PROCEDURE FOR OBTAINING A MODULATING COMPOSITION OF THE HUMAN INTESTINAL MICROBIOME (Machine-transla

专利摘要:
Procedure for obtaining a modulating composition of the human intestinal microbiome that comprises cultivating strains of probiotic microorganisms that include strains of the genera Saccharomyces, Lactobacillus, Bacillus, Streptococcus and Bifidobacterium, at least until they have reached their full logarithmic phase of growth, in broths of separate culture to obtain biomass broths, separately recover moist microbial biomass and subject them separately to cell lysis until reaching a percentage of cell breakage of at least 90%; to obtain respective biomasses of microbial lysates that are dried and mixed in certain proportions. (Machine-translation by Google Translate, not legally binding)
公开号:ES2783885A1
申请号:ES201930280
申请日:2019-03-27
公开日:2020-09-18
发明作者:Gracia Luis Usan
申请人:Igen Biolab Group AG；
IPC主号:

专利说明:

[0001] PROCEDURE FOR OBTAINING A MODULATING COMPOSITION OF THE HUMAN INTESTINAL MICROBIOME
[0003] TECHNICAL FIELD OF THE INVENTION
[0005] The present invention is included in the technical field of products and methods intended to modulate the composition of the human microbiome, to preserve and / or reestablish a balance of the microbiome in human patients affected or susceptible to be affected by diseases or disorders related to dysbiosis of the microbiome.
[0007] STATE OF THE ART PRIOR TO THE INVENTION
[0009] It has been proven that the imbalance of the human microbiome or human microbiota, also called dysbiosis, can have harmful effects on people's health, which not only affect systems such as the intestinal tract, the skin or the reproductive system, but can also have influences on other parts of the human body. Recent research suggests that dysbiosis can even participate in or even generate the appearance of various types of cancer, autism, etc.
[0011] This has resulted, on the one hand, in research aimed at developing methods based on the analysis of the human microbiome in order to detect balances for the diagnosis and early detection of a plurality of diseases and disorders and, on the other, in products such as, for example probiotic and prebiotic products as well as postbiotic products, intended to preserve and / or reestablish a balance of the microbiome in human patients affected or susceptible to being affected by diseases or disorders.
[0013] Thus, it has been described that the oral administration of a postbiotic extract that contains secondary metabolites of commensal bacteria present in the serum of patients suffering from cancer could have beneficial effects on the health of patients to whom said extract had been administered (J Jimeno et al. “A microbiome based model of anticancer intervention.” European Journal of Cancer, December 2016 Volume 69, Supplement 1, Page S151 Poster Session https://doi.org/10.1016/S0959-8049(16)33049- 0).
[0014] Probiotics are defined by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), as a mixture of live, non-pathogenic microorganisms, which when administered in adequate amounts provide or generate beneficial effects on the host's health [Reyes Esparza, Jorge A. and Rodríguez Fragoso, Lourdes: PROBIOTICS: HOW DOES A MIXTURE OF MICROORGANISMS DO A GREAT JOB . Rev. mex. science. farm [online]. 2012, vol.43, n.1, pp.7-17. ISSN 1870-0195.]. Probiotics are believed to enhance the properties of the intestinal microflora by preventing colonization of the tract by pathogenic bacteria. In relation to the above, there are three basic ways to achieve this effect: (i) the immune exclusion of a pathogenic bacteria, (ii) the exclusion of a pathogen by competitive adhesion and (iii) the synthesis of antimicrobial compounds that hinder colonization. from the gastrointestinal tract by the pathogen [Casula, Gabriella, and Simon M. Cutting. "BACILLUS PROBIOTICS: SPORE GERMINATION IN THE GASTROINTESTINAL TRACT." Applied and Environmental Microbiology 68.5 (2002): 2344-2352. PMC. Web. 14 Mar. 2018.].
[0016] The identification of different metabolic by-products produced by probiotic species, for which the term "postbiotics" has recently been coined, may represent an opportunity to develop new effective therapeutic strategies that would avoid the risks associated with the administration of these live bacteria [Cicenia A, Scirocco A, Carabotti M, Pallotta L, Marignani M, Severi C. POSTBIOTIC ACTIVITIES OF LACTOBACILLI-DERIVED FACTORS. J Clin Gastroenterol 2014; 48: S18e22].
[0018] In general, postbiotics include bacterial metabolic by-products, such as bacteriocins, organic acids, ethanol, diacetyl, acetaldehydes, and hydrogen peroxide, but it is also true that certain heat-killed probiotics retain important bacterial structures that can exert biological activity in the host. Postbiotics are non-toxic, non-pathogenic, and resistant to hydrolysis by mammalian enzymes, as these are non-viable bacterial products or metabolic by-products of probiotics [Islam SU. CLINICAL USES OF PROBIOTICS. Medicine (Baltimore) 2016; 95: 1e5]. Furthermore, recent studies suggest that these metabolic products would cause more beneficial effects than the probiotic microorganisms that produce them.
[0019] The large intestine of the human being is colonized by billions of microorganisms (grouped in about 1000 different species) that make up a very complex ecosystem, the human intestinal microbiota. Numerous studies have an impact on the important role that the intestinal microbiota plays in maintaining correct homeostasis and on how population variations in the microbiota can lead to pathological states that modify the homeostatic balance. These alterations in the intestinal microbial balance, called dysbiosis, can be due to both eating and physical habits, or infectious, among others.
[0021] The commensal intestinal microbiota contributes to the trophic functions of the intestine (the production of fermentation products and vitamins used by enterocytes), stimulates the immune system, favors the gastrointestinal tract, transforms / excretes toxic substances, protects the host against invasion of species pathogenic, regenerates and preserves the intestinal barrier, modulates intestinal motility, etc ...
[0023] In the progression of the disease in cancer patients, it is very common to observe a high picture of malnutrition that in many cases interferes with the correct treatment of the disease and recovery of the patients. Ultimately, this observed malnutrition can degenerate into cachexia.
[0025] The commensal microbiota ensures the mechanical integrity of the mucosal barrier, offering protection against harmful pathogenic microbes. Commensal bacteria can adhere to intestinal mucus and competitively inhibit the adhesion of enteropathogens; they also produce bacteriocins and SCFA, compounds that can inhibit the growth of other microorganisms. Furthermore, some antimicrobial metabolites, such as defensins secreted in the intestine, contribute to the host control of these microorganisms [Salzman NH, Underwood MA and Bevins CL. 2007. Paneth cells, defensins, and the commensal microbiota: a hypothesis on intimate interplay at the intestinal mucosa. Seminars in Immunology. 19.70-83].
[0027] The immune system fights pathogenic bacteria, but tolerates the presence of commensal species, despite the fact that their cellular structures are quite similar and they have common mechanisms of interaction with cell receptors host immune. This phenomenon is called immune tolerance. In this way, human immune cells differentiate between commensals and pathogens. This is carried out by the human innate immune system through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), transmembrane receptors that scan the external environment of the intestinal lumen, and intracellular receptors similar to Nod (NODLR), which protect the cytoplasmic space [Claes AK, Zhou JY and Philpott DJ. 2015. NOD-like receptors: guardians of intestinal mucosal barriers. Physiology (Bethesda) 30, 241-250; Sellge G and Kufer TA. 2015. PRR-signaling pathways-Learning from microbial tactics. Seminars in Immunology. 27, 75-84; Hevia A, Delgado S, Sanchez B et al. 2015. Molecular Players Involved in the Interaction Between Beneficial Bacteria and the Immune System. Frontiers in Microbiology 6: 1285].
[0029] Microbial imbalance in the gut, commonly referred to as dysbiosis, has been associated with a number of diseases such as inflammatory bowel disease, allergy to asthma, metabolic and cardiovascular diseases, as well as cancer [Klimesova K, Kverka M, Zakostelska Z et to the. 2013. Altered gut microbiota promotes colitis-associated cancer in IL-1 receptor-associated kinase M-deficient mice. Inflamm. Bowel Dis. 19: 1266-1277]. In fact, convincing evidence demonstrated that intestinal dysbiosis can lead to an overrepresentation of certain bacterial species that can promote colon carcinogenesis by promoting chronic inflammation or local immunosuppression [Grivennikov SI, Wang K, Mucida D et al. 2012. Adenomalinked barrier defects and microbial products drive IL-23 / IL-17-mediated tumor growth. Nature 491: 254-258]. It should be noted that experimental alterations of the intestinal microbiota even influence the appearance and progression of extraintestinal tumors, such as breast and hepatocellular carcinoma, probably through inflammatory and metabolic circuits [Yoshimoto S, Loo TM, Atarashi K et al. 2013 . Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 499: 97-101; Dapito DH, Mention A, Gwak GY et al. 2012. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell 21: 504-516; Marinelli L, Tenore GC and Novellino E. 2017. Probiotic species in the modulation of the anticancer immune response. Semin Cancer Biol 46: 182-190].
[0031] The human immune system has co-evolved with the microbiological ecosystem that it is found in the human body. In this way, commensal microorganisms are able to elude the immune response by attenuating the inflammatory response, for example, generating short-chain fatty acids, while the immune system controls concentration, location and types of microorganisms through a large number of PRR receptors (NLRs as cytosolic receptor that signals in case of infection or cell damage, and TLRs and CLRs as membrane receptors specialized in different molecules such as peptidoglycans, lipopolysaccharides, flagellin ...) capable of launching a strong innate immune response in case of be necessary [Maynard CL, Elson CO, Hatton RD et al.
[0032] 2012. Reciprocal interactions of the intestinal microbiota and immune system. Nature.
[0033] 489: 231-41], and to subsequently activate the adaptive immune response.
[0035] In situations of homeostasis (normal microbiota balance) the cells of the intestinal epithelium secrete cytokines TSLP, IL-33 and IL-25 that stimulate the expression of CD103 in dendritic cells, and these induce the development of regulatory T lymphocytes capable of containing the response immunological. Also epithelial cells are capable of producing abundant TGF-a and IL-10 which inhibits the pro-inflammatory effects of the NFkB signaling pathway.
[0037] The influence of the microbiota on the number of Natural Killer (NK) lymphocytes has also been described. This cell type is specialized in eliminating cells with viral infections or tumor cells, and it is observed in germ-free mouse models how a microbiota is necessary for the activation of these lymphocytes, in a process mediated by INF-I [Ganal SC, Sanos SL, Kallfass C et al. 2012. Priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota. Immunity. 37: 171-186]. In general, Natural Killer is activated by the presence of interferons and IL-2 secreted by T lymphocytes and constitutes an interesting target to activate the immune response of patients against tumors.
[0039] In recent years, studies have proliferated on the role of the microbiota as an immunomodulator at the systemic level [Rescigno M. 2014. Intestinal microbiota and its effects on the immune system. Cell Microbiol. 16: 1004-13]. Inflammatory processes can be modulated by the microbiota through the innate immune response via TLR / NOD signaling [Francescone R, Hou V, Grivennikov SI. 2014. Microbiome, Inflammation, and Cancer. Cancer Journal 20: 181-189], or variations in the concentration and differentiation of the different lymphocyte types have been observed in situations of dysbiosis or in germ-free animals. For example [Cheng M, Qian L, Shen G et al. 2014. Microbiota modulate tumoral immune surveillance in lung through a Y5T17 immune cell-dependent mechanism. Cancer Res. 74: 4030-41] shows how variations in the microbiota of mice caused by antibiotic treatments are capable of reducing the Th17 lymphocyte response to lung cancer by modifying the intestinal microbiota.
[0041] Epigenetic mechanisms - such as acetylation of histones, methylation of DNA promoters and the presence of large or small non-coding RNAs that affect the activity of transcription factors of genes by modulating their activity - are sensitive to metabolites. present in the environment generated by host bacteria. These metabolites generated will vary depending on environmental factors such as diet, tobacco or drugs [Passalacqua et al 2009]. There is a growing interest in diet, as an example, the impact of butyrate, which acts on epigenetic mechanisms leading to specific and effective therapeutic strategies in the prevention and treatment of different diseases, including cancer at the systemic level [Berni Canani R, Di Costanzo M and Leone L.
[0042] 2012. The epigenetic effects of butyrate: potential therapeutic implications for clinical practice. Clin epigenetics 4: 4].
[0044] The microRNAs -miRNA- are endogenous non-coding RNAs of about 22 nucleotides that regulate gene expression at the post-transcriptional level. Like protein-coding genes, the expression of miRNAs is also regulated by genes and epigenetic mechanisms [Liu et al 2009]. Currently, these types of non-coding RNA have been associated as markers of lung cancer and its evolution [Lin PY et al. 2010. MicroRNA in lung cancer. British Journal of cancer 103: 1144-48]. Furthermore, certain identifiable miRNAs have been described in early stages of disease [Lin PY et al. 2011. Circulating miRNA signature for early diagnosis of lung cancer. EMBO Molecular Medicine 3: 436-437, Ulivi P and Zoli W. 2014. miRNAs as Non-Invasive biomarkers for lung cancer diagnosis. Molecules, 19: 8220-8237].
[0046] Chromosomal acetylation and deacetylation of histones alter their interaction causing changes in regulation of gene expression. The main donor of acetyl groups for the formation of acetyl-CoA that participates in epigenomic acetylation reactions is the intestinal microbiota. Bacteria and eukaryotes share a common pathway for coenzyme A (CoA), pantothenate biosynthesis (vitamin B5), cysteine, and b-alanine. All of these are essential cofactors found in most foods in small amounts and are also generated by the gut microbiota [Shenderov BA. 2012. Gut indigenous microbiota and Epigenetics. Microbial Ecology in Health & Disease 23: 17195].
[0048] The microbiota provides signals to the intestinal epithelium that contribute to the effective function of the intestinal barrier. Neonatal exposure to a complex microbiota establishes the CpG methylation pattern in intestinal stem cells that is related to stem cell turnover and differentiation of IEC [Yu DH, Gadkari M, Zhou Q et al. 2015. Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome. Genome Biol 16: 211]. Short-chain fatty acids (SCFA) are absorbed as energy by the surface colonocytes that line the intestinal crypts and thus prevent diffusion to the stem cell niche, where these metabolites inhibit stem cell proliferation [Kaiko GE, Ryu SH, Koues OI et al. 2016. The colonic crypt protects stem cells from microbiota-derived metabolites. Cell 165: 1708-1720]. Interactions between IECs and the microbiota involve the regulation of histone deacetylases (HDACs) to modify the host epigenome and influence gene expression and barrier function [Alenghat et al 2013]. The expression of HDAC in IEC maintains intestinal homeostasis and barrier integrity [Alenghat T, Osborne LC, Saenz SA et al. 2013 Histone deacetylase 3 coordinates commensal-bacteria- dependent intestinal homeostasis. Nature 504: 153-157; Turgeon N, Gagne JM, Blais M et al.
[0049] 2014. The acetylome regulators Hdac1 and Hdac2 differently modulate intestinal epithelial cell dependent homeostatic responses in experimental colitis. Am J Physiol Gastrointest Liver Physiol 306: G594-G605; Zimberlin CD, Lancini C, Sno R et al.
[0050] 2015. HDAC1 and HDAC2 collectively regulate intestinal stem cell homeostasis. FASEB J 29: 2070-2080; Gonneaud A, Turgeon N, Boudreau F et al. 2016. Distinct roles for intestinal epithelial cell-specific Hdac1 and Hdac2 in the regulation of murine intestinal homeostasis. J Cell Physiol 231: 436-448].
[0052] It is known that, in homeostasis, the microbiota benefits from the warm and rich environment in nutrients, while humans benefit from a well-functioning metabolic engine that increases human capacity to extract nutrients from food [Maynard CL, Elson CO, Hatton RD et al. 2012. Reciprocal interactions of the intestinal microbiota and immune system. Nature. 489: 231-41]. In addition to a metabolic aid, the gut microbiota influences tissue development, inflammation, and immunity, promoting human health or disease [Lee YK, Mazmanian SK. 2010. Has the microbiota played a critical role in the evolution of the adaptive immune system Science 330: 1768-1773; Cho I & Blaser MJ. 2012. The human microbiome: at the interface of health and disease, Nat. Rev. Genet. 13: 260-270]. Beneficial bacteria, which promote human health, are commonly known as "probiotics" and are defined as living microorganisms that, when administered in adequate amounts, confer a benefit to the health of the host [FAO, WHO, 2001. Evaluation of Health and Nutritional Properties of Powder Milk and Live Lactic Acid Bacteria, Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation Report. 1-34]. As mentioned earlier, there is a mutualistic symbiosis between the gut microbiota and host immunity. In fact, the immune system has to strike a proper balance between tolerance to the microbiota and vigilance against infectious agents. Intestinal homeostasis is maintained as an inflammatory tone, allowing an adequate self-limited response to infectious agents or stress [Peterson CT, Sharma V, Elmén L et al. 2015. Immune homeostasis, dysbiosis and therapeutic modulation of the gut microbiota, Clin. Exp. Immunol. 179: 363-377]. The balance between tolerance and response to infections is the fruit of an extensive and cooperative cross-talk between the gut microbiota and the host that involves both innate and adaptive immunity [Klaenhammer TR, Kleerebezem M, Kopp MV et al. 2012. The impact of probiotics and prebiotics on the immune system. Nat. Rev. Immunol. 12: 728-734; Marinelli L, Tenore GC and Novellino E. 2017. Probiotic species in the modulation of the anticancer immune response. Semin Cancer Biol 46: 182-190].
[0054] Lactobacillus acidophilus isolated from traditional homemade yogurt induced a significant decrease in the mammary tumor growth pattern using a mouse model [Maroof H, Hassan ZM, Mobarez AM et al. 2012. Lactobacillus acidophilus could modulate the immune response against breast cancer in murine model, J. Clin. Immunol. 32: 1353-1359].
[0055] The administration of milk fermented by the probiotic strain Lactobacillus casei CRL431 in a preventive manner, or when the administration started after injection of the tumor cells, resulted in a delay or arrest of breast tumor development compared to the control group [ Aragon et al 2014]. Several lines of evidence suggest that part of the mechanism of action of L. casei CRL431 towards breast cancer would be the decrease of IL-6 in the blood serum and in the mammary glands.
[0057] A protective role for Lactobacillus reuteri ATCC-PTA-6475, originally isolated from human milk, against tumor in two different mouse models of breast cancer [Lakritz JR, Poutahidis T, Levkovich T et al. 2014. Beneficial bacteria stimulate host immune cells to counteract dietary and genetic predisposition to mammary cancer in mice, Int. J. Cancer 135: 529-540]. Regarding the mechanism of action, L. reuteri appeared to protect against cancer by supporting the immune system through the induction of anti-inflammatory CD4 CD25 T cells.
[0059] Lactobacillus casei BL23, known for its anti-inflammatory properties, was studied for its protective effects on colorectal cancer in mice [Lenoir M, Del Carmen S, Cortes-Perez NG et al. 2016. Lactobacillus casei BL23 regulates Treg and Th17 T-cell populations and reduces DMH-associated colorectal cancer, J. Gastroenterol. 51: 862-873]. The analysis of cytokines in the intestinal contents showed that the development of colorectal cancer was accompanied by an inflammatory state, with high levels of pro-inflammatory cytokines, such as monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor a (TNF- a), in the samples of the control group. In contrast, mice that received the probiotic appreciably showed decreased levels of the above-mentioned cytokines in intestinal samples and increased amounts of anti-inflammatories, such as IL-10.
[0061] In mice treated with the probiotic, lower levels of Tregs and a slight increase in Th17 cells were observed, suggesting that L. casei BL23 triggers a mixed type Th17 / Treg immune response [Lenoir M, Del Carmen S, Cortes-Perez NG et al. 2016. Lactobacillus casei BL23 regulates Treg and Th17 T-cell populations and reduces DMH-associated colorectal cancer, J. Gastroenterol. 51: 862-873].
[0062] In vivo experiments have shown that mice pre-inoculated orally with Lactobacillus acidophilus NCFM probiotics showed a 50.3% reduction in tumor growth, compared to untreated mice, after receiving colon cancer implantation (cells CT-26) [Chen CC, Lin WC, Kong MS et al. 2012. Oral inoculation of probiotics Lactobacillus acidophilus NCFM suppresses tumor growth both in segmental orthotopic colon cancer and extra intestinal tissue, Brit. J. Nutr. 107: 1623-1634].
[0064] Recently, Lactobacillus rhamnosus (JF414108.1) and L. plantarum (KC836552.1) were investigated in a mouse model of colorectal cancer (CT26 cells), for their ability to induce immune responses, including DC maturation, secretion of cytokines, the polarization of CD4 T cells and the activation of NK cell activity [Viaud S, Saccheri F, Mignot G et al. 2013. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide, Science 342: 971-976]. It was found that pre-inoculation with L. plantarnm significantly reduced tumor growth, prolonged survival time and activated innate immunity, increasing intratumoral levels of CD8 T and NK cells in the tumor microenvironment, while L. rhamnosus was somewhat less effective with the poor extended protective effects and a slight decrease in tumor volume in treated mice [Hu J, Wang C, Ye L et al. 2015. Anti-tumor immune effect of oral administration of Lactobacillus plantarum to CT26 tumor-bearing mice, J. Biosci. 40: 269-279].
[0066] L. rhamnosus GG has been shown to be more effective than Bacillus Calmette Guerin (BCG, Mycobacterium bovis) immunotherapy in reducing the incidence of recurrence in bladder cancer [Seow SW, Cai S, Rahmat JN et al. 2010. Lactobacillus rhamnosus GG induces tumor regression in mice bearing orthotopic bladder tumors, Cancer Sci. 101: 751-758]. ELISA analysis of bladder proteins revealed an increase in XCL1, a chemokine produced by activated T cells CD8 and yS, NK and master cells, which acts as a chemotherapeutic attractant for T and NK cells, thus assisting the process of tumor regression [Seow SW, Cai S, Rahmat JN et al. 2010. Lactobacillus rhamnosus GG induces tumor regression in mice bearing orthotopic bladder tumors, Cancer Sci. 101: 751-758].
[0067] Oral administration of Lactobacillus casei strain Shirota (LcS) has been found to enhance innate immunity by stimulating the activity of splenic NK cells. Oral feeding of dead LcS was able to stimulate the production of Th1 cytokines, which produced a suppressed production of IgE antibodies against ovalbumin in experimental mice. The ability to shift the mucosal immune response toward Th1 with probiotic bacteria provides a strategy for treating allergy disorders. METH A tumor cell growth in the lungs was also inhibited by intrapleural injection of LcS.
[0069] Oral administration of other probiotic bacteria, such as Streptococcus thermophilus (St), Lactobacillus fermentum (Lf), and yeast (Y), elicited different immune responses. The cofeeding antigen with probiotic bacteria can suppress both the cellular and antibody immune responses and can provide an effective protocol for attenuating autoimmune diseases, such as experimental allergic encephalomyelitis, by co-administration of myelin basic protein and probiotic bacteria Matsuzaki T & Chin J. 2000. Modulating immune responses with probiotic bacteria. Immunology and Cell Biology 78: 67-73].
[0071] A significant number of relevant studies have highlighted the immunomodulatory effects that Lactobacillus and Bifidobacterium strains exert on the host's immune system. For example, there is evidence that Bifidobacterium bifidum LMG13195 and Bifidobacterium breve IPLA20004 improve intestinal barrier function and preferentially cause Treg cell differentiation, which induces expression of anti-inflammatory cytokines, when co-cultured with the human colorectal adenocarcinoma cell line HT29 [ López P, González-Rodríguez I, Sánchez B et al. 2012. Interaction of Bifidobacterium bifidum LMG13195 with HT 29 cells influences regulatory-T-cell-associated chemokine receptor expression. Appl. Environ. Microbiol. 78. 2850-2857]. Lactobacillus rhamnosus GG interacts with macrophages in such a way that activated macrophages can discriminate between probiotic and pathogenic bacteria by regulating the TLR gene mediated by INF [Miettinen et al 2008] and the interaction between Lactobacillus casei CRL431 and immune cells associated with the intestine induce an increase in the number of CD-206 and TLR2 receptors [Aragón F, Carino S, Perdigón G. et al. 2014. The administration of milk fermented by the probiotic Lactobacillus casei CRL431 exerts an immunomodulatory effect against a breast tumor in a mouse model. Immunobiology 219: 457-464].
[0073] Soluble components produced by probiotic bacteria can also affect the interaction between bacteria and hosts. In Bifidobacterium longum, the secretion of serpin, a serine protease inhibitor, which specifically binds and inactivates human neutrophils and pancreatic elastase, has been shown to contribute to intestinal homeostasis [Ivanov et al 2006]. Furthermore, it has been observed that some proteins with characteristic biochemical motifs produced by both commensal and pathogenic bacteria can provoke specific functions and affect the immune cells of the intestinal lumen. This is the case of a family of proteins rich in serine threonine, which has been described in Lactobacillus and Bifidobacterium species , with a recently described kinase function [Zakharevich NV, Osolodkin DI, Artamonova II. et al. 2012. Signatures of the ATP-binding pocket as a basis for structural classification of the serine / threonine protein kinases of gram-positive bacteria. Proteins 80: 1363-1376; Nezametdinova VZ, Zakharevich NV, Alekseeva MG et al. 2014. Identification and characterization of the serine / threonine protein kinases in Bifidobacterium. Arch. Microbiol. 196,125-136]. In lactobacilli, a serine-threonine peptide, STp, which is contained in a protein secreted by L. plantarum, was found to be involved in bacterial aggregation [Hevia et al 2013]. Furthermore, this peptide can modulate the dendritic cell phenotype of patients with ulcerative colitis (UC) [Bernardo D, Sánchez B, Al-Hassi HO et al. 2012. Microbiota / host crosstalk biomarkers: regulatory response of human intestinal dendritic cells exposed to Lactobacillus extracellular encrypted peptide. PLoSONE 7: e36262; Al-Hassi HO, Mann ER, Sanchez B et al. 2014. Altered human gut dendritic cell properties in ulcerative colitis are reversed by Lactobacillus plantarum extracellular encrypted peptide STp. Mol. Nutr. FoodRes. 58: 1132-1143].
[0075] The immunomodulatory effect of Lactobacillus paracasei is mediated, at least in part, by the secreted protease lactozepine, which selectively degrades the IFN-g-inducible protein 10 (IP-10) that functions in lymphocyte recruitment [von Schillde MA, Hormannsperger G, Weiher M, et al. 2012. Lactocepin secreted by Lactobacillus exerts anti- inflammatory effects by selectively degrading proinflammatory chemokines. Cell Host Microbe 11: 387-396]. There are other examples of non-proteinaceous compounds that can exert certain effects on the host. Some species of Bifidobacterium possess unmethylated CpG motifs in their DNA that were capable of inducing the activation of TLR9, which is known to trigger a Th1 orientation of the immune system [Ménard O, Gafa V, Kapel N et al. 2010. Characterization of immune stimulatory CpG-rich sequences from different Bifidobacterium species. Appl. Environ. Microbiol. 76: 2846-255; Hevia A, Delgado S, Sanchez B et al. 2015. Molecular Players Involved in the Interaction Between Beneficial Bacteria and the Immune System. Frontiers in Microbiology 6: 1285]. Metabolites derived from the microbiota: short chain fatty acids (SCFA) Mammalian cells can detect microbes through pattern recognition receptors such as lipopolysaccharide (LPS) -recognizing toll-like receptors (TLRs). Recently, it has been discovered that microbe-derived short-chain fatty acids (SFCA) that are produced by commensal bacteria, such as Clostridia and Bifidobacteria, from the fermentation of carbohydrates and fiber, have become central players that mediate crosstalk between the microbiota and the host [Macfarlane S and Macfarlane GT. 2003. Regulation of short-chain fatty acid production. Proc Nutr Soc 62: 67-72; Bolognini D, Moss CE, Nilsson K et al. 2016. A novel allosteric activator of free fatty acid 2 receptor displays unique gi-functional bias. J Biol Chem 291: 18915-18931]. In particular, propionate, acetate, and butyrate, the three most abundant SCFAs in the intestinal lumen, have received increased attention in the field due to their potential beneficial impact on host physiology, including reduced inflammation and improved epithelial barrier function. although these effects have varied between studies [Brestoff JR and Artis D. 2013. Commensal bacteria at the interface of host metabolism and the immune system. Nat Immunol 14: 676-684; Rooks MG and Garrett WS. 2016. Gut microbiota, metabolites and host immunity. Nat Rev Immunol 16: 341-352; Suzuki T, Yoshida S and Hara H. 2008. Physiological concentrations of short-chain fatty acids immediately suppress colonic epithelial permeability. Br J Nutr 100: 297-305]. Germ-free mice express little or no SCFA, indicating that the production of these metabolites depends on the microbiota [Maslowski KM, Vieira AT, Ng A et al. 2009. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 461: 1282-1286].
[0077] Although their mechanism of action is not fully understood, SCFAs are thought to modulate host cellular processes through (1) direct inhibition of HDAC Activity and / or (2) activation of G protein-cou-pled-receptors (GPCR) [ Macia et al 2015; kim et al 2013]. HDACs remove acetyl protein residues Histones or non-histones and eighteen known mammalian HDACs are classified into four classes based on sequence homology. These epigenomic-modifying enzymes are often present in large protein complexes that target the target chromatin through transcription factor interactions. Since SCFAs, and particularly butyrate, have long been known to broadly inhibit the HDAC epigenomic family of enzymes, several recent studies have shown or suggested that SCFAs mediate interactions between the microbiota through epigenomic regulation [Woo V and Alenghat T. 2017. Hostmicrobiota interactions: epigenomic regulation. Current Opinion in Immunology 44: 52-60].
[0079] Some bacteria have the ability to metabolize derivatives of aromatic amino acids in the intestinal tract, giving rise to numerous compounds with antitumor properties (mainly antioxidants, as happened in the case of polyphenols) or tumorous, as in the case of phenol, indole or p-cresol, which they have mutagenic properties in addition to altering the composition of the microbiota. [Russell WR, Duncan SH, Scobbie L et al. 2013. Major phenylpropanoid-derived metabolites in the human gut can arise from microbial fermentation of protein. Mol Nutr Food Res. 57: 523-35, Louis P, Hold GL and Flint HJ. 2014. The gut microbiota, bacterial metabolites and colorectal cancer. Nature Reviews Microbiology 12: 661-72].
[0081] The commensal microbiota can influence spontaneous antitumor immunity, as well as therapy with mAb PD-L1. Within the intestinal commensal bacteria of mice, Bifidobacterium was identified as the main component of the beneficial antitumor immune effects observed in JAX mice. This mouse bacterium was found similar to B. breve, B. longum, and B. adolescentis (99% identity) that are commonly found in a healthy human intestine.
[0083] Bifidobacterium alters the activity of dendritic cells (DC), which in turn improves the function of tumor-specific CD8 T cells. The fact that the antitumor effect requires live bacteria implies that Bifidobacterium colonizes specific compartments within the intestinal section that allows interaction with host cells critical to modulate DC function or critical for the release of soluble factors that circulate systemically and leads to a improved DC function. It should be noted that these results suggest that there is another source of heterogeneity between subjects, with respect to the therapeutic effects of the anti PD-L1 mAb, and which is represented by the composition of the gut microbes, in addition to the immune status of the patient, the amount of infiltration of effector T cells at the tumor site, or the expression profile of T cell checkpoints.
[0085] There are experiments, which seem to clearly reveal that the gut microbiota is necessary for the anticancer effects of CTLA-4 blockade [Vétizou M, Pitt JM, Daillére R et al. 2015. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota, Science 350: 1079-1084]. These results were also confirmed in the Ret and MC38 melanoma models of colon cancer. Investigation of immune system cell function from antibiotic-treated mice reveals that Ab-induced activation of splenic CD4 effector T cells and tumor-infiltrating lymphocytes (TIL) against CTLA-4 was significantly decreased compared to SFP mice. untreated [Vétizou M, Pitt JM, Daillére R et al. 2015. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota, Science 350: 1079-1084].
[0087] The fact that the gut microbiota has a substantial influence on the immune response, both in animals and humans, has been amply demonstrated [Maynard CL, Elson CO, Hatton RD et al. 2012. Reciprocal interactions of the intestinal microbiota and immune system. Nature. 489: 231-41; Honda K and Littman DR. 2016. The microbiota in adaptive immune homeostasis and disease, Nature 535: 75-842016]. The gut microbiota affects the efficacy of checkpoint blocking against multiple tumor models, at least in mice [Sivan A, Corrales L, Hubert N et al.
[0088] 2015. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy, Science 350: 1084-1089, Vétizou M, Pitt JM, Daillére R et al. 2015. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota, Science 350: 1079-1084]. However, while an "optimized" microbiota increases the efficacy of the anti-PD-1 mAb, this was absolutely necessary for the activity of the anti-CTLA-4 mAb.
[0090] Clearly, all of these findings open up new avenues for cancer therapy. Experiments in mice would suggest that, in principle, the gut microbiota can be successfully manipulated to enrich itself with a particular beneficial bacteria or a Selected cocktail of bacteria to enhance antitumor immunity.
[0092] Three articles in Science support the idea that the composition of the gut microbiota modulates the response to immunotherapy by blocking antibodies against programmed cell death protein 1 (PD1) or PD1 ligand 1 (PDL1) in patients with epithelial tumors, including non-small cell lung cancer and renal cell carcinoma or melanoma. In the first of these studies, broad-spectrum antibiotic-induced dysbiosis was associated with failure of PD1- or PDL1-targeted immunotherapy in both patients and mice.In all three studies, fecal microbial analyzes of independent patient populations led to the identification of bacterial entities that are positively correlated with clinical response [Kroemer G and Zitvogel L. 2018. Cancer Immunotherapy in 2017: The breakthrough of the microbiota. Nature Rev Immunol 18: 87-88]
[0094] DESCRIPTION OF THE INVENTION
[0096] The present invention relates to a method for obtaining a modulating composition of the human intestinal microbiome, which is useful in the prevention and treatment of disorders caused or at least promoted by intestinal dysbiosis of the microbiota in humans.
[0098] The method of obtaining according to the invention comprises the steps of cultivating strains of probiotic microorganisms comprising strains of the genera Saccharomyces, Lactobacillus, Bacillus, Streptococcus and Bifidobacterium, in culture broths to obtain biomass broths of the probiotic microorganisms that comprise microbial biomass and culture broths, and recovering microbial biomass from microbial biomass broths to obtain wet recovered microbial biomass and is characterized in that
[0099] the strains of each microorganism are cultivated, at least until they have reached their full logarithmic phase of growth, in separate culture broths;
[0100] the microbial biomasses are recovered separately from the respective culture broths to obtain respective wet recovered microbial biomasses; each of the wet recovered microbial biomasses is subjected to cell lysis until reaching a percentage of cell breakage of at least 90% to obtain respective biomass of microbial lysates;
[0101] the respective biomasses of microbial lysates are dried to obtain respective dried microbial lysates;
[0102] the respective dry microbial lysates are mixed in proportions of 30% to 70% by volume of dry microbial lysates from cultures of strains of the genus Saccharomyces,
[0103] 10% to 30% by volume of dry microbial lysates from cultures of strains of the genus Lactobacillus,
[0104] 10% to 30% by volume of dry microbial lysates from cultures of strains of the genus Bacillus,
[0105] 3% to 10% by volume of dry microbial lysates from cultures of strains of the genus Streptococcus,
[0106] 3% to 10% by volume of dry microbial lysates from cultures of strains of the genus Bifidobacterium,
[0107] to obtain a mixture of dry microbial lysates.
[0109] From the mixture of microbial lysates, at least one sample of the mixture of dried microbial lysates is extracted and it is determined whether the sample comprises a minimum of 5000 copies / ml genomic sequences of each of the following genomic sequences:
[0114] and, when it is found that the sample comprises said minimum of genomic sequences, the mixture of the dried microbial lysates is selected to obtain the modulator composition.
[0115] In a preferred embodiment of the method, the minimum of genomic sequences further comprises at least 5000 copies / ml of additional genomic sequences matching at least 75% with each of the following additional genomic sequences
[0120] When it is found that the sample comprises said minimum of additional genomic sequences, the mixture of the dried microbial lysates is selected to obtain the modulator composition.
[0122] In one embodiment of the method, the strains of microorganisms of the genus Saccharomyces are strains of Saccharomyces cerevisiae.
[0124] The strains of microorganisms of the genus Lactobacillus puedeb selected from Lactobacillus gasseri, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus reuteri, and combinations thereof, strains of microorganisms of the genus Bacillus may be selected from strains of Bacillus subtilis, Bacillus coagulans, Bacillus clausii, and combinations thereof, and strains of microorganisms of the genus Bifidobacterium can be selected from strains of Bifidobacterium bifidum, Bifidobacterium infantis, and combinations thereof. In turn, the strains of microorganisms of the genus Streptococcus can be strains of Streptococcus thermophilus.
[0126] In a preferred embodiment, the process according to the invention, the microorganisms of the genus Lactobacillus are cultured at pH 6.4 10% in the absence of oxygen until an anaerobic culture broth is obtained in which the microorganisms of the genus Lactobacillus are in the full logarithmic phase of growth of its biomass and, when the microorganisms have reached their full logarithmic phase of growth has been reached, oxygen is introduced into the initial culture broth until it contains between 25% and 30% dissolved oxygen to obtain an oxygenated culture broth. Thus, microorganisms of the genus Lactobacillus can be cultured at pH 6.4% 0.1 in the absence of oxygen for 5.5 to 6.5 hours.
[0128] This oxygenated culture broth is seeded with, for example 4 to 6% v / v, of a culture of microorganisms of the genus Saccharomyces , and the microorganisms of the genus Lactobacillus and Saccharomyces are grown together, adjusting and keeping the culture broth with 20% to 30% of dissolved oxygen and maintaining the culture broth at pH 6.4 10%, particularly at pH 6.4 0.1, and more particularly during a first stage and allowing in a second stage that the pH drop to 4.5 10%. The culture is stopped when the pH has reached 4.5 10%, particularly at pH 4.5 0.1, to obtain broths of the microbial biomasses formed from the microorganisms of the Lactobacillus and Saccharomyces genera .
[0130] According to this preferred embodiment of the process, the microorganisms of the genus Lactobacillus are preferably cultured in the absence of oxygen for 5 to 7 hours and more preferably for 5.5 and 6.5 hours, and, in the first step, the microorganisms of the Lactobacillus and Saccharomyces genera for 7 to 9 hours, particularly for 7.5 to 8.5 hours.
[0132] Preferably, the microbial biomasses are recovered by centrifugation separately from the respective culture broths, and dried by spray drying or by lyophilization.
[0134] Microorganisms of the genus Bacillus can be cultured at 30 ° C 1 ° C for 20-24 hours, maintaining a dissolved oxygen concentration of at least 40% and a pH of 6.8 0.1, preferably keeping the pH stable by adding al minus one stabilizing agent which may comprise a 25% w / v aqueous ammonium solution and / or a 35% w / v aqueous tetraoxphosphoric acid solution.
[0136] Micro-organisms of the genus Streptococcus can be cultured at 37 ° C 1 ° C for approximately 24 hours under microaerophilic conditions and a pH of 6.8 0.1 without applying aeration to the culture broth, particularly at an initial oxygen saturation of less than 10 %, preferably at an initial oxygen saturation of less than 2%. Preferably, the microorganisms of the genus Streptococcus are cultured, keeping the pH stable by adding at least one stabilizing agent, which may comprise an aqueous solution of ammonia at 25% w / v and / or an aqueous solution of tetraoxphosphoric acid at 35% w / v .
[0138] Microorganisms of the genus Bifidobacterium can be cultured at 37 ° C 1 ° C for approximately 24 hours in the absence of oxygen and a pH of 6.2 0.1.
[0139] During the cultivation of the microorganisms of the genus Bifidobacterium, they are cultured, keeping the pH stable by adding at least one stabilizing agent, which may comprise an aqueous solution of ammonium at 25% w / v and / or an aqueous solution of tetraoxphosphoric acid at 35% w / v.
[0141] Preferably, each of the biomass broths obtained at the end of the culture are cooled, for example, to 4 ° C to 8 ° C to stop the cellular metabolism of the microorganisms present in the biomass broths.
[0143] The composition that can be obtained according to the method of the invention is a composition obtained from lysates of probiotic microorganisms that comprises genomic sequences from lysates of cultures of strains of microorganisms of the genera Saccharomyces, Lactobacillus, Bacillus, Streptococcus and Bifidobacterium, These lysates comprise less than 5000 copies / ml of each of the following genomic sequences:
[0148] On the other hand, the food supplement comprises the composition obtained from lysates of probiotic microorganisms.
[0150] According to a preferred embodiment, the lysates further comprise at least 5000 copies / ml of genomic sequences at least 75% coincident with each of the following genomic sequences:
[0154] The lysates obtained from cultures of strains of microorganisms of the genus Saccharomyces can come from cultures of Saccharomyces cerevisiae.
[0155] On the other hand, the lysates obtained from cultures of strains of microorganisms of the Lactobacillus genus can come from cultures of Lactobacillus gasseri, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus reuteri, or combinations thereof.
[0157] In turn, the lysates obtained from cultures of strains of microorganisms of the Bacillus genus may come from cultures of Bacillus subtilis, Bacillus coagulans, Bacillus clausii, or combinations thereof.
[0159] As for the lysates obtained from cultures of strains of microorganisms of the genus Bifidobacterium, these can come from cultures of Bifidobacterium bifidum, Bifidobacterium infantis, or combinations thereof.
[0161] Finally, the lysates obtained from cultures of strains of microorganisms of the genus Streptococcus can come from cultures of Streptococcus thermophilus.
[0163] The composition may comprise a relative abundance of at least 50% of proteins from Saccharomyces culture lysates. Likewise, the composition may comprise a relative abundance of at least 3% of proteins from culture lysates of each of the following microorganisms: Lactobacillus, Saccharomyces, Bacillus, Streptococcus and Bifidobacterium
[0165] In one embodiment of the invention, the composition comprises
[0166] proteins with enolase function classified in category KO K01689 at 0.5-5% relative abundance,
[0167] histone-like proteins with DNA binding function classified in category KO K03530 in a relative abundance of 0.5-5%,
[0168] proteins with alcohol dehydrogenase function classified in category KO K13953 in a relative abundance of 0.5-5%,
[0169] and proteins with thioredoxin function of total metabolic activity classified in category KO K03671 in a relative abundance of 0.5-5%.
[0171] The composition may also comprise relative abundances of
[0172] 0.1-5% of proteins with inorganic pyrophosphatase function classified in category KO K01507;
[0173] 0.5-5% proteins with phosphoglycerate mutase 2,3 function biophosphoglycerate-dependent classified in category KO K01834;
[0174] 0.1-5%, homocysteine 5-methyltetrahydropoeroylglutamate methyltransferase classified in category KO K00549;
[0175] 0.1-5%, S-adenosylmethionine synthetase classified in category KO K00789.
[0177] Also, the composition may comprise relative abundances of
[0178] 0.5-5% L24 large subunit ribosomal proteins classified in category KO K02895;
[0179] 0.5-5% molecular Dnak chaperone classified in category KO K04043; 0.1-5% S5 small subunit ribosomal protein classified in category KO K02988;
[0180] 0.1-5% ATP-dependent Dead RNA helicase classified in category KO K05592;
[0181] 0.1-5% S12 small subunit ribosomal protein classified in category KO K02950.
[0183] The KO categories correspond to the classification of the Kyoto Encyclopedia of Genes and Genomes (= KEGG Orthologies; KEGG = Kyoto Encyclopedia of Genes and Genomes - https://www.genome.jp/kegg/).
[0185] Preferably, the lysates of cultures of strains of microorganisms in the composition according to the present invention are dry microbial lysates, which have been obtained by atomization or lyophilization.
[0187] These dry microbial lysates may comprise a mixture of
[0188] 30% to 70% by volume of dry microbial lysates from cultures of strains of the genus Saccharomyces,
[0189] 10% to 30% by volume of dry microbial lysates from cultures of strains of the genus Lactobacillus,
[0190] 10% to 30% by volume of dry microbial lysates from cultures of strains of the genus Bacillus,
[0191] 3% to 10% by volume of dry microbial lysates from cultures of strains of the genus Bifidobacterium,
[0192] 3% to 10% by volume of dry microbial lysates from cultures of strains of the genus Streptococcus.
[0194] By means of the method according to the invention, a composition is obtained that comprises a combination of probiotic bacteria that after a growth and lysing process, from which an extract consisting of an amalgam of metabolites, proteins, genetic sequences and other compounds is generated. , which, when dosed efficiently, is capable of altering and modifying the host's microbiota through several mechanisms, in such a way that it confers an activation of the immune system, reversing dysbiosis in a natural way.
[0196] This set of parts of probiotic and symbiotic microorganisms, as well as their metabolites obtained from cultures in bioprocessors, supply the physiological functions of the microbiota, regulate dysbiosis and produce a clinical impact on pathologies associated with dysbiosis, such as inflammatory and neurodegenerative diseases. metabolic and cancer.
[0198] Oral administration of the product acts by regulating the function of the microbiota, through the administered amount of components with biological activity on the host.
[0200] The effects of taking the product are given by a mechanism of action based on an adjustment and reprogramming of the microbiota present in the intestinal tract. The effect exerted on the microbiota is not a local effect since the whole organism is benefited by this modulation of the microbiota, becoming a systemic effect.
[0202] Thus, for example, cancer patients present a dysbiosis of the microbiota, which translates into a reduction in a series of bacterial groups that live symbiotically and that provide beneficial effects on the host and a possible increase in pathogenic bacteria. In addition, this dysbiosis is also reflected in a lesser variety within the microbiota of cancer patients, this means that they have fewer different bacterial groups than healthy people.
[0204] The regulation of the microbiota is reflected in a multitude of physiological effects and a increase in the quality of life of patients. A correct microbiota produces a reduction of infections in the intestinal tract and a lesser possibility of translocation of pathogenic bacteria. All this is due in part to an improvement in the permeability of the intestinal epithelium together with a correct response of the immune system, together with an increase in the production and secretion of mucin and Tight Junction Proteins, fundamental proteins in the permeability of the intestinal epithelium. and other types of antimicrobial peptides such as defensins.
[0206] A global effect of the foregoing is a reduction in intestinal inflammation, a symptom suffered by cancer patients and which, when reversed, produces an improvement in the quality of life of patients.
[0208] Cancer patients have a series of metabolites and carcinogenic products that are significantly increased, such as amines, benzopyrenes ... among others. A regulation of the intestinal microbiota will reduce the production of these compounds, and on the contrary, increasing the intake of metabolites generated by beneficial microorganisms such as short chain fatty acids, will generate a correction of the functions of the immune system, decreasing carcinogenic metabolites, promoting an increase in the beneficial bacterial population in the microbiota and again generating interrupted connections with the immune system for the maintenance of homeostasis through epigenetic mechanisms among others.
[0210] One of the effectors of this chain of reactions is due to Short Chain Fatty Acids (SCFAs). The short chain fatty acids SCFA, refer to free fatty acids, with a small number of carbons. The most common are: formic acid, acetic acid, propionic acid and butyric acid. Because their hydrophobic chain is short and they have a carboxylic group, they are water-soluble compounds and can be easily absorbed and transported into cells.
[0212] Therefore, they reinforce the epithelial barrier of the intestine since they are a direct source of energy for the intestinal colonocytes, affecting the formation of intestinal mucus, increasing the survival of the epithelial cells, improving the protein junctions in the intestinal monolayer.
[0213] They have anti-inflammatory and anti-tumor activity: This improvement of the epithelial cells of the colon conditions them to have a better immune response and reduce the chronic inflammatory response that can be produced by cancer or by the invasion of pathogens. SCFAs are neutrophil chemotactics, and can regulate macrophages and dendritic cells. They have antitumor activity, promote response to stress, arrest in the cell cycle and apoptosis in tumor cells.
[0215] With the increase in these metabolites, an acidification occurs in the medium, generating an antimicrobial activity since it inhibits the growth of certain bacterial strains and therefore can modify the microbial population of the intestine.
[0217] Therefore, the composition obtained according to the process according to the invention exerts an imodulatory effect on the human organism through modulation of the microbiota that occurs when the human being ingests the composition.
[0219] BRIEF DESCRIPTION OF THE FIGURES
[0221] Below, some examples are described in which reference is made to the following figures that form an integral part of the present specification.
[0222] Figure 1 reflects the distribution of nemPAI values (%) of a Sample according to an embodiment of the composition according to the present invention.
[0223] Figure 2 shows the results of the different activities and production of cytokines obtained with the realization of the Sample.
[0224] Figures 3A and 3B represent the phagocytic activity of mouse splenocytes in the presence of different concentrations of the Sample.
[0225] Figures 4A and 4B illustrate the results of the relative phagocytosis (percentage;%) obtained from macrophages from the bone marrow of BalbC (Figure 4A) and C57BL / 6 (Figure 4B) mice grown in the presence of the Sample.
[0226] Figure 5 illustrates the results of relative phagocytosis (percentage;%) obtained from RAW macrophages cultured in the presence of the Sample.
[0227] Figures 6A and 6B illustrate the results of the relative production expressed in percentage (%) of reactive nitrogen species (RNS) derived from NO2-obtained from splenocytes from BalbC mice (figure 6A) and C57BL / 6. (Figure 6B) grown in the presence of the Sample.
[0228] Figures 7A and 7B illustrate the results of the relative production expressed in percentage (%) of reactive nitrogen species (RNS) derived from NO2-obtained from macrophages from the bone marrow of BalbC (figure 7A) and C57BL / 6 mice. (Figure 7B) grown in the presence of the Sample.
[0229] Figure 8 illustrates the relative production expressed in percentage (%) of reactive nitrogen species (RNS) derived from NO2- obtained from RAW macrophages grown in the presence of the Sample.
[0230] Figures 9A and 9B illustrate the results of the relative arginase activity expressed in percentage (%) obtained from splenocytes from BalbC (Figure 9A) and C57BL / 6 (Figure 9B) mice grown in the presence of the Sample.
[0231] Figures 10A and 10B illustrate the relative arginase activity expressed in percentage (%) obtained from macrophages from the bone marrow of BalbC (Figure 10A) and C57BL / 6 (Figure 10B) mice cultured in the presence of the Sample.
[0232] Figure 11 illustrates the results of the relative arginase activity expressed in percentage (%) obtained from RAW macrophages cultured in the presence of the Sample.
[0233] Figure 12 refers to a representative example of the graph and equation obtained with serial dilutions of the TNF-a standard, which was used to calculate the production of this cytokine by the different types of macrophages.
[0234] Figures 13A. and 13B illustrate the results of the production of TNF-a (pg / ml) by splenocytes obtained from BalbC (Figure 13A) and C57BL / 6 (Figure 13B) mice grown in the presence of the Sample.
[0235] Figures 14A and 14B illustrate the results of the production of TNF-α (pg / ml) by macrophages from the bone marrow of BalbC (Figure 14A) and C57BL / 6 (Figure 14B) mice cultured in the presence of the Sample.
[0236] Figure 15 illustrates the results of TNF-α production (pg / ml) by RAW macrophages cultured in the presence of the Sample.
[0237] Figure 16 illustrates a representative example of the graph and equation obtained with serial dilutions of the IFN- ^y standard.
[0238] Figures 17A and 17B illustrate the results of IFN-y production (pg / ml) by splenocytes obtained from BalbC (Figure 17A) and C57BL / 6 (Figure 17B) mice grown in the presence of the Sample.
[0239] Figures 18A and 18B illustrate the results of IFN- ^y production (pg / ml) by macrophages from the bone marrow of BalbC (Figure 18A) and C57BL / 6 (Figure 18B) mice grown in the presence of the Sample.
[0240] Figure 19 illustrates the IFN-y production (pg / ml) by RAW macrophages grown in the presence of the Sample.
[0241] Figure 20 illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-2 standard.
[0242] Figures 21A and 21B illustrate the production of IL-2 (pg / ml) by splenocytes obtained from BalbC (Figure 20A) and C57BL / 6 (Figure 20B) mice grown in the presence of the Sample.
[0243] Figures 22A and 22B illustrate the results of IL-2 production (pg / ml) by macrophages from the bone marrow of BalbC (Figure 22A) and C57BL / 6 (Figure 22B) mice cultured in the presence of the Sample.
[0244] Figure 23 illustrates the results of IL-2 production (pg / ml) by RAW macrophages cultured in the presence of the Sample.
[0245] Figure 24 illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-10 standard.
[0246] Figures 25A and 25B illustrate the results of IL-10 production (pg / ml) by splenocytes obtained from BalbC (Figure 23A) and C57BL / 6 (Figure 25B) mice grown in the presence of the Sample.
[0247] Figures 26A and 26B illustrate the results of IL-10 production (pg / ml) by macrophages from the bone marrow of BalbC (Figure 26A) and C57BL / 6 (Figure 26B) mice grown in the presence of the Sample.
[0248] Figure 27 illustrates the results of IL-10 production (pg / ml) by RAW macrophages cultured in the presence of the Sample.
[0249] Figure 28 illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-4 standard.
[0250] Figures 29A and 29B illustrate the results of IL-4 production (pg / ml) by splenocytes obtained from BalbC (Figure 29A) and C57BL / 6 (Figure 29B) mice grown in the presence of the Sample.
[0251] Figures 30A and 30B illustrate the results of IL-4 production (pg / ml) by macrophages from the bone marrow of BalbC (Figure 30A) and C57BL / 6 (Figure 30B) mice cultured in the presence of the Sample.
[0252] Figure 31B illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-13 standard.
[0253] Figures 32A and 32B illustrate the results of IL-13 production (pg / ml) by splenocytes obtained from BalbC (figure 32A) and C57BL / 6 (figure 32B) mice cultured in the presence of the Sample serial dilutions of the IL-13 standard.
[0254] Figures 33A and 33B. illustrate the results of IL-13 production (pg / ml) by macrophages from the bone marrow of BalbC (Figure 33A) and C57BL / 6 (Figure 33B) mice grown in the presence of the Sample.
[0256] MODES OF EMBODIMENT OF THE INVENTION
[0258] EXAMPLE 1 - PREPARATION OF A MODULATING COMPOSITION
[0260] Separate batches of respective lyophilisates of microorganisms of the Saccharomyces, Lactobacillus, Bacillus, Streptococcus and Bifidobacterium genera are prepared in Falcon tubes of 10 or 50 mL,
[0262] The starting microorganisms are stored at 4 ° C in the form of lyophilisates, identified at the species level. The conservation of the Bacillus and Streptococcus genera is carried out by cultivating small proportions of the lyophilizates in both Nutrient Broth and Trypticase Soy Broth inocula media for 15 hours at 250 rpm at 30 ° C and 150 rpm at 37 ° C.
[0264] In the case of Bifidobacterium and Lactobacillus, a small proportion of the respective lyophilisate is cultured in MRS Broth liquid medium for 48 hours at 37 ° C under anaerobic conditions. After the incubation time, the cells are concentrated by centrifugation and resuspended in 20% glycerol. The microbial suspensions are distributed into respective vials that are frozen at -80 ° C. In this way, the microorganisms can remain without loss of viability for 1 year.
[0266] In particular, the lyophilisates of microorganisms of the Bacillus and Streptococcus genus are aspirated with the help of an automatic pipette in a laminar flow hood, 100 ^ L of lyophilisate and resuspended in 2 mL of sterile milli-Q water. Next, a 500 mL flask is seeded, containing 100 mL of NB and TSB inoculum medium, respectively, with the 2 mL of the suspension. Flasks incubate at 250 rpm and 30 ° C and at 150 rpm and 37 ° C, respectively, for 15 h. Once the incubation time has elapsed, the growth obtained in the broth is evaluated by titration of optical density at 600 nm (OD600nm) and a pH reading. In addition, a plate of TSA medium, BCA and nutritive agar is carried out as sterility controls, which are incubated for 48 h at 30 ° C. The grown culture is supplemented with glycerol to a final concentration of 20%. The suspension is distributed in vials, at a rate of 1.8 mL per tube. Immediately, the vials with the microbial suspensions are frozen at -80 ° C and stored.
[0268] In the case of the Bifidobacterium and Lactobacillus genera , in a cabin for work under anaerobic conditions, 100 ^ L of the lyophilisate are resuspended in 2 mL of sterile milli-Q water. Next, a 500 mL flask is seeded with 100 mL of MRS Broth inoculum medium with the 2 mL of suspension. The culture incubates without shaking at 37 ° C for 48 hours in a Gas-Pak anaerobic jar. After the incubation time, the biomass production is evaluated by means of OD600nm and pH analysis. Plates of TSA, BCA and nutrient agar medium are seeded as sterility controls. The grown culture is supplemented with glycerol until reaching a final concentration of 20%. The suspension is distributed at a rate of 1.8 mL per vial, and the vials with the microbial suspensions are immediately frozen at -80 ° C.
[0269] For the fermentation process, an inoculation culture is prepared in a flask culture medium of each microorganism, in an adequate quantity and volume to be able to proceed to inoculate the fermenter with the respective incubated inocula.
[0271] For this, the vials stored at -80 ° C are immersed in a water bath at 30 ° C to allow a rapid and complete thawing of the inoculum of the respective microorganism. Without loss of time, we proceed to the sowing of individual inoculum flasks with the microorganisms.
[0273] The inoculum medium used for the growth of the genus Bacillus is Nutrient Broth (NB):
[0278] • Distribution into 500 mL flasks (foam stopper) with 100 mL of medium.
[0279] • pH before sterilization adjusted to 6.8.
[0280] • Sterility: 121 ° C for 20 min.
[0281] • pH after sterilization: 6.8 ± 0.2.
[0283] The inoculum medium for growth of the genus Streptococcus is Tryptic Soy Broth (TSB):
[0288] 1. Partition into 500 mL flasks (foam stopper) with 100 mL of medium. 2. pH before sterilization adjusted to 7.3.
[0289] 3. Sterility: 121 ° C for 20 min.
[0290] 4. pH after sterilization: 7.3 ± 0.2.
[0292] The inoculum medium used for the growth of the genus Bifidobacterium and Lactobacillus is De Man, Rogosa, Sharpe Broth (MRS Broth), a formulated and non-selective medium for the growth of lactic acid bacteria:
[0297] • Distribution into 500 mL flasks (foam stopper) with 100 mL of medium. • pH before sterilization adjusted to 6.2.
[0298] • Sterility: 121 ° C for 20 min.
[0299] • pH after sterilization: 6.2 ± 0.2.
[0301] The inoculums of the respective microorganisms are incubated in the inoculum media described above.
[0303] The incubation conditions to obtain inocula of microorganisms of the Bacillus genus are the following:
[0304] - Temperature: 30 ° C.
[0305] - Relative humidity: 40% approximately.
[0306] - Agitation: 250 rpm (2.5 cm eccentricity).
[0307] - Duration: 15 hours ± 2.
[0308] The incubation conditions to obtain inocula of microorganisms of the genus Streptococcus are the following:
[0309] - Temperature: 37 ° C.
[0310] - Relative humidity: 40% approximately.
[0311] - Agitation: 150 rpm (2.5 cm eccentricity).
[0312] - Duration: 24 hours ± 2.
[0314] The incubation conditions to obtain inocula of microorganisms of the genus Bifidobacterium and Lactobacillus:
[0315] - Temperature: 37 ° C.
[0316] - Duration: 24 hours ± 2.
[0318] Once the inoculums of the respective microorganisms have been obtained, the direct sowing of both fermenters is carried out separately. The seeding percentage is 3% for Bacillus and 5% each for Bifidobacterium, Lactobacillus and Streptococcus.
[0320] The culture medium used for the fermentation of the genus Bacillus is called MFIGEN. For each fermenter with a geometric volume of 30 L, prepare 20 L of culture broth (before sowing) with the following composition:
[0325] The components of the medium are dissolved in a 5 L plastic beaker and made up to the final volume with process water. The pH before sterilization is adjusted to 6.8 with H3PO435%. The sterility cycle in the fermenter is 121 ° C for 30 min with constant agitation at 150 rpm. After sterilization, the pH is readjusted to 6.8 ± 0.1 with NH4OH 25% and the level of medium is checked corresponds to the initial volume ± 1L. To carry out this adjustment under sterile conditions, a three-way addition system for: acid, base and defoamer will have been previously punctured in the fermenter.
[0327] The culture medium used for the fermentation of the genus Streptococcus is called MFSTREP. For each fermenter with a geometric volume of 30 L, 20 L of culture medium (before sowing) are prepared with the following composition:
[0332] The components of the medium are dissolved in a 5 L plastic beaker and made up to the final volume with process water. The pH before sterilization is adjusted to 7.3 with H3PO435%. The sterility cycle in the fermenter is 121 ° C for 30 min with constant agitation at 150 rpm. After sterilization, the pH is readjusted to 7.3 ± 0.1 with 25% NH4OH and it is verified that the level of medium corresponds to the initial volume ± 1L. To carry out this adjustment under sterile conditions, a three-way addition system for: acid, base and defoamer will have been previously punctured in the fermenter.
[0334] For both fermentations, when the medium is already at working temperature, a sample will be taken before sowing to corroborate the pH value in a benchtop pH meter, as well as to perform a sterility control on a solid TSA medium. and liquid TSB. Furthermore, before sowing, the oxygen probe must be calibrated. 95% of the dissolved oxygen value is adjusted in the initial aeration conditions supplying the minimum oxygen required for sampling.
[0335] For the genus Bifidobacterium and Lactobacillus, the culture medium has the following composition:
[0340] The final volume to which the medium is made up to volume is 20 L. The sterility cycle in the fermenter is 121 ° C for 30 min with a constant stirring of 150 rpm. The pH after sterile is adjusted to 6.2 ± 0.1 with NH4OH 25% or H3PO435%, according to requirements. To carry out this adjustment under sterile conditions, a three-way addition system for: acid, base and defoamer will have been previously punctured in the fermenter. After cooling to the working temperature, the sample corresponding to "before sowing" is taken to carry out a sterility control and external pH assessment.
[0342] After seeding the fermenter with the inoculum of the corresponding microorganism, the fermentation phase of each of the microorganisms is carried out separately for the generation of microbial biomass, establishing the best production conditions during the shortest possible process time. In the aerobic fermentation of Bacillus the main parameter that controls the fermentation is dissolved oxygen (DO), which should be kept above 40%. For the genus Streptococcus, the growth strategy is based on fermentation under microaerophilic conditions. Finally, in the case of Lactobacillus and Bifidobacterium an anaerobic fermentation develops.
[0343] For the fermentation of Bacillus the conditions are as follows:
[0345] - Temperature: 30 ° C ± 1 ° C throughout the process. At the end of the fermentation, it is lowered to 4-8 ° C to stop cellular metabolism and harvest the broth.
[0347] - Pressure: 0.5 atm.
[0349] - pH: 6.8 ± 0.1 during the whole process. Sterile addition of 25% NH4OH or 35% H3PO4 automatically, connecting the silicone gums to the peristaltic pumps of the fermenter and setting the set point on the equipment.
[0351] - Agitation: 150 rpm initially. Depending on the foam and the dissolved oxygen, this parameter increases by activating a cascade up to a maximum of 250 rpm.
[0353] - Aeration: 1 V / V / min. Depending on the foam and dissolved oxygen, manually increase from 0.1 V / V / min to a maximum of 1.5 V / V / min.
[0355] - Dissolved oxygen: it remains> 40%. If the% falls below the established limit, the modification of the physical parameters of the process begins: agitation and aeration. First the agitation cascade is activated until reaching the maximum and then, manually, the aeration begins to increase.
[0357] - Cycle: 20-24 hours approximately. The end of the fermentation is determined by a stop in the consumption of base (NH4OH).
[0359] - Foam control: manual addition of small amounts in case of high foam in the fermenter.
[0361] In the case of Streptococcus, microaerophilic incubation conditions are established:
[0362] - Temperature: 37 ° C ± 1 ° C throughout the process. At the end of the fermentation, it is lowered to 4-8 ° C to stop cellular metabolism and harvest the broth.
[0364] - Pressure: 0.5 atm.
[0366] - pH: 6.8 ± 0.1 during the whole process. Sterile addition of 25% NH4OH or 35% H3PO4 automatically, connecting the silicone gums to the peristaltic pumps of the fermenter and setting the set point on the equipment.
[0368] - Agitation: 150 rpm during the whole process.
[0370] - Aeration: 0 V / V / min. The minimum necessary for sampling is supplied.
[0372] - Dissolved oxygen: Free form advance.
[0374] - Cycle: 20-24 hours approximately.
[0376] - Foam control: manual addition of small amounts in case of high foam in the fermenter.
[0378] For the fermentation of Bifidobacterium the conditions are the following:
[0380] - Temperature: 37 ° C ± 1 ° C throughout the process. At the end of the fermentation, it is lowered to 4-8 ° C to stop cellular metabolism and harvest the broth.
[0382] - Pressure: 0.5 atm.
[0384] - pH: 6.2 ± 0.1 during the whole process. Sterile addition of 25% NH4OH or 35% H3PO4 automatically, connecting the silicone gums to the peristaltic pumps of the fermenter and setting the set point on the equipment.
[0386] - Agitation: 50 rpm during the entire process (the minimum value that guarantees the correct homogenization of the components of the medium).
[0387] - Dissolved oxygen: bubble nitrogen until the dissolved oxygen concentration is 0%. This value is controlled throughout the fermentation and although a continuous supply is not necessary, it can be occasionally bubbled when necessary.
[0389] - Cycle: 24 hours approximately. The end of the fermentation is determined by a stop in the consumption of base (NH4OH).
[0391] For the fermentation of Lactobacillus the conditions are as follows:
[0393] - Temperature: 37 ° C. At the end of the fermentation, it is lowered to 4 ° C to stop cellular metabolism and harvest the broth.
[0395] - Pressure: 0.5 atm.
[0397] - pH: 6.4 ± 0.1 during the first 18 hours. Sterile addition of 25% NH4OH or 35% H3PO4 automatically, connecting the silicone gums to the peristaltic pumps of the fermenter and setting the set point on the equipment.
[0399] - Agitation: 50 rpm during the entire process (the minimum value that guarantees the correct homogenization of the components of the medium).
[0401] - Dissolved oxygen: bubble nitrogen until the dissolved oxygen concentration is 0%. This value is controlled throughout the fermentation and although a continuous supply is not necessary, it can be occasionally bubbled when necessary.
[0403] - Cycle: 18-24 hours approximately. The end of fermentation is determined by a decrease in pH to a value close to 4.5.
[0405] At 6 hours of fermentation, when the Lactobacillus culture is expected to be in the full logarithmic phase of growth, the fermentation conditions are modified. Nitrogen from the fermenter begins to move with an air flow so that the% dissolved oxygen is in the range 25-30% (to maintain this value it may be necessary to increase the air flow input and agitation). In this way, an aerobic environment is created, tolerable for the growth of lactic acid bacteria and which, at the same time, allows the proliferation of another aerobic microorganism in the fermenter. In relation to the above, as soon as the above conditions are established in the fermenter, the bioreactor is reseeded with a percentage of a culture of S. cerevisiae of 5%. Said culture stock will have previously proliferated in YPD medium for 24 hours at 150 rpm and 37 ° C. The pH is maintained at 6.4 during the first 8 hours of co-cultivation and from that moment (14 hours of fermentation), the control of this parameter is deactivated and it is allowed to advance freely. Due to the acidification of the medium, the pH will reach an approximate value of 4.5, which will determine the end of the fermentation, due to the conversion of most residual sugars into short-chain organic acids (acetic acid, formic acid, acid lactic acid, citric acid, etc.) and other metabolites of interest. Productivity is increased when compared to simple monoculture of lactic acid bacteria, due to the modification of the fermentation conditions by the yeast.
[0407] Regarding the reasons for carrying out co-cultivation, it is well known that yeast has catalase activity, which would lead to a reduction in the amount of hydrogen peroxide in the broth, a radical that causes inhibition of the growth of the lactic bacteria. In this way, the growth rate of lactic acid bacteria is accelerated, with the consequent generation of metabolic by-products of great interest and with therapeutic potential, as has been demonstrated in later clinical stages.
[0409] The fermentation is stopped in the fermenter itself, lowering the temperature to a value between 4-8 ° C.
[0411] The recovery of the biomass obtained based on the fermentation of each of the microorganisms is carried out by means of centrifugation, obtaining each wet recovered biomass.
[0413] Subsequently, the rupture or lysis of the cells of each of the wet recovered biomass is carried out in a ball mill, obtaining respective biomasses of microbial lysates that are subsequently dried separately in an atomizer. The dried microbial lysates are mixed in a mixer to obtain modulating composition.
[0415] The list of raw materials required for the production of a batch of biomass of the Bacillus genus in a fermenter of 30 L of geometric volume is as follows:
[0417]
[0420] In the case of Streptococcus fermentation , the list of raw materials required for the production of a batch of biomass of the genus for a volume of 30 L of fermentation is:
[0422]
[0425] For the fermentation of Bifidobacterium and Lactobacillus, the list of raw materials required for the production of a batch of biomass of the genus for a volume of 30 L of fermentation is:
[0427]
[0428]
[0431] At the end of fermentation, biomass broths of probiotic microorganisms are obtained, comprising microbial biomass and culture broths.
[0433] To recover the microbial biomass from the microbial biomass broths and obtain wet recovered microbial biomass, the biomass broth is harvested from the fermenter, and the harvested broth is primed to a centrifuge with discs or conical dishes, operating under the following conditions:
[0435] - Feed flow: 5-10 L / h on average. At the beginning of the process, this flow rate can be significantly higher; however, at the end of the process when a certain degree of saturation may be being reached, the flow rate can be reduced to 1.5 L / h if the process is carried out continuously. If at any time the clarification comes out cloudy, it may be a consequence of an excess flow, in which case it would be necessary to reduce it, or a symptom that the bowl is full of solids and the dishes dirty, in which case it would be necessary to empty and clean the bowl.
[0437] - Centrifugation speed: 3,500 rpm.
[0439] The analyzes carried out during this procedure are as follows:
[0440] - PCV biomass (%). This assessment is carried out initially on the total broth and on different clarification samples throughout the centrifugation. If the separation is satisfactory, hardly any solids should remain in the clarification samples.
[0441] - Humidity (%). This parameter is determined on the final sample, to keep track of the humidity percentage throughout the recovery process. For this, 1 g of wet biomass is dried in a thermobalance at a temperature of 120 ° C.
[0443] The wet recovered biomass obtained after centrifugation is weighed and subjected to cell lysis. To do this, the wet biomass is resuspended in the same volume of water, and the breaking or lysis of the diluted biomass is carried out in a ball mill, due to the friction generated by zirconium silicate balls (2 mm). on the surface of the cell walls, within a grinding chamber with a horizontal axis.
[0445] The conditions for each grinding cycle are as follows:
[0446] - Ball loading and cell concentration. 1: 1 ratio of zirconium balls and diluted wet biomass, until the grinding chamber is filled.
[0447] - Agitator speed 3,000 rpm (with active cooling system).
[0448] - Residence time. 40 minutes of grinding.
[0449] - Temperature. Controlled environment with a cooling system of the equipment by recirculation of water through the jacket of the grinding chamber.
[0451] At this stage of the process the following is assessed:
[0452] - Cell breakage (%). By visualizing the optical microscope, the final percentage of breakage is evaluated, which must be greater than 90%. If the percentage of breakage is less than 90%, cycles of more than 20 minutes are carried out.
[0453] - Viscosity (cP). This evaluation is carried out in a rotational viscometer with a standard spindle for medium-high viscosity and allows to relate this high parameter with a greater degree of breakage, as a result of the interconnected structure that smaller dispersed particles usually form. - Coloring of broken biomass. Assessment of the tonality of the final sample, to associate it with a certain degree of cellular breakdown. In general, a whitish coloration is associated with a low percentage of breakage, whereas if the grayish coloration increases, this fact is indicative of a higher percentage of breakage.
[0454] The biomass of microbial lysates obtained in the previous step is dried in an atomizer using the spray drying technique , which allows the suspension of broken cells to be dried in water by spraying them in a stream of air at a high temperature. (Ta input). As a consequence of the evaporation of the water, the air stream cools down to a temperature that is a function of the amount of water evaporated (outlet Ta).
[0456] The atomization conditions are as follows:
[0457] - Inlet and outlet temperature 180 ° C ± 2 ° C and 80 ° C ± 10 ° C, respectively.
[0458] - Diameter of the spray needle 0.7 mm.
[0459] - Feeding speed. Due to the high viscosity of the broken biomass, this parameter is adjusted in such a way that the outlet temperature remains stable around 80 ° C, allowing complete drying of the sample. If liquid droplets are observed in the drying chamber or cyclone, then the feed rate should be reduced until the equipment completes drying of all supplied sample. In general, the viscosity of the mix should not exceed 300 cP viscosity for the peristaltic pump to feed the system.
[0461] The following analyzes are carried out on the final atomized sample:
[0462] - Humidity (%) and dry residue (%). For this, 1 g of atomized biomass is dried in a thermobalance at a temperature of 120 ° C.
[0463] - Dry weight (g / L). Titration in thermobalance at 120 ° C and oven at 65 ° C.
[0465] After spray drying, the dry mcirobial lysates are obtained, in four different batches of dry microbial lysates, namely, a first batch from the culture of microorganisms of the Lactobacillus and Saccharomyces genera , a second batch from the culture of microorganisms of the genus Bifidobacterium, a third batch from the culture of microorganisms of the genus Bacillus, and a fourth batch from the culture of microorganisms of the genus Streptococcus,
[0467] Microbial lysates from these four batches are mixed, in the following proportions:
[0468] 70% by volume of dry microbial lysates from cultures of strains of the genus Saccharomyces, and from cultures of strains of the genus Lactobacillus, 20% by volume of dry microbial lysates from cultures of strains of the genus Bacillus,
[0469] 5% by volume of dry microbial lysates from cultures of strains of the genus Streptococcus,
[0470] 5% by volume of dry microbial lysates from cultures of strains of the genus Bifidobacterium
[0472] Samples are extracted from the mixture of lysates and it is determined if the sample comprises a minimum of 5000 copies / ml genomic sequences of each of the genomic sequences previously identified in this specification:
[0477] and if it further comprises at least 5000 copies / ml of additional genomic sequences at least 90% matched with each of the following additional genomic sequences, also previously identified in this specification:
[0482] The determination of whether the sample comprises the minimum of genomic sequences of 5000 copies / ml of each of the genomic sequences previously identified is carried out by means of massive sequencing techniques known per se.
[0484] When it is verified that the sample comprises said minimum genomic sequences, the mixture of dry microbial lysates is collected to obtain the modulating composition.
[0486] EXAMPLE 2 - Analysis of the composition of the composition prepared in example 1
[0488] Sample preparation and protein digestion
[0490] 400 mg of the composition of Example 1 (hereinafter. Sample) were dissolved with a chaotropic buffer containing 8.4 M urea (USB Corporation, Cleveland, OH, USA), 2.4 M thiourea (Sigma Aldrich), 5% CHAPS (Sigma Aldrich), 5mM TCEP (Sigma Alrich) and a protease inhibitor cocktail (Sigma Aldrich) and incubated on ice for 15 minutes. Homogenization was performed by sonication by ultrasound application for 5 minutes in a Branson 2510 bath (Marshall Scientific, New Hampshire, USA). The homogenate was centrifuged at 20,000 xg at 4 ° C for 10 minutes, and the supernatant containing the solubilized proteins was used for further analysis. Next, 40 µg of proteins were precipitated by the methanol / chloroform method, and resuspended in 40 ^ g of a UTT buffer (7M urea, 2M thiourea, 10mM TEAB (sigma Aldrich) of multicaotropic sample solution.
[0491] The resuspended Sample was reduced with 2 ^ of 50mM TCEP at 37 ° C for 60 minutes, followed by the addition of 1 ^ L of MMTS reactant (SCIEX, Foster City, CA, USA) blocking cysteine for 10 minutes at room temperature. . The Sample was diluted to 140 µl with mM TEAB in order to reduce the urea concentration. Finally, the digestion was started by adding 2 µg of Pierce MS grade trypsin (Thermo-Fisher Scientific, Inc., Waltham, MA, USA) to each fraction in a ratio of 1:20 (w / w), and incubated at 37 ° C overnight on a shaker. The digested Sample was evaporated to dryness in a vacuum concentrator.
[0493] Liquid chromatography and mass spectrometry analysis (LC-MS)
[0495] The digested Sample was washed / desalted using Stage-Tips with 3M C18 Empore discs (Sigma Aldrich). A 1 ^ g aliquot of the resulting peptides was subjected to 1D-nano LC ESI-MS / MS (Liquid Chromatography Electrospray lonization Tandem Mass Spectrometric) analysis using an Eksigent Technologies nanoLC Ultra 1D plus (SCIEX, Foster City, CA, USA) coupled to a Triple TOF 5600 high speed mass spectrometer (SCIEX) with a Nanspray III source. The analysis column employed was an Acquity UPLC M-Class Peptide BEH C18 reverse phase silica-based column (Waters Corporation, Milford, MA, USA). The trap column was a C18 Acclaim PepMapTM 100 (Thermo-Fisher Scientific Inc.), 100 µm * 2 cm, 5 µm particle diameter, 100 A pore size, connected in line with the analysis column. The loading pump supplied a solution of 0.1% formic acid in water at t 2 ^ l / min. A flow rate of 250 nL was applied by a nano-pump operated under gradient elution conditions. The peptides were separated using a 250 min gradient with a B phase ranging from 2% to 90% (mobile phase A: 2% acetonitrile (Scharlab, SL, Spain), 0.1% formic acid (Sigma Aldrich); mobile phase B: 100% acetonitrile, 0.1% formic acid). The injection volume was 5 ^ l. The data were obtained using an Ionspray floating voltage of 2300 V, curtain gas 35, interface heater temperature 150, source gas 1 25 and ungrouping potential 150 V. For IDA parameters (Intelligent Data Analysis) a 0.25 s MS scan was followed in the mass range of 350 to 1250 Fa by 35 MS / MS scans of 100 ms in the mass range of 100 to 1800. The change criteria were established at ions greater than a mass / charge ratio (m / z) of 350 and less than m / z 1250, with a state of charge of 2-5 and an abundance threshold greater than 90 counts / second (cps). The above target ions were excluded for 15 s.
[0497] Proteomic data analysis and sequence search
[0499] The spectrometric data obtained were processed using PeakView v2.2 (SCIEX) software and exported to mgf files that were searched using Mascot Server v2.5.1 (Matrix Science, London, Reno United) against a protein database that contained protein sequences from microorganisms of the genera Bacillus, Lactobacillus, Bifidobacterium, Streptococcus and Saccharomyces from the Uniprot / Swissprot knowledge base (https://www.uniprot.org/statistics/Swiss-Prot - update: 20170412, 2,542,118 protein sequences), along with commonly existing contaminants. Obtaining data of the total number of matching peptide spectra for a given protein (referred to as Peptide-to-Spectrum-Matching (PSM) and exponetially modified Protein Abundance Index (emPAI)) were calculated for each of the proteins. PSM and emPAI, and can be used as a relative quantitative result of the proteins in a complex mixture based on the coverage of the proteins by peptide matches in a results database. The LC-MS analyzes were carried out at the proteomics facilities of the National Center for Biotechnology of the Higher Council for Scientific Research, which are part of ProteoRed.
[0501] Protein Abundances and Functions in the Sample
[0503] The Sample was thoroughly analyzed for expressed proteins, and 937 proteins were identified. For the 937 proteins identified, spectral counts were obtained to give an indication of their abundance. Normalized emPAI values (nemPAI%) were obtained based on the emPAI values by dividing each individual value by the sum of all emPAI values and multiplying each resulting value by 100%, to obtain the relative abundance of each protein with respect to total proteins. .
[0505] In addition, a functional analysis of the proteins in the Sample was performed by assigning k numbers using KEGG's internal annotation tool for KEGG Orthologies (KO), and looking for clusters of orthologous groups (COG) to analyze the Relative abundance of specific proteins in certain metabolic processes defined in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and COG (http://eggnogdb.embl.de/#/app/home).
[0507] Figure 1 reflects the distribution of nemPAI values. The most relevant results can be seen in tables 1A and 1B.
[0509] Table 1A
[0511]
[0512]
[0513]
[0515] Table 1B
[0516]
[0518]
[0519]
[0520]
[0522] In this table, the eggNOG indications have the meanings specified in Table 1C:
[0523] Table 1C
[0524]
[0525]
[0528] As can be seen in Figure 1, the distribution of nemPAI values obtained has an exponential shape, which indicates the existence of a great diversity of proteins in the Sample.
[0530] Only 10 out of 937 of the proteins were found to have a relative abundance of 1% or more. These proteins and their nemPAI values are identified in Figure 1D.
[0532] Table 1D
[0534]
[0535]
[0538] To identify the microbial origin of the proteins in the Sample, a comparative search was carried out between the mass spectrography data of the Sample against the proteins of microorganisms of the genera Bacillus, Lactobacillus, Bifidobacterium, Streptococcus and Saccharomyces from the base of Uniprot / Swissprot data. The microbial origins and contribution to the Sample proteome are summarized in Table 2:
[0539] Table 2
[0544] As can be seen, the microorganisms of the genus Saccharomyces appear to be the major contributors in terms of the total number of proteins and in terms of the expression level of proteins contained in the Sample.
[0546] The functional analysis based on the KEGG (KO) orthology carried out to verify the functions of the proteins in the different metabolic pathways that are defined by KEHH and COG revealed that 643 of the 937 proteins could be assigned to 352 functional categories. The nemPAI values for each KO category were obtained by summing all the nemPAI values for each protein that contributes to each KO category.
[0548] When the functional categories KO and the levels of abundance of the proteins in the Sample of functional categories and protein abundance levels from a proteome of a healthy adult human intestinal microbiota obtained using mass spectrometry and liquid chromatography, which resulted in the obtaining of more than 3,100 proteins assigned to 560 KO categories Table 3 summarizes this comparison:
[0550] Table 3
[0552]
[0555] The comparison of the KO categories of the Sample and the human microbiota also revealed that the respective proteomes only shared 78 common KO categories, that is, 9.4% of the total KO categories, approximately 32.8% of KO categories were only present in the Sample, and approximately 57.8% of KO categories were found in the gut microbiota. This suggests that the administration of the Sample contributes to the intestinal microbiota with proteins participating in metabolic activities that are not present or are only present at a lower level in the microbiota.
[0557] Notable functions are represented by those of the categories KO K01689 (enolase [EC: 4.2.1.11]), K03530 (histone-like DNA-binding protein) K13953 (alcohol dehydrogenase) and K03671 (thioredoxin) which have a contribution to the total metabolic activity of at least 1%.
[0559] Thus, enolase catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate and is essential for the degradation of carbohydrates through glycolysis. Furthermore, histones package and order eukaryotic genomes in chromatin which plays an important role in epigenetic regulation with variable covalent modifications. In turn, alcohol dehydrogenase serves to break down alcohols that would otherwise be toxic, while thioredoxin helps control reactive oxygen species and redox signaling in general.
[0561] Additionally, regarding the inherent metabolic capacities especially in the Sample, important differences were observed between the Sample and the intestinal microbiota. So. a total of 9 of the 78 KO categories were represented at least 10 times more in the Sample than in the intestinal microbiota, as summarized in Table 4:
[0563] Table 4
[0565]
[0566]
[0569] The following KO categories should be highlighted:
[0570] K01507: Inorganic pyrophosphatase plays a critical role in lipid metabolism, calcium absorption, and DNA synthesis
[0571] K01834: The phosphoglycerate 2,3 bisphosphoglycerate-dependent mutase participates in the sub-pathway that synthesizes pyruvate from 3-phosphate triglyceroldehyde, the pathway of glycolysis and in the degradation of carbohydrates.
[0572] K00549: 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine which results in the formation of methionine through the de novo pathway which is itself part of amino acid biosynthesis.
[0573] K00789. S-adenosylmethionine synthetase is an enzyme that creates S-adenosylmethionine (SAM) by reacting methionine and ATP; it enables DNA methylation and participates in gene transcription, cell proliferation, and the production of secondary methanolites.
[0575] EXAMPLE 3 - In vitro study of the immunomodulatory capacity of the composition analyzed in example 2
[0577] The immunomodulatory capacity of the Sample prepared according to Example 1 and analyzed in Example 2 was evaluated in vitro. Various cell types were used for the evaluation, including RAW 264.7 murine monocytes, differentiated mouse bone marrow macrophages (BMM) and splenocytes from mouse spleen explants, the latter two cell lines from two different types of mice; Balb / c (prototypical of Th2 immunity) and C57BL / 6 (prototypical of Th1 immunity).
[0579] The immunomodulatory capacity was determined in the mentioned cell types by performing various tests, including phagocytic capacity, the release of NO2- derived reactive nitrogen species (RNS), arginase activity, and the release of Th1 and Th2-type cytokines.
[0581] METHODOLOGY AND PROCEDURE
[0583] Sample Treatment
[0585] The Sample was subjected to treatment with ultraviolet light for 15 min in a laminar flow hood, in order to sterilize the content before introducing it into cell cultures. After the sterilization treatment was finished, it was dissolved at a concentration of 60 mg / ml in the same medium in which the cells to be tested were to grow (RPMI or DMEM, depending on the cell type). Although the Sample was soluble, it did not completely dissolve, revealing traces of precipitate in the stock solution. From the stock solution of the Sample, three serial 1:10 dilutions were made in the media corresponding to each cell type to obtain the working solutions, whose concentrations were 6 mg / ml, 0.6 mg / ml and 0 .06 mg / ml. The volume of each of these solutions added to each well was adjusted so that the concentration tested for each sample was 1 mg / ml (C1), 0.1 mg / ml (C2) and 0.01 mg / ml (C3).
[0587] Obtaining and maintaining cell lines
[0589] To evaluate the immunomodulatory response, three types of cells of the white series were used: RAW 264.7 monocytic cells, mouse bone marrow cells and splenocytes from mouse spleen explants.
[0591] RAW 264.7 monocytes were purchased from Sigma (Ref .: 91062702) and maintained in RPMI medium supplemented with 10% inactivated fetal bovine serum and 100 U / ml penicillin-100 µg / ml streptomycin at 37 ° C in the presence of 5 % CO2. Both the cell density and the subculture frequency were performed according to the supplier's instructions.
[0593] In order to obtain macrophages from bone marrow and splenocytes, 5 Balb / c mice (Th2 type immunity) and 5 C57BL / 6 mice (Th1 type immunity) were acquired through of the Animal Facility of the University of León, respecting the current regulations on the management and welfare of experimental animals. After euthanizing the animals, the spleen and femurs were extracted, proceeding to the recovery of splenocytes and bone marrow cells, respectively, following the protocols set up in the Toxicology laboratory of the University of León.
[0595] Splenocytes were resuspended in RPMI medium supplemented with 10% inactivated fetal bovine serum and 100 U / ml penicillin-100 ^ g / ml streptomycin and were used immediately to carry out the experiment, incubated at 37 ° C in the presence of 5% of CO2.
[0597] The bone marrow stem cells from the femur were resuspended in DMEM supplemented with 10% fetal bovine serum, 100 U / ml penicillin-100 ^ g / ml streptomycin and 20% L929 cell supernatants, which express the stimulating factor of macrophage colonies (MCSF), and incubated for 8 days at 37 ° C in the presence of 5% CO2. Next, the differentiated macrophages were collected and resuspended in DMEM supplemented with 10% fetal bovine serum, 100 U / ml penicillin-100 µg / ml streptomycin, proceeding immediately to the experiment. In some cases, the cell cultures were supplemented with lipopolysaccharides (LPS) from Eschenchia coli O111: B4 (Sigma, Ref .: L4391-1MG). This product was prepared at a concentration of 1 mg / ml in RPMI / DMEM medium and then its final concentration was adjusted to 60 µg / ml in the corresponding medium. The volume of this latter solution that was added to each well was adjusted so that the final concentration of LPS per well was 10 µg / ml (Tables 5a and 5B).
[0599] Experimental design
[0601] The design and experimental approach were the following depending on the tests to be carried out:
[0603] Arrangement of 48-well plates (0.95 cm2 of culture surface, total volume 600 l ^) for the evaluation of arginase activity and cytokine production
[0604] Cell lines were seeded at a density of:
[0605] - Bone marrow cells: 6x105 cells / well (6.3x105 cells / cm2).
[0606] - Splenocytes: 1.2x106 cells / well (1.26x106 cells / cm2).
[0607] - RAW. 2x105 cells / well (2.1x105 cells / cm2). Fewer RAW cells were used because they will multiply during the 48 hours of the experiment, unlike splenocytes and bone marrow cells.
[0609] Arrangement of 96-well plates (0.35 cm2 culture surface, total volume 150 l ^) for the evaluation of phagocytic capacity and RNS production
[0611] Cell lines were seeded at a density of:
[0612] - Bone marrow cells: 1.5x105 cells / well (4.28x105 cells / cm2).
[0613] - Splenocytes: 3x105 cells / well (8.57x105 cells / cm2).
[0614] - RAW: 5x104 cells / well (1.43x105 cells / cm2). Fewer RAW cells were used because they will multiply during the 48 hours of the experiment, unlike splenocytes and bone marrow cells.
[0616] Once the cells were added, the experiment presented the following arrangement:
[0618] - Negative control: cells with RPMI / DMEM medium.
[0620] - Positive control: cells with RPMI / DMEM medium and LPS (10 µg / ml final concentration in well).
[0622] - Sample test 1: cells with RPMI / DMEM medium and Sample at three different concentrations: 1 mg / ml (C1), 0.1 mg / ml (C2) and 0.01 mg / ml (C3) in RPMI / DMEM ( final concentration in well).
[0624] - Sample test 1 LPS: cells with RPMI / DMEM medium, LPS (10 ^ g / ml final concentration in well) and Sample at three different concentrations: 1 mg / ml (C1), 0.1 mg / ml (C2) and 0.01 mg / ml (C3) in RPMI / DMEM (final concentration in well).
[0626] The volumes were adjusted so that in the 48-well plates there was a final volume per well of 600 jl (Table 5.1) and in those of 96 wells of 150 jl (Table 5.2):
[0627] Table 5A
[0628] Contents of the different wells that were placed in 48-well plates to carry out the experiments to evaluate arginase activity and cytokine production.
[0630]
[0633] Table 5B
[0634] Contents of the different wells that were arranged in 96-well plates to carry out the experiments to evaluate the phagocytic capacity and NO production.
[0636]
[0637]
[0640] Three technical replications of each condition were carried out and in all cases the cells were incubated for 48 h at 37 ° C with 5% CO2. After this time, the pertinent analyzes were carried out to measure the different activities.
[0642] Determination of the phagocytic capacity of RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0644] Once the incubation time was over, the supernatant of the cultures grown in the 96-well plate was extracted, which was reserved for the assessment of NO production (see below).
[0646] The cells adhered to the wells were used to assess phagocytic capacity by means of the Neutral Red test. This dye is internalized in the lysosomes of macrophages, with which a greater phagocytic activity leads to a greater accumulation of neutral red inside the cell. To do this, immediately after extracting the supernatants, 150 ml of RPMI / DMEM medium (according to the cell line as indicated above) and 15 ml of 0.33% Neutral Red Solution (Sigma, Ref. N-2889) were added to each well previously. filtered to avoid the appearance of crystals. The cells were incubated at 37 ° C with 5% CO2 for 3 h in the dark.
[0648] Subsequently, the medium with the unincorporated dye was removed very carefully and the cells were washed twice with PBS, removing the remains of it well.
[0650] Next, 150 ml of breakdown and desorption solution (1% acetic acid, 50% ethanol, 49% water) was added to each well, pipetting said solution up and down. In this way the cell crushing, the release of the dye and the mixing of the same took place. This process ceased when a homogeneous reddish solution. Subsequently, 100 µL were removed from each well and placed in a new 96-well plate. At that time the absorbance at 540 nm and at 690 nm (background absorbance) was determined. The latter was subtracted from that obtained at 540 nm. Relative phagocytosis was estimated in percentage, setting the value provided by the Negative Control as 100%.
[0652] Determination of the production of NO ^2- derived reactive nitrogen species (RNS) by RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0654] The accumulation of NO2 was used as an indicator of NO production and was estimated by the Griess reaction. For this, the supernatants of the cultures grown in 96-well plates were used. For this purpose, 50 µl of the supernatants were extracted from each cell culture, which were placed in a new 96-well plate. They were then mixed with another 50 µl of the modified Griess reagent (Sigma, Ref .: G4410), which was prepared following the manufacturer's recommendations. The mixture was incubated for 15 min at room temperature and the absorbance at 540 nm was measured.
[0656] The relative NO production was estimated in percentage, setting the value provided by the Negative Control as 100%.
[0658] Determination of arginase activity in RAW 264.7 monocytes, mouse bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0660] After the end of the incubation time, the supernatant was extracted from the cultures grown in the 48-well plate, which was reserved for the assessment of cytokine production (see below).
[0662] The cells adhered to the wells were used to assess arginase activity, which was determined using the Arginase Activity Assay kit (Sigma, Ref .: MAK112) following the manufacturer's recommendations.
[0663] The relative arginase activity was estimated in percentage, setting the value provided by the Negative Control as 100%.
[0665] Determination of the production of immune response mediator molecules (cytokines) in RAW 264.7 monocytes, mouse bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0667] For the determination of the production of mediator molecules of the immune response by the tested cell lines, the supernatants of the cultures grown in 48-well plates were used.
[0669] The production of three cytokines of the Th1 type (cellular immunity: IL2, IFN-y and TNF-a) and 3 of the Th2 type (humoral immunity: IL10, IL4 and IL13) were analyzed by using commercial ELISA kits following the instructions manufacturer. Absorbance was measured on a Varioskan ™ LUX multimode microplate reader (Thermo Fisher) and cytokine production was expressed in pg / ml.
[0670] - Cytokines IFN ^and , IL2, IL4 and IL10: Th1 / Th2 Mouse Uncoated ELISA Kit with Plates (Thermo Fisher, eBiosciences, Ref .: 88-7711-44)
[0671] - TNF-a: TNF-alpha Mouse ELISA Kit (Thermo Fisher, eBiosciences, Ref .: BMS607HS)
[0672] - IL13: IL-13 Mouse ELISA Kit (Thermo Fisher, eBiosciences, Ref .: BMS6015)
[0674] Here are brief details of the cytokines that were tested:
[0676] * TNF-a: Stimulates the acute phase of the inflammatory reaction, producing local activation of the vascular endothelium, release of nitric oxide with vasodilation and increased vascular permeability, which leads to the recruitment of inflammatory cells, immunoglobulins and complement, causing activation of T and B lymphocytes.
[0677] * IFN-y: Its main function is the activation of macrophages, increasing their phagocytic capacity, both in the innate and adaptive responses. Another of its functions is to direct the differentiation of CD4 + T lymphocytes into Th1 lymphocytes, activation of dendritic cells and "natural killer" (NK) lymphocytes to induce greater secretion of IL-12, which promotes more differentiation to Th1, strongly amplifying the reaction.
[0678] * IL-2: Acts as a growth factor for T lymphocytes, induces all types of lymphocyte subpopulations and activates the proliferation of B lymphocytes. It intervenes in the inflammatory reaction by stimulating the synthesis of interferon, induces the release of IL-1, TNF-a and TNF
[0679] * IL-4: It acts as an anti-inflammatory by blocking the synthesis of IL-1, TNF-a, IL-6 and the inflammatory protein of the macrophage. Among other functions, it promotes the differentiation of Th2 lymphocytes, the proliferation and differentiation of B lymphocytes and is a potent inhibitor of apoptosis.
[0680] * IL-10: Cytokine capable of inhibiting the synthesis of pro-inflammatory cytokines by T lymphocytes and macrophages.
[0681] * IL-13: Modulates the production of IL-1, TNF, IL-8 and the inflammatory protein of the macrophage. It stimulates the growth and differentiation of B cells, and inhibits Th1 cells, as well as the production of inflammatory cytokines.
[0683] Statistical analysis of the results
[0685] The results were represented as the mean ± standard deviation of three replications. A statistically significant result was considered when the ANOVA test yielded a p value <0.05.
[0687] The results of the different activities and production of cytokines have been expressed graphically, always following the same scheme, which is indicated in Figure 2, which shows the results for the different activities and production of cytokines in the different cell lines.
[0689] Phagocytic capacity of RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0691] The phagocytic capacity was determined using the Neutral Red test as indicated in the section 'Determination of the phagocytic capacity of RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample "
[0692] Figure 3A and 3B represent the phagocytic activity of mouse splenocytes in the presence of different concentrations of the Sample. As can be seen, there is a significant dose-dependent phagocytosis-enhancing effect promoted by the highest concentration (1 mg / ml) of the sample in splenocytes from BalbC and C57BL / 6 mice. In all cases, the joint presence of LPS and samples produced a significant increase in phagocytic capacity to values similar to those provided by the positive control. You can see the relative phagocytosis expressed in percentage (%) obtained from splenocytes from BalbC (Figure 3A) and C57BL / 6 (Figure 3B) mice grown in the presence of the Sample, tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations of the Sample were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results.
[0694] Figures 4A and 4B illustrate the results of the relative phagocytosis (percentage;%) obtained from macrophages from the bone marrow of BalbC (Figure 4A) and C57BL / 6 (Figure 4B) mice grown in the presence of the Sample, which They were tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. The phagocytic activity of the macrophages from the bone marrow of BalbC and C57BL / 6 mice also increased significantly in a dose-dependent manner due to the two highest concentrations (1 and 0.1 mg / ml) of the Sample. In all cases, the joint presence of LPS and samples produced a significant increase in phagocytic capacity to values similar to those of the positive control.
[0696] Figure 5 illustrates the results of the relative phagocytosis (percentage;%) obtained from RAW macrophages cultured in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3 : 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. It can be seen that, in the case of RAW macrophages, although an increase in phagocytic capacity compared to the negative control was observed with the highest concentration of the Sample, this was not significant. In all cases, the joint presence of LPS and samples produced a significant increase in phagocytic capacity to values similar to those of the positive control.
[0698] Production of NO ² - derived reactive nitrogen species (RNS) by RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0700] The production of reactive nitrogen species (RNS) derived from NO2- was determined by the Griess reaction as indicated above.
[0702] Figures 6A and 6B illustrate the results of the relative production expressed in percentage (%) of reactive nitrogen species (RNS) derived from NO2- obtained from splenocytes from BalbC (figure 6A) and C57BL / 6 mice (figure 6B). ) grown in the presence of the tested Sample at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. As can be seen in these figures, only the highest concentration was able to significantly increase RNS production in splenocytes from BalbC and C57BL / 6 mice compared to the negative control. In the presence of LPS, only the highest concentration produced a significant increase, up to values similar to those provided by the positive control, in the production of RNS in splenocytes of the two types of mice. It should be noted that the signal provided by the enzymatic reaction was weak, although detectable by the spectrophotometer and above that provided by the blank wells.
[0704] Figures 7A and 7B illustrate the results of the relative production expressed in percentage (%) of reactive nitrogen species (RNS) derived from NO2- obtained from macrophages from the bone marrow of BalbC (Figure 7A) and C57BL / 6 (Figure 7B) mice grown in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. Regarding the production of RNS by macrophages from the bone marrow of BalbC and C57BL / 6 mice, a significant dose-dependent increase was observed, increasing with the two highest concentrations (1 and 0.1 mg / ml) of the sample. In all cases, the joint presence of LPS and sample produced a significant increase in RNS production to similar values, and even higher than those provided by the positive control.
[0706] Figure 8 illustrates the relative production expressed in percentage (%) of reactive nitrogen species (RNS) derived from NO2- obtained from RAW macrophages cultured in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. It can be seen that in the case of RAW macrophages (Figure 8), although an increase in RNS production was observed with the highest concentrations of the sample, it was not significant. In all cases, the joint presence of LPS and sample produced a significant increase in RNS production up to values similar to those provided by the positive control.
[0708] Arginase activity in RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0710] It is accepted that the specific expression of Arginase-1 by macrophages promotes inflammation, fibrosis and wound healing by increasing the levels of L-proline, polyamines, and the production of cytokines. of type Th2. For this reason, the analysis of arginase activity in these cells, which was carried out as indicated above, is interesting.
[0712] Figures 9A and 9B illustrate the results of the relative arginase activity expressed in percentage (%) obtained from splenocytes from BalbC (Figure 9A) and C57BL / 6 (Figure 9B) mice grown in the presence of the tested Sample at three concentrations. different (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. With respect to these figures, in which the arginase activity produced by splenocytes is indicated, it should be noted that the signal provided by the enzymatic reaction was weak, although detectable by the spectrophotometer and above that provided by the blank wells. In those cells from BalbC mice, no differences were observed between the positive and negative control or between these and the rest of the samples. On the contrary, with the cells from C57BL / 6 mice, significant differences were observed between the positive and negative control, and although an increase in arginase activity was observed only with the highest concentration (1 mg / ml) of the sample , this was not significant. In the presence of LPS, arginase activity increased in all cases, but not significantly.
[0714] Figures 10A and 10B illustrate the relative arginase activity expressed in percentage (%) obtained from macrophages from the bone marrow of BalbC (Figure 10A) and C57BL / 6 (Figure 10B) mice cultured in the presence of the sample tested at three concentrations. different (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. It can be seen that the arginase activity of macrophages from bone marrow of BalbC and C57BL / 6 mice was higher than that provided by splenocytes. On the other hand, as with splenocytes, in those bone marrow macrophages from BalbC mice, no differences were observed between the positive and negative control, with only a significant increase in arginase activity with respect to the negative control, in the case of the highest concentration (1 mg / m) in the presence of LPS. In the macrophages from the bone marrow of C57BL / 6 mice, significant differences were observed in arginase activity between the positive and negative control, following the same scheme provided by the splenocytes. In this specific case, all concentrations (1, 01 and 0.01 mg / ml) of the sample provided significant increases in arginase activity compared to the negative control. In all cases, the joint presence of LPS and samples produced a significant increase in arginase activity to values similar to, and even higher than, those provided by the positive control.
[0716] Figure 11 illustrates the results of the relative arginase activity expressed in percentage (%) obtained from RAW macrophages cultured in the presence of the sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The value provided by the Negative Control was set as 100%. The mean and standard deviation of three replicates are represented for each group of results. In this case of the RAW macrophages, it should be noted that the signal provided by the enzymatic reaction was weak, although detectable by the spectrophotometer and above that provided by the blank wells. With this cell line, a significant increase in arginase activity was observed compared to the negative control with the two highest concentrations (1 and 01 mg / ml). In all cases, the joint presence of LPS and sample produced an increase in arginase activity to values similar to those provided by the positive control, although it was only significant in the case of concentrations 0.1 and 0.01 mg / ml.
[0718] Production of immune response mediating molecules (cytokines) by RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample
[0720] In order to delve into the immunomodulatory properties of the sample provided by IGEN BIOLAB, its ability to modify cytokine secretion by various types of macrophages was evaluated. The Cytokine production was analyzed by ELISA as indicated in section 2.7, evaluating three typical Th1 response cytokines (cellular immunity: TNF-a, IFN- ^y and IL2) and three typical Th2 response cytokines (humoral immunity: IL10 , IL4 and IL13).
[0722] Figure 12 refers to a representative example of the graph and equation obtained with serial dilutions of the TNF-a standard, which was used to calculate the production of this cytokine by the different types of macrophages.
[0724] Th1 response
[0726] -TNF-a
[0728] To quantify the production of TNF-α by RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample, the equation of the curve provided by serial dilutions of the TNF-standard was used. to which was included in the TNF-alpha Mouse ELISA Kit (Thermo Fisher, eBiosciences). In all cases, R2 values greater than 0.99 were obtained. The assay range was 3.13-200 pg / ml, with the analytical sensitivity of this cytokine with this kit being 0.75 pg / ml. It should be noted that the sample had to be diluted (1: 3) in order to correctly quantify the results.
[0730] Figures 13A. and 13B illustrate the results of TNF-a production (pg / ml) by splenocytes obtained from BalbC (Figure 13A) and C57BL / 6 (Figure 13B) mice grown in the presence of the tested Sample at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. As can be seen in these figures, the production of TNF-a underwent a significant increase with respect to the negative control when the splenocytes from both BalbC and C57BL / 6 mice were cultured in the presence of any of the three concentrations of the sample. This increase was even higher than the values provided by the positive control. The joint presence of LPS and sample produced an increase in production of TNF-a to values similar or even higher than those provided by the positive control.
[0732] Figures 14A and 14B illustrate the results of the production of TNF-a (pg / ml) by macrophages from the bone marrow of BalbC (figure 14A) and C57BL / 6 (figure 14B) mice cultured in the presence of the tested Sample. at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. Regarding the production of TNF-a by macrophages obtained from the bone marrow of BalbC mice, small, although significant, differences were obtained between the positive and negative control. In this cell type, only the two highest concentrations (1 and 0.1 mg / ml) of the sample produced a slight increase compared to the negative control. The joint presence of LPS and samples produced a slight increase in the production of TNF-a to values similar to those provided by the positive control. In relation to the results obtained with the macrophages from the bone marrow of C57BL / 6 mice, a greater difference was observed between the values provided by both controls. In this case, the two highest concentrations (1 and 0.1 mg / ml) of the sample produced an increase in TNF-production with respect to the negative control. In all cases, the joint presence of LPS and samples produced an increase in the production of this cytokine to levels similar to those obtained with the positive control.
[0734] Figure 15 illustrates the results of TNF-α production (pg / ml) by RAW macrophages cultured in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. It can be observed that, when the production of TNF-a was analyzed in RAW macrophages (Figure 15), only the highest concentration (1 mg / ml) of the sample produced a significant increase in it compared to the negative control. In all cases, the joint presence of LPS and sample produced an increase in the production of this cytokine to levels similar to those obtained with the positive control.
[0735] -IFN-y
[0737] Figure 16 illustrates a representative example of the graph and equation obtained with serial dilutions of the IFN- ^y standard, which was used to calculate the production of this cytokine by the different types of macrophages. To quantitate the IFN-y by monocytes, macrophages marrow and splenocytes from explants of mouse spleen in the presence of Sample RAW 264.7 equation was used for curve provided by serial dilutions of the standard IFN ^and , which was included in the Mouse Uncoated ELISA Kit with Plates kit (Thermo Fisher, eBiosciences). In all cases, R2 values greater than 0.99 were obtained. The assay range was 15-2000 pg / ml, with the analytical sensitivity of this cytokine with this kit being 15 pg / ml.
[0739] Figures 17A and 17B illustrate the results of IFN-y production (pg / ml) by splenocytes obtained from BalbC (Figure 17A) and C57BL / 6 (Figure 17B) mice grown in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results.
[0741] Surprisingly, IFN-y production was much higher in splenocytes from BalbC mice than in splenocytes from C57BL / 6 mice (Figure 3.16). In the former, the addition of 1 mg / ml of sample produced a marked reduction in the production of this cytokine (below the limit of quantification), while the presence of 0.1 and 0.01 mg / ml of sample produced a large increase in IFN-levels and even above those observed with the positive control. Interestingly, the joint presence of LPS and 1 mg / ml of sample also caused a reduction in the values of this cytokine below the negative control and the limit of quantification, while with the rest of the concentrations values similar to those provided by the positive control. The results with splenocytes from C57BL / 6 mice were peculiar, the values being very close to the limit of quantification and there were no significant differences between the negative and positive controls. Only an increase in the production of IFN- and when 0.01 mg / ml sample was added to the cultures.
[0743] Figures 18A and 18B illustrate the results of the production of IFN-y (pg / ml) by macrophages from the bone marrow of BalbC (figure 18A) and C57BL / 6 (figure 18B) mice cultured in the presence of the tested sample. at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. In relation to the results obtained with the macrophages from the bone marrow of BalbC and C57BL / 6 mice, very low IFN-y values were obtained, these being below the limit of quantification and not allowing a correct analysis of the results.
[0745] Figure 19 illustrates the production of IFN-y (pg / ml) by RAW macrophages cultured in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. It can be seen that, with RAW macrophages, a similar situation occurred, obtaining very low values of IFN-y, these being below the limit of quantification and not allowing a correct analysis of the results.
[0747] -IL-2
[0749] Figure 20 illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-2 standard, which was used to calculate the production of this cytokine by the different types of macrophages. To quantify the production of IL-2 by RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample, the equation of the curve provided by serial dilutions of the IL2 standard was used, which was included in the Mouse Uncoated ELISA Kit with Plates kit (Thermo Fisher, eBiosciences). In all cases, R2 values greater than 0.99 were obtained. The assay range was 2-200 pg / ml, with the analytical sensitivity of this cytokine with this kit being 2 pg / ml.
[0750] Figures 21A and 21B illustrate the production of IL-2 (pg / ml) by splenocytes obtained from BalbC (figure 20A) and C57BL / 6 (figure 20B) mice cultured in the presence of the sample tested at three different concentrations. (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. A similar pattern of IL-2 production was observed in both types of splenocytes. The production of this cytokine was slightly higher in the positive control compared to the negative control, producing a significant decrease in its values when 1 mg / ml of the sample was added to the cultures. Interestingly, the joint presence of LPS and 1 mg / ml of the sample also caused a reduction in the values of this cytokine below the negative control, while with the rest of the concentrations, values similar to those provided by the controls.
[0752] Figures 22A and 22B illustrate the results of IL-2 production (pg / ml) by macrophages from the bone marrow of BalbC (figure 22A) and C57BL / 6 (figure 22B) mice cultured in the presence of the tested Sample. at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. In relation to the results obtained with the macrophages from the bone marrow of BalbC and C57BL / 6 mice, only very low IL-2 values were obtained, these being close to the limit of quantification and not allowing a correct analysis of the results.
[0754] Figure 23 illustrates the results of IL-2 production (pg / ml) by RAW macrophages cultured in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. It should be noted that only very low IL2 values were also obtained with RAW macrophages, being in some cases below the limit of quantification. This has not allowed a correct analysis of the results (Figure 23).
[0756] Th2 response
[0758] -IL-10
[0759] Figure 24 illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-10 standard, which was used to calculate the production of this cytokine by the different types of macrophages.
[0761] To quantify the production of IL-10 by RAW 264.7 monocytes, bone marrow macrophages, and splenocytes from mouse spleen explants in the presence of the Sample, the equation of the curve provided by serial dilutions of the IL-standard was used. 10, which was included in the Mouse Uncoated ELISA Kit with Plates kit (Thermo Fisher, eBiosciences). In all cases, R2 values greater than 0.99 were obtained. The assay range was 30-4000 pg / ml, with the analytical sensitivity of this cytokine with this kit being 30 pg / ml.
[0763] Figures 25A and 25B illustrate the results of IL-10 production (pg / ml) by splenocytes obtained from BalbC (Figure 23A) and C57BL / 6 (Figure 25B) mice grown in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. For both types of splenocytes, a similar pattern of IL-10 production was observed, although this was higher in the case of splenocytes from C57BL / 6 mice. A dose-dependent effect was observed with the sample. The addition of 1 and 0.1 mg / ml of sample produced a significant increase, in relation to the negative control, in IL-10 levels, although these were below those provided by the positive control (Figures 25A and 25B ). The joint presence of LPS and any of the three concentrations of the sample produced significant increases in IL-10 compared to the negative control, although in general these values were below those provided by the positive control.
[0765] Figures 26A and 26B illustrate the results of IL-10 production (pg / ml) by part of macrophages from the bone marrow of BalbC (figure 26A) and C57BL / 6 (figure 26B) mice cultured in the presence of the sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. It can be observed that, in relation to the results obtained with the macrophages from the bone marrow of BalbC and C57BL / 6 mice, lower IL-10 values were obtained compared to those obtained with splenocytes. It should be noted that in macrophages from the bone marrow of BalbC mice, a significant increase in IL-10 production was only obtained when the highest concentration (1 mg / ml) of the sample was added in the presence of LPS, reaching values above those provided by the positive control (Figures 26A and 26B). With the macrophages from the bone marrow of C57BL / 6 mice, a significant increase in IL-10 production was obtained compared to the negative control, with the highest concentration (1 mg / ml) of the sample. As in the case of bone marrow macrophages from BalbC mice, a significant increase in IL-10 production was also obtained. When the highest concentration (1 mg / ml) of the sample was added in the presence of LPS, reaching values above those provided by the positive control. The rest of the sample concentrations, when tested in the presence of LPS, resulted in IL-10 production similar to that provided by the positive control.
[0767] Figure 27 illustrates the results of IL-10 production (pg / ml) by RAW macrophages cultured in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. It can be seen that with RAW macrophages IL-10 values very close to the limit of quantification were obtained. Only the addition of 1 mg / ml of Sample in the presence of LPS produced a significant increase in the production of this cytokine, although the fact that such low values were obtained has not allowed a correct analysis of the results (Figure 27).
[0768] -IL-4
[0770] Figure 28 illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-4 standard, which was used to calculate the production of this cytokine by the different types of macrophages. To quantify the production of IL-4 by RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample, the equation of the curve provided by serial dilutions of the IL- standard was used. 4, which was included in the Mouse Uncoated ELISA Kit with Plates kit (Thermo Fisher, eBiosciences). In all cases, R2 values greater than 0.99 were obtained. The assay range was 4-500 pg / ml, with the analytical sensitivity of this cytokine with this kit being 4 pg / ml. The IL-4 production by splenocytes was not modified, neither by the presence of LPS, nor by the presence of any concentration of the sample. It should be noted that very low values were obtained in all cases, although above the limit of quantification.
[0772] Figures 29A and 29B illustrate the results of IL-4 production (pg / ml) by splenocytes obtained from BalbC (figure 29A) and C57BL / 6 (figure 29B) mice cultured in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. The IL-4 production by splenocytes was not modified, neither by the presence of LPS, nor by the presence of any concentration of the sample. It should be noted that very low values were obtained in all cases, although above the limit of quantification.
[0774] Figures 30A and 30B illustrate the results of IL-4 production (pg / ml) by macrophages from the bone marrow of BalbC (Figure 30A) and C57BL / 6 (Figure 30B) mice cultured in the presence of the tested Sample. at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. On the other hand, Figure 31A shows the results of IL-4 production (pg / ml) by cultured RAW macrophages in the presence of the Sample tested at three different concentrations (C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. It can be seen that, in the case of bone marrow macrophages (Figures 30A and 30B) and RAW (Figure 31), the production of this cytokine was not detected in any case as the values obtained were below the limit of quantification.
[0776] -IL-13
[0778] Figure 31B illustrates a representative example of the graph and equation obtained with serial dilutions of the IL-13 standard, which was used to calculate the production of this cytokine by the different types of macrophages, while Figures 32A and 32B illustrate the results of IL-13 production (pg / ml) by splenocytes obtained from BalbC (figure 32A) and C57BL / 6 (figure 32B) mice cultured in the presence of the sample tested at three different concentrations (C1 : 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results.
[0780] To quantify the production of IL-13 by RAW 264.7 monocytes, bone marrow macrophages and splenocytes from mouse spleen explants in the presence of the Sample, the equation of the curve provided by serial dilutions of the IL-standard was used. 13 (Figures 32A and 32B), which was included in the IL-13 Mouse ELISA Kit (Thermo Fisher, eBiosciences). In all cases, R2 values greater than 0.99 were obtained. The assay range was 7.8 pg / ml, and the analytical sensitivity of this cytokine with this kit was 2.8 pg / ml. The values obtained for this cytokine were generally very low, and cannot be quantified in RAW macrophages. In splenocytes, these values were also very low, being generally below the quantification index. Reliable values for splenocytes from BalbC mice were only observed in the case of concentration 2 (0.1 mg / ml) of the sample in the presence of LPS, these values being above those provided by the positive control (Figure 32A ). In splenocytes from C57BL / 6 mice IL-13 production could only be quantified in the positive control and in the case of concentration 1 (1 mg / ml) of the sample in the presence of LPS (Figure 32B). These low values, however, make it difficult to correctly interpret the results.
[0782] Figures 33A and 33B. illustrate the results of IL-13 production (pg / ml) by macrophages from the bone marrow of BalbC (figure 33A) and C57BL / 6 (figure 33B) mice grown in the presence of the sample tested at three different concentrations ( C1: 1 mg / ml; C2: 0.1 mg / ml and C3: 0.01 mg / ml). Those same concentrations were also tested in the presence of lipopolysaccharide (LPS). Each condition was tested in triplicate. The mean and standard deviation of three replicates are represented for each group of results. When the samples were tested on macrophages from the bone marrow of BalbC mice, quantifiable values were only observed in the case of concentrations 2 and 3 (0.1 and 0.01 mg / ml) of the sample For cells from bone marrow Bone from C57BL / 6 mice, values similar to those obtained by the positive control were observed in the case of the highest concentration (1 mg / ml) of the sample with and without the joint addition of LPS. The low values obtained, however, make it difficult to properly interpret the results.

权利要求:
Claims (35)
[1]
1. Procedure for obtaining a modulating composition of the human intestinal microbiome comprising
cultivating strains of probiotic microorganisms that comprise strains of the Saccharomyces, Lactobacillus, Bacillus, Streptococcus and Bifidobacterium genera , in culture broths to obtain biomass broths of the probiotic microorganisms that comprise microbial biomasses and culture broths;
recovering microbial biomass from microbial biomass broths to obtain wet recovered microbial biomass;
characterized because
the strains of each microorganism are cultivated, at least until they have reached their full logarithmic phase of growth, in separate culture broths;
the microbial biomasses are recovered separately from the respective culture broths to obtain respective wet recovered microbial biomasses; each of the wet recovered microbial biomasses is subjected to cell lysis until reaching a percentage of cell breakage of at least 90% to obtain respective biomass of microbial lysates;
the respective biomasses of microbial lysates are dried to obtain respective dried microbial lysates;
the respective dry microbial lysates are mixed in proportions of 30% to 70% by volume of dry microbial lysates from cultures of strains of the genus Saccharomyces ,
10% to 30% by volume of dry microbial lysates from cultures of strains of the genus Lactobacillus ,
10% to 30% by volume of dry microbial lysates from cultures of strains of the genus Bacillus,
3% to 10% by volume of dry microbial lysates from cultures of strains of the genus Streptococcus ,
3% to 10% by volume of dry microbial lysates from cultures of strains of the genus Bifidobacterium ,
to obtain a mixture of dry microbial lysates;
At least one sample is drawn from the mixture of the dried microbial lysates and it is determined whether the sample comprises a minimum of 5000 genomic sequences copies / ml of each of the following genomic sequences;

[2]
two.
[3]
3. Method according to claim 1 or 2, characterized in that the strains of microorganisms of the genus Saccharomyces are strains of Saccharomyces cerevisiae.
[4]
4. Process according to claim 1, 2 or 3, characterized in that the strains of microorganisms of the genus Lactobacillus are selected from strains of Lactobacillus gasseri, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus reuteri, and combinations thereof.
[5]
5. Method according to claim 1, 2, 3 or 4, characterized in that the strains of microorganisms of the genus Bacillus are selected from strains of Bacillus subtilis, Bacillus coagulans, Bacillus clausii, and combinations thereof.
[6]
6. Method according to any one of claims 1 to 5, characterized in that the strains of microorganisms of the genus Bifidobacterium are selected from strains of Bifidobacterium bifidum, Bifidobacterium infantis, and combinations thereof.
[7]
7. Method according to any one of claims 1 to 6, characterized in that the strains of microorganisms of the genus Streptococcus are strains of Streptococcus thermophilus.
[8]
8. Method according to any one of claims 1 to 7, characterized in that
the microorganisms of the genus Lactobacillus are cultivated at pH 6.4 10% in the absence of oxygen until obtaining an anaerobic culture broth in which the microorganisms of the genus Lactobacillus are in the full logarithmic phase of growth of their biomass,
When said full log phase of growth has been reached, oxygen is introduced into the initial culture broth until it contains between 25% and 30% of dissolved oxygen to obtain an oxygenated culture broth;
seeding the oxygenated culture broth with a culture of microorganisms of the genus Saccharomyces;
jointly culture the microorganisms of the Lactobacillus and Saccharomyces genera adjusting and maintaining the culture broth with 20% to 30% dissolved oxygen and maintaining the culture broth at pH 6.4 ^ 10% during a first stage and allowing in a second stage that the pH drops to 4.5 10%;
stop the culture when the pH has reached 4.5 10% to obtain broths of the microbial biomasses formed from the microorganisms of the Lactobacillus and Saccharomyces genera .
[9]
9. Process according to claim 8, characterized in that during the first stage the culture broth is adjusted and maintained at pH 6.4 0.1.
[10]
10. Process according to claim 8 or 9, characterized in that, in the second stage, the pH is allowed to fall to 4.5 0.1.
[11]
11. Process according to claim 8, 9 or 10, characterized in that the microorganisms of the genus Lactobacillus are cultured in the absence of oxygen for 5 to 7 hours.
[12]
12. The method according to claim 11, characterized in that the microorganisms of the genus Lactobacillus are cultured in the absence of oxygen for 5.5 and 6.5 hours.
[13]
13. Process according to any one of claims 8 to 12, characterized in that in the first stage the microorganisms of the Lactobacillus and Saccharomyces genera are jointly cultivated for 7 to 9 hours.
[14]
14. Method according to claim 13, characterized in that in the first stage the microorganisms of the Lactobacillus and Saccharomyces genera are jointly cultured for 7.5 to 8.5 hours.
[15]
15. Process according to any one of claims 8 to 14, characterized in that the oxygenated culture broth is seeded with 4 to 6% v / v of a culture of microorganisms of the genus Saccharomyces .
[16]
16. Process according to claim 15, characterized in that the oxygenated culture broth is seeded with 5% 0.1% v / v of a culture of microorganisms of the genus Saccharomyces.
[17]
17. Process according to any one of claims 8 to 16, characterized in that the microorganisms of the genus Lactobacillus are cultured at pH 6.4 10% in the absence of oxygen for 5.5 to 6.5 hours.
[18]
18. Process according to any one of the preceding claims, characterized in that the microbial biomasses are recovered by centrifugation separately from the respective culture broths.
[19]
19. Process according to any one of the preceding claims, characterized in that the microbial lysate biomasses are dried by spray drying.
[20]
20. Process according to any one of the preceding claims, characterized in that the microbial lysate biomasses are dried by lyophilization.
[21]
21. Process according to any one of the preceding claims, characterized in that the microorganisms of the Bacillus genus are cultured at 30 ° C 1 ° C for 20-24 hours, maintaining a dissolved oxygen concentration of at least 40% and a pH of 6 , 8 0.1.
[22]
22. Process according to claim 21, characterized in that the microorganisms of the genus Bacillus are cultured while maintaining the pH stable by adding at least one stabilizing agent.
[23]
23. Process according to claim 22, characterized in that the stabilizing agent comprises an aqueous ammonium solution at 25% w / v
[24]
24. Process according to claim 22 or 23, characterized in that the stabilizing agent comprises an aqueous solution of tetraoxphosphoric acid at 35% w / v.
[25]
25. Process according to any one of the preceding claims, characterized in that the microorganisms of the genus Streptococcus are cultured at 37 ° C 1 ° C for approximately 24 hours under microaerophilic conditions and a pH of 6.8 0.1 without applying aeration to the broth. cultivation.
26. Process according to claim 25, characterized in that the microorganisms of the genus Streptococcus are cultured at an initial oxygen saturation of less than 10%.
[26]
26. Method according to claim 25, characterized in that the microorganisms of the genus Streptococcus are cultured at an initial oxygen saturation of less than 2%.
[27]
27. Process according to claim 25, 26 or 27, characterized in that the microorganisms of the genus Streptococcus are cultured keeping the pH stable by adding at least one stabilizing agent.
[28]
28. Process according to claim 27, characterized in that the stabilizing agent comprises a 25% w / v aqueous ammonium solution
[29]
29. Process according to claim 27 or 28, characterized in that the stabilizing agent comprises an aqueous solution of tetraoxphosphoric acid at 35% w / v.
[30]
30. Process according to any one of the preceding claims, characterized in that the microorganisms of the genus Bifidobacterium are cultured at 37 ° C 1 ° C for approximately 24 hours in the absence of oxygen and a pH of 6.2 0.1.
[31]
31. Process according to claim 30, characterized in that the microorganisms of the genus Bifidobacterium are cultivated while maintaining the pH stable by adding at least one stabilizing agent.
[32]
32. Process according to claim 31, characterized in that the stabilizing agent comprises an aqueous ammonium solution at 25% w / v
[33]
33. Process according to claim 31 or 32, characterized in that the stabilizing agent comprises an aqueous solution of tetraoxphosphoric acid at 35% w / v.
[34]
34. Process according to any one of the preceding claims, characterized in that each of the biomass broths obtained at the end of the culture are cooled to stop the cellular metabolism of the microorganisms present in the biomass broths.
[35]
35. Process according to claim 29, characterized in that the biomass broths are cooled to 4 ° C to 8 ° C.

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同族专利:

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公开号 | 公开日

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ES2783723A1|2020-09-17|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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WO2016120320A1|2015-01-27|2016-08-04|Dupont Nutrition Biosciences Aps|Immunomodulatory composition comprising bifidobacteria|

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EP2512261A1|2009-12-18|2012-10-24|Hill's Pet Nutrition, Inc.|Pet food compositions including probiotics and methods of manufacture and use thereof|

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ES201930242A|ES2783723A1|2019-03-15|2019-03-15|COMPOSITION OBTAINED FROM BACTERIAL LISTS TO MODULATE THE HUMAN MICROBIOME, THERAPEUTIC FORMULATION AND FOOD SUPPLEMENT THAT INCLUDE THE COMPOSITION, AND USES OF THE COMPOSITION. |

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