CZ304791B6 - Strain of Bifidobacterium longum CCM 7952 bacteria and its use in human nutrition - Google Patents

Abstract

In the present invention, there is disclosed an industrial strain of Bifidobacterium longum bacteria deposited in the Czech Collection of Microorganisms, Tvrdého 14, Brno under the number CCM 7952 and composition containing this strain for modulation of immune response. The composition can further also comprise other probiotic strain, optionally various prebiotics, proteins, vitamins or mineral substances and can be in the form of a capsule, tablet or powder or food such as diary products, dressings or beverages. Preferably, the strain is comprised in concentration higher than 10e6 KTJ/g. The composition can be used for reducing level of proinflammatory cytokines, for prevention and/or treatment of undesired proinflammatory activity and for prevention and/or treatment of undesired proinflammatory allergic response to pollen.

Description

Bifidobacterium longum strain CCM 7952 and its use in human nutrition

Technical field

The present invention relates to living cells of a strain of Bifidobacterium longum CCM 7952 which is also a means for use in the preparation of anti-inflammatory agents for reducing the level of pro-inflammatory cytokines in preventing and / or treating unwanted pro-inflammatory activity. The invention provides the strain in the form of viable cells as a means for preventing and / or treating an undesirable allergic response to pollen.

The strain Bifidobacterium longum CCM 7952 was selected from a group of eighty bifidobacterial isolates from faeces of healthy breastfed babies based on favorable immunomodulatory properties.

BACKGROUND OF THE INVENTION

A number of strains of bacteria with probiotic effects used for human nutrition are known. The available probiotic strains were generally not tested for their ability to reduce the allergic response to different types of pollen. In the clinical study Ouwehand et al. Ouwehand AC ¹ . Nermes M, Collado MC, Rautonen N, Salminen S, Isolauri E, (2009) Specific probiotics alleviate allergic rhinitis during the birch pollen season. World J Gastroenterol. 15 (26): 3261-8, found that daily administration of two probiotic strains at a certain ratio (Lactobacillus acidophilus ATCC 700396 and Bifidobacterium lactis ATCC SD5219) reduced the inflammatory response of the nasal mucosa to birch pollen and alleviated symptoms such as runny nose and nasal congestion by approximately 20%. Since probiotic properties are known to be strain-specific from a number of studies, it is an object of the invention to provide a strain of human origin capable of favorably affecting the immune response of an organism.

SUMMARY OF THE INVENTION

The above mentioned deficiencies are eliminated by the strain of the bacterium Bifidobacterium longum CCM 7952, which was selected from the group of eighty bifidobacterial isolates from faeces of healthy breastfed babies on the basis of favorable immunomodulatory properties and deposited in the Czech Collection of Microorganisms, Tvrdého 14, Brno.

In another embodiment of the invention, the strain is included in the composition, which may contain other probiotic strains, optionally various prebiotics, proteins, vitamins or minerals. The strain can be supplied in the form of a capsule, tablet or powder or food such as dairy products, dressings or drinks. The strain is present at a concentration greater than 10 ⁶ cfu / g. The invention provides a strain of Bifidobacterium longum and its components for use in the preparation of anti-inflammatory agents for reducing the level of pro-inflammatory cytokines in preventing and / or treating unwanted pro-inflammatory activity.

The invention provides a strain in the form of viable cells for modulating an immune response. The invention provides a strain in the form of viable cells for the prevention and / or treatment of an undesirable allergic response to pollen.

Clarification of drawings

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the identification of bacterial strain CCM 7952 by amplified ribosomal DNA restriction analysis (ARDRA). Dendrogram shows results of cluster analysis (UPGMA, unweighted pair met-1 using arithmetic average) of DNA fragments produced by PCR product cleavage (genus-specific PCR according to Roy D, Sirois S. (2000) Molecular differentiation of Bifidobacterium species with amplified ribosomal DNA restriction analysis and alignment of short regions of the ldh gene FEMS Microbiol Lett. 191: 17-24), restriction enzymes 2α / wH1, Nci \, Sau3M, Taq ^and \ (Šrůtková et al., 2011) Efficiency of PCR-based methods in discriminating Bifidobacterium longum ssp. longum and Bifidobacterium longum ssp. infantis strains of human origin. J Microbiol Methods. 87 (1): 10-6. Giant. 2 shows the effect of B. longum ssp. Longum CCM 7952 in a model of acute bowel inflammation induced by administration of 2.5% sodium dextran sulfate in BALB / c mice. CCM 7952 (Group 2) or PBS (Control Group 1) were administered intragastrically for 10 days. Subsequently, inflammation of the intestine was induced by administration of 2.5% sodium dextran sulfate (DSS) in drinking water for 7 days. The experiment was then terminated and the inflammatory bowel parameters described below were monitored. Figure 3 shows the effect of strain CCM 7952 on the development of experimental bowel inflammation induced in mice by administration of a 2.5% sodium dextran sulfate solution. The group of mice treated with the strain CCM 7952 showed significantly lower weight loss (a parameter of intestinal inflammation development) compared to the control group without bacterial addition. Giant. 4 is a histological picture of colon descendens stained with hematoxilin-eosin. A-histological image of the colon of a healthy mouse, B - the colon of a control mouse that was not given CCM 7952, and that had mucosal inflammatory changes induced by DSS. C - colon of a mouse that was protected by administration of CCM 7952 against DSS-induced inflammatory mucosal changes. Giant. 5 is a schematic diagram of the situation to protect mice against intestinal inflammation by administering the strain CCM 7952 as the concentration of protective IgA antibodies also increases, which also contribute to the reduction of pro-inflammatory levels of the TNF-alpha and IFN-gamma cytokines in mesenteric lymph nodes. Giant. 6 shows the induction of birch pollen by B. longum CCM 7952 at specific antibody level. Systemic sensitization by the major component of birch pollen Bet v 1 in mice that were neonatally colonized with B. longum CCM 7952 and germ-free controls. Eight-week germ-free mice and mice colonized by birth by mother-to-mother transmission of CCM 7952 were sensitized (3 x sc) with the major component of birch pollen Bet v 1. Antibodies characterizing the induction of allergic sensitization (IgE, IgG2a, IgG1, IgA) ) in serum and intestinal washes of mice by ELISA. Results were expressed as means ± SEM. * P <0.05, ** P <0.01. It was confirmed that neonatal colonization of CCM 7952 bacteria protected mice from allergic sensitization. Giant. 7 is the induction of birch pollen by B. longum CCM 7952 at the level of the cytokine response. At the end of the experiment, the spleen cells of both control germ-free and neonatally colonized CCM 7952 bacteria were cultured (60 h) with the major component of birch pollen Bet 1 and the cytokine levels of IL-13 (A), IL-5 ( B), IL-4 (C), IL-10 (D), IFN-γ (E), IL-17 (F), and TNF-α (G) by ELISA. Results were expressed as means ± SEM. * P <0.05, ** P <0.01. It was confirmed that neonatal colonization of CCM 7952 bacteria protected mice from allergic sensitization.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

In one embodiment, the strain is in the form of a biologically pure culture.

Example 2

In another embodiment of the invention, the strain is contained in a composition comprising other probiotic strains, optionally various prebiotics, proteins, vitamins or minerals. The strain can be supplied in the form of a capsule, tablet or powder or food such as dairy products, dressings or drinks. The strain is present at a concentration greater than 10 ⁶ cfu / g.

-2GB 304791 B6

Example 3

The invention provides a strain or compositions comprising a strain of Bifidobacterium longum and components thereof for use in the preparation of anti-inflammatory agents for reducing the level of pro-inflammatory cytokines in preventing and / or treating unwanted pro-inflammatory activity.

Example 4

The invention provides a live cell strain as a means of modulating an immune response.

Example 5

The invention provides the strain in the form of viable cells as a means for preventing and / or treating an undesirable allergic response to pollen.

Workflow:

1) Sampling

One gram of faeces of breast-fed infants was resuspended in 9 ml PBS and decimally diluted in TPY agar with mupirocin (100 mg / L) and acetic acid (1 ml / L) and anaerobically cultured at 37 ° C for 72 hours. Colonies of typical morphological appearance were tapped, purified on TPY agar and subsequently cultured in TPY broth. The grown isolates were stored in TPY (MRS) broth with 20% glycerol and stored at -70 ° C.

2) Identification and typing of the strain

The affinity of the strain to the genus Bifidobacterium was determined by the reaction of the enzyme fructose-6-phosphate ketolase (Orban JI, Patterson JA (2000)), by the molar ratio produced by Bifidobacterium (J. Microbiol Methods. 40: 221-4). lactic acid and acetic acid detected by capillary isotachophoresis (ZKL 01, Slovakia) and by the FISH method with a probe specific for the genus Bifidobacterium (Ribo technologies, Netherlands). All three tests confirmed that the strain belonged to the genus Bifidobacterium (F6FK positive test, molar acetic / lactic acid ratio 1.96).

The API 50CH test (BioMérieux, France) was used for species identification based on the fermentation profiles of carbohydrates and sugar alcohols with the following results:

0 0 0: + + + 0 0 0 ++ + + 0 0 0 C 0 3 C +! C 0 0 ♦ * (+) * + + + Ϊ 0i ..... + '+ D ...... O ...... 0' ....... + '... ............. + ......... IL 0 '..... 6' ....... 0 ..... Di ... ... 0 -....... 0 ........... D ......... 0 ............... ............ ...................................... .................................................. ...

The APIZYM assay (bioMérieux, France) with positive results for leucinarylamidase, cystinarylamidase, acid phosphatase, naphthol-AS-BIphosphohydrolase, alpha and beta galactosidase, alpha and beta glucosidase, N-acetyl-beta-glucosaminidase and alpha activity was used to determine enzymatic activities. -mannosidases.

Identification by molecular biological methods (genus and species specific PCR, rep-PCR, ARDRA) (Fig. 1).

3) The possibility of using bacteria as a probiotic is conditioned by its survival in the environment of the gastrointestinal tract. The ability to survive the acidic environment of the stomach (pH 3) and bile acids was detected.

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Determination of resistance to bile salts was performed on plates with co-solid media containing different concentrations of bile salts (0.3; 0.7 and 1.0%, OXOID L55). The numbers of colony forming units (cfu) on the individual bile salt media were compared with those on control agar (cultivation 72 to 96 hours under the same conditions). Under the above conditions, 27.0, 1.2 and 0.3% of cells survived at a concentration of 0.3, 0.7 and 1.0% of bile salts.

The determination of low pH resistance was performed as follows. The test strain was cultured to a stationary growth phase at 37 ° C in MRSC broth with 0.05% (w / v) Lcysteine hydrochloride. Subsequently, the cells were collected by centrifugation at 4000 g for 15 minutes. The supernatant was separated by decantation and cells were flooded with MRS broth with 0.05% (w / v) L-cysteine hydrochloride and adjusted to pH 3.0 with HCl. After mixing, the sample was inoculated by decimal dilution with saline on MRS57 agar and the initial cfu count was determined. After incubating the MRS pH 3 for 3 hours at 37 ° C, cfu counts were again determined in the same manner. The percentage of surviving cells was determined by dividing the number of cfu on MRS agar at 3 and 0 hours. Under the above conditions, 0.2% of the cells survived.

4) The possibility of using bacteria as probiotics also depends on its ability to adherence to the intestinal wall.

4.1. Adherence of lactic acid bacteria strains

A suspension of probiotic prokaryotic cells under investigation in vitro was added to the grown layer of eukaryotic cells. After the application period, the cells were rinsed and preparations were prepared for both optical microscopy and electron microscopy. All laboratory operations were carried out in standard controlled mode, the workplace owns ISO 2001 standards.

4.2. Used cells

Eukaryotic model cells were selected as heteroploid cell lines of epithelioid character, especially HeLa and Caco2.

4.3. Culture media

Eukaryotic cells were cultured in MEM medium with conventional fetal bovine serum supplements, antibiotics and non-essential amino acids. Co-cultivation was performed in MEM medium supplemented with fetal bovine serum and L-glutamine. Prokaryotic cells were cultured in LB or MRSC medium.

4.4. Conditions of cultivation

Cultivation of eukaryotic cells was standard in an atmosphere of 5% CO ₂ at 37 ° C. Passaging was performed with trypsin. Cells were regularly checked microscopically in phase contrast. Cultivation of prokaryotic cells was again standard in tubes at 37 ° C. For application to the eukaryotic layer, the culture was in an exponential phase of growth.

4.5. Application of lactic acid bacteria

Lactic acid bacteria strains were added to the eukaryotic cell culture and this mixture was cultured at 37 ° C for 24 hours. The cell layer was then washed with PBS, fixed and ready for further preparation.

4.6. Preparation for microscopy

Cell-opaque lenses were fixed for optical microscopy in alcohol-acetic fixation and subsequently stained in May-Griinwald / Giemza). After drying, the glasses were prepared into a Canadian balsam and prepared for evaluation. For electron microscopy, the cell layer on the cover glass was fixed in glutaraldehyde and, after dewatering, prepared for preparation for scanning electron microscopy.

The adherence to the eucaryotic line Caco2 was 26.4%.

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5) The safety of the bacteria for the host was verified in a germ-free mouse colonization model. Colonization stability (10 ¹⁰ CFU / g faeces) was also confirmed in subsequent generations of mice.

6) Immunomodulatory effects of CCM 7952 were tested under in vitro system conditions. Bacteria of strain CCM 7952 were cultured with immune cells (spleen, dendritic and lineal macrophages) and the ability of strain CCM 7952 to stimulate the production of anti-inflammatory and inflammatory cytokines was studied. Testing on immune cells revealed that the addition of CCM 7952 bacteria stimulated the production of the anti-inflammatory cytokine IL-10.

7) In a subsequent step, the effect of strain CCM 7952 on the development of experimental intestinal inflammation induced in mice by administration of sodium dextran sulfate was investigated. Inflammatory parameters (weight loss, shortening of colon length, rectal bleeding, rectal prolapse, stool consistency change, bowel morphology, inflammatory cytokine production) were significantly lower in animals receiving preventive CCM 7952 bacteria prior to induction of experimental inflammation. compared to the control group.

8) The most prominent probiotic protective effect of strain CCM 7952 was demonstrated in the birch pollen allergy model. It has been shown that germ-free mice with neonatal maternal CCM 7952 bacteria were protected from the development of allergic sensitization to the major component of birch pollen Bet v 1. Whereas germ-free controls induced allergic sensitization at the level of antibody production (IgE, IgG1) and proallergic cytokines (IL). -4, IL-5, IL-13, IL-17), mice seeded with CCM 7952 were free from allergic reactions and levels of endpoints were significantly reduced. This bacterial colonization was found to induce the formation of regulatory Fox P3-positive cells.

Basic results of the study

bíM »® BstóttWní kigenJ kontrója IFN-gamma IL-β TNF-α IL-10 rieWLQÍQi SJCUftlO * zántton Ríossibí skuDlna Stove group Sfcumiu «· KftD & ftlDi group ttwini »· sanatam CCDM 218 1864.8 * 421.9 “ 1540.0 * 900.9 * 650.2 * 62.0 “ 952.6 * 190 6 934.B * 197 9 “ 2090 6 * 596.0 “ 187.4 * 13.5 “ 159 0 * 17.9 CCDM 366 552 * 23 1 240.0 ± 221.5 229.0 * 13.0 613.0 228.2 200.2 * 21.7 330.4 * 108 268.2 * 13.7 " 280.6 * 41 8 B. longum CCDM 219 185.2 * 59.3 * 103.2 511.6 * 79.8 * 419.4 * 160.7 181.3 * 18.3 122.6 * 24.3 173.9 * 17.9 * 70.2 * 11.3 CCM 7952 172.8 ± 79.7 66.6 ± 27.1 452.3 ± 36.1 279.8 ± 77.0 138.0 ± 124 114 ^ ± 174 270.2 ± 24.5 “ 98.0 ± 16.7 CCDM 372 743.6 * 182 0 1150.6 * 403.2 637.6 * 80.1 “ 1030.1 * 70.9 * 451.0 * 37.4 “ 612.8 * 22.1 531.8 * 38.6 " 371 8 * 76.5 “ S. Infanti * CCDM 389 108.0 * 58.9 260.0 * 126.3 492.8 * 116.9 832.4 * 105.3 384.3 * 55 5 ' 476 6 * 77.5 542.3 * 85.0 “ 357.4 * 81.3 * B. CCDM 368 153.5 ± 90 9 33 8 17.2 334.5 51.5 191.0 * 133.4 166.0 * 16.5 162.5 * 93.0 93 0 4.2 123.0 * 102 8 CCDM 370 668.8 * 151.2 · 269.3 * 86 15 923.3 * 114.3 " 704.8 * 98.8 357.2 ± 35.9 “ 404.0 * 65.2 243.2 * 16.1 " 192 3 * 53.0 CCDM 371 145.0 * 65.3 264.0 * 109.25 675.0 * 69 9 “ 639.5 * 87.5 290.4 * 0.1 ’ 363.0 37.5 605.0 * 52.2 " 310.5 * 37.4 CCDM 373 1819.3 * 275.2 ” 1261.8 * 349.23 987.0 * 87.0 “ 725.8 * 245.2 494.5 * 59.7 " 497.8 * 171 7 185 0 * 14.2 * 164.3 * 56.2 trnut medivm 11.8H0.7 131.5 + 88.5 13.0 + 4.1 114.5 + 70 63 + 2 7 74.5 * 43.0 7.3 ± 0.6 28.3 + 18 2 LPS 508.3 * 164.1 552.6 * 377.5 397.3 ± 47.6 581.2 * 320.3 166.0 * 14.3 157.0 ± 64.4 177.0 * 29 1310 * 39

tab. and

Tab. 1 Determination of immunomodulatory properties of ten selected strains of the genus Bifidobacterium (including strain CCM 7952) under in vitro conditions. The bacteria of the selected strains were inactivated with 1% formaldehyde and then cultured with spleen cells isolated from healthy (untreated) mice and with spleen cells from a group of mice that were induced inflammatory bowel disease by administering a 2.5% sodium dextran sulfate solution after drinking for 48 hours. in culture medium at 37 ° C, in a humidified atmosphere enriched with 5% CO ₂ . The ratio of bacteria to immune cell was 10: 1 (Amrouche et al., 2006).

Cytokine levels were determined in the supernatant after cell culture by ELISA. The strain CCM 7952 significantly stimulated the production of the anti-inflammatory cytokine IL-10, while the production of pro-inflammatory cytokines INF-γ, IL-6 and TNF-α by spleen cells in CCM 7952 was significantly lower in both groups of mice compared to other bacterial strains and positive control (LPS 1 pg / ml). Values are presented as mean ± SEM, statistical evaluation of individual cytokine levels was performed by ANOVA compared to cytokine production of unstimulated cells (shown in the table as medium) * p <0.05, ** p <0.01, *** p <0.001.

- see Fig. 2 and Fig. 3

Experimental group Severity severity index Diseased Colon length Degree of mucosal damage (cm) (Cooper et al 1993)

CCM -952

1.24 ± 0.29

-, 86 ± 0.12 ^:

0-1

Control (PBS)

2.5 ± 0.18 ± 0.18

3-4

Both groups of mice saw a 2.5% DSS solution for 1 week. The first group of mice received a dose of 2x10 ⁸ CFU in 0.2 ml of buffered saline 10 days before drinking the solution and during the seven-day drinking of the DSS solution daily. Control mice received this solution without the addition of bacteria.

Disease severity index: mouse body weight decrease (0 = no weight loss, 1 = 15% weight loss, 2 = 5 to 10% weight loss, 3 points 10 to 20% weight loss), stool consistency (0 = solid stool, 2 = lower bowel stool, 4 = diarrhea), rectal and colon bleeding (0 = no rectal and colon bleeding, 2 = positive hemocult, 4 = severe bleeding). The sum of individual parameter scores for each mouse is divided by 3 (the number of endpoints) to produce a disease severity score from 0 (healthy mouse) to 4 (maximum disease severity) (Cooper et al., 1993) Clinicopathologic study of dextran sulfate sodium Experimental murine colitis. Lab Invest, 69 (2): 238-49. The results were statistically evaluated by Student's t-test.

Claims (7)

PATENT CLAIMS 1. Industrial strain of Bifidobacterium longum RB25-P deposited in the Czech Collection of Microorganisms, Tvrdého 14, Brno under the number CCM 7952. 2. A composition for modulating an immune response comprising the bacterial strain of claim 1. Composition according to claim 2, characterized in that it is in the form of a capsule, tablet or powder or a foodstuff such as dairy products, dressings or drinks. Composition according to claim 2, characterized in that the strain is present in a concentration higher than 10 ⁶ CFU / g. The composition according to claim 2, further comprising further probiotic strains, optionally prebiotics, proteins, vitamins or minerals. The composition of claim 2 for reducing the level of pro-inflammatory cytokines in preventing and / or treating undesired pro-inflammatory activity. The composition of claim 2 for preventing and / or treating an undesirable allergic pollen response.