WO2017009676A1 - Compositions for maintaining and restoring microbiota-host symbiosis - Google Patents

Abstract

This invention is related to compositions containing combinations of microbial lysates and micronized polysaccharides and use thereof for maintaining and restoring microbiota-host symbiosis in humans or animals or plants.

Description

Compositions for Maintaining and Restoring

Microbiota-Host Symbiosis

Technical Field

[0001] The present invention is related to compositions containing combinations of microbial lysates and micronized polysaccharides for maintaining and restoring symbiosis between microbiota-hosts in humans, animals and plants. The scientific rationale of this invention is that administration of microbial lysate instead of live microbes and administration of micronized polysaccharides instead of natural forms of polysaccharides may enhance benefits of these substances and decrease their po- tential drawbacks.

[0002] The novelty of the present invention is the addition of micronized polysaccharides to microbial lysates. No prior art mentions or uses the combination of killed microbes with micronized polysaccharides.

[0003] The inventive step of the present invention is the surprising benefit of the combination of microbial compounds (DNA/RNA and LPS/PG) and micronized polysaccharides on basic mechanisms of the microbiota-host symbiosis. Experimental studies clearly demonstrated that the biological effects of these compounds are not their mathematical sum but significantly higher for maintaining or restoring microbiota-host symbiosis of human, animal and plant organisms. No prior art mentions and profits from the synergism between microbial lysate and micronized polysaccharides.

[0004] The industrial applicability of the present invention is that a line of products containing microbial lysates and micronized polysaccharides may be formulated and manufactured for human and veterinary medicines and nutrition as well as for plant nutrition and fertilization of plant cultures. No prior art mentions or manufactures these combinations.

Definitions

[0005] Microbiota or microflora: This is the community of microbes (bacteria, ar- chaea, viruses and yeasts) which lives in symbiosis with the human, animal and plant organisms. The human microbiota is formed by approximately 10¹⁴ microbes.

[0006] Microbiome: Genome of the microbiota is denominated 'microbiome'. In humans it is formed by 3 million genes, which is 100 times higher than the human genome. [0007] Microbiota-host symbiosis: This is a mutually advantageous correlation between the microbial community and the host organism. Release of microbial compounds, such as deoxyribonucleic acid/ribonucleic acid (DNA/RNA), lipopolysaccha- ride (LPS) and peptidoglycan (PG) to stimulate the host neuroimmune and neuroho- monal mechanisms represents the microbial contribution to symbiosis. Formation a mucus gel for colonization and nutrition of microbes represents the cardinal host contribution to the symbiosis. Microbiota-host symbiosis plays a central role in the host survival and adaptation to the continuously changing environment.

[0008] Microbiota-host dysbiosis: This is a disorder of the symbiosis characterized by quantitative and/or qualitative changes in the bioavailability of microbial compounds as well as of the mucus. Dysbiosis is directly involved in the development of various disorders or diseases.

[0009] Microbial lysates: They are killed and fragmented microbes containing cytoplasmic DNA/RNA as well as cell-wall LPS and PG. Synonymous terms are "cell-free extract of bacteria", "killed bacteria", and "ghost bacteria". From a functional point of view these compounds are nutrients of microbial origin, shortly 'microbial compounds' or 'microbial nutrients' when administered orally.

[0010] Mucous membranes: Anatomically, it comprises the gastro-intestinal tract, airways, urogenital apparatus and the ocular surface (conjunctiva and cornea). In humans, they represent approximately 400 square meter surface. Histologically, they contain an uppermost epithelial layer and an underlying connective tissue layer, in which immune cells, nerve cells and endings, as well as capillaries are distributed. Functionally, they represent a selective barrier between the organism and the environment. Mucous membranes represent the largest neuro-immune and neuro- endo- crine organ of humans.

[0011] Polysaccharides: The terms polysaccharide and glycan are defined as synonyms meaning "compounds consisting of a large number of monosaccharides linked glycosidically". The most common forms of polysaccharides are starch, pectin, cellulose, chitin and inulin. However, in practice the term glycan may also be used to refer to the carbohydrate portion of glycoconjugates, such as glycoproteins, petidoglycans, glucosaminoglycans (mucopolysaccharides) and glycolipids. [0012] Mucus: This is a layer of polysaccharides located at the interface of microbiota and host epithelium. The main components of the mucus are secreted by the host epithelium to which polysaccharides from the food are attached. The quality and quantity of mucus are critical to the maintenance of a homeostatic relationship (sym- biosis) between the microbiota and their hosts.

[0013] Micronization: This is a procedure of reducing the average diameter of solid material's particles, and subsequently, of enhancing the digestions and absorption of these materials. Usually, the term micronization is used when the particles that are produced are only a few micrometers in diameter. Disclosure of Invention

[0014] The current invention is based on recent scientific findings that the symbiosis between microbes (prokaryotes) and higher-order organisms (eukaryotes) is an essential biological condition for the survival and adaptation of any living organism in the Earth. For example the human organism evolutionarily lives in symbiosis with a community of approximately 10¹⁴ (100 trillion) microbes, called microflora or microbiota. It located mainly in the gut, but less extensively on the surface of other mucous membranes (oral cavity, airways, urogenital apparatus, conjunctiva, cornea) and on the skin. The genome of microbiota, called microbiome, is approximately 100 times larger than the human genome. [0015] The microbiota of the host organism is established and maturated early in the life, as well as it is continuously reshaped by environmental microbes. For example, the gut microbiota is repeatedly influenced by microbes normally found in the food. Recently, the whole microbiota of three different dietary patterns was characterized in order to estimate the average total amount of daily microbes ingested via food and beverages. Three different dietary patterns were analyzed: (1) the average AMERICAN diet: focused on convenience foods, (2) recommended diet: emphasizing fruits and vegetables, lean meat, dairy, and whole grains, and (3) vegan diet: excluding all animal products. According to Lang et al microbial analysis showed, that the recommended diet had the highest total amount of microbes at 1.3 ^χ 10⁹ CFU per day, fol- lowed by the vegan diet at 6 * 10⁶ and the AMERICAN diet 1.4 * 10⁶ CFU per day respectively (Lang JM, Eisen JA, Zivkovic AM. The microbes we eat: abundance and taxonomy of microbes consumed in a day's worth of meals for three diet types. Peer J. 2014 Dec 9;2:e659). [0016] Another recent study identified in human several "foreign" genes which derived from bacteria by horizontal gene transfer. Many horizontally acquired genes code for enzyme activities: 2 genes of the amino-acid metabolism, 15 genes of the macromolecule modification, 13 genes of the lipid metabolism, 5 genes are involved in the antioxidant activities, and 7 genes in the innate immune responses. These observations strongly suggested that foreign genes of bacterial origin are involved in basic physiologic functions such as metabolism and immune defense of hosts.

[0017] The symbiosis represents a balanced cross-talk between microbial and human genome. Here we summarize the main mechanisms of microbiota host symbio- sis in the gut as a paradigm.

[0018] Role of microbial compounds: The cytoplasmic fraction of microbial lysate contains genomic nucleic acids (DNA/RNA) which bind to specific receptors (TLR7-9) of the epithelial cells, immune cells, nerve endings and capillary cells and it exerts anti-inflammatory effects. The cell-wall fraction of killed microbes contains lipopoly- saccharides (LPS) and/or peptidoglycans (PG), which binds to another types of receptors (TLR2-4) of the same host cells and exerts pro-inflammatory effects. It should be emphasized that these regulatory effects of microbial compounds are not restricted to the gut or other mucous membranes, but through blood stream and nerve fibers they exert systemic neuro-immune and neuro-hormonal effects. Furthermore, after binding to receptors, these microbial compounds are internalized by phagocytosis into the epithelial cells, immune cells, nerve endings and capillary cells and they are catabolized by the phago-lysosomal system of these cells. Both phagocytosis and catabolism are energy consuming processes. Thus microbial compounds may also up-regulate or down-regulate local and systemic metabolic processes, and in this way they are crucial mediators in the physiologic regulation of both local and systemic life-functions. It should be kept in mind, that almost all microbial compounds released from killed, but not live cell. This is a crucial process for the bioavailability of microbial compounds.

[0019] Role of host compounds:

(i) Mucus layer forms a selective barrier for microbes, briefly: non-pathogenic may adhere to the mucus, while pathogens do not. The mucus layer at the interface of microbiota and host plays a critical role in the cross-talk between the microbiota and host organism. Both host-derived glycocalyx and secreted mucins as well as food- derived polysaccharides constitute a glycosylated environment for microbiota colonization. Furthermore, many of the polysaccharides, even those, which are not- digestible for the host, are metabolized by the microbes.

(ii) Epithelial barrier: The normal barrier function of the gut epithelial cell layer is se- lective and dynamic. It facilitates the appropriate absorption of macronutrients (proteins, carbohydrates, lipids) and micronutrients (vitamins, salts and trace elements), but it blocks the translocation of toxic substances and live microorganisma. Glycans are integral components of cell-junctions which are the anatomical regulator epithelial barrier functions;

(Hi) Antibacterial substances: Epithelial cells, in addition to secretion of mucins, also produce some substances (lysozyme, antibacterial peptides) to selectively kill microbes resulting in physiological lysate of microbes. The balance between these two processes (i.e. promoting or inhibiting colonization of microbes) regulates the stability of the microbial community and of the adaptation to the continuously changing envi- ronmental conditions (food intake, physical and psychical stress, medicines).

[0020] Genetic disposition, life-style and dietary factors may cause perturbation of the microbiota-host symbiosis, called dysbiosis. Main consequences of dysbiosis are (i) impaired digestion and absorption of nutrients, and (ii) break-down of the mucosal barrier resulting in enhanced and not-selective translocation of microbial compounds or even whole microbes.

[0021] While certain levels of microbial compounds are essential for physiological stimulation of the neuroimmune and neurohormonal systems of the host, several observations showed lower or higher than normal amount of microbial compounds in some human tissues and organs (liver, muscle, blood) in dysbiosis.

[0022] It is widely accepted that a poor microbial community is causally involved in several diseases. For example: Animals grown in germ-free environment are more susceptible to both infections and neurologic or psychic stress. Delayed contact of new-borns to microbes (due to Caesarean delivery and formula feeding instead of vaginal delivery and breast feeding) causes delayed maturation of the neuroimmune and neurohormonal system, retarded physical and mental development and life-long increased susceptibility to various diseases.

[0023] In contrast, excessive and abnormal microbial compounds were found in various organs related to common pathologies. For example, in metabolic syndrome, which refers to disorders of the lipid, carbohydrate and protein metabolism, and it is a well-known risk for several common diseases like type 2 diabetes, obesity, fatty liver, atherosclerosis, hypertension and neurodegenerative diseases- in acute coronary syndrome in sclerosis multiplex and in some non-inflammatory neurological diseases, in cancer-related loss of muscle and adipose tissue, in Parkinson disease, , in major depressive disorder in peptic ulcer and gastritis , in primary biliary cirrhosis. [0024] Recent studies revealed that certain aerobic bacteria may influence aerobic metabolism of cellular energy production in mitochondria. It should be kept in mind that according to the current concept, mitochondria, the aerobic "powerhouse" of the cells, originated from aerobic bacteria during the evolution, i.e. mitochondria are evolutionary endosymbionts of bacterial origin which became cellular organelle.

[0025] Furthermore, microbiota may communicate with the host mitochondria and an association between microbiome community structures with its own host mtDNA variants was found. Microbes release membrane vesicles which contain DNA compo- nents such as 16s rRNA gene, that vesicles could be engulfed by host macrophages, causing release of mediators to physiologically regulate host organism in normal conditions. However, excessive amount of bacterial DNA RNA may up-regulate host organism resulting in pathological conditions.

[0026] These observations indicate that under certain conditions the epithelial barrier is leaky, the catabolism of translocated bacteria is incomplete, and bacterial

DNA/RNA may permanently or at least for long time persist in human organism. The subsequent upregulation of the neuroimmune and neurohormonal systems results in enhanced generation of pro-inflammatory signaling molecules, first of all lipid peroxides, cytokines and neuropeptides which are responsible for the development of sys- temic subclinical ("low-grade") or manifest inflammation.

[0027] The main embodiment of the current invention are compositions for maintaining and restoring symbiosis between microbiota-hosts in humans, animals and plants, where said composition contains a combination of

(i) at least one microbial lysate containing DNA/RNA and/or LPS/PG derived from at least one species of bacteria and/or archaea, and/or yeasts and/or viruses residing in the human, or in the animal or/and in the plant microbiota, or from their combinations, or derived from their synthetic or semisynthetic analogs; and the solid content of the microbial lysate having a particle size from 0,001 to 5,0 micron; and (ii) at least one micronized polysaccharide compatible for human and/or animal nutrition and/or plant nutrition and/or fertilization of plants, derived from natural sources comprising at least one species of bacteria, algae, mushrooms, plants or from their combination, or derived from their synthetic or semisynthetic analogs, and the said micronized polysaccharide having a particle size between 0,1 micron to 5 micron or a fibril length of 0,1 micron to 10 mm while

(iii) the ratio of microbial lysate to polysaccharide has to be higher for polysaccharide by their dry weight content.

[0028] It is well known that both live, microbes and polysaccharides are habitual components of the food for humans or animals, and of the fertilizers for plants, and they are digested, absorbed and utilized by host in normal conditions (in symbiosis), However, in abnormal conditions (in dysbiosis) the bioavailability of both microbial compounds and polysaccharides is compromised, and administration of live bacteria (probiotics) and polysaccharides (prebiotics) may results in poor or uncertain benefits in these conditions. Compositions formulated according to the present invention containing a combination of microbial lysate and micronized polysaccharide, which can avoid this drawback of dysbiosis, as fragmentation of microbes and polysaccharides improves bioavailability of both compounds. While combinations of probiotics (live bacteria) and prebiotics (polysaccharides) are usually denominated as "symbiotics", for better identification of compositions according to the current invention, the new term "prosymbiotics" shall be introduced for a combination of microbial lysate and micronized polysaccharides. This prosymbiotic composition is the novelty oi the current invention.

[0029] Another embodiment of the current invention are compositions according to the main embodiment wherein

(i) the microbial lysates are derived from beneficial and/or saprophyte-commensal and/or pathogenic microorganism, or from the combination thereof, i.e. regardless from biological characteristics of live (not-lysed) microbes, and

(ii) the micronized polysaccharides are derived from polysaccharides digesti- ble/absorbable and/or non-digestible/non-absorbable for humans and/or animals, as well as from polysaccharides degradable/absorbable and/or non-degradable/non- absorbable for the host plants, regardless of biological characteristics of natural (non-micronized) forms of polysaccharides. [0030] Microbial lysates according to the invention may derive from bacteria, arche- ae, viruses, and yeasts. Only main tools for preparation of microbial lysate are outlined hereunder: (i) physical fragmentation comprises mechanical, ultrasonic, ultra- centrifuge, high pressure, osmotic-stress, gamma irradiation, UV-light, microwave, cryo-fracturing, or (ii) biological fragmentation by enzymes comprises proteases, lipases, nucleases, amylases, lactases, xylanases, or combination thereof. Some of these technologies may be combined with previous exposure of microbes to heat, known as tyndallization or pasteurization. It should be kept in mind, that the tyndalli- zation kills all microbes, i.e. it is sterilization, while pasteurization kills most but not all of the microbes. Microbial lysates may be standardized to their nucleic acid and/or protein content related to the dry-weight.

[0031] Microbial lysates have at least three advantages as compared to their live counterparts: (i) they have better bioavailability, as the killing and fragmentation facilitate both engulfment (phagocytosis) and intracellular catabolism of microbes; (ii) no risk of infection, which may happen even if "non-pathogenic" bacteria (probiotics) are administered; and (iii) safer and longer efficacy as no need for special storage condition (low temperature, humidity, darkness).

[0032] Polysaccharides orglycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes. In general, they are found on the cell surface and frequently called as 'glycocalix' or 'surface-coat'. Thus, polysaccharides play an important role in cell-cell interactions. For example, glycans are involved in "self and "non self discrimination, which may be relevant to the patho-physiology of various diseases. Furthermore, glycans on an immune cell's surface will help dictate that migration pattern of the cell, for example the response of cells to certain bacterial molecules, such as LPS and PG. Glycans, in particular mucin of the mucous membranes, have been found to be important in developing normal intestinal and other mucus membrane microflora. For example, certain strains of intestinal bacteria bind specifically to mucin, allowing them to colonize the intestine. A functional mucus layer is a key requirement for gastrointestinal health as it serves as a barrier against bacte- rial invasion and subsequent inflammation. Recent developments showed the biological significance a glycans in plants specifically their impact on plant physiology.

[0033] Polysaccharides according to the present invention may readily be available from sources such as algae (e.g. alginates), plants (e.g. starch, cellulose, pectin, guar gum, mannan), mushrooms, microbes (e.g. dextran, xanthan gum) and animals (e.g. chitosan, glycosaminoglycans) and they can also be produced by means of recombinant DNA techniques.

[0034] Compositions according to the present invention may also contain some natu- ral polysaccharides, which have been found to show particular biological or even medical benefits. For examples but not exclusively:

(i) Mushrooms: Fungal bioactive polysaccharides deriving mainly from the Basidio- mycetes family approximately 700 species and medicinal mushrooms have been well known and widely used in far Asia as part of traditional diet and medicine, and in the last decades have been the core of intense research for the understanding and the utilization of their medicinal properties in naturally produced pharmaceuticals.

(ii) Algae: They have numerous commercial applications in products such as stabilizers, thickeners, emulsifiers, food, feed, beverages etc. The total polysaccharide concentrations in the seaweed species of interest range from 4-76 % of the dry weight.

(iii) Aloe vera: The aloe parenchyma tissue or pulp, in addition to the different carbohydrates, has been shown to contain proteins, lipids, amino acids, vitamins, enzymes, inorganic compounds and small organic compounds.

(iv) Chicory and Jerusalem artichoke: both are rich in inulin (fructan) which is widely used as a component of functional foods, and for prebiotics.

[0035] For extraction of polysaccharides from their natural sources any of the biologically compatible (non-toxic) solvent may be used or may be released by high- pressure at any temperature, and (ultra)centrifugation. For preparation of insoluble fragmentation of polysaccharides, any biologically compatible technique of microniza- tion may be used. Both, extractable and insoluble fragments of polysaccharides have a particle size between 0,1 micron to 5 micron or a fibril length of 0,1 micron to 10 mm. The ratio of microbial lysate to polysaccharide has be higher for polysaccharide by their dry weight content..

[0036] Own studies showed (see Biological examples): (i) both in vitro and in vivo studies clearly demonstrated that both, microbial lysate and micronized polysaccharide exert their biological effects on the mucosal epithelial and immune cells, these two basic structure of the microbiota-host symbiosis; (ii) these effects were significant even if they were administered alone; (iii) their combination used in the same model resulted in surprising synergistic benefits, significantly higher than the mathematical sum of their effects. Although, exploration of the exact mechanism of this synergy is out of the scope of this invention, observations made strongly suggested, that coadministration of microbial lysate and micronized polysaccharides enhances the phagocytosis and intracellular metabolism of these macromolecular aggregates. This synergy in the bioavailability is an unexpected effect coming from the combination of these compounds. This is the inventive step of the current invention.

[0037] Another embodiment of the current invention is that

- for human use the microbial lysate is selected from the human microbiota and com- bined with polysaccharides prepared from any sources compatible for human nutrition;

- for animal use the microbial lysate is selected from the microbiota of the given animal combined with polysaccharides from any sources compatible for animal nutrition; and

- for plant nutrition/fertilization the microbial lysates contain microbes selected from the microbiota of the given herb, and polysaccharides should be compatible for plant nutrition/fertilization.

[0038] Another embodiment of the current invention is that benefits of compositions containing combinations of microbial lysates and polysaccharides may be further en- hanced and customized by addition of at least one pharmacologically compatible ingredient such as omega-3 fatty acids in a natural form (fish oil, cod liver oil, vegetable oil or their combination), or sources of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) or an ester thereof; triglyceride, or phospholipids preferably phosphatidyl-inositol, phosphatidyl-choline, phosphatidyl- ethanolamine, phosphatidyl-serine and sphingomyelin, or combinations thereof; or as omega-3 fatty acids in the form of one of its precursors derived from fish-oil, plant-oil or a combination thereof, as vitamin A, B, C, D, E, F, K and ubiquinone/ubiquinol; and/or hormones (corticosteroids, sex-steroid), and /or metal ions (sodium, calcium, magnesium, potassium, phosphor, zinc, iron, selenium), and/or L-carnitine, ami- nocarnitines, alpha-lipoic acid, glutathion, amino acids, fatty acids, glycosaminogly- cans, bioflavonoids, polyphenols, curcumin, terpenes, alkaloides (berberine), volatile oils, and antibiotics. [0039] A further embodiment of the current invention is that compositions containing microbial lysates and micronized polysaccharides may further contain at least one formulation ingredient (water, glycerin, beeswax, sorbitol, lecithin, anti-aggregates, emulgens, thickeners, buffers, preservatives, etc.) for improving specific delivery, or for increasing bioavailability of the active and additive ingredients.

[0040] Another embodiment of the current invention is that compositions containing microbial lysates and micronized polysaccharides may be manufactured for human and animal use

(i) for enteral administration (oral, intra-gastric, trans-nasal, rectal); or

(ii) for parenteral administration such as intravenous, intraocular, intramuscular, intradermal, intra-articular, intraocular, intra-lesional, para-lesional, or subcutaneous application; or

(iii) for topical administration in the form of eye-drops, ear-drops, gel, spray, ointment, lotion; or

(iv) for liposome encapsulated delivery and

(v) for use as a soft-gel.

[0041] Another embodiment of this invention is the use of combinations of microbial lysates and micronized polysaccharides for plant nutrition/fertilization as well as for laboratory sciences.

[0042] Combinations of microbial lysates and micronized polysaccharides with the mentioned additive and formulation ingredients may result in very high variability of compositions and subsequently a wide range of application. Some of the most common - but not exclusive - targets to which the composition can be addressed are the following:

(i) bacterial, viral, fungal and parasitic infections,

(ii) vaccination against influenza, hepatitis, Bacillus Calmette-Guerin (BCG), poliomyelitis, Di-Per-Te (diphtheria-pertussis-tetanus), epidemic parotitis, measles or an antiallergic vaccine,

(iii) mucous membrane diseases, selected from the group of conjunctivitis, otitis, per- iodotitis, oesophagitis, reflux disease, gastritis, enteritis, colitis, cholecystitis, cystitis, pyelo-nephritis, sinusitis, bronchitis, vaginitis, prostatitis, or diseases selected from the group of hepatitis, cirrhosis, nephritis, pleuritis and fibrosis cystica, (iv) autoimmune diseases wherein the autoimmune inflammation manifests as rheumatoid arthritis, juvenile (type I) diabetes, Crohn's disease, ulcerous colitis, psoriasis, lupus erythematous or multiple sclerosis,

(v) neuropsychiatric diseases, selected from schizophrenia, depression, anxiety and panic-disease,

(vi) allergenic diseases, selected from bronchial asthma, atopic dermatitis, hay-fever, allergic conjunctivitis and allergic rhinitis,

(vii) metabolic syndrome, which comprises dyslipidemia, atherosclerosis, arterial hypertension, type 2 diabetes, obesity, fatty liver,

(viii) proliferative neovascular and neoplastic disease, wherein the neovascular disease is proliferative retinopathy, retinopathy of prematurity (ROP) or malignant tumors,

(ix) age-related degenerative disease wherein the age-related degenerative disease is Alzheimer's disease, Parkinson's disease, amyotropic lateral sclerosis, otosclero- sis, osteoporosis, osteoarthritis, sarcopenia, hairlessness or age-related skin changes,

(x) soft tissue damage, wherein the soft tissue damage is postoperative inflammation, sport injuries, extreme sport activities, contusions, burnings, cancer, lung cancer, colon cancer, lymphomas or chemotherapy associated inflammations,

(xi) eye diseases, wherein the eye disease is uveitis, diabetic retinopathy, age-related macular degeneration, glaucoma or cataract,

(xii) arterial hypertension and atherosclerosis-related diseases: coronary heart disease, cardiac arrhythmia, chronic heart failure, nephrosis syndrome, peripheral vascular diseases,

(xiii) ischemia-reperfusion, acute cerebral ischemia or chronic cerebral ischemia, stroke,

(xiv) skin diseases: sensitive or irritable skin, cellulitis, and

(xv) special medical conditions: to gene-transfer using natural, semi-synthetic and/or synthetic DNA and/or RNA; to support survival of transplanted stem cells and organs; to culture medium for cell-culture, tissue-culture and bacterial-culture; to feed infants, and to feed critically ill and convalescent persons. [0043] Clinical studies reported at least 500 cases of successful fecal transplantation to cure severe chronic gastrointestinal infections resistant to antibiotics. In these cases microbiota (live bacteria) from healthy donors were used to restore or at least to attenuate severe disorders of the microbiota-host symbiosis. The approach taken according to the current invention, namely using microbial lysate in combination with polysaccharides, may also be applied as an alternative for fecal transplantation, as only the biologically active ingredients (nucleic acids and proteins) of the microbiota are administrated.

[0044] Another embodiment of the current invention is the use of microbial lysates in combination with polysaccharides for maintaining and restoring microbiota-animal symbiosis. Similarly to humans, animals also live in symbiosis with a community of microbes. HOWEVER, changes in their life style and nutrition, like domestication cause perturbation of this symbiosis and subsequent disorders or diseases similar to humans. Consequently, administration of probiotics and microbial lysates is recom- mended for maintaining and restoring microbiota-host symbiosis. It should be kept in mind that several aspects of human diseases related to the microbiota came from animal model of diseases, thus the pathogenic mechanism is well studied and is essentially similar for both humans and animals. Combination of selected polysaccharides with microbial lysate for animal nutrition improves the bioavailability of microbial DNA/RNA and LPS/PG, likely in humans.

[0045] Another embodiment of the current invention is the use of microbial lysates in combination with polysaccharides for maintaining and restoring microbiota-plant symbiosis. The plant microbiota is associated with roots (rhisosphere) leaf (phyllo- sphere) and crop (endosphere) and contains mainly Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Both the leaf and root microbiota contain bacteria that provide pathogen protection, but root microbiota members appear to serve additional host functions through the acquisition of nutrients from soil for plant growth. Accumulating evidence suggests that the plant microbiota emerges as a fundamental trait that includes mutualism enabled through diverse biochemical mechanisms, as revealed by studies on plant growth-promoting and plant health-promoting bacteria.

[0046] Bacterial fertilizers containing bacteria such as cellulose decomposing bacteria (Pseudomonas putida), nitrogen-fixing bacteria (Azotobacter chroococcum), and phosphorus mobilization bacteria (Bacillus megaterium) are widely used for maintain- ing and restoring microbiota-plant symbiosis. These products contain live bacteria and their benefits on plant are similar to those of probiotics on human or animal health. However, the limitations of their benefits are also similar to those of live probiotics. Thus, administration of microbial lysate may have the same advantages for both, humans and plants. Selection of microbes for lysate may come from any member of plant microbiota. Addition of micronized polysaccharides enhances adherence and utilization, i.e. the bioavailability of microbial compounds, similarly to those in humans and animals.

[0047] The current invention is different from EP 2205253 in the following aspects: (i) different composition: active ingredients form an obligate combination of microbial lysate and micronized polysaccharides of habitual nutrients instead of an obligate combination of probiotic lysate with omega-3 fatty acids; (ii) different mode of action: the target of these compositions is microbiota-host symbiosis instead of the Plasma Membrane Redox System, (iii) different target for application: for humans or for ani- mals or for plants instead of the previous compositions which are recommended for humans and mammals only, and (iv) different composition of microbial lysate: the present invention claim the use of lysate prepared from all kinds of bacteria, archaea, yeasts and viruses instead of probiotics alone.

[0048] Several symbiotic products (i.e. combination of probiotics and prebiotic) are on the market recommended for maintaining and restoring normal microflora or the microbiota-host symbiosis or to prevent or even to treat some related disorders or diseases. All of them use exclusively live probiotics in contrast to the present invention.

Examples

I. Biological Examples

Example I: In vitro study was performed on the biological effects of microbial lysate and micronized polysaccharide.

Methods: Inflammatory cells were obtained from the peritoneal cavity of mice 4 h after i.p. lipopolysaccharide (LPS) injection. The abdominal cavity was washed by 2.5 ml cell culture medium supplemented with 10% foetal calf serum under sterile conditions. Then 100 μΙ samples of the lavage fluid were injected into 800 μΙ culture medium in a 24-well plate. Microbial lysate was prepared from heat-killed Lactobacillus acidophilus (LBL) exposed to sonication using S220 High Performance Ultrasonicator to reach <5,0 micron particle size. Micronized polysaccharide was prepared from freeze dried extract of a mushroom {Cordyceps pruinosa) sonicated (Branson Ultrasonic Corporation) for 8 min at room temperature to reach <1mm particle size. For studies 100μg/ml LBL and 1mg/ml crude unfiltered extract were used.

Results: LBL in 100 μg/ml dose has shown significant inhibitory effect on LPS- induced IL-1 β and TNF-a release from peritoneal macrophages of mice as compared to the control group, which was treated with physiological salt solution. Micronized extract of mushroom at 1mg/ml dose has also showed inhibitory effects on LPS- induced production of both IL-1 β and TNF-a from peritoneal macrophages of mice as compared to the control group. Although, these effects were approximately 35% lower than those of the LBL. The mixture of LBL and micronized extract of mushroom showed significantly higher inhibitory effects in the same model system as compared to either of them or even the sum of their effects.

Example II: In vivo animal studies were performed on the effects of microbial lysate and micronized polysaccharide on injured corneal epithelium and on the related inflammation.

Methods: Standardized alkali burnings on the cornea of 2 New-Zealand rabbits by paper 5 mm diameter filter paper disc containing 0,1 normal NaOH were performed. Rabbits were divided into 3 groups: Group I: One eye of 4 rabbits was treated 5 times daily with eye-drops containing microbial lysate prepared from Lactobacillus casei (0,1 % DNA of the dry weight), while the contralateral eye received physiological salt solution as an eye-drops 5 times daily. Group II: One eye of 4 rabbits was treated 5 times daily with a micronized polysaccharide containing eye-drops prepared from rose petals of Rosa centifolia (36,5% polysaccharide of dry weight) while the contralateral eye received physiological salt solution as an eye-drops 5 times daily. Group III: One eye of 4 rabbits was treated 5 times daily with eye-drops containing a mixture microbial lysate (prepared from Lactobacillus casei) and micronized polysaccharide (prepared from rose petals of Rosa centifolia), according to Formulation Example W./2 while the contralateral eye received physiological salt solution.

Results: Treated eyes in each group showed shorter time of epithelial regeneration as compared to the control eyes (12,45 +/- 2,33 days vs 16,6+/-3,26 days). The difference is statistically significant (<0,001). There was no significant difference be- tween microbial lysate and micronized polysaccharide treated groups. However, the time of epithelial regeneration was significantly shorter in the microbial lysate- micronized polysaccharide treated group as compared to either microbial lysate or micronized polysaccharide treated groups (<0,01). After 6 weeks of treatment, each treatment groups showed significantly smaller scar formation of the cornea as com- pared to those of the control eyes. While no significant difference was found between the microbial lysate and micronized polysaccharide groups, the scar formation was significantly smaller in the microbial lysate micronized polysaccharide groups as compared to either microbial lysate or micronized polysaccharide groups. Importantly, histopathology of the corneas using electron microscopy showed intacellular ag- gregates the microbial lysate-micronized polysaccharide complexes in the macrophages, indicating that these compounds are internalized and catabolized together in the target cell. This synergy in bioavailability may explain the in vivo synergy in epithelial healing and reduced inflammation (scar formation). This is a completely new observation, not disclosed in any prior art so far.

II. Composition Examples

In these examples particle size of microbial lysate varies from 0,001 to 5,0 micron, the particle size of micronized polysaccharides varies from 0,1 micron to 5 micron or fibril length of 0,1 micron to 1000 micron

Example 1: Composition containing: Lactobacillus acidophilus Iysate10 mg, micronized polysaccharide prepared from mushroom Cordyceps pruinos) 15 mg, physiologic salt solution for injection 5 ml. This composition is indicated as an im- munostimulant administered in combination of flu vaccine.

Example 2: Composition containing: Lactobacillus casef lysate 10 mg; micronized polysaccharide prepared from rose petals of Rosa centifolia 15 mg, physiologic salt solution for eyedrops 15 ml. This composition is indicated for topical use in eye 'dry- syndrome'.

Example 3: Composition containing: Bifidobacterium animalis lysate 25 mg; Helicobacter pylori lysate 5 mg; micronized polysaccharide prepared from aloe vera 100 mg; cod liver oil 500mg; lecithin 2 mg, distilled water 5 ml, and cyclodextrine quantum satis for soft gel. This composition is indicated for topical use to treat 'sensitive skin' and after superficial skin-burnings.

Example 4: Composition containing: Sacharomyces cerevisiae lysate 30mg; Lactobacillus rhamnosus Iysate15mg; Porphirinomas gingivalis lysate 0,5mg; micronized polysaccharide prepared from mushroom Coriolus versicolor 100mg; fish oil 500mg; L-carnitine 100 mg; lecithin 2 mg; glycerol 10 mg; Distilled water 10 ml, and gelatine quantum satis for soft gel. This composition is indicated for oral administration to enhance immune function in osteoarthritis.

Example 5: Composition containing: Lactobacillus rhamnosus Iysate 5 mg; mi- cronized polysaccharide prepared from mushroom Agaricus blazei Murill 100mg; beeswax 10mg; Distilled water for injection 10ml. This composition is indicated for parenteral administration to enhance immune function in chronic urogenital infections.

Example 6: Composition containing: Bifidobacterium longum lysate 25 mg; Listeria monocytogenes lysate 5 mg; micronized polysaccharide prepared from algae 100 mg; gelatine quantum satis for soft gel. This composition is indicated for oral use in treating chronic airway inflammations. Example 7: Composition containing: Lactobacillus plantarum lysate 100 mg; Staphylococcus thermophiles Iysate100 mg, micronized polysaccharide prepared from tart- cherry 100 mg; Smoke tree (Cotinus coggygria 'Royal Purple') extract 100 mg; Vehicles quantum satis for tooth paste.

Example 8: Composition containing: Escherichia coli lysate 50 g; micronized polysaccharide prepared from Jerusalem artichoke 100g; distilled water lOOOml.This preparation is indicated for treating chronic diarrhoea of chicken.

Example 9: Composition containing Clostridium perfringens lysate 5 mg, micronized polysaccharide prepared from maize 100 g; micronized polysaccharide prepared from grain 100 g; distilled water 1000 ml. This preparation is indicated for treating chronic diarrhoea of domestic animals (swine, cow, and sheep).

Example 10: Composition containing: Pseudomonas putida lysate 50g; Agrobacte- rium vitis lysate 50 g; Xanthomonas citri lysate 50g; micronized polysaccharide prepared from chicory 100g; distilled water 1000ml. This preparation is indicated for bio fertilization either on leaves or in the soil.

Example 11. Composition containing Lactobacillus acidophilus lysate 12,5mg;

Bifidobacterium bifidum lysate 12,5mg; Jerusalem artichoke powder 100,0mg,; thiamine 0,5mg; niacin 5,0mg; piridoxin 0,5mg; folate 75mcg; Vitamin B12 5 meg; Vitamin A 0,15 mg; Vitamin D 1 ,1 meg; flax seed powder 1000,0 mg and vehicles for hard capsule or tablet quantum satis. This composition is indicated for treating metabolic diseases, like type 2 diabetes, dyslipidemia, obesity, and atherosclerosis.

Example 12. Composition containing Lactobacillus rhamnosus lysate 12,5 mg; Lactobacillus reuteri lysate 12,5 mg: cod liver oil 500 mg; CoQ10 10 mg; acetyl-L- carnitine 100,0 mg, micronized polysaccharide prepared from chicory 100 mg, rape lecithin 15mg and vehicles for hard capsule or tablet quantum satis. This composition is indicated for treating neurodegenerative diseases, like Alzheimer's disease, Parkinson's disease, dementia.

Example 13. Composition containing Lactobacillus rhamnosus 12,5 mg; Bifidobacterium bifidum lysate 12,5 mg; Jerusalem artichoke powder 100,0 mg,; thiamine 0,5 mg; niacin 5,0 mg; piridoxine 0,5 mg; folate 75mcg; Vitamin B12 5 meg; Vitamin A 0,15 mg; Vitamin D 1 ,1 meg; Lutein 10,0 mg, Coenzyme Q10 10,0 mg, acetyl-l- carnitine 100,0 mg, fish oil 500,0 mg and vehicles for softgel quantum satis. This composition is indicated for treating age-related macular degeneration.

Claims

Claim 1. Composition for maintaining and restoring symbiosis between microbiota- hosts in humans, animals and plants, whereby said composition contains a combination of

(i) at least one microbial lysate containing deoxyribonucleic acid/ribonucleic acid and/or lipopolysaccharide/peptidoglycan derived from at least one species of bacteria and/or archaea, and/or yeasts and/or viruses residing in the human microbiota, the animal microbiota or the plant microbiota, or from their combinations, or derived from their synthetic or semisynthetic analogs; and the solid content of said microbial lysate having a particle size from 0,001 to 5,0 micron; and

(ii) at least one micronized polysaccharide compatible for human and/or animal nutrition and/or fertilization of plants, derived from natural sources comprising at least one species of bacteria, algae, mushrooms, plants, or any combination thereof, or from their synthetic or semisynthetic analogs; and said micronized polysaccharide having a particle size from 0,1 micron to 5 micron or fibril length of 0,1 micron to 10 mm and

(iii) the ratio of the microbial lysate to the polysaccharide has to be higher for the polysaccharide as measured by their dry-weight content.

Claim 2. The composition according to Claim 1 wherein

(i) the microbial lysate is derived from beneficial and/or saprophyte-commensal and/or pathogenic microorganism, or from any combination thereof, regardless from biological characteristics of not-lysed microbes, and

(ii) the micronized polysaccharide is derived from polysaccharides digesti- ble/absorbable and/or non-digestible/non-absorbable for humans and/or animals, from polysaccharides degradable/absorbable and/or non-degradable/non-absorbable for host plants, regardless from biological characteristics of natural, non-micronized forms of polysaccharides.

Claim 3. The composition according to any of Claims 1 and 2, containing at least one pharmacologically compatible additive ingredient selected from the group of omega-3 fatty acids in natural form such as fish oil, cod liver oil, vegetable oil or any combination thereof; sources of alpha-linolenic acid, eicosapentaenoic acid or docosahex- aenoic acid or any ester thereof; triglyceride; phospholipids, preferably phosphatidyl- inositol, phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidyl-serine and sphingomyelin, or any combination thereof; omega-3 fatty acids in form of one of its precursors derived from fish-oil, plant-oil, or any combination thereof; vitamin A, B, C, D, F, K and ubiquinone; hormones, such as corticosteroids and sex-steroid; metal ions; L-carnitine, aminocarnitines, alpha-lipoic acid, glutathion, amino acids, bioflavonoids, polyphenols, curcumin, terpenes, alkaloides, volatile oils, antibiotics, glycosa- minoglycans; or any combination thereof.

Claim 4. The composition according to any of Claims 1 to 3, containing at least one formulation ingredient selected from the group of water, glycerin, beeswax, sorbitol, lecithin, anti-aggregates, emulgens, buffers, preservatives, or any combination thereof; for improving delivery and increasing bioavailability.

Claim 5. The composition according to any of Claims 1 to 4; manufactured for human and animal use for

(i) enteral application such as oral, intra-gastric, trans-nasal, rectal application;

(ii) parenteral application such as intravenous, intraocular, intramuscular, intradermal, intraarticular, intralesional, paralesional, subcutaneous application;

(iii) topical application in form of eye-drops, gel, spray, ointment, lotion;

(iv) liposome encapsulated delivery;

(v) soft-gel.

Claim 6. The composition according to any of Claims 1 to 5 used for prevention and treatment in:

(i) bacterial, viral, fungal and parasitic infections;

(ii) vaccination against influenza, hepatitis, Bacillus Calmette-Guerin, poliomyelitis, diphtheria-pertussis-tetanus, epidemic parotitis, measles, anti-allergic vaccine;

(iii) mucous membrane diseases selected from conjunctivitis, periodontitis, caries, oesophagitis, reflux disease, gastritis, enteritis, colitis, cholecystitis, cystitis, pyelo- nephritis, sinusitis, bronchitis, vaginitis, prostatitis; or selected from hepatitis, cirrhosis, nephritis, pleuritis, fibrosis cystica;

(iv) autoimmune diseases, selected from rheumatoid arthritis, juvenile or type I diabetes, Crohn's disease, ulcerous colitis, psoriasis, lupus erythematous, multiple sclerosis;

(v) neuropsychiatric diseases, selected from schizophrenia, depression, anxiety, panic-disease;

(vi) allergenic diseases selected from bronchial asthma, atopic dermatitis, hay-fever, allergic conjunctivitis, allergic rhinitis;

(vii) metabolic diseases and syndrome, selected from dyslipidemia, atherosclerosis, arterial hypertension, type 2 diabetes, obesity, fatty liver;

(viii) proliferative neovascular diseases, selected from proliferative retinopathy, retinopathy of prematurity, malignant tumors;

(ix) age-related degenerative diseases, selected from Alzheimer's disease, Parkinson's disease, amyotropic lateral sclerosis, otosclerosis, osteoporosis, osteoarthritis, sarcopenia, hairlessness, age-related skin changes;

(x) soft tissue damages, selected from postoperative inflammation, sport injuries, extreme sport activities, contusions, burnings, cancer, lung cancer, colon cancer, lymphomas and chemotherapy associated inflammations;

(xi) eye diseases, selected from uveitis, diabetic retinopathy, age-related macular degeneration, glaucoma, cataract;

(xii) arterial hypertension and atherosclerosis-related diseases, selected from coronary heart disease, cardiac arrhythmia, chronic heart failure, nephrosis syndrome, peripheral vascular diseases; (xiii) skin diseases, selected from sensitive or irritable skin, cellulitis;

(xiv) ischemia-reperfusion, selected from acute cerebral ischemia, chronic cerebral ischemia, stroke;

(xv) special medical conditions, selected from gene-transfer using natural, semisynthetic and/or synthetic deoxyribonucleic acid and/or ribonucleic acid; supporting survival of transplanted stem cells and organs; culturing medium for cell-culture, tissue-culture and bacterial-culture; feeding infants; feeding critically ill and convalescent persons; substituting fecal transplantation; or any combination thereof;

(xvi) animal nutrition; or

(xvii) plant nutrition, plant fertilization.

Claim 7. The composition according to any of Claims 1 to 6, where the at least one microbial lysate is derived from bacteria selected of the group Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium animalis, Sacharomyces cerevisiae, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus plantarum, Escherichia coli, Clostridium perfringens, Pseudomonas putida