WO2022020971A1 - Souches de l'espèce lactobacillus plantarum et lactobacillus brevis et composition probiotique les comprenant - Google Patents

Souches de l'espèce lactobacillus plantarum et lactobacillus brevis et composition probiotique les comprenant Download PDF

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WO2022020971A1
WO2022020971A1 PCT/CL2020/050083 CL2020050083W WO2022020971A1 WO 2022020971 A1 WO2022020971 A1 WO 2022020971A1 CL 2020050083 W CL2020050083 W CL 2020050083W WO 2022020971 A1 WO2022020971 A1 WO 2022020971A1
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composition
strain
strains
rgm
probiotic
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PCT/CL2020/050083
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Sandra Rayen QUILODRÁN VEGA
Julio Cesar VILLENA
Jorge Roberto Toledo Alonso
Oliberto SÁNCHEZ RAMOS
Leonardo Miguel ALBARRACÍN
Eduardo RAMOS DELGADO
Nelson Roberto ROJAS VELIS
Roberto Guillermo VEGA FERNANDEZ
Carlos Sunkel Letelier
Humberto Italo BERNASCONI MUÑOZ
Alonso Leandro JEREZ MORALES
Camilo Alfredo HERNÁNDEZ PIRUL
Sindy Tatiana DÍAZ CASTILLO
Pablo Ignacio FASCE SAENGER
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Laboratorio Pasteur S.A.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to the technical field of medicine, particularly to probiotic bacterial strains of the Lactobacillus genus and a composition comprising at least one or a mixture of these.
  • Probiotics are live microorganisms that, administered in adequate amounts, provide health and nutritional benefits to the host (Food and Agricultural Organization of the United Nations and World Health Organization. 2001). It has been reported that probiotics may play an important role in the immune, digestive, and respiratory systems, and that they may have a significant effect in alleviating infectious diseases in children and other high-risk populations (Food and Agricultural Organization of the United Nations and World Health Organization. 2006).
  • probiotic strains of the genera Lactobacillus and Bifidobacterium have been shown to exert a function in one or more components of the immune system, for example, in the humoral, cellular, or non-specific immunity (Erickson KL, Hubbard NE. Probiotic Immunomodulation in Health and Disease J Nutr 2000;130(2S Suppl):403S-409S).
  • probiotic compositions that favor the immune system.
  • the probiotic described in patent document US 10,064,426 B2 describes the use of probiotics in pregnant women to increase the immune system of their children, where said probiotic contains Lactobacillus rhamnosus CGMCC 1.3724.
  • Patent document US 10,251,407 B2 describes a probiotic comprising Lactobacillus paracasei LMG P-24778 for the treatment of gastrointestinal diseases associated with, for example, an immature immune system, food allergies or irritable bowel syndrome, Crohn's disease or enterocolitis necrotizing
  • Patent document US 9,931,363 B2 describes the probiotic strains Lactobacillus salivarius DSM 22775, Bifidobacterium breve DSM 16604, Lactobacillus pentosus DMS 21980, Streptococcus thermophilus DSM 18616, Lactobacillus casei ssp. Rhamnosus DSM 16605, and Lactobacillus acidophilus DSM 21717, which have a useful immunomodulatory effect for the treatment of pathologies associated with alterations in the immune system.
  • a first object of the present invention corresponds to a probiotic composition to increase the immune response in mammals with digestive disorders that comprises at least one isolated and purified probiotic bacterial strain of the genus Lactobacillus sp.
  • said strain is selected from the group consisting of Lactobacillus plantarum and Lactobacillus brevis, or a mixture of these; and an appropriate excipient.
  • said strain is selected from the group consisting of Lactobacillus plantarum deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) under the RGM access code 2608, and Lactobacillus brevis deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) under the RGM access code 2611.
  • the probiotic composition comprises a range between 1.0 x 10 6 to 1.0 x 10 14 CFU of the probiotic strain per gram of composition, more preferably it comprises a range between 1.0 x 10 9 and 1.0 x 10 11 CFU of the probiotic strain per gram of composition.
  • the probiotic composition comprises 2 x 10 9 CFU of the Lactobacillus plantarum strain (RGM 2608) per gram of composition.
  • the probiotic composition comprises 2 x 10 9 CFU of the Lactobacillus brevis strain (RGM 2611) per gram of composition.
  • the probiotic composition comprises 2 x 10 9 CFU of the Lactobacillus plantarum strain (RGM 2608) per gram of composition and 2 x 10 9 CFU of the Lactobacillus brevis strain (RGM 2611) per gram of composition.
  • the appropriate excipient is a mixture of fructooligosaccharides (FOS) and glucose.
  • FOS fructooligosaccharides
  • the fructooligosaccharides are found in a concentration between 40-80% w/w of the total composition, and the glucose is found in a concentration between 25-45% w/w of the total composition.
  • the fructooligosaccharides are in a concentration between 55-65% w/w, and the glucose is in a concentration between 35-40% w/w.
  • a second object of the present invention corresponds to an isolated and purified bacterial probiotic strain, which consists of the Lactobacillus plantarum strain deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) under the access code RGM 2608; and the Lactobacillus brevis strain deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) under the access code RGM 2611. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention refers to new probiotic strains with demonstrated immunomodulatory activity, which are useful for the manufacture of a probiotic composition for human and/or animal consumption.
  • This probiotic composition is useful for the treatment of pathologies associated with alterations in the immune and/or gastrointestinal system.
  • the present invention also comprises a probiotic composition containing at least one of said probiotic strains or a mixture thereof.
  • One of the objects of the present invention corresponds to a probiotic composition
  • a probiotic composition comprising at least one strain of the genus Lactobacillus sp., preferably at least one strain selected from the group consisting of the species Lactobacillus plantarum, Lactobacillus brevis, or a mixture of are; and an appropriate excipient.
  • the strain of Lactobacillus plantarum is the one deposited in the Chilean Collection of Microbial Genetic Resources
  • the Lactobacillus brevis strain is the one deposited in the Chilean Collection of Microbial Genetic Resources
  • the present invention also considers any mutant strain of those previously mentioned, as long as they maintain a biological activity similar to those presented in this invention.
  • said mutant has a similarity percentage with the genome of the Lactobacillus plantarum RGM 2608 strain or with the genome of the Lactobacillus brevis RGM 2611 strain of at least 99.5%.
  • the present invention also considers any mutant of said strains that have been transformed with plasmids.
  • the probiotic composition comprises a range between 1.0 x 10 6 CFU/g and 1.0 x 10 14 CFU/g of one or a mixture of the previously mentioned strains.
  • the composition comprises between 1.0 x 10 9 and 1.0 x 10 11 CFU/g.
  • the probiotic composition comprises approximately 2 x 10 9 CFU of the Lactobacillus plantarum strain (RGM 2608) per gram of composition.
  • the probiotic composition comprises approximately 2 x 10 9 CFU of the Lactobacillus brevis strain (RGM 2611) per gram of composition.
  • the probiotic composition comprises approximately 2 x 10 9 CFU of the Lactobacillus plantarum strain (RGM 2608) per gram of composition and approximately 2 x 10 9 CFU of the Lactobacillus brevis strain (RGM 2611) per gram of composition.
  • the present probiotic composition additionally comprises an excipient suitable for the administration of said composition orally.
  • the composition is administered in water, or as part of a food, beverage, or nutritional supplement.
  • the composition may also contain other ingredients such as stabilizers, flavors, sweeteners, colorants, preservatives, antioxidants, or other ingredients for nutritional purposes (for example, carbohydrates, fats, proteins, vitamins and/or minerals).
  • the probiotic composition comprises monosaccharides, disaccharides or oligosaccharides as excipient, preferably a mixture of these.
  • monosaccharides that can be used are glucose, dextrose (D-glucose), fructose, galactose, tagatose, among others.
  • the probiotic composition comprises a mixture of fructooligosaccharides with glucose.
  • the fructooligosaccharides are found in the composition in a concentration between 40-80% w/w of the total composition, more preferably between 55-65% of the total composition.
  • the concentration of fructooligosaccharides is 60% w/w of the composition.
  • the glucose is found in the composition in a concentration between 25-45% w/w of the total composition, more preferably between 35-40% w/w of the total composition.
  • the glucose concentration is between 36% - 38% w/w of the composition.
  • the probiotic composition can be administered to a subject who has an impaired or immature immune system, or a subject with a risk of or currently having a gastrointestinal disease.
  • the composition is capable of preventing, reducing and/or treating pathologies associated with those previously mentioned, such as acute diarrhea, chronic diarrhea, food hypersensitivity, necrotizing enterocolitis, intestinal mucositis, among others.
  • Said subject can be a human, preferably infants and children, or an animal for the use of the probiotic composition in veterinary medicine.
  • the present invention also relates to the isolated and purified probiotic strains Lactobacillus plantarum RGM 2608 and Lactobacillus brevis RGM 2611. Both strains were isolated from milk samples, specifically Lactobacillus plantarum RGM 2608 was isolated from goat milk samples, and Lactobacillus brevis RGM 2611 was isolated from pig milk samples. Since both strains come from milk, they have a greater affinity to oligosaccharide structures, intestinal mucosa and/or sialic acid (present in children).
  • the Lactobacillus plantarum RGM 2608 strain has a broad spectrum of pathogen inhibition action that encompasses both enterobacteria and bacteria that infect the respiratory tract.
  • the Lactobacillus brevis RGM 2611 strain which also has an effect on enteric pathogens that cause diarrhea, has an immunomodulatory capacity that activates macrophages and increases IgA levels, which makes it a very suitable strain to act at the intestinal level.
  • Lactobacillus plantarum RGM 2608 and Lactobacillus brevis RGM 2611 strains when analyzed together in the in vitro pathogen inhibition assays, so they can be mixed in a probiotic composition.
  • a hemolysin detection assay was performed on the potentially probiotic strains previously isolated. For this, the strains were seeded on sheep blood agar and incubated for 24 to 48 h in microaerophilia, at 37°C. After this period, the appearance of hemolysis halos was observed. There are complete hemolysis, called beta hemolysis and partial called alpha. Strains that were beta hemolytic were discarded. The strains identified as TUCO-2 and TUCO-5 did not present beta-type hemolysis.
  • a gelatinase detection assay was performed on the potentially probiotic strains previously isolated. To do this, the strains were seeded with a straight loop in a bacteriological gelatin medium, which were then incubated for 24 h in microaerophilia, at 37°C. Gelatin liquefaction was then verified. For this, the tubes were placed in the refrigerator to rule out liquefaction due to temperature and, after 4 h, if the gelatin was in its gel form, then the strain lacked gelatinases. The strains whose gelatin was liquid were discarded because they had gelatinases. The gelatins with the strains identified as TUCO-2 and TUCO-5 maintained their gel form.
  • An antibiogram of the potentially probiotic strains was performed in MRS agar medium. Each strain was planted on grass and the sensidiscs of each antibiotic were deposited (gentamicin, ampicillin, erythromycin, tetracycline, vancomycin, amikacin, amoxicillin, ciprofloxacin, because, for the Lactobacillus strains, these are the antibiotics that have shown low levels of potency over those strains). The cultures were carried out for 24 h at 37°C and under microaerophilic conditions.
  • the potentially probiotic strains were transferred from their normal development under the previously mentioned conditions (37°C for 24 - 48°C under microaerophilia), from MRS agar to skim milk agar and incubated at 37 Q C in microaerophilia for 24 h.
  • the developments of the strains identified as TUCO-2 and TUCO-5 presented degradation halos, that is, they degrade casein, giving them an advantageous technological characteristic.
  • the potentially probiotic strains were transferred from their normal development under the previously mentioned conditions (37°C for 24 - 48°C under microaerophilia), from MRS agar to MRVP broth, and were incubated for 24 hours at 37°C.
  • the strains identified such as TUCO-2 and TUCO-5 are producers of acids from glucose, since they were positive methyl red and negative Voges Proskauer.
  • the identification of the selected strains TUCO-2 and TUCO-5 was carried out by amplification of the 16S ribosomal DNA through a polymerase chain reaction.
  • primers 27 F SEQ ID NO: 1
  • 1492R SEQ ID NO: 2
  • the product of this amplification was sequenced and SEQ ID NO: 3 was obtained for the strain identified as TUCO-2 and SEQ ID NO: 4 for the TUCO-5 strain.
  • the BLAST bioinformatic algorithm and the "16S ribosomal RNA sequences" database were used. The analyzes showed that the TUCO-2 strain belongs to the Lactobacillus plantarum species, and the TUCO-5 strain belongs to the Lactobacillus plantarum species. to the species Lactobacillus brevis Both strains were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM), under the access codes Lactobacillus plantarum RGM 2608 and Lactobacillus brevis RGM 2611.
  • ChRGM Collection of Microbial Genetic Resources
  • Strain aggregation assays are divided into two: autoaggregation (between strains of the same species) and coaggregation (between different strains).
  • Is aggregation refers to or provides clues to the mechanism of action of the strains. For example, good self-aggregation is related to the ability of the strain to bind and deposit itself on some surface or tissue and, if it also has an effect on pathogens, then it would have an exclusion (prevention) effect.
  • the strain coaggregates well with a pathogenic strain and also has an effect on it, then it can be related to a displacement mechanism, that is, curative.
  • the autoaggregation and coaggregation capacity of the selected probiotic strains Lactobacillus plantarum RGM 2608 and Lactobacillus brevis RGM 2611 was evaluated.
  • each strain was brought to 0.5 McFarland and incubated at 37°C for 6 h, without shaking. After 6 h, the McFarland values were measured again. Since these strains tend to settle at the bottom of the tube, the McFarland value was measured without shaking the tube and said value gradually decreased.
  • the percentage of autoaggregation was calculated using the following formula:
  • McFarland values 0.5 McFarland
  • volumes of the probiotic strains were mixed with each of the reference or pathogenic strains, incubated at 37°C without shaking, and the readings were made. McFarland values at 6 h.
  • the percentage of coaggregation was calculated using the following formula: Where:
  • McFmezcia McFarland value mixture of the probiotic strain and the reference or pathogenic strain
  • McFpat McFarland value of the reference or pathogenic strain
  • McFprob McFarland value of the probiotic strain
  • the self-aggregation of the strains at 6 hours of interaction corresponds to 19% for the Lactobacillus plantarum RGM 2608 strain, and 15% for the Lactobacillus brevis RGM 2611 strain, a value similar to that of the commercial probiotic strain L. Casei Shirota analyzed (11% ).
  • Table 1 Percentage of coaggregation of the strains of the present invention in comparison with a commercial probiotic strain at 6 h of interaction. Additionally, the probiotic strains presented coaggregation at 24 hours with other pathogens, as shown in Table 2.
  • Example 5 Inhibitory action of the strains on pathogens
  • the strains were adjusted to 0.5 MacFarland each and were mixed in a 1: 1 ratio. stand for 30 minutes until the drops were absorbed and incubated at 37°C, in microaerophilia for 24 hours.
  • the pathogenic strains Salmonella sp. and E. coli were seeded on nutrient agar and incubated aerobically at 37°C for 24 hours.
  • the values of each pathogenic strain were adjusted to 0.5 McFarland, from which a volume of 1 ml was taken and added to a tube with 9 ml of melted semi-solid BHI agar and kept at 48°C.
  • the mixture of the molten agar with the pathogenic strain was poured over the spot cultures of already developed strains and incubated aerobically at 37°C for 24 hours. The results were obtained by measuring the diameters of the inhibition halos produced.
  • the probiotic strains were able to inhibit the growth of the pathogens listed below:
  • Strain L. plantarum RGM 2608 Salmonella sp., Escherichia coli, enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), Pseudomonas aerug inosa, Klebsiella pneumoniae, Clostridium perfringens, Acinetobacter baumanii (by diffusion of the supernatant in agar) , Listeria monocytogenes, S. aureus, E. coli, Salmonella sp., ETEC and EHEC (for soft spot agar, that is, the action of the potential live probiotic).
  • EHEC enterohemorrhagic E. coli
  • ETEC enterotoxigenic E. coli
  • Pseudomonas aerug inosa Klebsiella pneumoniae
  • Clostridium perfringens Clostridium perfringens
  • Acinetobacter baumanii by diffusion of the
  • Strain L. Brevis RGM 2611 Salmonella sp., Escherichia coli, enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), Pseudomonas aerug inosa, Klebsiella pneumoniae, Clostridium perfringens, Acinetobacter baumanii (by diffusion of the supernatant in agar) , Listeria monocytogenes, S. aureus, E. coli, Salmonella sp., ETEC and EHEC (for soft spot agar, that is, the action of the potential live probiotic).
  • EHEC enterohemorrhagic E. coli
  • ETEC enterotoxigenic E. coli
  • Pseudomonas aerug inosa Klebsiella pneumoniae
  • Clostridium perfringens Clostridium perfringens
  • Acinetobacter baumanii by diffusion of the super
  • the inhibitory effect of the strains on each other was analyzed using the soft Spot Agar technique. For this, from a 48 h culture of the strains, a suspension was prepared in a buffer solution adjusted to 0.5 McFarland of each strain. Inoculums of 2 pl were taken from this suspension and deposited on 10 ml of MRS agar, left to stand for 30 minutes until the drops were absorbed and incubated at 37°C, in microaerophilia for 24 hours. After this period, from a culture of each strain adjusted to 0.5 McFarland in buffer solution, a volume of 1 ml of each strain was taken and added to a tube with 9 ml of melted semi-solid MRS agar and maintained at 48°C. .
  • the mixture of the molten agar with one of the strains was poured over the spot cultures of the other already developed strain and vice versa.
  • the strains were incubated under the conditions already described. The results were obtained by measuring the diameters of the inhibition halos produced.
  • the probiotic strains Lactobacillus plantarum RGM 2608 and Lactobacillus brevis RGM 2611 showed slight inhibition, the Lactobacillus brevis RGM 2611 strain being inhibited mostly by the Lactobacillus plantarum RGM 2608 strain with medium power (halos averaged in diameter: 1.9). However, this may mean that they will probably colonize different niches in the consumer, ensuring protection in a larger tissue area, which is an advantage over other probiotic products known to date.
  • Example 7 In vitro immunomodulation assays The ability of the strains to activate pattern recognition receptors (PRRs), involved in the recognition of Gram-positive bacteria by the immune system, and which have been associated with the beneficial immunomodulatory effects of some probiotic bacteria including Toll-like receptor 2 (TLR2) and NOD1 and NOD2 receptors.
  • PRRs pattern recognition receptors
  • TLR2 Toll-like receptor 2
  • NOD1 and NOD2 receptors Toll-like receptor 2
  • studies were carried out in transfected cells that individually overexpress the mentioned RRPs (HEK RRP cells). The immunomodulatory activity was studied using the NF-KB factor.
  • the NF-kB factor reporter assay was performed using HEK TLR2 , FIEKNOD I , and HEK N0D2 cells.
  • the cells were transfected with the pGLM-ENH-luc ⁇ vector, which includes the A1 and A2 sites of the human NF-kB gene.
  • the different HEK RRP cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, and were stimulated with the strains L. plantarum RGM 2608 and L. Brevis RGM 2611 for 24 hours.
  • Example 8 In vivo immunomodulation assays The ability of the strains to stimulate the activation of peritoneal macrophages and increase IgA levels, two common properties of probiotics with an immunomodulatory effect, was evaluated.
  • mice of the Balb/c strain were fed with a composition of the probiotic strains in water for five consecutive days at doses of 10 8 cells/day/mouse.
  • doses of 10 8 cells/day/mouse One day after the last administration, the animals were sacrificed and samples of the peritoneal cavity and intestinal fluid were taken for the corresponding determinations.
  • Peritoneal macrophages were separated and divided into two groups to evaluate their phagocytic capacity against yeasts and their microbicidal capacity by NBT analysis.
  • the intestinal fluid samples were used for the determination of total IgA by means of the ELISA technique. The results obtained are shown in Table 4.
  • mice of the Balb/c strain were sacrificed, samples of the peritoneal cavity were taken and the macrophages were separated for their culture.
  • Example 10 Preparations of the probiotic composition.
  • Formulation 1 a) 0.1 g of Lactobacillus brevis RGM 2611 with a count of 1.0 x 10 11 CFU/g, equivalent to a count of 2 x 10 9 CFU/ g of the composition; b) 3 g of fructooligosaccharides; and c) 1.9 g of glucose.
  • Formulation 3 a) 0.1 g of Lactobacillus plantarum RGM 2608 with a count of 1.0 x 10 11 CFU/g, equivalent to a count of 2 x 10 9 CFU/g of the composition; b) 0.1 g of Lactobacillus brevis RGM 2611 with a count of 1.0 x 10 11 CFU/g, equivalent to a count of 2 x 10 9 CFU/g of the composition; c) 3 g of fructooligosaccharides; and d) 1.8 g of glucose.

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Abstract

La présente invention concerne de nouvelles souches probiotiques du genre Lactobacillus qui ont une activité immunomodulatrice, lesquelles souches sont utiles pour la préparation d'une composition probiotique qui accroit la réponse immunitaire chez les mammifères atteints de troubles digestifs. La présente invention se rapporte également à une composition probiotique qui comprend au moins une souche probiotique ou un mélange desdites souches probiotiques. Les souches probiotiques de la présente invention sont de préférence Lactobacillus plantarum RGM 2608 et Lactobacillus brevis RGM 2611. Cette composition probiotique est utile pour le traitement de pathologies associées à des altérations du système immunitaire et/ou gastro-intestinal.
PCT/CL2020/050083 2020-07-29 2020-07-29 Souches de l'espèce lactobacillus plantarum et lactobacillus brevis et composition probiotique les comprenant WO2022020971A1 (fr)

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Publication number Priority date Publication date Assignee Title
US7354757B2 (en) * 2002-07-22 2008-04-08 Agtech Products, Inc. Lactobacillus brevis strain
WO2017034480A1 (fr) * 2015-08-27 2017-03-02 Mastercard Asia/Pacific Pte.Ltd Procédé de gestion de portefeuilles électroniques

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