BG112923A - Method for propagating antimicrobial cultures - Google Patents

Abstract

The present invention is related to the development of a method for 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 for the accumulation of antimicrobial metabolites. The present invention also relates to a propagated antimicrobial culture and its application as an antimicrobial agent thereof.

Description

METHOD FOR PREPARATION OF HA ANTIMICROBIAL CULTURES

[1] The present invention provides a method for propagating an antimicrobial culture. In particular, the present invention relates to a method for propagating an antimicrobial culture, comprising incubating a viable microorganism in the presence of a microbial lysate under suitable conditions that allows the accumulation of antimicrobial metabolites. The present invention also relates to propagated antimicrobial culture and its use as an antimicrobial agent.

INTRODUCTION

[2] Infectious diseases, including bacterial infections, continue to be a serious health problem worldwide. Probiotics are defined as "living micro-organisms which, when administered in sufficient quantities, are beneficial to the health of the host". In the last few years, the use of probiotics in humans has been the subject of increasing attention, as the amount of scientific evidence for the properties of probiotic bacteria, their functionality and their beneficial effects is growing. The drive to find more new probiotics is driven by the growing demand for functional probiotic foods and beverages, as well as nutritional supplements, due to the growing awareness of health and the growing awareness of consumers about 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-associated diarrhea, are associated with deficiencies or compromised intestinal microflora, and certain probiotics are thought to have a beneficial effect in controlling such disorders. .

[3] In particular, lactic acid bacteria, especially Lactobacillus, are the most commonly used microorganisms as probiotics - "Generally recognized as safe (GRAS). Acidity, the presence of bile salts, and pancreatic enzymes in the gastrointestinal tract (GIT) are some of the major stresses that orally administered probiotics face in the gastrointestinal tract. In addition to being able to survive, a probiotic strain must also be able to attach to the intestinal tract after

Attorney Ref: 9999 / P001-SAR to colonize him (at least temporarily). Because IBD is a dynamic environment, the flow of food that is processed in the gut can wash away any bacteria that is not attached to the intestinal mucosa. Therefore, probiotic strains that have the ability to attach are more likely to colonize IBDs and have positive effects.

[4] Interest in the use of probiotics in industry has increased dramatically. It is important that these probiotics and probiotic-containing products can be produced on an industrial scale, reproducibly and without compromising their beneficial effects.

[5] Various methods for the production of probiotics are described and well documented. Some of these methods describe the preparation of mixed microbial cultures in a liquid culture medium. A major challenge in the production of such mixed crops is the microbial competition between different populations in the culture medium. One way to produce mixed microbial cultures is to propagate the different strains individually and then combine the propagated strains in the desired ratio. However, this is very time consuming and expensive. Therefore, there is still a need to develop efficient and cost-effective methods for the production of antimicrobial cultures such as probiotics.

BRIEF SUMMARY OF THE INVENTION

[6] The inventors have developed a method for the reproducible production of an antimicrobial culture by propagation of a microorganism in the presence of a microbial lysate.

[7] According to one aspect, the method of the invention comprises the following steps:

a. inoculating an aqueous culture medium with an inoculum containing a microorganism to obtain an inoculated medium containing about 10 O ^{2 -5x 10} O 10 viable cells / ml;

B. mixing the inoculated medium with a microbial lysate to form a mixture;

incubating the mixture at the first incubation temperature (1 Tc);

d. cooling the mixture from step c. to a second incubation temperature (2Tc) which is at least about 0.5 ° C lower than

Attorney Ref: 9999 / P001-SAR first incubation temperature (ITc) and incubation of the cooled mixture at 2Tc to obtain a propagated antimicrobial culture; and optional

f. collection of the propagated antimicrobial culture.

[8] The method according to the present invention allows industrial or large scale production of a propagated antimicrobial culture.

[9] According to one aspect, the method according to the invention comprises the following steps:

f. Addition to a large-scale aqueous production medium of a propagated antimicrobial culture according to claim 1 as an inoculum;

g. incubating the inoculated medium at the first incubation temperature (1Tc) to obtain a propagated antimicrobial culture; and optional

h. collection of propagated antimicrobial culture.

[10] The present method is perfectly suitable for the production of propagated antimicrobial cultures which contain a large number of different antimicrobial metabolites.

[11] In addition, this method can be applied using only food-grade materials.

[12] Although the inventors do not wish to be bound by theory, it is believed that the method according to the present invention favors the ability to communicate (quorum sensing) between microorganisms, and in particular between bacteria, such as lactic acid bacteria.

[13] The present inventors have made the surprising and unexpected observation that when a microbial culture is propagated according to the method of the present invention, the propagated microorganisms, and in particular bacteria, such as lactic acid bacteria, are released as a result of metabolic stress. collide, increased amounts of various antimicrobial metabolites. In particular, the authors of

Attorney Ref: 9999 / P001-SAR of the present invention have surprisingly and unexpectedly found that when the microbial culture is propagated according to the method of the present invention, the multiplied microorganisms, and in particular bacteria, such as lactic acid bacteria, as a result of the combination of metabolic stress, they encounter, and the ability of microorganisms to quorum sensing secrete the maximum amount of different antimicrobial metabolites.

[14] In some embodiments, the propagated antimicrobial culture includes Lactobacilli and a microbial lysate. In some embodiments, the propagated antimicrobial Lactobacilli are Lactobacillus delbrueckii. In some embodiments, the propagated antimicrobial Lactobacilli are Lactobacillus delbrueckii subsp. bulgaricus. In some embodiments, Lactobacilli are Lactobacillus delbrueckii subsp. bulgaricus, strain GLB 44.

115] 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 various diseases or conditions in humans, animals and plants, such as microbial diseases or conditions. In various embodiments, the propagated antimicrobial culture is useful for treating various diseases or conditions in humans, animals and plants, such as bacterial diseases or conditions.

[17] In some embodiments, the propagated antimicrobial culture is useful for the treatment of diseases or conditions associated with bacterial pathogens, such as E. coli and Helicobacter pylori.

BRIEF DESCRIPTION OF THE FIGURES

[18] FIG. 1 illustrates the antibacterial activity of L. bulgaricus GLB44 against H. pylori - Zones of inhibition as a function of fermentation time.

DETAILED DESCRIPTION OF THE INVENTION

[19] An antimicrobial culture propagation method is presented in which the incubation of a microorganism under appropriate conditions in the presence of an optimized amount of microbial lysate results in increased levels of antimicrobial metabolites in the propagated culture.

Attorney Ref: 9999 / P001-SAR

[20] In the present disclosure, various publications, patents and published descriptions of patents are indicated by identifying citations. Thus, the findings in these publications, patents and published patent descriptions are incorporated by reference in this disclosure to describe more fully the achievements in the field to which this disclosure relates.

R1] According to one aspect, a method for propagating an antimicrobial culture is provided, the method comprising the following steps:

a. inoculating an aqueous plant medium with a microorganism-containing inoculum to obtain an inoculated medium that contains about 10 O ^{2 -5x10 10} viable cells / ml;

B. mixing the inoculated medium with a microbial lysate to form a mixture;

c. incubating the mixture at the first incubation temperature (1Tc);

d. cooling the mixture according to step c. to a second incubation temperature (2Tc), which is at least about 0.5 ° C lower than the first incubation temperature (1Tc) and incubating the cooled mixture at the second incubation temperature to obtain propagated antimicrobial culture; and optional

f. collection of the propagated antimicrobial culture.

[22] A method for propagating an antimicrobial culture is also presented, the method comprising the following steps:

f. Addition to a large-scale aqueous culture medium of a propagated antimicrobial culture according to claim 1 as an inoculum;

g. incubating the inoculated medium at the first incubation temperature (1 Tc) to obtain a propagated antimicrobial culture; and optional

h. collection of propagated antimicrobial culture.

[23] In the context of this document, some terms may have the following definitions.

Attorney Ref: 9999 / P001-SAR

[24] In the context of this document, the singular with a short and a full article are synonyms and can be used interchangeably - "one / one / one or more" or "at least one / one / "at least one" - unless the wording and / or context clearly indicate otherwise. For example, the term "a cell" means both a single cell and a plurality of cells, including mixtures thereof.

[25] As used in the English text, the term "comprising" means including, composed of, consisting of, containing and representing.

[26] All figures and numbers used in this text to denote quantities, proportions of materials, physical properties of materials and / or uses are to be understood as modified or qualified by the term "about". , unless explicitly stated otherwise.

[27] The term "about" used in this text includes the given number or number and +/- 10% of the given number or digit. As a non-limiting example, the term "about ten (10)" will cover from nine (9) to eleven (11) or 9-11.

[28] As used herein, the term 'antimicrobial' refers to the destruction or inhibition of the growth of micro-organisms, in particular pathogenic micro-organisms, such as pathogenic bacteria.

[29] As used herein, the term "propagating an antimicrobial culture" means a method of multiplying or increasing the number of microorganisms, such as bacteria, by reproduction, growth or proliferation in a predefined culture medium, such as for example an aqueous medium, under controlled incubation conditions. The incubation conditions vary with the various microorganisms known to those skilled in the art and are adapted as necessary to carry out the methods of the present invention.

[30] In some embodiments, the propagated antimicrobial culture obtained by the method of the present invention is lyophilized. In some variants reproduced

Attorney Ref: 9999 / P001-SAR antimicrobial culture obtained by the method of the present invention is lyophilized (freeze dried).

[31] In some embodiments, the inoculum is lyophilized.

[32] In some embodiments, the inoculum is lyophilized (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 harvested antimicrobial culture is lyophilized. In some embodiments, the harvested antimicrobial culture is lyophilized (freeze dried).

[34] In some embodiments, the inoculum is collected for further processing. In some embodiments, the harvested inoculum is lyophilized (freeze dried).

[35] The term 'aqueous medium', as used herein, refers to a nutrient medium or propagation medium prepared with, with or with the aid of water that promotes the growth of micro-organisms.

[36] In some embodiments, the aqueous medium is inoculated with microorganisms. As used herein, the term "inoculated" refers to the introduction or addition to the aquatic environment of microorganisms, cells, lysates or combinations thereof.

[37] In some embodiments, the aqueous medium used in the present method typically contains at least 70 wt.% Water. Preferably, the aqueous culture medium contains at least 80 wt.%, And most preferably contains 90 wt.% Water. In addition to water, the aquatic culture medium contains a source of carbon and nitrogen, and optionally all other ingredients that are necessary for growth organisms, such as salts, which supply essential elements such as magnesium, phosphorus and sulfur.

[38] In some embodiments, the aquatic environment is animal milk or dairy milk. In some embodiments, animal milk or dairy milk may be derived from a cow, sheep, goat, camel, deer, buffalo, yak, mare, donkey, etc.

[39] In some embodiments, the aquatic environment is non-animal milk or non-dairy milk. In some embodiments, non-dairy milk or non-dairy milk may be derived from almonds, soy, rice, cashews, coconut, oats, flax, hemp, etc.

Attorney Ref: 9999 / P001-SAR

[40] In some embodiments, the aquatic environment is largely free of animal or dairy products or animal 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, blood, fish, crustaceans, shellfish and more. As used herein, the term "animal by-product" is a product obtained or derived from animals that is different from muscle meat.

[42] The term 'substantially free' as used herein means that the content is sufficiently low or negligible so that human contact with the cultures described here will not pose a significant risk. In some embodiments, the aquatic environment is a plant environment.

[43] In some embodiments, the plant medium may be acai, agave, almond, aloe, apple, apricot, arugula, avocado, beet, pepper, blackberry, blue-green algae, blueberry, branded, cayenne, celery juice or extract. , chia, coriander, cloves, coconut, cucumber, dandelion, dates, dill, garlic, ginger, ginkgo, grapefruit, guava, hemp, jalapeno peppers, kale, kiwi, lemon, lemongrass, lime, lime, macaque , orange, parsley, peach, pear, pineapple, raspberry, mint, spinach, spirulina, strawberries, sweet potatoes, tomatoes, turmeric root, watermelon, young wheat germ or the plant environment is soy milk, rice milk, almond milk, coffee, linseed oil, herbal tea, or maple syrup, or honey.

[44] In some embodiments, the plant environment includes 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 aquatic nutrient medium.

[45] In some embodiments, the aquatic environment is a vegetable medium. In some embodiments, the vegetable medium may be carrot juice, pea juice, tomato peel, artichoke, broccoli, broccoli blossoms, capers, cauliflower, asparagus, red beets, henna, cassava, ginger, parsnips, yams or turmeric.

Attorney Ref: 9999 / P001-SAR

[46] In some embodiments, the vegetable environment includes 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 aquatic nutrient medium.

| 47] In some embodiments, the vegetable environment includes 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 embodiments, the aqueous nutrient medium comprises a combination of one or more plant-based media and one or more vegetable-based media in different ratios.

[49] In some embodiments, the aqueous culture medium may contain carrot juice and pea juice in a ratio of about 50-50. In some embodiments, the aqueous medium may be 50-50 spirulina extract and carrot juice. In some embodiments, the aqueous culture medium may be from tomato peel extract and carrot juice in a ratio of 50-50.

[50] In some embodiments, the aquatic environment is a vegan environment. As used herein, the term 'vegan' means that it does not contain components (such as meat, eggs or dairy products) derived from animals or animal products.

[51] As used herein, the term "inoculum" means a microbial material or cell culture that is added to another material or other substance, such as an aqueous nutrient medium. In some embodiments, the inoculum includes living or viable microbial cells. In some embodiments, the inoculum used in the present method comprises at least one microorganism. Microorganisms that may be used may include prokaryotes or eukaryotes. Microorganisms can be selected from viruses, bacteria or fungi.

[52] Preferably, the microorganism is selected from bacteria. The world of bacteria consists of over 70,000 species and each of them has many different characteristics. Samples of micro-organisms propagated by this method may be taken, for example, from complex cultures for the fermentation of

Attorney Ref: 9999 / P001-SAR food for humans or animals, mixed crops for biosecurity, complex probiotics, microbiome.

[53] In some embodiments, the inoculum contains about 10 ² to 5x10 ¹⁰ viable cells / ml or more. According to some embodiments, the inoculum can be prepared using microfluidic systems.

[54] In some embodiments, the inoculum used in the present method includes lactic acid bacteria.

[55] Probiotics typically include one of three genera of bacteria: Lactobacillus, Bifidobacteria, and Streptococcus. There are many types of bacteria in each of these three genera.

[56] The genus Lactobacillus contains over 180 species. In some embodiments, 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 variants, Lactobacilli are of the species L. delbrueckii. In some embodiments, the Lactobacilli are Lactobacilli delbrueckii bulgaricus, Lactobacilli delbrueckii lactis, Lactobacilli delbrueckii delbrueckii, or Lactobacilli delbrueckii indicus. In some embodiments, the Lactobacilli are 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, Lactobacilli are Lactobacillus delbrueckii bulgaricus, strain GLB 44. Lactobacillus delbrueckii subsp. bulgaricus GLB 44 were deposited with the National Bank for Industrial Microorganisms and Cell Cultures in Sofia, Bulgaria, on April 17, 2014, access number NBIMCC № 8814.

[61] The different strains of Lactobacillus delbrueckii subsp. bulgaricus have different morphology. For example, some are straight, others are curved, some are a single cell, others are a pair or a line of three. The following length of Lactobacillus delbrueckii subsp. bulgaricus GLB 44 is from about 14 pm to about 16 pm. Lactobacillus delbrueckii subsp. bulgaricus GLB 44 is also often found as short chains of two or three straight rods that are glued together.

Attorney Ref: 9999 / P001-SAR

[62] In some embodiments, Lactobacillus delbrueckii subsp. bulgaricus can be used as bacteria in the present invention. Lactobacillus delbrueckii subsp. bulgaricus are found in nature in Bulgaria in four different plants: Cornus mas (common dogwood), Galanthus nivalis (common snowdrop), Calendula officinalis (calendula) and Prunus spinosa (thistle).

[63] In some embodiments, the inoculated aqueous medium contains about 1O ^{2 -5x10 10} viable cells / ml. In some embodiments, the inoculated aqueous medium contains about 10 ² -5x10 ⁹ viable cells / ml. In some embodiments, the inoculated aqueous medium contains about 10 ² -5x10 ⁸ viable cells / ml. In some embodiments, the inoculated aqueous medium contains about 10 ² -5x10 ⁷ viable cells / ml. In some embodiments, the inoculated aqueous medium contains about 10 ² -5x10 ⁶ viable cells / ml. In some embodiments, the inoculated aqueous medium contains about 10 ² -5x10 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 environment contains about Yu 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.

165] Those skilled in the art know that the viability of microbial cells can be assessed or determined by the number of colony-forming units in milliliters (CFU / mL, if colony-forming units are tested), or in grams (CFU / g). ) if solid material is tested.

[66] If the propagated antimicrobial culture is liquid, the concentration of viable cells can be determined as the number of colony-forming units per milliliter (CFU / mL or culture medium). In some embodiments, the concentration of viable cells in the composition is from 0.5 million to 1 billion CFU / mL, from 0.5 million to 500 million CFU / mL, from 0.5 million to 400 million CFU / mL, from 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,

Attorney Ref: 9999 / P001-SAR from 0.5 million to 75 million CFU / mL, from 0.5 million to 50 million CFU / mL, from 0.5 million to 10 million CFU / mL, from 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, from 5 million to 400 million CFU / mL, from 5 million to 300 million CFU / mL, from 5 million to 200 million CFU / mL, from 5 million to 150 million CFU / mL, from 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 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 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 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 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 million 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 million CFU / mL, from 100 million to 125 million CFU / mL, from 125 million to 1 million yard 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 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 million CFU / mL, 200 million to 1 billion CFl J / 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, out of 300

Attorney Ref: 9999 / P001-SAR 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, 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 viable cells can be estimated by colony-forming units in one gram (CFU / g). In some embodiments, the concentration of the propagated antimicrobial culture, such as the propagated antimicrobial agent, is from 0.5 million to 1 billion CFU / g, from 0.5 million to 500 million CFU / g, from 0.5 million to 400 million CFU / g. g, from 0,5 million to 300 million CFU / g, from 0,5 million to 200 million CFU / g, from 0,5 million to 150 million CFU / g, from 0,5 million to 125 million CFU / g, from 0.5 million to 100 million CFU / g, from 0.5 million to 75 million CFU / g, from 0.5 million to 50 million CFU / g, from 0.5 million to 10 million CFU / g, from 0 , 5 million to 5 million CFU / g, from 0.5 million to 1 million CFU / g, from 1 million to 1 billion CFU / g, from 1 million to 500 million CFU / g, from 1 million to 400 million CFU / g / g, from 1 million to 300 million CFU / g, from 1 million to 200 million CFU / g, from 1 million to 150 million CFU / g, from 1 million to 125 million CFU / g, from 1 million to 100 million CFU / g g, from 1 million to 75 million CFU / g, from 1 million to 50 million CFU / g, from 1 million to 10 million CFU / g, from 1 million to 5 million CFU / g, 5 million up 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 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, from 10 million up to 1 billion CFU / g, from 10 million to 500 million CFU / g, from 10 million to 400 million CFU / g, from 10 million to 300 million CFU / g, from 10 million to 200 million CFU / g, from 10 million up to 150 million CFU / g, from 10 million to 125 million CFU / g, from 10 million to 100 million CFU / g, from 10 million to 75 million CFU / g, from 10 million to 50 million CFU / g, from 50 million up to 1 billion CFU / g, from 50 million to 500 million CFU / g, from 50 million to 400 million CFU / g, from 50 million to 300 million CFU / g, from 50 million to 200 million CFU / g, from 50 million up to 150 million CFU / g, from 50 million to 125 million CFU / g, from 50 million to 100 million CFU / g, from 50 million to 75 m CFU / g, from 100 million to 1 billion CFU / g, from 100 million to 500 million CFU / g, from 100 million to 400 million CFU / g, from 100 million to 300 million

Attorney Ref: 9999 / P001-SAR

CFU / g, from 100 million to 200 million CFU / g, from 100 million to 150 million CFU / g, from 100 million to 125 million CFU / g, from 125 million to 1 billion CFU / g, from 125 million to 500 million CFU / g, from 125 million to 400 million CFU / g, from 125 million to 300 million CFU / g, from 125 million to 200 million CFU / g, from 125 million to 150 million CFU / g, 150 million to 1 billion CFU / g, from 150 million to 500 million CFU / g, from 150 million to 400 million CFU / g, from 150 million to 300 million CFU / g, from 150 million to 200 million CFU / g, from 200 million to 1 billion CFU / g, from 200 million to 500 million CFU / g, from 200 million to 400 million CFU / g, from 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

Microbial lysate

[68] As used herein, the term 'microbial lysate' or 'lysate' means a mixture of microbial antigens, peptides or metabolites derived from various inactivated, damaged or destroyed microbes. Antigens, peptides or metabolites are obtained by chemical or mechanical lysis of microorganisms and the collection of their extract from cultured microbial strains. In some embodiments, the microbial lysate used in the present method is derived from at least one microorganism. The microbial lysate that can be used may contain prokaryotes or eukaryotes. The microbial lysate can be obtained from viruses, bacteria or fungi. In some embodiments, the lysate is learned 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 belonging 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.

Attorney Ref: 9999 / P001-SAR

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

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

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

[74] In some embodiments, the microbial lysate contains about 2% nutrient medium.

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

[76] In some embodiments, the microbial lysate is derived from a microorganism that is different from the propagated microorganism. In some embodiments, the microbial lysate comprises a set of lysates derived from various microorganisms. In some embodiments, the microbial lysate is a set of different microbial lysates in which at least one of the mixed microorganisms is propagated.

177] It is believed that the addition of microbial lysate to the aqueous culture medium contributes to the release of antimicrobial metabolites from multiplied microorganisms. The addition of a microbial lysate to the aqueous culture medium is thought to enhance the release of antimicrobial metabolites from the multiplied microorganisms.

Incubation equipment and systems

Attorney Ref: 9999 / P001-SAR

[78] In order to overcome some of the limitations of existing systems, such as the growth rate of microorganisms and the quorum effect, the methods described here apply appropriately adapted incubation apparatus and techniques that use different types and forms of physical force, such as pseudo-forces. Various types and forms of physical forces, such as pseudoforces, can be generated, for example, as a result of a rotating platform that results in enhanced advection and chaotic mixing of microorganisms, such as bacteria, including an aqueous culture medium and a lysate. In some embodiments, the gas-permeable membranes cover the microbial culture chambers (e.g., microfluidic systems) in which the cultures are enclosed to allow passive but efficient aeration of the sample, and thus optimal production of antimicrobial metabolites.

[79] Without prejudice to theoretical assumptions, in a rotating platform incubation system, Euler pseudoforce (which is perpendicular to the centrifugal pseudoforce) can be used to generate turbulent flow and ensure uniform mixing, for example in a microfluidic microfluidic system chamber. Euler pseudoforces are inertial forces that occur when a microfluidic system is subjected to cycles of one-way rotation with positive and negative acceleration. Mixing depends on the geometry of the chamber, the positive / negative acceleration and the angular spin angle.

180] For liquid microbial cultures, such as bacterial cultures, rapid and healthy growth depends on a variety of factors, including (1) aeration so that bacteria have access to fresh oxygen for their growth, (2) the availability of nutrients when the samples are completely mixed to deliver nutrients homogeneously throughout the culture, (3) minimizing biofilms and aggregates when shaking and stirring prevents bacterial culture from settling to the bottom of the chamber and the formation of biofilms or aggregates that impede reproduction and (4) optimal interaction of cultured bacteria with lysis metabolites or antigens, resulting in increased expression of antimicrobial metabolites by cultured bacteria.

Attorney Ref: 9999 / P001-SAR

[81] Low Reynolds microfluidic systems have laminar flow regimes that 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 or reproduction of microbial cultures compared to the same microorganism when incubated using standard laboratory equipment. In some embodiments, the multiplication rate of the microorganism may 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% higher than the rate of reproduction of the same micro-organism when incubated using standard laboratory equipment.

[82] In some embodiments, the antimicrobial culture is propagated using a microfluidic system. In some embodiments, the inoculum is prepared using a microfluidic system.

[83] In some embodiments, the method relies on the propagation of antimicrobial cultures in commercial or large-scale production facilities. In some embodiments, the method may rely on fermentation systems consisting of large fermentation tanks, bioreactors, or large-scale purification systems.

184] The term "large scale" (largescale) used herein is synonymous with "commercial scale" and means about 5 to 500 L or a larger amount of antimicrobial culture.

Methods for incubation of microbial culture

[85] In some embodiments, the incubation of the microbial culture is performed at a temperature such as the first incubation temperature (1Tc) or the second incubation temperature (2Tc) in the range of 20 ° C to 60 ° C. In some embodiments, the microbial culture is incubated at a temperature in the range of 30 ° C to 50 ° C. In some embodiments, the microbial culture is incubated at a temperature between 35 and 39 ° C. In some embodiments, the incubation of the microbial culture is preferably performed at a temperature in the range between

Attorney Ref: 9999 / P001-SAR about 35.5 and 38.5 ° C. In some embodiments, the incubation of the microbial culture is preferably performed at a temperature in the range between about 36 and 38 ° C. In some embodiments, the incubation of the microbial culture is preferably performed at a temperature in the range between about 36.5 and 38.25 ° C. In some embodiments, the incubation of the microbial culture is preferably performed at a temperature in the range between about 37 and 38 ° C. In some embodiments, the incubation of the microbial culture is performed at a temperature of about 38 ° C.

[86] In some embodiments, the microbial mixture is incubated at the first incubation temperature (1Tc) for at least about 30 minutes, preferably for at least about 45 minutes, preferably for at least about 1 hour. , preferably for at least about 1.5 hours, preferably for at least about 2 hours, preferably for at least about 2.5 hours, preferably for at least about 3 hours, preferably for at least about 4 hours, preferably for at least about 5 hours, preferably for at least about 6 hours, preferably for at least about 6 hours, 5 hours, preferably for at least about 7 hours, preferably for at least about 7.5 hours, preferably for at least about 8 hours, preferably for at least about 7 hours 8.5 hours, preferably for at least about 9 hours, preferably for at least about 9.5 hours, preferably for at least about 10 hours, preferably for at least about 10.5 hours, preferably for at least about 11 hours, preferably for at least about 11.5 hours, preferably for at least about 12 hours, preferably for at least about 12.5 hours, preferably for most at least about 13 hours, preferably for at least about 13.5 hours, preferably for at least about 14 hours or longer.

[87] In one embodiment, the mixture is incubated at the first incubation temperature (1Tc) for 12 hours.

188] One of the steps of the method described herein involves cooling a microbial culture. In some embodiments, cooling is performed on an incubated microbial culture

Attorney Ref: 9999 / P001-SAR at first incubation temperature (1Tc). In some preferred embodiments, the cooling is performed on an antimicrobial culture incubated at the first incubation temperature (1 Tc) according to step c. Of the present method.

[89] According to the method described herein, a step of cooling the microbial culture to the second incubation temperature (2Tc) is performed before the subsequent incubation. In some embodiments, a microbial culture that has been pre-incubated at the first incubation temperature (1Tc) prior to any additional incubation at the second incubation temperature (2Tc) is cooled to the second incubation temperature (2Tc). In some embodiments, an antimicrobial culture that has been incubated at the first incubation temperature (1Tc) as described in point c of the method is cooled to the second incubation temperature (2Tc) before subsequent incubation at the second incubation temperature (2Tc).

[90] The second incubation temperature (2Tc) is 3 ° C-7 ° C lower than the first incubation temperature 1Tc. In some embodiments, the second incubation temperature of 2Tc is 3 ° C-7 ° C lower than the first incubation temperature of 1Tc.

[91] In some embodiments, the second incubation temperature (2Tc) is at least about 0.6 ° C lower than the first incubation temperature (1Tc), preferably at least about 0.75 ° C lower than the first incubation temperature. temperature (1Tc), preferably at least about 1 ° C lower than the first incubation temperature (1Tc), preferably at least about 1.25 ° C lower than the first incubation temperature (1Tc), for preferably at least about 1.5 ° C lower than the first incubation temperature (1Tc), preferably at least about 1.75 ° C lower than the first incubation temperature (1Tc), preferably with the most slightly about 2 ° C lower than the first incubation temperature (1Tc), preferably by at least about 2.25 ° C lower than the first incubation temperature (1Tc), preferably by at least about 2.5 ° C below -lower than the first incubation temperature (1Tc), preferably at least about 2.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 3 ° C lower than the first incubation temperature (1Tc), preferably at least about 3.25 ° C lower than the first incubation temperature (1Tc), preferably at least about 3.5 ° C below -low from the first incubation temperature

Attorney Ref: 9999 / P001-SAR (ITc), preferably at least about 3.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 4 ° C lower than the first incubation temperature (1Tc), preferably at least about 4.25 ° C lower than the first incubation temperature (1Tc), preferably at least about 4.5 ° C lower than the first incubation temperature (1Tc), preferably at least about 4.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 5 ° C lower than the first incubation temperature (1Tc), preferably at least about 5 , 25 ° C lower than the first incubation temperature (1Tc), preferably at least about 5.5 ° C lower than the first incubation temperature (1Tc), preferably at least about 5.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 6 ° C lower than the first incubation temperature (1Tc), preferably at least about 6.25 ° C lower and from the first incubation temperature (1Tc), preferably at least about 6.5 ° C lower than the first incubation temperature (1Tc), preferably at least about 6.75 ° C lower than the first incubation temperature ( 1 Tc), preferably at least about 7 ° C lower than the first incubation temperature (1 Tc) or more.

[92] In some embodiments, the microbial mixture is incubated at 2Tc before incubated at 1Tc.

[93] In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for at least about 30 minutes. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for at least 45 minutes. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for at least 1 hour. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for at least 1.5 hours. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for 2 hours, preferably about 2.5 hours. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for about 3 hours. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for about 3.5 hours. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for about 4 hours. In some embodiments, the microbial mixture is incubated at

Attorney Ref: 9999 / P001-SAR second incubation temperature (2Tc) for about 4.5 hours. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for about 5 hours. In some embodiments, the microbial mixture is incubated at the second incubation temperature (2Tc) for about 5.5 hours or longer.

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

[95] According to a preferred embodiment, the mixture of Lactobacillus delbrueckii bulgaricus GLB 44 strain is incubated at the first incubation temperature (1Tc) of about 38 ° C for about 12 hours and the cooled mixture is incubated at the second incubation temperature (2Tc) of about 33 ° C. With for about 2 hours.

Quorum sensing

[96] Without wishing to be bound by theoretical assumptions, quorum sensing (intercellular signaling) is generally considered to be a response to fluctuations in cell population density. Quorum sensing microorganisms, such as bacteria, produce and release chemical signaling molecules called autoinducers, the concentration of which increases as a function of cell density. Detection of the minimum threshold stimulating concentration of an autoinductor leads to a change in gene expression. Microorganisms such as Gram-positive and Gram-negative bacteria use quorum sensing communication chains to regulate a wide range of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, spore formation and biofilm formation. In general, Gram-negative bacteria use homoserine lactones as autoinducers, and Gram-positive bacteria use processed oligopeptides for communication. As a result of advances in the field, it has recently been found that communication between cells through autoinducers takes place both within and between bacterial species.

[97] It is believed that when a microbial culture is propagated according to the method of the present invention, the multiplied microorganisms and in particular bacteria, such as lactic acid bacteria, due to the metabolic stress they encounter during reproduction, release elevated levels of various antimicrobials.

Attorney Ref: 9999 / P001-SAR metabolites. In particular, when the microbial culture is propagated according to the method of the present invention, the multiplied microorganisms, and in particular bacteria, such as lactic acid bacteria, due to the combination of metabolic stress and quorum sensing ability release high levels of various antimicrobial metabolites.

[98] Exposure to microbial cultures, such as cultures of bacteria or lactic acid bacteria, to a combination of stressful growth conditions, such as suboptimal incubation temperature (2Tc) after incubation at the optimum temperature (1Tc) and in the presence of microbial lysate, may lead to maximization of antimicrobial metabolites from propagated antimicrobial culture.

[99] In some embodiments, exposure to microbial cultures, such as bacterial or lactic acid cultures, to a combination of stress growth conditions, such as suboptimal incubation temperature (2Tc) and in the presence of a microbial lysate, may maximize antimicrobial metabolites from propagated antimicrobial culture. In some embodiments, the propagated antimicrobial culture can be used as an antimicrobial inoculum to inoculate an aqueous culture medium obtained in large-scale production.

[100] In some embodiments, exposure of microbial cultures, such as bacterial cultures or lactic acid bacteria, comprising large-scale aqueous culture medium and antimicrobial inoculum, to a combination of stressful growth conditions, such as suboptimal incubation temperature (2Tc ) after incubation at the optimum temperature (1 Tc) can lead to maximization of antimicrobial metabolites from the propagated antimicrobial culture.

[101] As will be known to those skilled in the art, when levels of antimicrobial metabolites in antimicrobial cultures reach a certain desired concentration or amount, as shown in FIG. 1, the growth or reproduction of the microbial culture must be significantly reduced or completely stopped. There are various methods for significantly reducing or stopping the growth of microbial cultures and they are well known to those skilled in the art. As an example of methods for significantly reducing or stopping the growth of microbial cultures, which, however, do not exhaust the possibilities,

Attorney Ref: 9999 / P001-SAR to provide rapid cooling of the propagated antimicrobial culture, freeze drying of the propagated antimicrobial culture, etc.

[102] In some embodiments, the growth or reproduction rate of the microbial culture is significantly reduced or stopped when the desired level of antimicrobial metabolites is reached.

[103] The desired level of antimicrobial metabolites may depend on the preferred subsequent uses of the antimicrobial culture.

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

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

[106] In some embodiments, the antimicrobial metabolites may 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 that can induce an immune response, and specifically to the production of antibodies.

[107] In various embodiments, the propagated antimicrobial culture may be useful for treating microorganism-caused diseases, such as bacteria, in mammals, and in particular humans, by inhibiting the bacterial quorum sensing cascade and converting the pathogen to avirulent. These diseases include endocarditis, respiratory and lung infections (predominantly in immunocompromised patients), bacteremia, skin conditions, vaginal infections, infections of the colon and central nervous system, ear infections, including otitis externa, eye infections, bone infections and joints, urinary tract infections, gastrointestinal infections and skin infections, including wound infections, pyoderma and atopic dermatitis, all of which can be caused by Helicobacter pylori or E. coli.

Antimicrobial cultures and their use

Attorney Ref: 9999 / P001-SAR

[108] Generally speaking, the present invention provides a method for producing propagated antimicrobial cultures that can be used to reduce the virulence of bacterial pathogens. It will be apparent to those skilled in the art that the propagated antimicrobial culture obtained by the present methods can be used in a wide variety of fields, such as ecology, industry and medicine, to prevent and / or treat injuries or diseases caused by bacteria. In some embodiments, the propagated antimicrobial culture obtained by the present methods can be used to preserve meat, food for humans and animals, in cosmetics as antimicrobials for the skin, in nutraceuticals, food supplements and drugs.

[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 embodiments, the antimicrobial agent may be used for topical cleaning or treatment solutions, such as disinfectants, detergents, household cleaners, and washing powder in the form of a spray or dispersant. In a preferred form, these solutions can be applied to windows, containers, floors, clothes, surfaces in the kitchen and bathroom, as well as on other surfaces in food preparation and personal hygiene. In addition, the multiplied antimicrobial culture can be used as an antibacterial ingredient in personal care products, toilets and cosmetics such as toothpaste, mouthwash, soaps, shampoos, shower gels, ointments, creams, lotions, nebulizers, deodorant disinfectants, as well as and in solutions for storing contact lenses, mouthwash, beverages and animal feed.

[112] In some embodiments, the propagated antimicrobial culture is lyophilized. As used herein, the term 'lyophilised' means the preservation of the propagated antimicrobial culture by rapid freezing and subsequent removal of the ice in vacuum or by sublimation. In some embodiments, the lyophilized antimicrobial culture is preserved for long-term storage. When

Attorney Ref: 9999 / P001-SAR Some variants of lyophilized antimicrobial culture include viable microbial cells. In some embodiments, lyophilization can be used to prepare individual doses, which are then reconstituted for injection.

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

[114] In some embodiments, the lyophilized antimicrobial culture is a lyophilized antibacterial agent.

[115] In some embodiments, a propagated antimicrobial culture is provided in combination with one or more pharmaceutically acceptable ingredients. In some embodiments, a propagated antimicrobial culture is provided in combination with one or more pharmaceutically acceptable excipients or additives.

[116] In the present context, a "pharmaceutically acceptable excipient" refers to an inert substance that is added to a pharmaceutical composition in order to further facilitate the administration of a propagated antimicrobial culture, for example a lyophilized culture. Examples of, but not limited to, excipients include calcium carbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[117] In some embodiments, the pharmaceutical compositions comprise an antimicrobial agent. In some embodiments, the pharmaceutical compositions include an antibacterial agent.

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

[119] Therefore, a propagated antimicrobial culture, such as a lyophilized culture or agent, will be therapeutically effective if it is able to affect the concentration of the target microorganism, where it is able to treat or prevent a disease caused by the microorganism, such as

Attorney Ref: 9999 / P001-SAR bacterial disease or disorder in humans after a propagated antimicrobial culture, such as a lyophilized culture, has been applied to them. In some embodiments, the disease or disorder is associated with a pathogen. In some embodiments, the pathogen is a serotype of Salmonella Enterica. 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 1 monophasic, Salmoratyla or salmonella. 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, Y Shingellasis.

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

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

[123] In another embodiment, the propagated antimicrobial culture, e.g., a lyophilized culture according to the present invention, can be administered directly to animals, preferably mammals, and especially to humans as an antibiotic per se, as a mixture with another or in the form of pharmaceutical preparations. which allow enteral or parenteral administration and which contain as active ingredient an effective dose of the multiplied antimicrobial culture, for example a lyophilized culture, in addition to conventional pharmaceutical excipients and additives.

[124] As mentioned above, in addition to the propagated antimicrobial culture, the culture may also contain conventional, usually inert carrier materials, additives or excipients. Thus, the culture may contain additives or excipients commonly used in galenic dosage forms, for example fillers, diluents, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, preservatives, sweeteners, colorants, flavors, buffers, in addition, solvents or solubility enhancers or depot agents, as well as salts for modifying the osmotic pressure, tablet coatings or antioxidants. They may also contain two or

Attorney Ref: 9999 / P001-SAR more cultures, as well as other active drugs, such as antivirals, antifungals or antibiotics.

[125] The cultures according to the present invention can be used alone, in combination with other compounds according to this invention or in combination with other active compounds, for example with active ingredients which are already known for the treatment of the above-mentioned diseases. case a favorable additive effect is observed.

In another aspect, the present invention provides a process for the manufacture of a product selected from food, beverages, food, nutraceuticals and animal feed, the process comprising combining one or more ingredients with a propagated antimicrobial culture.

[127] In some embodiments, propagated antimicrobial culture products may contain inert inorganic or organic excipients.

[128] In some embodiments, for example, lactose, corn starch or derivatives thereof, talc, stearic acid and its salts, etc. may be used for the manufacture of pills, tablets, film-coated tablets and hard gelatin capsules. Excipients for soft gelatin capsules and suppositories are, for example, fats, waxes, semi-solid and liquid polyols, natural or hardened oils, etc. Suitable excipients for the production of solutions and syrups are, for example, water, alcohol, sucrose, invert sugar, glucose, polyols, etc ..

[129] The dose may vary widely and should be adjusted according to individual circumstances. For the above applications, the appropriate dose will vary depending on the route of administration, the specific condition to be treated, and the desired effect. In principle, however, satisfactory results are obtained at daily doses of about 0.1 to 100 mg / kg body weight of the animals, preferably 1 to 50 mg / kg. Suitable dosages for larger mammals, for example humans, are in the range of 10 mg to 3 g / day, taken for convenience once, divided into doses of 2 to 4 doses per day or in the form of depot preparations.

Attorney Ref: 9999 / P001-SAR

[130] In general, a daily dose of approximately 0.1 mg to 5000 mg, preferably between 10 and 500 mg, is suitable for mammals and in particular for humans for oral administration, which is the preferred route of administration according to the invention. In the case of other routes of administration, the daily dose is also within similar limits. The compounds of formula I or formula II may also be used in the form of drug precursors or in a suitably modified form which releases the active ingredient in vivo.

[131] In another use, the compounds of the present invention may be used as pharmacologically active components or ingredients of medical devices, instruments and articles with an effective dose of a propagated antimicrobial culture, for example a lyophilized culture according to the present invention. The amount of cultures used, for example, to cover the surfaces of medical devices varies to some extent depending on the method of application and the field of application. In general, however, the concentration varies from about 0.01 mg per cm ² to about 100 mg per cm ² . Similarly, the amount of cultures must be adapted to the mode of administration if the cultures according to the invention are used as components or ingredients of cleaning agents, treatment solutions or ointments. In general, effective doses are in the range of about 0.1 μΜ to about 1000 mM.

Protocols and data exchange

[132] In some embodiments, the methods and systems disclosed herein further involve the generation of one or more protocols. In some embodiments, the methods disclosed herein include the storage of one or more protocols. In some embodiments, the methods disclosed herein involve the exchange of one or more protocols. In some embodiments, the protocol includes information about the ability of a microorganism to multiply and produce a propagated antimicrobial culture. In some embodiments, the protocol contains recommendations for a therapeutic regimen using the propagated antimicrobial culture as an antimicrobial agent. In some embodiments, the protocol contains recommendations for the dosage of a propagated antimicrobial culture when used as an antimicrobial agent.

Attorney Ref: 9999 / P001-SAR

[133] In order to make the present invention understandable and practical, some variants will be described in the present text by means of the following examples, which, however, do not exhaust the possibilities.

EXPERIMENTAL EXAMPLE

In vitro test for antimicrobial activity - FIG. 1

1134] Vegan nutrient medium: - 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 - Without maximization of antimicrobial metabolites

[137] Inoculum preparation: a one-step process

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

Fermentation and lyophilization.

[139] Cultivation of L. bulgaricus GLB44 in a vegan culture medium at a constant temperature of 38 ° C for 24 hours in a bioreactor without aeration.

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

[141] Cultivation of L. bulgaricus GLB44, L. bulgaricus GLB3 and L. bulgaricus GLB27 with maximization of antimicrobial metabolites or growth of the culture.

Obtaining inoculum from propagated antimicrobial culture: a two-step process

1142] The first step of culturing the inoculum lasts 12 hours at 38 degrees Celsius.

Attorney Ref: 9999 / P001-SAR

[143] The second step begins at 12 o'clock, when the exponential phase is at its peak, the temperature is lowered from 38 to 33 degrees C for 2 hours to create additional stress on the bacterial culture. Ultrasonic disintegration of part of this sample.

[144] An inoculation mixture (10% (v / v) of the final fermentation mixture) was then prepared - 8% GLB44 culture in the exponential phase and 2% lysate from disintegrated GLB44 cells.

Fermentation and lyophilization:

[145] Cultivation of L. bulgaricus strains in a bioreactor without aeration of the vegan nutrient medium for 12 hours at 38 degrees Celsius, after lowering the temperature to 33 ° C for another 12 hours. Sampling after the 4th hour of fermentation, every 2 hours. Neutralization to pH = 5.90 and lyophilization of the culture samples.

[146] Comparative study of the antimicrobial activity of samples of L. bulgaricus with respect to a clinical isolate of H. Pylori.

Samples of L. bulsaricus from each time point of the studied fermentation process

[147] 1. L. bulgaricus GLB44 Without antimicrobial maximization: liquid sample - reconstituted lyophilised product (1:10, product: sterile saline) | 148] 2. L. bulgaricus GLB44 Antimicrobial maximization: liquid sample - reconstituted lyophilized product (1 : 10, product: sterile saline)

[149] 3. L. bulgaricus GLB3 Antimicrobial maximization: liquid sample - reconstituted lyophilised product (1:10, product: sterile saline)

[150] 4. L. bulgaricus GLB18 Antimicrobial maximization: liquid sample - reconstituted lyophilised product (1:10, product: sterile saline))

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

The test strain was cultured under anaerobic conditions in Brucella medium with 5% fetal bovine serum (B-FBS).

In vitro antibacterial test.

Attorney Ref: 9999 / P001-SAR

[152] The assay was performed by agar diffusion using wells [References 1,2]. The final concentration of Helicobacter pylori in Brucella agar plates with hemin, vitamin K and 5% sheep blood (HBV) was 3x10 ⁵ CFU / mL

[153] Samples of L. bulgaricus were prepared as described above and 100 microliters (μΐ) in 3 replicates (3x) were placed in each well of Petri dishes.

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

[155] Inhibition zones were measured after incubation for 120 hours.

CONCLUSION

[156] In our experiments, we observed that when an inoculum was maximized by a microbial lysate, such as inactivated bacteria and temperature shock, the production of unexpectedly high levels of various antimicrobial metabolites was observed. In addition, we observed that when the maximized inoculum is used to inoculate large-scale fermentation, a second temperature shock on the corresponding fermentation results in unexpectedly high levels of various antimicrobial metabolites. Accordingly, the effects we observed are temperature shocks and the accumulation of inoculum peptides in the main fermentation as a result of quorum sensing. Together they lead to the cumulative effect in the main fermentation. In essence, we believe that we create bacteriocin in the inoculum, which then, as a result of quorum sensing, produces higher levels of the same bacitracin in large-scale or final fermentation.

[157] In the final fermentation stage, the growth or multiplication of microbes must be stopped to the maximum, otherwise the levels of antimicrobial metabolites such as bacteriocin decrease significantly as the multiplied microorganisms reabsorb the metabolites and then process them as food. It is clearly seen from FIG. 1 that antimicrobial levels begin to decline after the peak in the log phase. By abruptly cooling or freezing the propagated antimicrobial culture when the antimicrobial

Attorney Ref: 9999 / P001-SAR metabolites are at their peak, it is possible to maximize antimicrobial levels in the final solution / product.

* * *

[158] The disclosure illustratively described herein may accordingly be applied in practice in the absence of any element or elements, limitation or limitations not specifically disclosed herein. For example, the terms "comprising", "including", etc., must be interpreted in a broad sense and without restrictions. Furthermore, the terms and expressions have been used in this text as descriptive terms and not to limit and the use of such terms and expressions is not intended to exclude equivalents of features shown and described or parts thereof, but are considered to be many variants in the scope of disclosure are possible.

REFERENCES:

[1] S. Magaldi, S. Mata-Essayag, S. 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 determine the bacterial activity of natural products, Braz. J. Microbiol. 38 (2007) 369-380.

Claims (25)

Claims 1. A method for propagating an antimicrobial culture, the method comprising the following steps: a. inoculating an aqueous culture medium with an inoculum comprising a microorganism to obtain an inoculated medium containing about 10 O ^{2 -5x 10} O 10 viable cells / ml; B. mixing the inoculated medium with a microbial lysate to form a mixture; c. incubating the mixture at the first incubation temperature (1Tc); d. cooling the mixture from point c) to a second incubation temperature (2Tc) that is at least about 0.5 ° C lower than the first incubation temperature and incubating the cooled mixture at 2Tc to obtain a propagated antimicrobial culture; and optional f. collection of the propagated antimicrobial culture. 2. A method for propagating an antimicrobial culture, the method further comprising the following steps: f. adding to a large-scale aqueous nutrient medium a propagated antimicrobial culture according to claim 1 as an inoculum; g. incubating the inoculated medium at the first incubation temperature (1Tc) to obtain a propagated antimicrobial culture; and optional h. collection of propagated antimicrobial culture. A method for propagating an antimicrobial culture according to any one of claims 1 or 2, wherein the inoculum is derived from a microbiome. The method for propagating an antimicrobial culture according to claim 3, wherein the microorganism is a bacterium. The method for propagating an antimicrobial culture according to claim 4, wherein the bacterium is selected from lactic acid bacteria, Bifidobacteria or combinations thereof. The method for propagating an antimicrobial culture according to claim 5, wherein the lactic acid bacterium is Lactobacillus delbrueckii subsp bulgaricus. Attorney Ref: 9999 / P001-SAR The method for propagating an antimicrobial culture according to claim 6, wherein Lactobacillus delbrueckii subspecies bulgaricus and Lactobacillus bulgaricus GLB44. A method for propagating an antimicrobial culture according to any one of the preceding claims, wherein the lysate is obtained from a microorganism according to any one of claims 3 to 7. The antimicrobial culture propagation method of claim 8, wherein the microbial lysate comprises about 0.1% v / v culture medium, preferably about 0.2% nutrient medium, preferably about 0.4% nutrient medium, preferably about 0.8% nutrient medium, preferably about 1% nutrient medium, preferably about 1.2% nutrient medium, preferably about 1.4% nutrient medium, preferably about 1.8% nutrient medium, preferably about 2% nutrient medium, preferably about 3% nutrient medium, preferably about 4% nutrient medium, preferably about 5% nutrient medium, preferably about 6% nutrient medium, preferably about 7% nutrient medium, preferably about 8% nutrient medium , preferably about 9% nutrient medium, preferably about 10% nutrient medium, preferably about 11% nutrient medium, preferably about 12% nutrient medium, preferably about 13% nutrient medium, preferably about 14% food nutrient medium, preferably about 15% nutrient medium or more. A method for propagating an antimicrobial culture according to any one of claims 8 or 9, wherein the microbial lysate contains about 2% v / v nutrient medium. A method for propagating an antimicrobial culture according to any one of the preceding claims, wherein the first incubation temperature (1Tc) is in the range between about 20 ° C to 60 ° C, preferably between about 30 ° C to 50 ° C, preferably between about 35-39 ° C, preferably between about 35.5-38.5 ° C, preferably between about 36-38 ° C, preferably between about 36.5-38.25 ° C, preferably between about 37-38 ° C, preferably about 38 ° C. A method for propagating an antimicrobial culture according to any one of the preceding claims, wherein the first incubation temperature (1 Tc) is about 38 ° C. A method for propagating an antimicrobial culture according to any one of the preceding claims, wherein the mixture is incubated at the first incubation temperature (1Tc) for at least about 30 minutes, preferably for at least about 45 minutes, preferably in for at least about 1 hour, preferably for at least about 1.5 hours, preferably in Attorney Ref: 9999 / P001-SAR for at least about 2 hours, preferably for at least about 2.5 hours, preferably for at least about 3 hours, for at least about 4 hours , preferably for at least about 5 hours, preferably for at least about 6 hours, preferably for at least about 6.5 hours, preferably for at least about 7 hours, for preferably for at least about 7.5 hours, preferably for at least about 8 hours, preferably for at least about 8.5 hours, preferably for at least about 9 hours, for preferably for at least about 9.5 hours, preferably for at least about 10 hours, preferably for at least about 10.5 hours, preferably for at least about 11 hours, for preferably for at least about 11.5 hours, preferably for approx for at least about 12 hours, preferably for at least about 12.5 hours, preferably for at least about 13 hours, preferably for at least about 13.5 hours, preferably in for at least about 14 hours or longer. A method for propagating an antimicrobial culture according to any one of the preceding claims, wherein the mixture is incubated at the first incubation temperature (1Tc) for about 12 hours. A method for propagating an antimicrobial culture according to any one of the preceding claims, wherein the incubated mixture of step c) is cooled to a second incubation temperature (2Tc) that is at least about 0.6 ° C lower than the first incubation temperature. (1Tc), preferably at least about 0.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 1 ° C lower than the first incubation temperature (1Tc), preferably at least about 1.25 ° C lower than the first incubation temperature (1Tc), preferably at least about 1.5 ° C lower than the first incubation temperature (1Tc), preferably at least about 1.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 2 ° C lower than the first incubation temperature (1Tc), preferably at least about 2.25 ° C lower than the first incubation temperature (1 Tc), preferably at least about 2.5 ° C lower than the first incubation temperature (1 Tc) ), preferably at least about 2.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 3 ° C lower than the first incubation temperature (1Tc), preferably with the most at least about 3.25 ° C lower than the first incubation temperature (1Tc), preferably at least about 3.5 ° C lower than the first incubation temperature (1Tc), preferably at least about 3.75 ° C lower than the first incubation temperature (1 Tc), for Attorney Ref: 9999 / P001-SAR preferably at least about 4 ° C lower than the first incubation temperature (1Tc), preferably at least about 4.25 ° C lower than the first incubation temperature (1Tc) , preferably at least about 4.5 ° C lower than the first incubation temperature (1Tc), preferably at least about 4.75 ° C lower than the first incubation temperature (1Tc), preferably at most slightly about 5 ° C lower than the first incubation temperature (1Tc), preferably at least about 5.25 ° C lower than the first incubation temperature (1Tc), preferably at least about 5.5 ° With a lower than the first incubation temperature (1Tc), preferably at least about 5.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 6 ° C lower than the first incubation temperature (1Tc), preferably at least about 6.25 ° C lower than the first incubation temperature (1Tc), preferably at least about 6.5 ° C lower than the first and an incubation temperature (1Tc), preferably at least about 6.75 ° C lower than the first incubation temperature (1Tc), preferably at least about 7 ° C lower than the first incubation temperature (1Tc). or more. A method for propagating an antimicrobial culture according to any one of the preceding claims, wherein the cooled mixture is incubated at the second incubation temperature (2Tc) for at least about 30 minutes, preferably for at least about 45 minutes, preferably for at least about 1 hour, preferably for at least about 1.5 hours, preferably for at least about 2 hours, preferably about 2.5 hours, preferably about 3 hours, preferably about 3.5 hours, preferably about 4 hours, preferably about 4.5 hours, preferably about 5 hours, preferably about 5.5 hours or longer. 17. A method according to any preceding claim wherein the inoculated medium contained about 10 ⁹ viable cells / ml, preferably about 10 ⁸ viable cells / ml, preferably 10 ⁷ viable cells / ml, preferably 10 ⁶ viable cells / ml. A method of propagating an antimicrobial culture according to any one of the preceding claims, wherein the cooled mixture is incubated at the second incubation temperature (2Tc) for about 2 hours. The antimicrobial culture propagation method of claim 7, wherein the mixture is incubated at the first incubation temperature (1Tc) at about 38 ° C for about 12 hours and the cooled mixture is incubated at the second incubation temperature (2Tc) at about 33 ° C. With for about 2 hours. Attorney Ref: 9999 / P001-SAR Propagated antimicrobial culture obtained by a method according to any one of the preceding claims. A propagated antimicrobial culture according to claim 20, wherein the culture is lyophilized. Propagated antimicrobial culture according to any one of claims 20 or 21 for use as an antimicrobial agent. Propagated antimicrobial culture according to claim 22 for use in the treatment of Helicobacter pylori. A propagated antimicrobial culture according to claim 22 for use in the treatment of E. coli. A process for the production of a product selected from foodstuffs, beverages, foodstuffs, nutraceuticals and animal feed, the process comprising combining one or more ingredients with a propagated antimicrobial culture obtained by a method according to any one of claims 1 to 19.