200 million CFU/mL, 100 million to 150 million CFU/mL, 100 million to 125 million CFU/mL, 125 million to 1 billion CFU/mL, 125 million to 500 million CFU/mL, 125 million to 400 million CFU/mL, 125 million to 300 million CFU/mL, 125 million to 200 million CFU/mL, 125 million to 150 million CFU/mL, 150 million to 1 billion CFU/mL, 150 million to 500 million CFU/mL, 150 million to 400 million CFU/mL, 150 million to 300 million CFU/mL, 150 million to 200 million CFU/mL, 200 million to 1 billion CFU/mL, 200 million to 500 million CFU/mL, 200 million to 400 million CFU/mL, 200 million to 300 million CFU/mL, 300 million to 1 billion CFU/mL, 300 million to 500 million CFU/mL, 300 million to 400 million CFU/mL, 400 million to 1 billion CFU/mL, 400 million to 500 million CFU/mL, or 500 million to 1 billion CFU/mL or more such as 10 billion CFU/mL

[67] If the propagated antimicrobial culture is a solid such as a propagated antimicrobial agent, the concentration of the viable cells can be estimated via colony-forming units per gram (CFU/g). In some embodiments, the concentration of the propagated antimicrobial culture such as a propagated antimicrobial agent is from 0.5 million to 1 billion CFU/g, 0.5 million to 500 million CFU/g, 0.5 million to 400 million CFU/g, 0.5 million to 300 million CFU/g, 0.5 million to 200 million CFU/g, 0.5 million to 150 million CFU/g, 0.5 million to 125 million CFU/g, 0.5 million to 100 million CFU/g, 0.5 million to 75 million CFU/g, 0.5 million to 50 million CFU/g, 0.5 million to 10 million CFU/g, 0.5 million to 5 million CFU/g, 0.5 million to 1 million CFU/g, 1 million to 1 billion CFU/g, 1 million to 500 million CFU/g, 1 million to 400 million CFU/g, 1 million to 300 million CFU/g, 1 million to 200 million CFU/g, 1 million to 150 million CFU/g, 1 million to 125 million CFU/g, 1 million to 100 million CFU/g, 1 million to 75 million CFU/g, 1 million to 50 million CFU/g, 1 million to 10 million CFU/g, 1 million to 5 million CFU/g, 5 million to 1 billion CFU/g, 5 million to 500 million CFU/g, 5 million to 400 million CFU/g, 5 million to 300 million CFU/g, 5 million to 200 million CFU/g, 5 million to 150 million CFU/g, 5 million to 125 million CFU/g, 5 million to 100 million CFU/g, 5 million to 75 million CFU/g, 5 million to 50 million CFU/g, 5 million to 10 million CFU/g, 10 million to 1 billion CFU/g, 10 million to 500 million CFU/g, 10 million to 400 million CFU/g, 10 million to 300 million CFU/g, 10 million to 200 million CFU/g, 10 million to 150 million CFU/g, 10 million to 125 million CFU/g, 10 million to 100 million CFU/g, 10 million to 75 million CFU/g, 10 million to 50 million CFU/g, 50 million to 1 billion CFU/g, 50 million to 500 million CFU/g, 50 million to 400 million CFU/g, 50 million to 300 million CFU/g, 50 million to 200 million CFU/g, 50 million to 150 million CFU/g, 50 million to 125 million CFU/g, 50 million to 100 million CFU/g, 50 million to 75 million CFU/g, 100 million to 1 billion CFU/g, 100 million to 500 million CFU/g, 100 million to 400 million CFU/g, 100 million to 300 million CFU/g, 100 million to 200 million CFU/g, 100 million to 150 million CFU/g, 100 million to 125 million CFU/g, 125 million to 1 billion CFU/g, 125 million to 500 million CFU/g, 125 million to 400 million CFU/g, 125 million to 300 million CFU/g, 125 million to 200 million CFU/g, 125 million to 150 million CFU/g, 150 million to 1 billion CFU/g, 150 million to 500 million CFU/g, 150 million to 400 million CFU/g, 150 million to 300 million CFU/g, 150 million to 200 million CFU/g, 200 million to 1 billion CFU/g, 200 million to 500 million CFU/g, 200 million to 400 million CFU/g, 200 million to 300 million CFU/g, 300 million to 1 billion CFU/g, 300 million to 500 million CFU/g, 300 million to 400 million CFU/g, 400 million to 1 billion CFU/g, 400 million to 500 million CFU/g, or 500 million to 1 billion CFU/g, or more such as 10 billion CFU/ml.

Microorganism lysate

[68] The term“microorganism lysate” or“lysate” as used herein, means a mixture of microbial antigens, peptides or metabolites derived from different inactivated, disrupted or disintegrated microbes. Antigens, peptides or metabolites are obtained by either chemical or mechanical lysis of microorganisms and their extract collected from culturing microbial strains. In some embodiment, the microorganism lysate employed in the present method is obtained from at least one microorganism. The microorganism lysate that can be employed can include prokaryote or eukaryote. The microorganism lysate can be derived from viruses, bacteria or fungi. In some embodiments the lysate is derived from bacteria. In some embodiments the lysate is derived from lactic acid bacteria.

[69] In some embodiments the lysate is derived from lactic acid bacteria which belong to the genus Lactobacillus. In some embodiments, the lysate is derived from L. acidophilus, L. brevis, L. buchneri, L. casei, L. curvatus, L. delbrueckii, L. fermentum, L. helveticus, L. plantarum, L. reuteri, L. sakei, or L. salivarius. [70] In some embodiments, the lysate is derived from L. delbrueckii. In some embodiments, the lysate is derived from Lactobacilli delbrueckii bulgaricus, Lactobacilli delbrueckii lactis, Lactobacilli delbrueckii delbrueckii, or Lactobacilli delbrueckii indicus. In some embodiments, the lysate is derived from Lactobacilli delbrueckii bulgaricus.

[71] In some embodiments, the lysate is derived from Lactobacillus plantarum GLP3.

[72] In some embodiments, the lysate is derived from Lactobacillus delbrueckii bulgaricus strain GLB 44.

[73] In some embodiments, the microorganism lysate comprises about 0.1% v/v of the growth medium, preferably about 0.2% of the growth medium, preferably about 0.4% of the growth medium, preferably about 0.8% of the growth medium, preferably about 1% of the growth medium, preferably about 1.2% of the growth medium, preferably about 1.4% of the growth medium, preferably about 1.8% of the growth medium, preferably about 2% of the growth medium, preferably about 3% of the growth medium, preferably about 4% of the growth medium, preferably about 5% of the growth medium, preferably about 6% of the growth medium, preferably about 7% of the growth medium, preferably about 8% of the growth medium, preferably about 9% of the growth medium, preferably about 10% of the growth medium, preferably about 11% of the growth medium, preferably about 12% of the growth medium, preferably about 13% of the growth medium, preferably about 14% of the growth medium, preferably about 15% of the growth medium or more.

[74] In some embodiments, the microorganism lysate comprises about 2% of the growth medium.

[75] In some embodiments, the microorganism lysate is derived from the same microorganism which is propagated.

[76] In some embodiments, the microorganism lysate is derived from an altogether different microorganism than the microorganism which is propagated. In some embodiments the microorganism lysate comprises a pool of lysates derived from different microorganisms. In some embodiments the microorganism lysate comprises a pool of different microorganism lysates where at least one of the pooled microorganisms is propagated. [77] It is believed that the addition of microbial lysate in the aqueous growth medium contributed to the release of antimicrobial metabolites by the propagated microorganisms. It is believed that the addition of microbial lysate in the aqueous growth medium enhances the release of antimicrobial metabolites by the propagated microorganisms.

Incubation apparatus and systems

[78] In order to try and address some of the limitations of the current systems, for example speed of growth of the microorganism and quorum effect, the methods described herein can employ suitably adapted incubation apparatus and techniques which utilise different types and forms of physical forces, such as for example pseudo forces. The different types and forms of physical forces, such as for example pseudo forces can be generated for example as a result of a rotating platform resulting in increased advection and chaotic mixing of microorganism, e.g. bacteria, comprising aqueous growth medium and lysate. In some embodiments, gas permeable membranes covering microorganism culture chambers (e.g., microfluidic systems) within which the cultures are enclosed to allow for passive, yet effective aeration of the sample and thus optimal antimicrobial metabolite production.

[79] Without wishing to be bound by theory, in a rotating incubation platform system, Euler pseudo force (which is perpendicular to centrifugal pseudo force), may be used to generate vortical flow and provide uniform mixing within for example a microfluidic chamber of a microfluidic system. Euler pseudo forces are inertial forces that are produced when a microfluidic system experiences cycles of unidirectional acceleration-and-deceleration rotation. Thus, mixing is dependent on chamber geometry, acceleration/deceleration rate, and angular spin.

[80] For liquid microbial cultures, such as bacterial cultures, rapid and healthy growth, depends on factors including (1) aeration, so that bacteria have access to fresh oxygen for growth, (2) nutrient availability, where samples are thoroughly mixed to provide nutrients homogenously throughout the culture, (3) minimization of biofilms and clumping, where shaking and agitation prevents bacteria culture from settling to the bottom of a chamber and forming biofilms or clumps that hinder reproduction, and (4) optimal interaction of cultured bacteria with metabolites or antigens of the lysate which leads to an enhanced expression of antimicrobial metabolites by the cultured bacteria. [81] Microfluidic systems having low Reynolds numbers exhibit laminar flow regimes, which are dominated by viscous, rather than inertial forces. Thus, without turbulent mixing, microfluidic devices must rely on either passive molecular diffusion or external energy sources. In some embodiments, the microfluidic system accelerates the growth rate or proliferation rate of the cultured microorganism compared to the same microorganism incubated using standard laboratory equipment. In some embodiments, proliferation rate of the microorganism can be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% faster than the proliferation rate of the same microorganism incubated using standard laboratory equipment.

[82] In some embodiment the antimicrobial culture is propagated using a microfluidic system. In some embodiment the inoculum is produced using a microfluidic system.

[83] In some embodiments the method relies on propagating antimicrobial cultures in a commercial or largescale manufacturing facilities. In some embodiments, the method can rely on fermentation systems consisting of large fermentation tanks, bioreactors and large-scale purification systems.

[84] As used herein the term“largescale” is synonymous with“commercial scale” and means about 5 to 500L or more of total antimicrobial culture.

Methods of incubating a Microorganism culture

[85] In some embodiments, incubating the microorganism culture is conducted at temperature such as first incubation temperature (ITc) or second incubation temperature (2Tc) in the range of 20 °C to 60 °C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of 30°C to 50°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of between about 35-39°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of preferably between about 35.5-38.5°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of preferably between about 36- 38°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of preferably between about 36.5-38.25°C. In some embodiments, incubating the microorganism is conducted at a temperature in the range of preferably between about 37-38°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the about 38°C.

[86] In some embodiments, the microorganism mixture is incubated at the first incubation temperature (ITc) for at least about 30 mins, preferably at least about 45 mins, preferably at least about 1 hour, preferably at least about 1.5 hours, preferably at least about 2 hours, preferably at least about 2.5 hours, preferably at least about 3 hours, preferably at least bout 4 hours, preferably at least about 5 hours, preferably at least about 6 hours, preferably at least about 6.5 hours, preferably at least about 7 hours, preferably at least about 7.5 hours, preferably at least about 8 hours, preferably at least about 8.5 hours, preferably at least about 9 hours, preferably at least about

9.5 hours, preferably at least about 10 hours, preferably at least about 10.5 hours, preferably at least about 11 hours, preferably at least about 11.5 hours, preferably at least about 12 hours, preferably at least about 12.5 hours, preferably at least about 13 hours, preferably at least about

13.5 hours, preferably at least about 14 hours or more.

[87] In one embodiment the mixture is incubated at the first incubation temperature (ITc) for 12 hours.

[88] One of the steps of the method described herein involves cooling of a microorganism culture. In some embodiments the cooling is carried out on a microbial culture incubated under first incubation temperature (ITc). In some preferred embodiments the cooling is carried on an antimicrobial culture incubated under the first incubation temperature (ITc) as per step c. of the method.

[89] According to the method described herein, a step of cooling of a microorganism culture to a second incubation temperature (2Tc) before further incubation is carried out. In some embodiments the cooling to a second incubation temperature (2Tc) is carried out on a microbial culture that was previously incubated under a first incubation temperature (ITc) before any further incubation at the second incubation temperature (2Tc). In some embodiments the cooling is carried on an antimicrobial culture incubated under a first incubation temperature (ITc) as per step c. of the method to a second incubation temperature (2Tc) before further incubation at the second incubation temperature (2Tc). [90] The second incubation temperature (2T c) is between 3°C-7°C below the first incubation temperature ITc. In some embodiments the second incubation temperature of 2Tc is between 3°C- 7°C less than the first incubation temperature ITc.

[91] In some embodiments, the second incubation temperature (2Tc) is at least about 0.6°C below the first incubation temperature (ITc), preferably at least about 0.75°C below the first incubation temperature (ITc), preferably at least about 1 °C below the first incubation temperature (ITc), preferably at least about 1 25°C below the first incubation temperature (ITc), preferably at least about 1.5°C below the first incubation temperature (ITc), preferably at least about 1.75°C below the first incubation temperature (ITc), preferably at least about 2°C below the first incubation temperature (ITc), preferably at least about 2.25°C below the first incubation temperature (ITc), preferably at least about 2.5°C below the first incubation temperature (ITc), preferably at least about 2.75°C below the first incubation temperature (ITc), preferably at least about 3°C below the first incubation temperature (ITc), preferably at least about 3.25°C below the first incubation temperature (ITc), preferably at least about 3.5°C below the first incubation temperature (ITc), preferably at least about 3.75°C below the first incubation temperature (ITc), preferably at least about 4°C below the first incubation temperature (ITc), preferably at least about 4.25°C below the first incubation temperature (ITc), preferably at least about 4.5°C below the first incubation temperature (ITc), preferably at least about 4.75°C below the first incubation temperature (ITc), preferably at least about 5°C below the first incubation temperature (ITc), preferably at least about 5.25°C below the first incubation temperature (ITc), preferably at least about 5.5°C below the first incubation temperature (ITc), preferably at least about 5.75°C below the first incubation temperature (ITc), preferably at least about 6°C below the first incubation temperature (ITc), preferably at least about 6.25°C below the first incubation temperature (ITc), preferably at least about 6.5°C below the first incubation temperature (ITc), preferably at least about 6.75°C below the first incubation temperature (ITc), preferably at least about 7°C below the first incubation temperature (ITc) or more.

[92] In some embodiments, the microorganism mixture is incubated at 2Tc before it is incubated at ITc.

[93] In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 30 mins. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 45 mins. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 1 hour. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 1.5 hours. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 2 hours, preferably about 2.5 hours. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 3 hours. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 3.5 hours. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 4 hours. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 4.5 hours. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 5 hours. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 5.5 hours or more.

[94] According to a preferred embodiment, the microorganism mixture is incubated at the second incubation temperature (2Tc) for 2 hours.

[95] According to a preferred embodiment the Lactobacillus delbrueckii bulgaricus strain GLB 44 mixture is incubated at the first incubation temperature (ITc) at about 38°C for about 12 hours and the cooled mixture is incubated at the second incubation temperature (2Tc) at about 33°C for about 2 hours.

Quorum sensing

[96] Without wishing to be bound by theory, quorum sensing is generally considered to represent a response to fluctuations in cell-population density. Quorum sensing microorganisms for instance bacteria, produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Microorganism such as Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers, and Gram- positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species.

[97] It is believed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, particularly bacteria such as lactic acid bacteria, due to the metabolic stress they experience during propagation, the microorganisms release increased levels of different antimicrobial metabolites. In particular, when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, particularly bacteria such as lactic acid bacteria, due to the combination of the metabolic stress and quorum sensing capabilities of the propagated microorganisms, the microorganisms release high levels of different antimicrobial metabolites.

[98] By exposing the microorganism cultures such as for example bacterial or lactic acid bacterial cultures to a combination of stress propagation conditions such as suboptimal incubation temperature (2Tc) after incubation at optimal temperature (ITc) and in the presence of microbial lysate, can lead to antimicrobial metabolite maximisation of the propagated antimicrobial culture.

[99] In some embodiments, exposing the microorganism cultures such as bacterial or lactic acid bacterial cultures to a combination of stress propagation conditions such as suboptimal incubation temperature (2Tc) and in the presence of microbial lysate, can lead to antimicrobial metabolite maximisation of the propagated antimicrobial culture. In some embodiments the propagated antimicrobial culture can be used as an antimicrobial inoculum for large scale aqueous growth medium inoculation.

[100] In some embodiments, exposing a microorganism cultures such as for example bacterial or lactic acid bacterial cultures comprising a largescale aqueous growth medium and an antimicrobial inoculum to a combination of stress propagation conditions such as suboptimal incubation temperature (2Tc) after incubation at optimal temperature (ITc) can lead to antimicrobial metabolite maximisation of the propagated antimicrobial culture.

[101] It would be understood by the skilled person that when antimicrobial metabolites levels in the antimicrobial cultures reach a certain desirable concentration or amount, as illustrated for example in FIG. 1, the growth or propagation of the microbial culture has to be substantially reduced or stopped completely. Different methods of substantially reducing or stopping the growth of microbial cultures and these would be well known to the skilled person. By way of non-limiting examples of methods of substantially reducing or stopping the growth of microbial cultures include rapid cooling down of the propagated antimicrobial culture, freeze drying of the propagated antimicrobial culture and others.

[102] In some embodiments, the growth rate or propagation rate of the microbial culture is substantially reduced or stopped when a desirable level of antimicrobial metabolites is reached.

[103] A desirable level of antimicrobial metabolites can depend on the preferred subsequent uses of the antimicrobial culture.

[104] In some embodiments the desirable level of antimicrobial metabolites is at the peak of log phase.

[105] In some embodiments the growth or propagation of the antimicrobial culture is substantially reduced or stopped upon reaching the peak of log phase as shown in FIG. 1.

[106] In some embodiments the antimicrobial metabolites can be toxic to pathogenic microorganisms such as bacterial pathogens. As used herein the term“toxic” refers to a toxin or other microbial substance or metabolite which induces an immune response especially the production of antibodies.

[107] In various embodiments, the propagated antimicrobial culture can be useful for the treatment of mammalian in particular human diseases caused by microorganisms such as bacteria through the inhibition of the bacterial quorum sensing cascade rendering the pathogen avirulent. Such diseases include endocarditis, respiratory and pulmonary infections (preferably in immunocompromised patients), bacteraemia, skin conditions, vagina, colon, central nervous system infections, ear infections including external otitis, eye infections, bone and joint infections, urinary tract infections, gastrointestinal infections and skin and soft tissue infections including wound infections, pyoderma and atopic dermatitis which all can be triggered by Helicobacter pylori or E. coli.

Antimicrobial cultures and uses thereof

[108] In general, the present invention provides a method for producing propagated antimicrobial cultures which can be used in reducing the virulence of bacterial pathogens. It is clear to persons skilled in the art, that the propagated antimicrobial culture obtained by the present methods can be applied in a wide variety of different fields such as environmental, industrial and medical applications in order to prevent and/or treat damages or diseases caused by bacteria. In some embodiments the propagated antimicrobial culture obtained according to the present methods can be used in meat preservation, food and feed preservation, cosmetics as skin anti-microbial, nutraceuticals, dietary supplement and pharmaceuticals.

[109] In some embodiments, the propagated antimicrobial culture can be used as an antimicrobial agent.

[110] In some embodiments the propagated antimicrobial culture can be used as an antibacterial agent.

[111] In some embodiment the antimicrobial agent can be used for topical cleaning and treatment solutions such as disinfectants, detergents, household cleaner and washing powder formulations in the form of a spray or a dispensable liquid. In a preferred form, these solutions can be applied to windows, containers, floors, clothes, kitchen and bathroom surfaces and other surfaces in the area of food preparation and personal hygiene. In addition, the propagated antimicrobial culture can be used as antibacterial ingredients in personal hygiene articles, toiletries and cosmetics such as dentifrices, mouthwashes, soaps, shampoos, shower gels, ointments, creams, lotions, nebulisers, deodorants and disinfectants and storage solutions for contact lenses, mouthwash, beverages and animal feed.

[112] In some embodiments the propagated antimicrobial culture is lyophilised. As used herein the term“lyophilised” means preserving the propagated antimicrobial culture by freezing it very quickly and then subjecting it to a vacuum or sublimation to remove the ice. In some embodiments, the lyophilised antimicrobial culture is preserved long-term. In some embodiment the lyophilised antimicrobial culture comprises viable microbial cells. In some embodiment, lyophilisation can be used to prepare a dosage form that is to be reconstituted for injection.

[113] In some embodiments the lyophilised antimicrobial culture is a lyophilised antimicrobial agent.

[114] In some embodiments the lyophilised antimicrobial culture is a lyophilised antibacterial agent. [115] In some embodiments there is provided a propagated antimicrobial culture combined with one or more pharmaceutically acceptable ingredients. In some embodiments there is provided a propagated antimicrobial culture combined with one or more pharmaceutically acceptable excipients or additive.

[116] In the present context, a "pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the propagated antimicrobial culture, for example a lyophilised culture. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatine, vegetable oils and polyethylene glycols.

[117] In some embodiments the pharmaceutical compositions comprise the antimicrobial agent. In some embodiments the pharmaceutical compositions comprised the antibacterial agent.

[118] Thus, the present invention also relates to compositions including pharmaceutical compositions comprising a therapeutically effective amount of a propagated antimicrobial culture, for example a lyophilised culture, as mentioned herein. As used herein a propagated antimicrobial culture, for example a lyophilised culture, will be therapeutically effective if it is able to affect the target microorganism concentration.

[119] Preferably, a propagated antimicrobial culture, for example a lyophilised culture or agent, will be therapeutically effective if it is able to affect the target microorganism concentration where it is able to treat or prevent a microorganism related disease such as bacteria- related disease or disorder in a subject after the propagated antimicrobial culture, for example a lyophilised culture, has been administered to a subject. In some embodiments the disease or disorder is associated with a pathogen. In some embodiments the pathogen is a Salmonella Enterica serotype. In some embodiments, the pathogen is Salmonella typhimurium, Salmonella enteritidis, Salmonella newport, Salmonella hadar, Salmonella oranienburg, Salmonella javiana, Salmonella saintpaul, Salmonella muenchen, Salmonella agona, Salmonella I monophasic, Salmonella montevideo, or Salmonella paratyphi. In some embodiments, the pathogen is Salmonella typhimurium. In some embodiments, the pathogen is Salmonella enteritidis.

[120] In some embodiments, the pathogen is Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cryptosporidium parvum, Escherichia coli, Giardia lamblia, Hepatitis A, Listeria monocytogenes, Norwalk virus, Staphylococcus, Shingella, Toxoplasma gondii, Vibrio, or Yersiniosis.

[121] In some embodiments, the pathogen is Helicobacter pylori.

[122] In some embodiments, the pathogen is Escherichia coli.

[123] In a further embodiment, the propagated antimicrobial culture, for example a lyophilised culture of the present invention can be administered directly to animals, preferably to mammals, and in particular to humans as antibiotics per se, as mixtures with one another or in the form of pharmaceutical preparations which allow enteral or parenteral use and which as active constituent contain an effective dose of the propagated antimicrobial culture, for example a lyophilised culture, in addition to customary pharmaceutical excipients and additives.

[124] As mentioned above, in addition to the propagated antimicrobial culture, the culture can contain further customary, usually inert carrier materials, additives or excipients. Thus, the culture can also contain additives or adjuvants commonly used for instance in galenic formulations, such as, e.g., fillers, extenders, disintegrants, binders, glidants, wetting agents, stabilizers, emulsifiers, preservatives, sweetening agents, colorants, flavourings or aromatisers, buffer substances, and furthermore solvents or solubilizers or agents for achieving a depot effect, as well as salts for modifying the osmotic pressure, coating agents or antioxidants. They can also contain two or more cultures and also other therapeutically active substances such as antivirals, antifungals or antibiotics.

[125] Thus, the cultures of the present invention can be used alone, in combination with other compounds of this invention or in combination with other active compounds, for example with active ingredients already known for the treatment of the afore mentioned diseases, whereby in the latter case a favourable additive effect is noticed.

[126] In another aspect, the present invention provides a process of preparing a product selected from food products, beverages, nutritional products, nutraceuticals and animal feed, the process comprising combining one or more ingredients with a propagated antimicrobial culture.

[127] In some embodiment, the propagated antimicrobial culture products can incorporate inert, inorganic or organic excipients. [128] In some embodiments, to prepare pills, tablets, coated tablets and hard gelatine capsules, e.g., lactose, com starch or derivatives thereof, talc, stearic acid or its salts, etc. can be used. Excipients for soft gelatine capsules and suppositories are, e.g., fats, waxes, semi-solid and liquid polyols, natural or hardened oils etc. Suitable excipients for the production of solutions and syrups are, e.g., water, alcohol, sucrose, invert sugar, glucose, polyols etc.

[129] The dose can vary within wide limits and is to be suited to the individual conditions in each individual case. For the above uses the appropriate dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired. In general, however, satisfactory results are achieved at daily dosage rates of about 0.1 to 100 mg/kg animal body weight preferably 1 to 50 mg/kg. Suitable dosage rates for larger mammals, e.g., humans, are of the order of from about 10 mg to 3 g/day, conveniently administered once, in divided doses 2 to 4 times a day, or in sustained release form.

[130] In general, a daily dose of approximately 0.1 mg to 5000 mg, preferably 10 to 500 mg, per mammalian in particular human individual is appropriate in the case of the oral administration which is the preferred form of administration according to the invention. In the case of other administration forms too, the daily dose is in similar ranges. The compounds of Formula I or Formula II can also be used in the form of a precursor (pro-drug) or a suitably modified form, that releases the active compound in vivo.

[131] In a further embodiment, the compounds of the present invention can be used as pharmacologically active components or ingredients of medical devices, instruments and articles with an effective dose of propagated antimicrobial culture, for example a lyophilised culture of the present invention. The amount of the cultures used to coat for example medical device surfaces varies to some extent with the coating method and the application field. In general, however, the concentration range from about 0.01 mg per cm ² to about 100 mg per cm. In a similar way the amount of the cultures has to be adjusted to the application mode if the cultures of the invention are used as components or ingredients in cleaning, treatment solutions or ointments. In general, effective dosages range from about 0.1 mM to about 1000 mM.

Reports and Data Transmission

[132] In some embodiments, the methods and systems disclosed herein further comprise generating one or more reports. In some embodiments, the methods disclosed herein further comprise storing one or more reports. In some embodiments, the methods disclosed herein further comprise transmitting one or more reports. In some embodiments, the report includes information on the capability of a microorganism to propagate and generate a propagated antimicrobial culture. In some embodiments, the report provides recommendations on a therapeutic regimen of the propagated antimicrobial culture as an antimicrobial agent. In some embodiments, the report provides recommendations on the dosage of a propagated antimicrobial culture as an antimicrobial agent.

[133] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples.

EXPERIMENTAL EXAMPLE

In vitro antimicrobial activity test - FIG. 1

[134] Vegan nutrient media: - 100% carrot juice.

[135] Lactobacillus strains used in the experiments: 1. Lactobacillus bulgaricus GLB44; 2. Lactobacillus bulgaricus GLB3; 3. Lactobacillus bulgaricus GLB27

[136] Cultivation of L. bulgaricus GLB44 - No Antimicrobial metabolite Maximization

[137] Preparing of the inoculum: One-step process

[138] Cultivation of L. bulgaricus GLB44 in the vegan nutrient media at 38°C for 22 hours to reach exponential phase. Then use only this live culture to inoculate the fermentation vegan media - 10% (v/v) of the final fermentation media.

Fermentation and lyophilization·.

[139] Cultivation of L. bulgaricus GLB44 in the vegan nutrient media at constant temperature of 38°C for 24 hours in bioreactor with no aeration.

[140] Taking samples after the 4th hour of the fermentation, every 2 hours. Neutralization to pH=5.90 and lyophilization of the taken culture samples.

[141] Cultivation of L. bulgaricus GLB44, L. bulgaricus GLB3 and L. bulgaricus GLB27 - with Antimicrobial metabolite maximization or expansion of the culture.

Preparing of a propagated antimicrobial culture inoculum: Two-step process [142] The first step of the inoculum cultivation lasts 12 hours at 38 degrees Celsius.

[143] The second step starts at the 12th hour when the exponential phase is at its peak, we drop the temperature from 38 to 33 degrees C for 2 hours to create a further stress to the bacterial culture. Ultrasonic disintegration of part of this sample.

[144] Then preparing an inoculation mix (10% (v/v) of the final fermentation media) - 8% GLB44 culture in exponential phase and 2% disintegrated GLB44 cells lysate.

Fermentation and lyophilization:

[145] Cultivation of the L. bulgaricus strains in bioreactor with no aeration on the vegan nutrient media for 12 hours at 38 degrees Celsius, then drop the temperature to 33°C for another 12 hours. Taking samples after the 4th hour of the fermentation, every 2 hours. Neutralization to pH=5.90 and lyophilization of the taken culture samples.

[146] Comparative study of the antimicrobial activity of L. bulgaricus samples against clinical isolate of H. Pylori.

L. bulsaricus samples from every test time point of the fermentation process

[147] 1. L. bulgaricus GLB44 No Antimicrobial Maximization: liquid sample - reconstituted lyophilized product (1 : 10, productsterile saline)

[148] 2. L. bulgaricus GLB44 Antimicrobial Maximization: liquid sample - reconstituted lyophilized product (1 : 10, productsterile saline)

[149] 3. L. bulgaricus GLB3 Antimicrobial Maximization: liquid sample - reconstituted lyophilized product (1 : 10, productsterile saline)

[150] 4. L. bulgaricus GLB18 Antimicrobial Maximization: liquid sample - reconstituted lyophilized product (1 : 10, productsterile saline)

[151] Helicobacter pylori - clinical isolate, culture in exponential phase of growth.

The test strain was cultivated under anaerobic conditions in Brucella broth with 5% Fetal bovine serum (B-FBS).

In vitro antibacterial test: [152] The test was done by agar well diffusion method [References 1, 2] The final concentration of Helicobacter pylori in the Brucella agar plates with hemin, vitamin K and 5% sheep blood (BMB) was 3x10 ⁵ CFU/ml.

[153] The L. bulgaricus samples were prepared as described above and were administered in a volume of 100 microliters (mΐ) in each well in the petri dishes in 3 replicates (3x).

[154] The agar plates were placed in Gas-Pak jars containing microaerophilic gas generating sachets and incubated at 37°C.

[155] The zones of inhibition were then measured after 120 hours of incubation.

CONCLUSION

[156] From our experiments we observed that when an inoculum is maximized with the use of microbial lysate such as deactivated bacteria and the temperature shock we observe the production of unexpectedly high levels of different antimicrobial metabolites. In addition, we observed that when using the maximised inoculum for inoculation of a largescale fermentation, a second temperature shock on the actual fermentation leads to unexpectedly high levels of different antimicrobial metabolites in the fermentation. Accordingly, the effects we observed are both the temperature shocks and the accumulation of the peptides from the inoculum to the main fermentation due to the quorum sensing. Together they form the cumulative effect in the main fermentation. Effectively, we believe we are creating bacteriocin in the inoculum, which then creates through quorum sensing higher levels of the same bacitracin in the largescale or final fermentation.

[157] The microbial growth or propagation in the final fermentation has to be stopped at the peak otherwise the levels of antimicrobial metabolites such as bacteriocin drop significantly because the propagating microorganisms reabsorb the metabolites and proceed to metabolise these as food. It is clear from FIG. 1 that the antimicrobial levels start dropping down after the peak of the log phase. By dramatically cooling down or freeze drying the propagated antimicrobial culture when the antimicrobial metabolites are at the peak it is possible to maximize the antimicrobial levels in the final solution/product.

[158] The disclosure illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms“comprising”,“including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.

* * *

REFERENCES:

[1] S. Magaldi, S. Mata-Essayag, C. Hartung de Capriles, et al., Well diffusion for antifungal susceptibility testing, Int. J. Infect. Dis. 8 (2004) 39-45.

2] C. Valgas, S.M. De Souza, E.F.A. Smania, et al., Screening methods to determine antibacterial activity of natural products, Braz. J. Microbiol. 38 (2007) 369-380.