WO2023173181A1 - Bifidobacterium longum ssp. longum strain and a bifidobacterial preparation having hypolipidemic, anti-inflammatory immunomodulating and insulin resistance decreasing effects - Google Patents
Bifidobacterium longum ssp. longum strain and a bifidobacterial preparation having hypolipidemic, anti-inflammatory immunomodulating and insulin resistance decreasing effects Download PDFInfo
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- WO2023173181A1 WO2023173181A1 PCT/BG2023/000009 BG2023000009W WO2023173181A1 WO 2023173181 A1 WO2023173181 A1 WO 2023173181A1 BG 2023000009 W BG2023000009 W BG 2023000009W WO 2023173181 A1 WO2023173181 A1 WO 2023173181A1
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- longum
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Classifications
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- 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
- C12N1/205—Bacterial isolates
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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
- A61K35/745—Bifidobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/533—Longum
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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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Definitions
- This invention is in the field of biotechnology and relates to a probiotic Bifidobacterium strain in particular, characterized by a complex of beneficial properties regarding human health and its use in probiotic preparations for the prevention of metabolic, cardiovascular and inflammatory diseases.
- Bifidobacteria are considered probiotics because they are living microorganisms that have a beneficial effect on human health when taken in sufficient quantity.
- a minimum level of approximately 10 6 -10 7 v ’ a ⁇ ' e bifidobacteria cells per gram of intestinal contents is suggested.
- Bifidobacteria are one of several dominant culturable bacterial groups present in the colonic microflora, playing an important role in maintaining a balanced healthy intestinal microflora. There is compelling evidence of the relationship between gut microflora, obesity, the endocannabinoid system, systemic low-grade inflammation, lipopolysaccharides, intestinal permeability, and arterial stiffness.
- Probiotics in addition to improving the composition of the intestinal microflora, also improve immunity, favourably affecting inflammatory and metabolic diseases, and reducing the risk of developing atherosclerosis.
- One of the most important prerequisites for the manifestation of their beneficial effects is the adhesive properties of bacterial cells, as well as their survival and ability to colonize the gastrointestinal tract.
- probiotics The beneficial effects on health demonstrated by probiotics are strainspecific and even strains of the same genus and species exhibit different properties at varying degrees.
- the beneficial effect of bifidobacteria against intestinal inflammatory diseases such as ulcerative colitis and Crohn’s disease has already been proven, and treatment with probiotic preparations accompanies drug therapy.
- the mechanisms of this beneficial effect on intestinal inflammations are prevention of the adhesion of pathogens on the intestinal epithelium; immunomodulatory effect through the induction of the synthesis of antiinflammatory cytokines IL-10 (interleukin 10) and/or TGF-p (transforming growth factor beta), reduction of synthesis of proinflammatory cytokine IL-8 (interleukin 8) by epithelial cells, absence or reduced induction of TNFa (Tumour Necrosis Factor alpha), IL-1 (Interleukin 1) and/or IL-6 (Interleukin 6); proteolytic degradation of antigens with proinflammatory properties; elimination of toxins, etc.
- IL-10 interleukin 10
- TGF-p transforming growth factor beta
- TNFa Tumor alpha
- IL-1 Interleukin 1
- IL-6 Interleukin 6
- proteolytic degradation of antigens with proinflammatory properties elimination of toxins, etc.
- the persistently increased activity of neutrophils may lead to the formation of ulcers or the occurrence of intestinal bleeding [4], conditions that are concomitant with inflammatory bowel disease.
- the probiotic strains lower the concentration of bile salts through their BSH activity, which is compensated by the synthesis of new amounts of bile acids from cholesterol and the conjugation of these bile acids with taurine or glycine leading to the production of fresh reserves of bile salts, replacing the deconjugated bile salts.
- the main feature in the selection of bacterial strains for the development of probiotic preparations with a beneficial effect on cholesterol metabolism is the presence of significant hydrolase activity towards bile salts.
- a strain of Bifidobacterium longum ssp. longum strain has been described in a Korean patent KR102224072B1 [6], possessing a combination of two main functional characteristics: direct reduction of total serum cholesterol and immune regulation by lowering the secretion of pro- inflammatory cytokines TNFa and IL-6 and increasing the secretion of antiinflammatory cytokine IL- 10. These effects were confirmed by in vitro functional assessment of the strain and in vivo experiments in mice.
- cardiovascular diseases are a leading cause of morbidity and mortality worldwide, and the main reason for their occurrence, development and complications is atherosclerosis.
- the main risk factors the combination of which leads to the appearance and accelerated development of the atherosclerotic process, are hypertension, dyslipidemia, obesity and pro- inflammatory status, diabetes mellitus or impaired glucose metabolism, and immobility. Often these factors are combined in a single patient, which sharply increases the risk of cardiovascular disease.
- the phenomenon is well- known in patients with metabolic syndrome. The more risk factors that exist in a patient, the greater the likelihood of cardiovascular disease, because their harmful effects are multiplied when they are combined. Therefore, it is important to reduce the combined influence of risk factors by influencing their totality simultaneously, rather than individually.
- An actual problem is the selection of a probiotic strain of Bifidobacterium longum ssp. longum, which, in addition to immune regulation and direct cholesterol reduction, also exhibits hydrolase activity against bile salts.
- Another current issue is proving hypolipidemic and anti-inflammatory immunomodulatory activities of the strain Bifidobacterium longum ssp. longum through a clinical trial of the application of the strain in patients at risk of developing atherosclerosis.
- the subject of the present invention relates to the new strain Bifidobacterium longum ssp. longum 3/15 filed under No. NBIMCC 9085 on 26 January 2022 with the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) of the Republic of Bulgaria, possessing a complex of health-beneficial properties: hypolipidemic activity, anti-inflammatory immunomodulating activity, ability to reduce insulin resistance, strong adhesion to epithelial cells and high survival rate in the gastrointestinal tract.
- NBIMCC National Bank for Industrial Microorganisms and Cell Cultures
- the subject of the invention is also a bifidobacterial preparation obtained from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and possessing a clinically proven set of health benefits: improvement of lipid parameters, reduction of insulin resistance, beneficial effect on inflammatory status markers.
- the subject of the invention is also the use of the new strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and the bifidobacterial preparation obtained from Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 for simultaneous beneficial influence on multiple health risk factors and prevention of a specific inflammatory or metabolic disease, but also of diseases caused by these risk factors in their totality, in the specific case for the prevention of atherosclerosis and cardiovascular diseases.
- NBIMCC 9085 was isolated from faecal samples of a healthy 28-y ear-old volunteer who had not used antibiotics in the past 1 year and had not used probiotics in the past six months.
- the sample taken is serially diluted in anaerobic peptone water in an anaerobic box to a dilution of 10' 6 and inoculated on a non-selective medium for anaerobes “Blood Liver agar” with added 5.0% defibrinated horse blood. Cultivation is anaerobic at 37°C for 72 hours.
- Colonies with a typical Bifidobacterium genus morphology are inoculated into MRS broth with L- cysteine and lithium chloride and cultured in an anaerobic environment at a temperature of 37°C for 24 hours.
- Genus-specific PCR is used to identify the gram-positive cultures, which are of a rod shape that is typical of the Bifidobacterium genus.
- Species affiliation and strain identity of the strain was established using precise DNA-based methods (Figure 1) such as 16S rDNA sequencing, Pulsed Field Gel Electrophoresis.
- the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is gram-positive, catalase-negative, and rod-shaped.
- a microscopic photo of the strain is presented in Figure 2.
- the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is capable of significantly lowering serum cholesterol concentration.
- the strain exhibits two main activities related to reducing the level of cholesterol - a direct lowering of the cholesterol level, as well as its indirect reduction as a consequence of the hydrolase activity of the strain towards bile salts (BSH activity). The extent of both strain activities is determined in vitro.
- BSH activity of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is performed by help of reverse-phase liquid chromatography (RP- HPLC) with UV detection at 210 nm.
- RP- HPLC reverse-phase liquid chromatography
- the strain cultured in a medium with added bile salts sodium taurocholate (TCA) and sodium glycocholate (GCA) achieve 72.0% ⁇ 1.9% deconjugation of bile salts that are present in the medium.
- the strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 possesses anti-inflammatory immunomodulatory activity, which is manifested by the ability of the strain to favourably alter the induction of pro-inflammatory and anti-inflammatory cytokines.
- the anti-inflammatory immunomodulatory ability of the strain was determined in vitro by quantifying the expression of signal peptides TNF-a, IL-8, IL-6, TGF-P and IL-10 from a combined system of two cell lines: human monocyte cell line THP-1 and epithelial cell line HT-29, separated by a semipermeable membrane. (Example 1.3)
- the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 exhibits strong adhesion to epithelial cells and high survival rates in the gastrointestinal tract, which enhances the impact of the beneficial effects of the strain on the human organism.
- the survival of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was determined in the clinical monitoring on the administration of the bifidobacterium preparation of the strain. Faecal samples from nine volunteers, patients in the clinical trial, were examined after 30 days of daily administration of the bifidobacterial preparation. It has been determined an average number of colonies of 2.2x10 6 CFU/g. (Example 3.4)
- the bifidobacterial preparation subject of this invention is obtained by cultivating the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. (Example 2) It can be administered in liquid or dry (lyophilized) form.
- the lyophilized bifidobacterial preparation can be applied both in powder form and also in the form of capsules.
- the liquid bifidobacterial preparation contains live cells of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 - at least 1x10 9 CFU/g.
- the lyophilized bifidobacterial preparation contains live cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 - at least 5x10 9 CFU/g. Residual moisture content is 4% ⁇ 1%.
- the bifidobacterial preparation can be used as an additive to products in dry and/or liquid form. When adding it to lactic acid products, it is added at a quantity of 0.5% - 2%.
- the lyophilized bifidobacterial preparation can be encapsulated. Before encapsulation, an anti-caking agent, microcrystalline cellulose, is added to it. Each capsule contains no less than 2xl0 9 viable cells of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085
- vascular risk markers respectively cytokines and inflammatory factors, whose pathophysiological role is of great importance in the light of considering metabolic syndrome and obesity as a state of low-grade inflammation leading to endothelial dysfunction.
- results of the medical research within the framework of the conducted clinical trial show simultaneous favourable influence on three risk factors leading to the development of atherosclerosis, namely lipoproteinemia, the state of insulin resistance and the inflammatory status.
- the clinical trial of effects after administration of the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 confirmed the in vitro findings on the functional characteristics of the strain, namely hypolipidemic and anti-inflammatory immunostimulatory properties, as well as establishing a novel functional effect - a reduction of diabetes risk.
- the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is recommended for successful application in medical practice in the primary and secondary prevention and therapy of atherosclerosis, cardiovascular and inflammatory diseases, and diabetic risk.
- Figure 1 Pulsed field gel electrophoretic profile of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 with the AsnI. M enzyme and m-molecular markers. Unfolding conditions: initial pulse 2s, final pulse 15s, voltage 5V/cm, duration 24h.
- the molecular masses of DNA fragments of Bifidobacterium longum ssp. longum LBB 3/15 NBIMCC 9085 in kbp are: 199, 136, 112, 99, 93, 73, 61, 57, 51, 47, 38.7, 26, 22.2, 20.5, 17, 9, 14.2, 13.5, 11.6, 9.0, 8.5, 8.0, 7.5, 6.8.
- Figure 3 Change in cytokine level TNF-a when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to the placebo group.
- Figure 4 Change in cytokine level IL-ip when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to the placebo group.
- Figure 5 Change in cytokine level IL-6 when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to the placebo group.
- Example 1 In vitro functional assessment of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
- the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is cultivated anaerobically at 37°C for 18 hours in MRS L-cysteine broth.
- the medium contains 0.3% bile salts and 100 pg/ml water-soluble cholesterol (cholesterol-PEG600).
- the inoculum is 0.5% and contains 1x10 9 CFU /ml. After incubation, the cells are pelleted by centrifugation and 0.5 ml of the supernatant is treated with 4 ml of 2M KOH in methanol at 80°C for 20 minutes.
- the process is followed by extraction with 5 ml of hexane and 4 ml of the hexane layer is transferred to test tubes and evaporated with a stream of nitrogen at a temperature of 60°C.
- the dry residue is dissolved in 0.5 ml of isopropanol and the amount of cholesterol contained is determined by gas chromatography. The degree of cholesterol reduction is determined based on an uninoculated control medium.
- Table I presents the results of the direct adsorption of cholesterol, expressed as a percentage of cholesterol reduction.
- the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is cultivated anaerobically at 37°C for 18 hours in MRS broth with L-cysteine, adding the two most important bile salts in the medium: sodium taurocholate (TCA) and sodium glycocholate (GCA) at a concentration of 1 mM, each.
- TCA sodium taurocholate
- GCA sodium glycocholate
- the inoculum is 0.5% and contains 1x10 9 CFU /ml, and the uninoculated medium is used as a control.
- BSH activity is performed by reverse-phase liquid chromatography (RP-HPLC) with UV detection at 210 nm. Free and conjugated bile acids are detected in reverse-phase gradient mode.
- Solvent A is 65% methanol in 0.03 M sodium acetate at pH 4.3 adjusted with phosphoric acid.
- Solvent B is methanol with HPLC quality.
- the eluent program is an isocratic step for 8 min at 15% eluent B and then a linear gradient to 85% for 17 min. This is followed by an isocratic step of 5 minutes at 85%. The injected amount is 5 pl.
- Table II presents the results of the determination of BSH activity of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 as a percentage of hydrolysis of the bile salts present in the medium, after testing in triplicate and averaging the results.
- the anti-inflammatory immunomodulatory activity is evaluated by quantifying the expression of pro-inflammatory and anti-inflammatory signal peptides from a combined system of two cell lines: the human monocyte cell line THP-1 and the epithelial cell line HT-29, separated by a semipermeable membrane.
- the system of the two cell lines is appropriate to assess the induction of both pro- inflammatory cytokines TNF-a, IL-8 and IL-6, as well as to assess the induction of anti-inflammatory cytokines IL- 10 and TGF-p. Both cell lines are maintained at 37°C and 5% CO2 on RPMI medium 1640 for cell line THP-1 and on DMEM medium for cell line HT-29.
- Both media were supplemented with 10% fetal bovine serum, 4.5 g/1 glucose, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1.5 g/1 sodium bicarbonate, 100 U/ml penicillin, 100 pg/ml streptomycin.
- lxl0 6 per millilitre THP-1 cells 5xl0 5 per well
- PMA phorbol 12- myristate- 13 -acetate
- Epithelial HT-29 cells were grown as a monolayer in semipermeable membrane inserts and matured for 14 days with a daily change of DMEM medium.
- the differentiated adhered macrophages were washed twice with PBS buffer (Dulbecco’s phosphate-buffered saline) and incubated with fresh medium without PMA for 24 hours.
- Lipopolysaccharide of Salmonella is added at a concentration of 1 pg/ml in RPMI 1640 medium and incubation continued for another 4 hours.
- 0.5 ml of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 at a concentration of 1x10 6 CFU/ml per well were added to the differentiated macrophages suspended in RPMI 1640 medium. The cells are incubated for 24 hours in a CO2 incubator.
- the epithelial cell lines Caco-2 and HT-29 are used, which are cultured as a monolayer of mature epithelial cells in DMEM (Dilbecco's modified Eagle's medium) medium supplemented with 10% fetal bovine serum. Incubation is performed at a temperature of 37°C in a water-saturated atmosphere containing 5% CO2. When about 90% of a monolayer is formed, cells were passaged using 0.25% trypsin and 10 mM EDTA solution at a temperature of 37°C for 10 minutes.
- Eukaryotic cells were seeded in 6-well clusters at a concentration of 2x10 5 cells/ml. The medium was changed every 2 days for a total of 14 days. During this period, the monolayer is formed (for about 3-4 days) and cell receptors maturate. The monolayers were then washed twice with PBS buffer and the bacterial culture of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was added to them at a concentration of lxlO 8 CFU/ml in 2 ml DMEM without antibiotics. Bacterial and eukaryotic cells were incubated together for 60 minutes at 37°C in a water-saturated atmosphere containing 5% CO2.
- the process is followed by a fivefold washing of the wells with PBS, fixing with methanol for 3 minutes, Gram staining and microscopic assessment of the adhesion.
- the number of adhered bacterial cells per 20 epithelial cells was determined by counting 10 microscopic fields.
- the adhesion assay was performed in triplicate on both Caco-2 and HT-29 cell lines, and the results were averaged.
- the bifidobacterial preparation is obtained by cultivating the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
- the strain is cultivated in a medium of MRS broth containing lithium chloride and L-cysteine, in anaerobic conditions at a temperature of 37°C for 18-24 h.
- a specially prepared medium is inoculated in an industrial fermenter to carry out the fermentation process of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
- the medium includes 8% milk powder, 2% whey powder, 2% lactose, 0.5% yeast extract, 0.5% peptone casein and 2% fructooligosaccharide.
- the pH of the medium is adjusted to pH 6.8 and the medium is sterilized in a fermenter at 120°C for 15 minutes. Then cooled to a temperature of 37°C and inoculated with 5% of the starter culture. Fermentation is carried out at a temperature of 37°C for 14-16 hours with continuous blowing with carbon dioxide. After reaching pH 4.9- 5.0, the fermentation process is stopped by cooling with ice water and subsequently adjusting the active reaction to pH 5.8-6.2.
- the resulting biomass is a liquid bifidobacterial preparation containing not less than 1 xlO 9 CFU/g viable cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
- the cryoprotective medium with 10% sucrose and 0.5% pectin is added to the fermented medium in a ratio of 1 : 10 and loaded in an industrial lyophilizer.
- the sublimation is performed at a temperature of minus 28°C to minus 30°C with a layer height of no more than 1.2 cm. Secondary drying is at a temperature of 30°C. After the process is completed, the vacuum in the sublimation chamber is compensated with nitrogen.
- the lyophilized bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is packaged in an inert (nitrogen) atmosphere.
- the final product contains at least 5xl0 9 CFU/g viable cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and its water content is 4% ⁇ 1%.
- the lyophilized bifidobacterial preparation can be encapsulated on an encapsulation machine by first adding an anti-caking agent, for example, microcrystalline cellulose.
- an anti-caking agent for example, microcrystalline cellulose.
- Each capsule contains not less than 2x10 9 CFU/g viable cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
- Example 3 A clinical trial of the effects upon the administration of a bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 as a probiotic
- the objective of the clinical trial is to evaluate the hypolipidemic and anti-inflammatory immunomodulating effect of the bifidobacterial preparation in patients with dyslipoproteinemia and high atherosclerotic vascular risk or atherosclerotic disease, and development of recommendations for its use in primary and secondary prevention of cardiovascular diseases.
- Clinical studies were performed in the conditions of a double-blind, placebo-controlled clinical trial in a group of 60 patients, 42 women and 18 men, with mild to moderate dyslipoproteinemia, respectively with increased cholesterol, low-density lipoproteins and/or triglycerides.
- the patients were not administered hypolipidemic drugs or other nutritional supplements having metabolic effects.
- the age of the patients is between 18 and 75 years. They were divided into two groups - a group of 30 patients who took the probiotic bifidobacterial preparation, tentatively called the “probiotic group”, and a group of 30 patients who received a placebo product, tentatively called the “placebo group”.
- the clinically observed product is a bifidobacterial preparation of the probiotic strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085, with a number of viable cells not less than 5xl0 9 CFU/g.
- the bifidobacterial preparation is administered in the form of capsules, each of which contains no less than 2x10 9 viable cells of the probiotic strain.
- the placebo product is a capsule containing dry milk without bacterial cells of the probiotic strain Bifidobacterium, longum ssp. longum 3/15 NBIMCC 9085.
- the monitored and evaluated parameters were lipid panel, serum levels of vascular inflammation markers (C-reactive protein, cytokines TNF-a, IL-ip, IL-6, TGF-p), as well as fasting serum insulin level and insulin resistance by HOMA-IR index.
- C-reactive protein cytokines TNF-a, IL-ip, IL-6, TGF-p
- fasting serum insulin level and insulin resistance by HOMA-IR index as well as fasting serum insulin level and insulin resistance by HOMA-IR index.
- results were processed statistically with the methods of descriptive statistics, Wilcoxon Signed Ranks Test and parametric T-test. Analysis of patient groups showed a similar gender and age distribution. Patients in the “probiotic group” were 23 women and 7 men, aged 21 to 70 years, with a mean age of 54.37 ⁇ 11.91 years. Patients in the “placebo group” were 19 women and 11 men, aged 22 to 75 years, with a mean age of 49.60 ⁇ 13.79 years. All patients had mild and moderate lipid disorders - increases in the levels of total and LDL cholesterol, lower values of HDL cholesterol, and an increase in triglycerides. All had metabolic syndrome, overweight or obesity, two-thirds had arterial hypertension under therapeutic control, 35 patients had type 2 diabetes under therapy, and 29 patients had proven hepatic steatosis.
- Table V shows the dynamics of the changes in the lipid indicators of patients in the “probiotic group”.
- Table VI shows the dynamics of the changes in the lipid indicators of patients in the “placebo group”.
- Table VII presents the results of other investigated biochemical and metabolic indicators of patients in the “probiotic group”. Table VII.
- Table VIII. presents the results of other studied biochemical and metabolic indicators of patients in the “placebo group”. Table VIII.
- the changes in serum uric acid were similar, with its values significantly decreasing by 3.45% in the patients of the “probiotic group”, while the patients of the “placebo group” had insignificant changes in the level of the uric acid.
- the monitored levels of serum transaminases, GGTP and CRP were within reference values in both groups and remained without significant changes.
- Results of the conducted clinical trial confirm the anti-inflammatory immunomodulating effect of the bifidobacterial preparation - lowering of TNF-a, IL- 1 P, IL-6 and moderate increase of TGF-pi.
- Faecal samples were taken from 9 volunteer patients from the “probiotic group” and processed according to the described procedure for the isolation of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
- Colonies of the Bifidobacterium genus in faecal samples that show the strainspecific DNA profile of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 obtained by the Pulse Electrophoresis method ( Figure 1) averaged 2.2xl0 6 CFU/g.
- the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and the bifidobacterial preparation of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 have a unique combination of probiotic properties, suitable for the prevention of atherosclerosis and cardiovascular diseases, inflammatory diseases, as well as for reducing the diabetes risk.
- Patent KR102224072B Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application.
Abstract
The subject of the present invention relates to the new strain Bifidobacterium longum ssp. longum 3/15, filed under No. NBIMCC 9085 with the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) of the Republic of Bulgaria, possessing a complex of health beneficial properties: hypolipidemic activity, anti-inflammatory immunomodulatory activity, ability to reduce insulin resistance, strong adhesion to epithelial cells and high survival rate in the gastrointestinal tract. The subject of the present invention is also a bifidobacterial preparation obtained by cultivating the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 with a clinically proven set of functional effects, including improvement of lipid indicators, reduction of insulin resistance, beneficial effect on markers of inflammatory status. The invention also relates to the use of the new strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and the bifidobacterial preparation for the prevention and therapy of atherosclerosis, cardiovascular and inflammatory diseases and to reduce the diabetes risk.
Description
Bifidobacterium longum ssp. longum strain and a bifidobacterial preparation having hypolipidemic, anti-inflammatory immunomodulating and insulin resistance decreasing effects
TECHNICAL FIELD
This invention is in the field of biotechnology and relates to a probiotic Bifidobacterium strain in particular, characterized by a complex of beneficial properties regarding human health and its use in probiotic preparations for the prevention of metabolic, cardiovascular and inflammatory diseases.
BACKGROUND ART
Bifidobacteria are considered probiotics because they are living microorganisms that have a beneficial effect on human health when taken in sufficient quantity. A minimum level of approximately 106 -107 v’a^'e bifidobacteria cells per gram of intestinal contents is suggested. [1]
Bifidobacteria are one of several dominant culturable bacterial groups present in the colonic microflora, playing an important role in maintaining a balanced healthy intestinal microflora. There is compelling evidence of the relationship between gut microflora, obesity, the endocannabinoid system, systemic low-grade inflammation, lipopolysaccharides, intestinal permeability, and arterial stiffness.
Probiotics, in addition to improving the composition of the intestinal microflora, also improve immunity, favourably affecting inflammatory and metabolic diseases, and reducing the risk of developing atherosclerosis. One of the most important prerequisites for the manifestation of their beneficial effects is the adhesive properties of bacterial cells, as well as their survival and ability to colonize the gastrointestinal tract.
The beneficial effects on health demonstrated by probiotics are strainspecific and even strains of the same genus and species exhibit different properties at varying degrees.
The beneficial effect of bifidobacteria against intestinal inflammatory diseases such as ulcerative colitis and Crohn’s disease has already been proven, and treatment with probiotic preparations accompanies drug therapy. The mechanisms of this beneficial effect on intestinal inflammations are prevention of the adhesion of pathogens on the intestinal epithelium; immunomodulatory effect through the induction of the synthesis of antiinflammatory cytokines IL-10 (interleukin 10) and/or TGF-p (transforming growth factor beta), reduction of synthesis of proinflammatory cytokine IL-8 (interleukin 8) by epithelial cells, absence or reduced induction of TNFa (Tumour Necrosis Factor alpha), IL-1 (Interleukin 1) and/or IL-6 (Interleukin 6); proteolytic degradation of antigens with proinflammatory properties; elimination of toxins, etc. [2]
The increased induction of pro-inflammatory cytokines, especially TNFa, stimulates epithelial cells to produce interleukin IL-8, which stimulates the activity of neutrophil cells. [3] The persistently increased activity of neutrophils may lead to the formation of ulcers or the occurrence of intestinal bleeding [4], conditions that are concomitant with inflammatory bowel disease.
The immunomodulatory effect is strictly strain-specific. Strains of lactobacilli and bifidobacteria have been studied, some of which lead to an increase in the level of TNFa and IL-6, while others decrease TNFa and increase IL- 10. [5]
It has been established that some strains of bifidobacteria can lower serum cholesterol. High levels of serum cholesterol are the prime cause of several diseases such as hyperlipidemia, atherosclerosis, and lipid metabolic syndrome. The cholesterol-lowering mechanism is due to the direct adsorption of cholesterol to lipoprotein fractions of the bacterial cell walls or through the significant hydrolase activity towards bile salts (BSH bile salt hydrolase activity) of some probiotic strains. The probiotic strains lower the concentration of bile salts through their BSH activity, which is compensated
by the synthesis of new amounts of bile acids from cholesterol and the conjugation of these bile acids with taurine or glycine leading to the production of fresh reserves of bile salts, replacing the deconjugated bile salts. The main feature in the selection of bacterial strains for the development of probiotic preparations with a beneficial effect on cholesterol metabolism is the presence of significant hydrolase activity towards bile salts.
It is also known that good levels of the enzyme “bile salt hydrolase” in bifidobacteria are associated not only with its ability to reduce cholesterol, but also to reduce inflammation.
A strain of Bifidobacterium longum ssp. longum strain has been described in a Korean patent KR102224072B1 [6], possessing a combination of two main functional characteristics: direct reduction of total serum cholesterol and immune regulation by lowering the secretion of pro- inflammatory cytokines TNFa and IL-6 and increasing the secretion of antiinflammatory cytokine IL- 10. These effects were confirmed by in vitro functional assessment of the strain and in vivo experiments in mice.
It is known that cardiovascular diseases are a leading cause of morbidity and mortality worldwide, and the main reason for their occurrence, development and complications is atherosclerosis. The main risk factors, the combination of which leads to the appearance and accelerated development of the atherosclerotic process, are hypertension, dyslipidemia, obesity and pro- inflammatory status, diabetes mellitus or impaired glucose metabolism, and immobility. Often these factors are combined in a single patient, which sharply increases the risk of cardiovascular disease. The phenomenon is well- known in patients with metabolic syndrome. The more risk factors that exist in a patient, the greater the likelihood of cardiovascular disease, because their harmful effects are multiplied when they are combined. Therefore, it is important to reduce the combined influence of risk factors by influencing their totality simultaneously, rather than individually.
An actual problem is the selection of a probiotic strain of Bifidobacterium longum ssp. longum, which, in addition to immune regulation and direct cholesterol reduction, also exhibits hydrolase activity against bile salts.
Another current issue is proving hypolipidemic and anti-inflammatory immunomodulatory activities of the strain Bifidobacterium longum ssp. longum through a clinical trial of the application of the strain in patients at risk of developing atherosclerosis.
DISCLOSURE OF INVENTION
The subject of the present invention relates to the new strain Bifidobacterium longum ssp. longum 3/15 filed under No. NBIMCC 9085 on 26 January 2022 with the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC) of the Republic of Bulgaria, possessing a complex of health-beneficial properties: hypolipidemic activity, anti-inflammatory immunomodulating activity, ability to reduce insulin resistance, strong adhesion to epithelial cells and high survival rate in the gastrointestinal tract.
The subject of the invention is also a bifidobacterial preparation obtained from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and possessing a clinically proven set of health benefits: improvement of lipid parameters, reduction of insulin resistance, beneficial effect on inflammatory status markers.
The subject of the invention is also the use of the new strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and the bifidobacterial preparation obtained from Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 for simultaneous beneficial influence on multiple health risk factors and prevention of a specific inflammatory or metabolic disease, but also of diseases caused by these risk factors in their totality, in the specific case for the prevention of atherosclerosis and cardiovascular diseases.
The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was isolated from faecal samples of a healthy 28-y ear-old volunteer who had not used antibiotics in the past 1 year and had not used probiotics in the past six months. The sample taken is serially diluted in anaerobic peptone water in an anaerobic box to a dilution of 10'6 and inoculated on a non-selective medium for anaerobes “Blood Liver agar” with added 5.0% defibrinated horse blood. Cultivation is anaerobic at 37°C for 72 hours. Colonies with a typical Bifidobacterium genus morphology are inoculated into MRS broth with L- cysteine and lithium chloride and cultured in an anaerobic environment at a temperature of 37°C for 24 hours. Genus-specific PCR is used to identify the gram-positive cultures, which are of a rod shape that is typical of the Bifidobacterium genus. Species affiliation and strain identity of the strain was established using precise DNA-based methods (Figure 1) such as 16S rDNA sequencing, Pulsed Field Gel Electrophoresis. The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is gram-positive, catalase-negative, and rod-shaped. A microscopic photo of the strain is presented in Figure 2.
Isolation of the strain from the intestinal tract of a healthy volunteer proves its GRAS (generally recognized as safe) status.
The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is capable of significantly lowering serum cholesterol concentration. The strain exhibits two main activities related to reducing the level of cholesterol - a direct lowering of the cholesterol level, as well as its indirect reduction as a consequence of the hydrolase activity of the strain towards bile salts (BSH activity). The extent of both strain activities is determined in vitro.
Direct elimination of cholesterol is mostly due to its adsorption on components of the cell wall of a strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. In determining this activity, residual cholesterol is measured in a medium with added water-soluble cholesterol. Measurements are performed by gas chromatography and showed an average of 62.7% ± 1.6% direct cholesterol lowering. (Example 1.1)
The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 demonstrates high bile salt hydrolase activity, thus indirectly affecting serum cholesterol levels. As a result of the deconjugation of bile salts, their concentration decreases, which is compensated by the synthesis of new bile acids and the conjugation of these bile acids to new bile salts. The participation of serum cholesterol in the synthesis of new bile acids reduces its concentration.
The determination of BSH activity of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is performed by help of reverse-phase liquid chromatography (RP- HPLC) with UV detection at 210 nm. The strain cultured in a medium with added bile salts sodium taurocholate (TCA) and sodium glycocholate (GCA) achieve 72.0% ± 1.9% deconjugation of bile salts that are present in the medium. (Example 1.2)
The ability of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 to lower cholesterol was also demonstrated by the decrease in the values of lipid indicators in the clinical trial after administration of the bifidobacterial preparation obtained from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. (Example 3.1)
In addition to the above, the strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 possesses anti-inflammatory immunomodulatory activity, which is manifested by the ability of the strain to favourably alter the induction of pro-inflammatory and anti-inflammatory cytokines.
The anti-inflammatory immunomodulatory ability of the strain was determined in vitro by quantifying the expression of signal peptides TNF-a, IL-8, IL-6, TGF-P and IL-10 from a combined system of two cell lines: human monocyte cell line THP-1 and epithelial cell line HT-29, separated by a semipermeable membrane. (Example 1.3)
The results of the in vitro assessment of the induction of signal peptides by Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 showed downregulation of the expression of pro-inflammatory cytokines IL-8, IL-6, TNF-a,
and an increase of the synthesis of anti-inflammatory cytokines TGF-p and IL- 10. It has been demonstrated the ability to significantly lower the level of interleukin IL-8 from 2734 ± 149 pg/ml in the control to 112 ± 13 pg/ml in the assessment of the strain, which is an essential factor in the prevention and therapy of inflammatory diseases of the intestinal tract.
A beneficial effect on indicators of inflammatory status was also found during the clinical trial with the administration of the bifidobacterium preparation obtained from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085: levels of pro-inflammatory cytokines TNF-a, IL-1 p and IL-6 are decreased, and the level of anti-inflammatory cytokine TGF-P is increased. (Example 3.3)
Simultaneously, the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 exhibits strong adhesion to epithelial cells and high survival rates in the gastrointestinal tract, which enhances the impact of the beneficial effects of the strain on the human organism.
The ability of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 to adhere to epithelial cells was determined in vitro using the epithelial cell lines Caco-2 and HT-29. The results showed that the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 possesses very strong adhesion with an average number of more than 15 adhered bacterial cells per eukaryotic cell. (Example 1.4)
The survival of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was determined in the clinical monitoring on the administration of the bifidobacterium preparation of the strain. Faecal samples from nine volunteers, patients in the clinical trial, were examined after 30 days of daily administration of the bifidobacterial preparation. It has been determined an average number of colonies of 2.2x106 CFU/g. (Example 3.4)
The bifidobacterial preparation subject of this invention is obtained by cultivating the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. (Example 2)
It can be administered in liquid or dry (lyophilized) form. The lyophilized bifidobacterial preparation can be applied both in powder form and also in the form of capsules.
The liquid bifidobacterial preparation contains live cells of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 - at least 1x109 CFU/g.
The lyophilized bifidobacterial preparation contains live cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 - at least 5x10 9 CFU/g. Residual moisture content is 4% ± 1%.
The bifidobacterial preparation, lyophilized or liquid, can be used as an additive to products in dry and/or liquid form. When adding it to lactic acid products, it is added at a quantity of 0.5% - 2%.
The lyophilized bifidobacterial preparation can be encapsulated. Before encapsulation, an anti-caking agent, microcrystalline cellulose, is added to it. Each capsule contains no less than 2xl09 viable cells of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085
A double-blind placebo-controlled clinical trial on the administration of the bifidobacterial preparation from the specially selected strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was conducted. During the clinical trial, the hypolipidemic and anti-inflammatory effects of the bifidobacterial preparation were evaluated in patients with a high atherosclerotic vascular risk or atherosclerotic disease - all of them had mild and moderate lipid disorders, overweight or obesity, more than half of them had arterial hypertension under therapeutic control, type 2 diabetes under therapy, as well as proven hepatic steatosis. (Example 3)
Very good tolerability and lack of unwanted side effects were observed during the course of administration of the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
The performed blood tests on a number of biochemical, metabolic and hormonal indicators prove statistically significant beneficial metabolic effects
on the lipid metabolism after 30 days of administration of the probiotic bifidobacterial preparation: lowering of total serum cholesterol; lowering of LDL cholesterol (cholesterol in low-density lipoproteins) and non-HDL cholesterol, which confirmed the hypolipidemic effect of the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. (Example 3.1)
An important part of the clinical studies is the analyses of the levels of vascular risk markers, respectively cytokines and inflammatory factors, whose pathophysiological role is of great importance in the light of considering metabolic syndrome and obesity as a state of low-grade inflammation leading to endothelial dysfunction.
The results of the clinical trial show a favourable change in the investigated cytokines and confirm the anti-inflammatory immunomodulating effect of the bifidobacterial preparation. (Example 3.3)
At the same time, an additional effect is reported during the clinical trial, namely a statistically significant improvement in the state of insulin resistance in the patients receiving the bifidobacterial preparation. A decrease in basal glycemia and insulinemia has been reported, as well as a decrease the index of insulin resistance, thus establishing a new effect - a reduction of diabetes risk and improving diabetes control. (Example 3.2)
Results of the medical research within the framework of the conducted clinical trial show simultaneous favourable influence on three risk factors leading to the development of atherosclerosis, namely lipoproteinemia, the state of insulin resistance and the inflammatory status. Thus, the clinical trial of effects after administration of the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 confirmed the in vitro findings on the functional characteristics of the strain, namely hypolipidemic and anti-inflammatory immunostimulatory properties, as well as establishing a novel functional effect - a reduction of diabetes risk.
Based on the above, the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is recommended for successful application in medical practice in the primary and secondary prevention and therapy of atherosclerosis, cardiovascular and inflammatory diseases, and diabetic risk.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. Pulsed field gel electrophoretic profile of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 with the AsnI. M enzyme and m-molecular markers. Unfolding conditions: initial pulse 2s, final pulse 15s, voltage 5V/cm, duration 24h. The molecular masses of DNA fragments of Bifidobacterium longum ssp. longum LBB 3/15 NBIMCC 9085 in kbp are: 199, 136, 112, 99, 93, 73, 61, 57, 51, 47, 38.7, 26, 22.2, 20.5, 17, 9, 14.2, 13.5, 11.6, 9.0, 8.5, 8.0, 7.5, 6.8.
Figure 2. Microscopic picture of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085
Figure 3. Change in cytokine level TNF-a when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to the placebo group.
Figure 4. Change in cytokine level IL-ip when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to the placebo group.
Figure 5. Change in cytokine level IL-6 when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to the placebo group.
Figure 6. Change in cytokine level TGF- i when taking the bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 compared to the placebo group.
MODES FOR CARRYING OUT THE INVENTION
Example 1. In vitro functional assessment of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
1.1. Assessment of direct cholesterol reduction capacity
The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is cultivated anaerobically at 37°C for 18 hours in MRS L-cysteine broth. The medium contains 0.3% bile salts and 100 pg/ml water-soluble cholesterol (cholesterol-PEG600). The inoculum is 0.5% and contains 1x109 CFU /ml. After incubation, the cells are pelleted by centrifugation and 0.5 ml of the supernatant is treated with 4 ml of 2M KOH in methanol at 80°C for 20 minutes. The process is followed by extraction with 5 ml of hexane and 4 ml of the hexane layer is transferred to test tubes and evaporated with a stream of nitrogen at a temperature of 60°C. The dry residue is dissolved in 0.5 ml of isopropanol and the amount of cholesterol contained is determined by gas chromatography. The degree of cholesterol reduction is determined based on an uninoculated control medium.
The measurements are performed three times and the results are averaged. Table I presents the results of the direct adsorption of cholesterol, expressed as a percentage of cholesterol reduction.
Table I
1.2. Assessment of hydrolase activity against bile salts (BSH activity)
The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is cultivated anaerobically at 37°C for 18 hours in MRS broth with L-cysteine, adding the two most important bile salts in the medium: sodium taurocholate
(TCA) and sodium glycocholate (GCA) at a concentration of 1 mM, each. The inoculum is 0.5% and contains 1x109 CFU /ml, and the uninoculated medium is used as a control.
The determination of BSH activity is performed by reverse-phase liquid chromatography (RP-HPLC) with UV detection at 210 nm. Free and conjugated bile acids are detected in reverse-phase gradient mode. Solvent A is 65% methanol in 0.03 M sodium acetate at pH 4.3 adjusted with phosphoric acid. Solvent B is methanol with HPLC quality. The eluent program is an isocratic step for 8 min at 15% eluent B and then a linear gradient to 85% for 17 min. This is followed by an isocratic step of 5 minutes at 85%. The injected amount is 5 pl.
Table II. presents the results of the determination of BSH activity of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 as a percentage of hydrolysis of the bile salts present in the medium, after testing in triplicate and averaging the results.
Table II.
1.3. Assessment of anti-inflammatory immunomodulatory activity
The anti-inflammatory immunomodulatory activity is evaluated by quantifying the expression of pro-inflammatory and anti-inflammatory signal peptides from a combined system of two cell lines: the human monocyte cell line THP-1 and the epithelial cell line HT-29, separated by a semipermeable membrane. The system of the two cell lines is appropriate to assess the induction of both pro- inflammatory cytokines TNF-a, IL-8 and IL-6, as well as to assess the induction of anti-inflammatory cytokines IL- 10 and TGF-p. Both cell lines are maintained at 37°C and 5% CO2 on RPMI medium 1640
for cell line THP-1 and on DMEM medium for cell line HT-29. Both media were supplemented with 10% fetal bovine serum, 4.5 g/1 glucose, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 1.5 g/1 sodium bicarbonate, 100 U/ml penicillin, 100 pg/ml streptomycin. In a 48-well plate for cell lines, in the lower wells, lxl06 per millilitre THP-1 cells (5xl05 per well) were differentiated into macrophages using 5 pg/ml PMA (phorbol 12- myristate- 13 -acetate), added to the growth medium, for 24 hours in a CO2 incubator. Epithelial HT-29 cells were grown as a monolayer in semipermeable membrane inserts and matured for 14 days with a daily change of DMEM medium. The differentiated adhered macrophages were washed twice with PBS buffer (Dulbecco’s phosphate-buffered saline) and incubated with fresh medium without PMA for 24 hours. Lipopolysaccharide of Salmonella is added at a concentration of 1 pg/ml in RPMI 1640 medium and incubation continued for another 4 hours. After aspirating the medium, 0.5 ml of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 at a concentration of 1x106 CFU/ml per well were added to the differentiated macrophages suspended in RPMI 1640 medium. The cells are incubated for 24 hours in a CO2 incubator.
Assessment of the induction of signal peptides TNF-a, IL-8, IL-6, TGF-P and IL- 10 from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is done by sandwich ELISA. After final incubation, the supernatant was collected and centrifuged to eliminate bacterial and human cells. For determining the signal peptides, 100 pl of the obtained supernatants are used. Assessment of the induction of each cytokine was performed in triplicate. Table III. shows the averaged results of the triplicate induction test for the five cytokines.
Table III.
1.4. Assessment of adhesion to epithelial cells
To determine the adhesion of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 to epithelial cells, the epithelial cell lines Caco-2 and HT-29 are used, which are cultured as a monolayer of mature epithelial cells in DMEM (Dilbecco's modified Eagle's medium) medium supplemented with 10% fetal bovine serum. Incubation is performed at a temperature of 37°C in a water-saturated atmosphere containing 5% CO2. When about 90% of a monolayer is formed, cells were passaged using 0.25% trypsin and 10 mM EDTA solution at a temperature of 37°C for 10 minutes. Eukaryotic cells were seeded in 6-well clusters at a concentration of 2x105 cells/ml. The medium was changed every 2 days for a total of 14 days. During this period, the monolayer is formed (for about 3-4 days) and cell receptors maturate. The monolayers were then washed twice with PBS buffer and the bacterial culture of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 was added to them at a concentration of lxlO8 CFU/ml in 2 ml DMEM without antibiotics. Bacterial and eukaryotic cells were incubated together for 60 minutes at 37°C in a water-saturated atmosphere containing 5% CO2. The process is followed by a fivefold washing of the wells with PBS, fixing with methanol for 3 minutes, Gram staining and microscopic assessment of the adhesion. The number of adhered bacterial cells per 20 epithelial cells was determined by
counting 10 microscopic fields. The adhesion assay was performed in triplicate on both Caco-2 and HT-29 cell lines, and the results were averaged.
The results of determining the adhesion of Bifidobacterium longum ssp. longum 3/15, NBIMCC 9085, expressed as the average number of adhered bacterial cells per eukaryotic cell are presented in Table IV.
Table IV.
Example 2. Production of bifidobacterial preparation
The bifidobacterial preparation is obtained by cultivating the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
To obtain a starter culture, the strain is cultivated in a medium of MRS broth containing lithium chloride and L-cysteine, in anaerobic conditions at a temperature of 37°C for 18-24 h. With the resulting starter culture containing a biomass of about 1x109 CFU/ml, a specially prepared medium is inoculated in an industrial fermenter to carry out the fermentation process of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085. The medium includes 8% milk powder, 2% whey powder, 2% lactose, 0.5% yeast extract, 0.5% peptone casein and 2% fructooligosaccharide. The pH of the medium is adjusted to pH 6.8 and the medium is sterilized in a fermenter at 120°C for 15 minutes. Then cooled to a temperature of 37°C and inoculated with 5% of the starter culture. Fermentation is carried out at a temperature of 37°C for 14-16 hours with continuous blowing with carbon dioxide. After reaching pH 4.9- 5.0, the fermentation process is stopped by cooling with ice water and subsequently adjusting the active reaction to pH 5.8-6.2.
The resulting biomass is a liquid bifidobacterial preparation containing not less than 1 xlO9 CFU/g viable cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
To obtain a lyophilized bifidobacterial preparation, the cryoprotective medium with 10% sucrose and 0.5% pectin is added to the fermented medium in a ratio of 1 : 10 and loaded in an industrial lyophilizer.
The sublimation is performed at a temperature of minus 28°C to minus 30°C with a layer height of no more than 1.2 cm. Secondary drying is at a temperature of 30°C. After the process is completed, the vacuum in the sublimation chamber is compensated with nitrogen. The lyophilized bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 is packaged in an inert (nitrogen) atmosphere. The final product contains at least 5xl09 CFU/g viable cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and its water content is 4% ± 1%.
The lyophilized bifidobacterial preparation can be encapsulated on an encapsulation machine by first adding an anti-caking agent, for example, microcrystalline cellulose. Each capsule contains not less than 2x109 CFU/g viable cells of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
Example 3. A clinical trial of the effects upon the administration of a bifidobacterial preparation from the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 as a probiotic
The objective of the clinical trial is to evaluate the hypolipidemic and anti-inflammatory immunomodulating effect of the bifidobacterial preparation in patients with dyslipoproteinemia and high atherosclerotic vascular risk or atherosclerotic disease, and development of recommendations for its use in primary and secondary prevention of cardiovascular diseases.
Clinical studies were performed in the conditions of a double-blind, placebo-controlled clinical trial in a group of 60 patients, 42 women and 18 men, with mild to moderate dyslipoproteinemia, respectively with increased
cholesterol, low-density lipoproteins and/or triglycerides. During the clinical trial, the patients were not administered hypolipidemic drugs or other nutritional supplements having metabolic effects. The age of the patients is between 18 and 75 years. They were divided into two groups - a group of 30 patients who took the probiotic bifidobacterial preparation, tentatively called the “probiotic group”, and a group of 30 patients who received a placebo product, tentatively called the “placebo group”.
The clinically observed product is a bifidobacterial preparation of the probiotic strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085, with a number of viable cells not less than 5xl09 CFU/g. The bifidobacterial preparation is administered in the form of capsules, each of which contains no less than 2x109 viable cells of the probiotic strain.
The placebo product is a capsule containing dry milk without bacterial cells of the probiotic strain Bifidobacterium, longum ssp. longum 3/15 NBIMCC 9085.
All patients took 1 capsule 3 times a day with a bifidobacterial preparation or a placebo product, respectively, for 30 days as an adjunct to a recommended hypolipidemic dietary regimen under the conditions of a double-blind placebo-controlled trial. All patients underwent a clinical examination before and after the completion of the trial. Twice, before and after the clinical trial, a fasting venous blood sample was collected for the study of haematological, biochemical, and metabolic, including lipid and hormonal indicators, as well as specific markers for vascular risk assessment, incl. cytokines. The monitored and evaluated parameters were lipid panel, serum levels of vascular inflammation markers (C-reactive protein, cytokines TNF-a, IL-ip, IL-6, TGF-p), as well as fasting serum insulin level and insulin resistance by HOMA-IR index.
The obtained results were processed statistically with the methods of descriptive statistics, Wilcoxon Signed Ranks Test and parametric T-test.
Analysis of patient groups showed a similar gender and age distribution. Patients in the “probiotic group” were 23 women and 7 men, aged 21 to 70 years, with a mean age of 54.37 ± 11.91 years. Patients in the “placebo group” were 19 women and 11 men, aged 22 to 75 years, with a mean age of 49.60 ± 13.79 years. All patients had mild and moderate lipid disorders - increases in the levels of total and LDL cholesterol, lower values of HDL cholesterol, and an increase in triglycerides. All had metabolic syndrome, overweight or obesity, two-thirds had arterial hypertension under therapeutic control, 35 patients had type 2 diabetes under therapy, and 29 patients had proven hepatic steatosis.
All patients tolerated the administered capsules very well and none of them reported adverse effects. During clinical monitoring, no adverse side effects were detected.
3.1. Assessment of lipid indicators
Table V. shows the dynamics of the changes in the lipid indicators of patients in the “probiotic group”.
Table V.
* Statistical confidence
Table VI. shows the dynamics of the changes in the lipid indicators of patients in the “placebo group”.
Table VI.
The summary results of the study of lipid indicators showed that in the “probiotic group” compared to the “placebo group” a significant favourable reduction of total cholesterol by 5.57%, by 11.71 % of LDL, and by 3.86% of non-HDL. VLDL cholesterol in the “placebo group” increased by 0.167 mM/L, while the increase in the “probiotic group” was only 0.018 mM/L. Average levels of HDL cholesterol and triglycerides before the clinical trial were at target levels and did not change significantly after the one-month administration of the bifidobacterium preparation.
The analysis of individual changes in the lipid indicators of patients in the “probiotic group” shows that more than half of them have favourable changes - total cholesterol decreased in 53.33% of the patients, LDL cholesterol decreased in 50% of the patients, VLDL cholesterol decreased in 56.66% of the patients, and HDL cholesterol increased in 26.66% of the patients.
3.2. Assessment of other biochemical and metabolic indicators
Table VII. presents the results of other investigated biochemical and metabolic indicators of patients in the “probiotic group”.
Table VII.
* Statistical confidence
Table VIII. presents the results of other studied biochemical and metabolic indicators of patients in the “placebo group”. Table VIII.
* Statistical confidence
The analysis shows that the “probiotic group” exhibits a statistically significant reduction in the state of insulin resistance accompanying the metabolic syndrome and decreasing of blood glucose by 6.41%. basal insulinemia by 9.81% and HOMA-IR index by 6.53%, while the changes of these indicators in the “placebo group” were not statistically significant. These results add a very significant beneficial effect of the application of the
bifidobacterial preparation regarding the improvement of the state of insulin resistance. The changes in serum uric acid were similar, with its values significantly decreasing by 3.45% in the patients of the “probiotic group”, while the patients of the “placebo group” had insignificant changes in the level of the uric acid. The monitored levels of serum transaminases, GGTP and CRP were within reference values in both groups and remained without significant changes.
CRP levels at baseline in both groups were within the reference range and remained within the reference range, with no significant change after 30 days of administration of the bifidobacterial preparation and the placebo product (Table VII and Table VIII).
3.3. Assessment of the levels of cytokines and inflammatory factors
Results of the studied cytokines are presented in Figure 3, Figure 4, Figure 5 and Figure 6. The changes in their levels are statistically significant for the “probiotic group”, and after 30 days of administration of the bifidobacterial preparation, it is reported a decrease in the level of pro- inflammatory cytokines TNF-a by 6.56% (Figure 3), IL- Ip by 3.34% (Figure 4) and IL-6 by 7.60% (Figure 5), as well as an increase in anti-inflammatory cytokine TGF- i by 14.87% (Figure 6). These data point to a beneficial effect of the bifidobacterial preparation on the proinflammatory status in metabolic syndrome by modulating the cytokine profile.
Results of the conducted clinical trial confirm the anti-inflammatory immunomodulating effect of the bifidobacterial preparation - lowering of TNF-a, IL- 1 P, IL-6 and moderate increase of TGF-pi.
3.4. of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 in the gastrointestinal tract
Faecal samples were taken from 9 volunteer patients from the “probiotic group” and processed according to the described procedure for the isolation of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085.
Colonies of the Bifidobacterium genus in faecal samples that show the strainspecific DNA profile of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 obtained by the Pulse Electrophoresis method (Figure 1) averaged 2.2xl06 CFU/g.
The results presented in Table IX. show a high survival rate of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 in the gastrointestinal tract.
Table IX.
INDUSTRIAL APPLICABILITY
The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 and the bifidobacterial preparation of the strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 have a unique combination of probiotic properties, suitable for the prevention of atherosclerosis and cardiovascular diseases, inflammatory diseases, as well as for reducing the diabetes risk.
REFERENCES
• Paineau D, Carcano D, Ley er G, Darquy S, Alyanakian M, Simoneau G, Bergmann J, Brassart D, Bomet F & Ouwehand A (2008) Effects of seven potential probiotic strains on specific immune responses in healthy adults:
a double-blind, randomized controlled trial. FEMS Immunol Med Microbiol 53:107-113
• Hedin, M. Mullard, E. Sharratt et al., (2010). Probiotic and prebiotic use in patients with inflammatory bowel disease: a case-control study. Inflammatory Bowel Diseases, vol. 16, no. 12, pp. 2099-2108
• Papadopoulos, J., Karpouzis, A., Tentes, J., Kouskoukis, C. (2014). Assessment of Interleukins IL-4, IL-6, IL-8, IL-10 in Acute Urticaria. J Clin Med Res. 6(2): 133 - 137
• Belkaid Y, and Hand T. (2014) Role of the microbiota in immunity and inflammation. Cell, vol. 157, no. 1, pp. 121-141
• O'Flaherty S. & Klaenhammer TR, (2010) The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host, Int. DJ2Q-. 262-268
• Patent KR102224072B1. Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application.
Claims
Claim 1. The strain Bifidobacterium longum ssp. longum 3/15, filed at NBIMCC under No. NBIMCC 9085 with a strain-specific DNA profile with molecular masses of its DNA fragments in kbp: 199, 136, 112, 99, 93, 73, 61, 57, 51, 47, 38.7, 26, 22.2, 20.5, 17, 9, 14.2, 13.5, 11.6, 9.0, 8.5, 8.0, 7.5, 6.8.
Claim 2. The strain Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085, according to Claim 1, simultaneously exhibiting hypolipidemic activity by directly reducing cholesterol and by hydrolysis of bile salts, anti-inflammatory immunomodulatory activity, and ability to reduce insulin resistance, high adhesion to epithelial cells and a high survival rate in the gastrointestinal tract.
Claim 3. The bifidobacterial preparation containing a strain of Bifidobacterium longum ssp. longum 3/15 NBIMCC 9085 with a viable cell count at least lxlO9 CFU/g.
Claim 4. The bifidobacterial preparation, according to Claim 3, simultaneously exhibiting a hypolipidemic effect, an anti-inflammatory immunomodulatory effect and an insulin resistance-reducing effect.
Claim 5. The bifidobacterial preparation, according to Claim 3, in the form of a lyophilized or a liquid culture.
Claim 6. The use of the bifidobacterial preparation, according to Claim 3, as a probiotic.
Claim 7. The use of the bifidobacterial preparation, according to Claim 6, as a food additive and/or in any form in food for humans and animals.
Claim 8. The use of the bifidobacterial preparation, according to Claim 6, as an additive to a starter culture for food production.
Claim 9. The use of the bifidobacterial preparation, according to Claim 6, in the composition of functional products and/or pharmaceutical preparations, intended to influence health indicators in humans or animals.
Claim 10. The bifidobacterial preparation, according to Claim 3, for prevention and/or therapy of inflammatory diseases and/or atherosclerosis and/or cardiovascular diseases and/or for reducing the diabetes risk.
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| WO2017097987A1 (en) * | 2015-12-11 | 2017-06-15 | Alimentary Health Limited | Bifidobacterium longum for treating obesity and associated metabolic disorders |
| WO2019038449A1 (en) * | 2017-08-25 | 2019-02-28 | University College Cork - National University Of Ireland, Cork | Bifidobacterium longum for treating obesity and weight management |
| KR20210022221A (en) * | 2019-08-19 | 2021-03-03 | 주식회사 빙그레 | Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application |
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| WO2017097987A1 (en) * | 2015-12-11 | 2017-06-15 | Alimentary Health Limited | Bifidobacterium longum for treating obesity and associated metabolic disorders |
| WO2019038449A1 (en) * | 2017-08-25 | 2019-02-28 | University College Cork - National University Of Ireland, Cork | Bifidobacterium longum for treating obesity and weight management |
| KR20210022221A (en) * | 2019-08-19 | 2021-03-03 | 주식회사 빙그레 | Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application |
| KR102224072B1 (en) | 2019-08-19 | 2021-03-10 | 주식회사 빙그레 | Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application |
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| BELKAID YHAND T: "Role of the microbiota in immunity and inflammation", CELL, vol. 157, no. 1, 2014, pages 121 - 141, XP028601900, DOI: 10.1016/j.cell.2014.03.011 |
| GOTOVA IRINA ET AL: "Selected Lactobacillus bulgaricus and Streptococcus thermophilus Strains from Bulgarian Yogurt Demonstrate Significant Anti-Inflammatory Potential", ACTA MICROBIOLOGICA BULGARICA, vol. 33, no. 3, 1 September 2017 (2017-09-01), pages 137 - 142, XP093057475, Retrieved from the Internet <URL:https://actamicrobio.bg/archive/september-2017/amb-september-2017-article-7.pdf> * |
| GOTOVA IRINA MARINOVA ET AL: "Clinical Assessment of Lactobacillus gasseri Strain, Selected by In-Vitro Tests", INTERNATIONAL JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, 16 June 2017 (2017-06-16), pages 156 - 160, XP093057476, Retrieved from the Internet <URL:https://article.sciencepublishinggroup.com/pdf/10.11648.j.ijmb.20170204.11.pdf> [retrieved on 20230623], DOI: 10.11648/j.ijmb.20170204.11 * |
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| JAROCKI PIOTR ET AL: "A New Insight into the Physiological Role of Bile Salt Hydrolase among Intestinal Bacteria from the Genus Bifidobacterium", PLOS ONE, vol. 9, no. 12, 3 December 2014 (2014-12-03), pages e114379, XP093057979, Retrieved from the Internet <URL:https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0114379&type=printable> DOI: 10.1371/journal.pone.0114379 * |
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