JP2017515799A - Lactobacillus plantarum Inducia strain DSM 21379 as an antimicrobial agent against cholesterol lowering agents and Clostridium difficile - Google Patents

Abstract

The present invention relates to a microbial strain, Lactobacillus plantarum Inducia DSM 21379, used as a cholesterol-lowering agent, an antimicrobial agent, and an antioxidant, and to enhance the natural defense function of a subject. L. Plantarum Inducia is used to prevent cholesterol metabolism disorders and ongoing cardiovascular disease to reduce LDL-cholesterol levels in the blood. L. Plantarum Inducia enhances the innate defense function and cellular immunity, as well as the antioxidant activity of the human body by reducing the level of oxidized low density lipoprotein (ox-LDL) as an antioxidant Strengthen. This strain can also be used as an antimicrobial agent to reduce the risk of Clostridium difficile-associated diarrhea (CDAD) by preventing the germination of Clostridium difficile spores and inhibiting the growth of Clostridium difficile vegetative cells. used. L. A composition comprising Plantarum Inducia reduces cholesterol and ox-LDL levels in the blood, enhancing the innate defense function and reducing the risk of CDAD. The composition can further comprise xylitol.

Description

  The present invention relates to the fields of microbiology and nutrition relating to the Lactobacillus plantarum Inducia (DSM 21379) strain having various uses in the field of biotechnology.

(LDL-cholesterol)
One factor that causes cardiovascular disease (CVD) is abnormally high cholesterol levels. In recent years, the view of high cholesterol as a hazardous substance is returning to the anomaly of its particles, particularly low density lipoprotein cholesterol (LDL-c) and apolipoprotein B (apo B). LDL-c accounts for 60-70% of total cholesterol. LDL-c particles carry cholesterol, triglycerides, fat-soluble vitamins and antioxidants. LDL-cholesterol is an important modulator for prevention of atherosclerosis and maintenance of cardiovascular health. Oxidized low density lipoprotein (Ox-LDL) is a marker of oxidative stress specific to LDL particles. Ox-LDL can be considered as one of the biochemical risk markers for coronary heart disease. Thus, LDL-c is widely recognized as a cardiovascular risk marker established according to the results of numerous clinical trials that demonstrate the ability of LDL-c to independently induce the development and progression of coronary heart disease (For example, refer nonpatent literature 1, March 14, 2014 search).

  The bile salt hydrolase (BSH) activity of probiotics has been regarded as one of the main mechanisms of cholesterol lowering action (see, for example, Non-Patent Document 2, Non-Patent Document 3, and Non-Patent Document 4). BSH (EC 3.5.1.24; cholylglycine hydrolase) is an enzyme that catalyzes the hydrolysis of glycine and / or taurine-conjugated bile salts to amino acid residues and free bile acids. BSH activity in lactic acid bacteria is strongly correlated with the natural habitat, and is mainly observed in species related to the digestive tract such as Lactobacillus species. However, there are various variations between strains.

  The close relationship between the host gastrointestinal mucosal epithelial cells and the microbiota is of greatest importance for health. Among the microflora unique to the gastrointestinal tract (GIT), lactic acid bacteria digest food-derived cholesterol (see, for example, Non-Patent Document 5). In Cune Castellana, 2009 patents (see, for example, US Pat. No. 6,057,031; AB Probiotics SA), Lactobacillus plantarum strains CECT7528, CECT7526 and CECT7529, either alone or in composition, are Both showed the ability to reduce cholesterol. These strains have bile salt hydrolase (BHS) activity and are also pathogenic strains (Salmonella enterica Enteritidis), Salmonella enterica Typhimurium, Yersinia sp. It also has antagonist activity to inhibit the growth of Clostridium perfringens, Clostridium ramnosus, Enterococcus faecalis) and can be used as a probiotic bacterium.

  Pereira et al. (See, for example, Non-Patent Document 6). Demonstrating the role of short chain fatty acid concentrations, specifically the molar ratio of propionate and / or bile salt deconjugation, as a major mechanism involved in the properties of L. fermentum that are said to reduce cholesterol did.

  However, the effect of strains of Lactobacillus species on serum cholesterol levels (and cholesterol percentage) is strain-specific and depends on the origin and characteristics of the particular strain (see, for example, Non-Patent Document 7). .

(Probiotics)
Probiotics are viable microbial food ingredients that have been shown to benefit human health when given in appropriate amounts (a joint FAO / WHO drafting guideline for the assessment of probiotics in food (See Section, Non-Patent Document 8, March 14, 2014 search).

  In a narrower definition, probiotics are accepted as living microorganisms that favorably affect the host by improving the gut microbial balance. At present, probiotic products provide numerous host functions (anti-infective properties) by either supporting the physiological activity of the host or reducing the risk of disease to improve health. Strain-specific preparations targeting morphological, immunological and metabolic functions). In general, it has been recognized that the probiotic potential of different strains of the same species may have different probiotic effects (see, for example, Non-Patent Document 9 and Non-Patent Document 10). ).

The most widely applied probiotics belong to the species Bifidobacterium and Lactobacillus. Probiotics are generally consumed as part of fermented foods such as yogurt, kefir, cheese or dietary supplements. The colonic microflora is well stabilized and does not change easily due to the mucosal microflora. However, if given in a sufficient amount (a daily dose of at least 10 ⁹ CFU), it may show the effect of probiotic intervention.

  Treatment concepts with probiotics reduce or eliminate potential pathogens and toxins, release nutrients, antioxidants, growth factors, stimulate gastrointestinal motility, and innate and adaptive It is to regulate immunity.

  Lactobacillus plantarum is widely distributed from naturally fermented plant materials, dairy products and meat products, and also in human GIT (see, for example, Non-Patent Document 11).

  L. Plantarm strains are available as probiotic foods or supplements (L. plantarum 299v, DSM9843, Probi AB, Sweden), which are multispecies probiotic supplements VSL # 3 (VSL Pharmaceuticals, Inc. USA).

  Lactobacillus plantarum Inducia (DSM 21379) strain enhances natural cell immunity and barrier function of gastrointestinal mucosa through secretion of plantaricin, an antimicrobial peptide, induction of cytokine IL-6, production of putrescine However, it has been disclosed as probiotics that increase the amount of lymphoid tissue in the intestine (see Mikesaar et al., Patent Document 2, 2009, Bio-Competence Center of Health Dairy Products).

(Disturbance of microbial ecology of the digestive tract)
It is well known that the application of broad spectrum antimicrobial preparations to treat infections and inflammatory complications can cause serious imbalances among the GI microflora.
(Infection with Clostridium difficile)
In the 1970s, Clostridium difficile was identified as a causative factor for antibiotic-related diarrhea. Clostridium difficile, an anaerobic spore-forming enteric pathogen, is prevalent in hospitals and nursing homes (see, for example, Non-Patent Document 12). C. of exogenous sources Difficile infection is Infection in the form of a difficile spore is triggered. C. Difficile induces disease through the action of secreted toxins produced by vegetative cells rather than spores. Endogenous spore production is essential for the spread of Clostridium difficile infection. These spores must germinate and return to vegetative cell growth in order to cause disease (see, for example, Non-Patent Document 13).

  C. Severe sequelae such as pseudomembranous enteritis (PMC) develop in a quarter (25%) of patients infected with difficile. Clostridium difficile associated diarrhea (CDAD) increases mortality, prolongs hospitalization, and dramatically increases overall health care costs. Previous treatment guidelines have focused on the use of vancomycin in the case of moderate and severe pseudomembranous enterocolitis (see, for example, Non-Patent Document 14, March 14, 2014).

Clostridium difficile infection recurs in approximately 20% of patients, with subsequent recurrence increasing from 40% to 60% (see, eg, Non-Patent Document 15). Antimicrobial treatment disrupts the complex balance of diverse microorganisms, and C.I. It is a major factor in the pathogenesis of difficile colonization and endogenous-derived diseases. Recurrent C.I. Patients with difficile infection (CDI) have significantly reduced bacterial diversity compared to controls (see, eg, Non-Patent Document 16). The maintenance and restoration of microbial diversity is representative of a new strategy. The critical moment for the prevention and treatment of this disease is to find the possibility of reverting gut microbiota changes during and after antibiotic therapy with various regimens including probiotic administration. Most probiotics temporarily settle in the gastrointestinal tract and produce bactericidal acids and peptides that promote “competition” between microorganisms through competition for attachment to nutrients and epithelium. These effects are observed in C.I. It seems to reduce the preference of the environment for difficile. Previous studies have shown that probiotics for CDI prevention are acidophilus and L. L. casei, S. et al. S. bouulardi, or L. It is suggested to include a combination of L. rhamnosus. In addition, more than 10 ⁹ CFU / day dosing is more effective than lower doses.

  The antimicrobial activity of probiotic strains is C.I. This is one of the suggested mechanisms for competition with difficile. Lactic acid bacteria lower the pH of the local gastrointestinal environment and in addition C.I. Produces short-chain fatty acids that prevent adherence of difficile (see, for example, Non-Patent Document 17). Secondly, the possibility of protecting the intestinal barrier by probiotics has been demonstrated by C.I. It may result in stabilization of gastrointestinal permeability by interfering with the binding of difficile toxins A and B, and inhibition of pseudomembrane development on the gastrointestinal epithelium.

  However, not all probiotics have the ability to produce the mechanism of action described above.

  The strain specificity of lactobacilli is C.I. Difficile infection or C.I. It is an important factor to consider when looking for probiotics that are promising in the prevention of difficile toxin binding (see, eg, Non-Patent Document 18).

  In addition, some clinical trials failed to obtain statistical evidence demonstrating the effect of certain probiotics on CDAD prevention. Those who have received probiotics (multistrain preparations of Lactobacillus acidophilus and Bifidobacterium bifidum), including 2941 elderly adults exposed to antibiotics Authors of large randomized trials with known risk did not show a reduced risk for CDI (RR 0.71; 95% CI 0.34-1.47; p = 0.35) (eg, non-patented (Ref. 19). Even after testing with various probiotics to treat CDI for several years, probiotic strains, doses, and durations are still under discussion (see, eg, Non-Patent Document 20). ).

(Application of xylitol)
Xylitol is a 5-C sugar alcohol such as pentitol and is found in plants, fungi and algae. Xylitol is an important intermediate product in mammalian carbohydrate metabolism, ie human blood contains up to 8 × 10 ⁻⁵ M xylitol.

  The consumed xylitol is not completely absorbed, and the unabsorbed part can be used as dietary fiber for bacterial fermentation to convert xylitol into short fatty acid chains utilized in the energy pathway. Xylitol affects the growth of some species of gut microbiota in the large intestine and stimulates the growth and activity of the inherent microbiota. One gram of xylitol has 2.4 kcal, compared to 1 gram of glucose having 3.87 kcal. Xylitol is advertised as “safe” for individuals with diabetes and hyperglycemia (see, for example, Non-Patent Document 21). In our previous study on the CACO-2 cell line, we found that 1% xylitol was C.I. It was discovered that vegetative cell attachment of difficile reference strain VPI10463 seems to prevent attachment of the cells on the cells. In an applied hamster model (see, eg, Non-Patent Document 22), 1 ml of a 20% xylitol solution together with Lactobacillus rhamnosus GG significantly protected the animal from the development of severe enterocolitis (eg, (See Non-Patent Document 22.) These probiotics L. In an empirical study using xylitol in combination with rhamnosus GG, Clostridium difficile vegetative cells were pre-cultured in an anaerobic laboratory and applied to cell culture or hamster inoculation.

  Conversely, in an actual clinical setting or nursing home, C. cerevisiae survives in the aerobic environment of these facilities. Infection occurs by contact with the highly resistant spores of difficile. Spores begin to germinate inside the host intestine.

  Some authors have noted that in animal models, some of the sugars are C.I. It is hypothesized that the expression of difficile toxins A and B can be blocked (see, for example, Non-Patent Document 23).

  Accordingly, there remains a need for probiotic strains that are effective in reducing LDL-cholesterol, especially ox-LDL, as well as reducing the risk of Clostridium difficile infection.

European Patent No. 2,485,743 European Patent No. 2288360

http: // www. mayometicallaboratories. com / articles / communique / 2011/11. htmh Pereira, D.A. I. McCartney, A .; L. Gibson, G .; R An in vitro study of the probabilistic potential of a bill-salt-hydrating Lactobacillus fermentum strain, and deter- mination of the waters. Appl Environ Microbiol. 2003: 69: 4743-4852 Liong, M .; T. T. Shah, N .; P. Bile salt deconjugation abilities, bill salt hydrolase activity and cholesterol co-precipitation abilities of lactobacillus strains. Int Daily J.M. 2005: 15: 391-398 Lye, H .; S. Rahmat-Ali, G .; R. , Liong, M .; T. T. Mechanisms of cholesterol removal by lactobacilli under conditions, that mimic the human gastrointestinal tract, Int Dairy J. 2010: 20 (3): 169-175 Gillian, S.M. E. , Nelson, C .; R. Maxwell, C .; , Association of cholesterol by Lactobacillus acidophilus. Appl Environ Microbiol. 1985: 49, 377-381 Pereira, D.A. I. McCartney, A .; L. Gibson, G .; R. , An in vitro study of the probabilistic potential of a bill-salt-hydrolizing Lactobacillus fermentum strain, and deter- mination of the waters of the water. Appl Environ Microbiol. 2003: 69,4743-4752. Tanaka, H .; K. Doesburg, T .; Iwasaki and I. Mireau, Screening of practical acid bacteria for bill salt hydrolase activity. J. et al. Dairy Sci. 1999: 82: 2530-2535 ftp: /// ftp. fao. org / docrep / fao / 009 / a0512e / a0512e00. pdf Ouwehand, A.M. C, Salminen, S .; , Isolauri, E .; , Probiotics: an over of of the beneficial effects. Antonio Van Leeuwenhoek. 2002: 82, 279-289 Ljungh, A .; , Wadstrom, T .; , Lactic acid bacteria as probiotics. Curr Issues Intest Microbiol. 2006: 7, 73-89 Hammes, W.M. P. , Weiss, N .; and Hertel, The genera Lactobacillus and Carnobacterium. Prokaryotes. 2006: 4: 320-403 Britton, R.A. A. Young, V .; B. , Interaction bethethe the intestinal microbiota and host in Clostridium difficile colonization resistance. Trends Microbiol. 2012: 20, 313-319 Burns D.C. A, Heap J. et al. T. T. Minton N.M. P. Clostridium difficile sporation: an update. Res Microbiol. 2010; 161 (9): 730-4 http: // www. update. com / contents / clostridium-difficile-in-adults-treatment Kelly, C.I. P. LaMont, J .; T. T. , Clostridium difficile-more differential tan ever. N Engl J Med. 2008: 359, 1932-1940 Chang, J. et al. Y. , Antonopoulos, D .; A. , Kalra, A .; Tonelli, A .; Khalife, W., et al. T. T. Schmidt, T .; M.M. Young, V .; B. Decreased diversity of the fecal microbiome in recurrent C.I. difficile-associated dialrhea. J Infect Dis. 2008: 197, 435-438 McFarland, L.M. V. Beneda, H .; W. , Clarridge, J .; E. Raugi, G .; J. et al. Implications of the changing face of C.I. difficile disease for health care practitioners. Am J Infect Control. 2007: 35, 237-253 Tejero-Sarinena S. Barlow J .; , Costabile A, Gibson G. R. , Rowland I .; Antipathogenic activity of probiotics against Salmonella Typhimurium and Clostridium difficile in anaerobic batch culture systems: Is it due to synergies in probiotic mixtures or the specificity of single strains? Anarobe. 2013: 24; 60-65 Allen, S .; J. et al. , Waleham, K .; , Wang, D .; Bradley, C. Hutchings, H .; , Harris, W .; , Dhar, A .; Brown, H .; , Foden, A .; Gravenor, M .; B. Mack, D .; Lactobacilli and bifidobacteria in the prevention of antibiotic-associated diarhoea and clostridium divalente birefringent in ID (PL). Lancet. 2013: 382, 1249-1257 Naber, P.A. , Mikesaar, M .; , Interactions between, Lactobacilli and antibiotic-associated diarhea. Adv Appl Microbiol. 2004: 54 231-260 Talbot J.M. M.M. K. P. Fisher The Need for Special Foods and Sugar Substitutes by Individuals with Diabetes Mellitus. Diabetes Care 1978: 1; 231-240 Naber, P.A. Lehto, E .; Salminen, S .; , Mikesaar, M .; Inhibition of adherence of Clostridium differential to Caco-2 cells. FEMS Immunol Med Microbiol. 1996: 14,205-209 Karlsson, S.M. Burman, L .; G. Aklerund, T .; Induction of toxins in Clostridium difficile is associated with dramatic changes of it's metabolism. Microbiology. 2008: 154, 3430-3436 WHO. The International Task Force. Obesity: Presenting and Managing the Global Epidemi. Report of a WHO Consultation on Obesity. Geneva, Switzerland. WHO / Nut / NCD / 98.1998; 1 NORIP, Rustad P.M. , Felding P .; , Francon L. , Kairisto V .; Lahti A. Martinsson A. , Hytoft Petersen P .; , Simonsson P.M. , Steinsland H, Uldall A .; The Nordic Reference Interval Project 2000: recommended reference intervals for 25 common biochemical properties. Scan J Clin Lab Invest. 2004: 64: 271-284 http: // www. r-project. org

  The present invention is an oxidative stress marker, oxidized low density lipoprotein (ox-LDL), as a cholesterol-lowering agent in preventing cholesterol metabolism disorders by lowering blood LDL-cholesterol levels and ongoing cardiovascular disease. ), 2008 4 for use as an antioxidant for use in enhancing the antioxidant activity of the human body by reducing the level of oxidative stress (OSI) and total peroxide count (TPX). L. deposited on the 16th of March in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH with the registration number DSM21379. A composition comprising Plantarum Inducia strain DSM21379 is provided.

  Therefore, L. Compositions and foodstuffs containing plantarum Inducia DSM 21379 have a cardioprotective effect.

  Another object of the present invention is to prevent endogenous and / or exogenous Clostridium difficile-related diarrhea (CDAD) by preventing germination of Clostridium difficile spores and inhibiting the growth of Clostridium difficile vegetative cells. L. as an antimicrobial agent in reducing the risk of It is to provide a composition comprising Plantarum Inducia strain DSM21379. L. Plantarum Inducia can be used concurrently with antibiotic treatment.

  The composition having resistance to Clostridium difficile may further contain xylitol.

  L. The composition comprising plantarum Inducia can be used for the production of foods, food supplements or pharmaceutical or veterinary products. The food product can be a dairy or meat product, a sweetener, and the like. The dairy product can be a fermented dairy product, cheese, yogurt, and the like.

  Dietary supplements may be used in the form of powders (capsules, lozenges, tablets, powder sachets, etc.) or liquids (ampoules). The strain may be used in lyophilized form.

L. An effective dose of Plantarum Inducia strain DSM21379 is about 1 × 10 ⁹ CFU to 1 × 10 ¹⁰ CFU, preferably 5 × 10 ⁹ CFU per day.

  The term “comprising” as used herein includes “including” as well as “consisting”, eg A composition that includes plantarum Inducia may consist of the strain alone or may include additional ones.

FIG. 5 shows pH values of Inducia in xylitol-containing and control-modified MRS media in a microaerobic and anaerobic environment. By reverse transcription (RT) and real-time PCR amplification (qPCR), C.I. It is a figure which shows presence of the toxA and B gene in a difficile VPI10643. By reverse transcription (RT) and real-time PCR amplification (qPCR), ampicillin and C.I. FIG. 6 shows the presence of the toxB gene in the liver and small intestine of hamsters treated with difficile. a) normal mucosa of the large intestine after probiotic administration and b) C.I. It is a figure which shows the mucous membrane which the difficile infected. FIG. 6 shows morphological changes in experimental CDAD infection. a) moderate hyperemia in the small intestine and b) large intestine, and c) strong hyperemia in the pancreas and d) spleen. e) PMN infiltration at the pseudomembrane in the small intestine and f) at the pseudomembrane in the large intestine.

[Example]
The invention is further illustrated in the following examples, which are not to be construed as limiting the scope of the invention.
I. L. as a cholesterol-lowering agent by reducing LDL-cholesterol in the blood. Use of Plantarum Inducia DSM 21379

Example 1
L. Plantarum Inducia DSM 21379 Bile Salt Hydrolase Activity The purpose of the following in vitro tests is It was to evaluate the presence of plantarum Inducia DSM 21379 bile salt hydrolase activity.

  Method. L. The assessment of the bile salt hydrolase (BSH) activity of Plantarum Inducia DSM 21379 was performed according to Cone Castellana, 2009 (see, for example, US Pat.

Three Lactobacillus strains were cultured overnight on MRS agar at 37 ° C. under microaerobic conditions. After incubation, the culture was standardized to McFarland 3.0. Single strain cultures were assayed for BSH activity. Cultures around paper discs sterilized on MRS agar plates supplemented with 4% (w / v) sodium taurodeoxycholic acid (TDCA, Sigma, USA) and 0.37 g / l CaCl ₂ Impregnated. The plate was incubated anaerobically at 37 ° C. for 72 hours and the diameter of the precipitation zone around the disc was measured. The BSH activity was then calculated according to the formula BSH activity = (IZD-DD) / 2 by subtracting the disc diameter (DD, mm) from the inhibition zone diameter (IZD, mm) and dividing this difference by 2 (Table 1).

  L. The BSH activity of Plantarum Inducia DSM 21379 is described in Reference L. Plantar strains BAA793 and L. It was higher than L. gasseri DSM23882 (Table 1).

(Example 2)
L. Plantar Inducia DSM 21379's ability to reduce LDL-cholesterol Human-intervented study ISRCTN 79645828 “The effect of probiotic yogurt on blood index and microflora of healthy volunteers” was consumed with yogurt containing the strain L. in the blood when It was to assess the ability of Plantarum Inducia DSM 21379 to reduce LDL-cholesterol in the short term (3 weeks).

Yogurt preparation. As an auxiliary starter (inoculation dose is 1 g / t of milk) Probiotic yoghurt was obtained from conditioned pasteurized (+ 92- + 95 ° C. for 5 min) milk using Plantarum Inducia DSM 21379 (2 × 10 ¹¹ CFU / g). Pasteurized milk was quickly cooled to +35 to + 43 ° C. and then mixed with starter cultures and probiotic strains. Milk was fermented to reach pH 4.2-4.5 and cooled to + 23- + 27 ° C. Yogurt was sugared with 5% sugar, placed in a plastic cup and cooled to +2 to + 6 ° C. Yogurt without supplemental probiotics was used as a control.

  Human volunteer trial design. A double blind placebo controlled (DBPC) cross-exploratory study was performed according to the guidelines of the Declaration of Helsinki. The study was approved by the Ethics Review Board (Protocol No. 190T-11, 2010) for human research at Tartu University, Estonia. All participants signed their written informed consent at enrollment and were given the opportunity to withdraw from the study at any time. L. for biomarkers of health status in healthy adults (n = 49). A study was conducted to investigate the effects of yogurt including plantarum Inducia DSM 21379. Within one month prior to the study, participants were asked to continue their regular diet and avoid probiotic products (eg food supplements, yogurt, cheese, kefir, etc.).

Two groups of participants simultaneously received 150 g of test yogurt daily, ie L.P. The consumption of yogurt containing plantarum Inducia DSM 21379 (4 × 10 ⁷ CFU / g) or control yogurt was started for 3 weeks. Probiotic L. The daily dose of Plantarum Inducia was 6 × 10 ⁹ CFU. After a 2-week washout period, volunteers were crossed and consumed probiotic yogurt or control yogurt for an additional 3 weeks.

(Clinical investigation)
Subjects were clinically investigated and plasma samples were collected after an overnight fast to refuse any drugs, tobacco, alcohol and tea or coffee. Each participant was evaluated for anthropometric indicators. Body mass index (BMI) was calculated as weight (kg) divided by height squared (m ² ). Use BMI, the range of normal body weight in healthy volunteers (18.5~24.9kg / m ^2), overweight (≧ 25.0kg / m ²⁾ and obese (≧ 30.0kg / m ² (See, for example, Non-Patent Document 24).

  Samples of fasting blood were taken 4 times, ie at the time of enrollment. Collected after administration of either a product containing Plantarum Inducia DSM 21379 or a control product, after a washout period, and after administration of a control or probiotic product at the end of the study.

  Hematological indicators: plasma lipids: total cholesterol, LDL-cholesterol (LDL-c), HDL-cholesterol (HDL-c) and triglycerides certified in the local clinical laboratory (United Laboratories of Clinical Clinics, Estonia) Determined by standard experimental methods using the assay described. The interval for routine laboratory testing proposed by the Nordic Reference Interval Project (see, eg, Non-Patent Document 25) was used as a reference.

(Statistical analysis)
Statistical analysis by using R2.10.1 (eg, see Non-Patent Document 26, searched March 25, 2014) and GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA) Went. All data were expressed as mean and standard deviation (mean ± SD). Baseline and intervention data were compared by paired t test or Wilcoxon rank sum test according to the data distribution.

  Differences were considered statistically significant when the value was p <0.05.

  The two groups of healthy volunteers in the cross-test were not different in terms of their clinical data (Table 2).

L. The major change after consumption of plantarum Inducia DSM 21379 was revealed by the content of LDL-cholesterol particles. L. After consumption of yogurt containing plantarum Inducia DSM 21379, LDL-cholesterol content decreased (Table 3).

  Therefore, L. Consumption of Plantarum Inducia DSM 21379 yogurt reduced the LDL-cholesterol level in the blood by 9.1%.

(Example 3)
For oxidative stress markers oxidized low density lipoprotein (oxLDL), degree of oxidative stress (OSI) and total peroxide count (TPX). Effect of yogurt containing plantarum Inducia DSM 21379.

During a parallel intergroup double-blind study IRCTN 26344255, adults with mild hypercholesterolemia (LDL-cholesterol> 3.0 mmol / L) (n = 136) were treated with 150 g of probiotics (L Plantarum Inducia DSM 21379 daily dose was 5 × 10 ⁹ CFU) or control yogurt was consumed. The short term effect of the probiotic product was measured 4 weeks after the intervention and the sustained effect was measured at the end of the study, ie 8 weeks after the intervention.

  The study was approved by the Ethics Review Board (Protocol No. 207 / M-11 19.09.2011) for human research at the University of Tartu, Estonia.

L. at an average daily dose of 5 × 10 ⁹ CFU. Consumption of 150 g yogurt containing plantarum Inducia DSM 21379 was between 4th and 8th week (−0.12 mmol / L; p = 0.04) and from baseline to 8th week (−0.09 mmol / L) P = 0.029) resulted in stable LDL-cholesterol reduction in the probiotic group (Tables 4a and 4b).

  Since ox-LDL is a marker of oxidative stress specific to LDL particles, ox-LDL can be regarded as one of the biochemical risk markers for coronary heart disease. L. Administration of yogurt with plantarum Inducia DSM 21379 resulted in a stable intra-ox reduction of ox-LDL in the probiotic yogurt group throughout the 8-week intervention (Tables 5a and 5b). From baseline to week 8, the change was statistically significant (-4.10 U / L; p = 0.003). The change was also significant for placebo (p = 0.026). No effect on oxLDL was recorded in the placebo group.

  L. Administration of probiotic yogurt containing plantarum Inducia DSM 21379 also resulted in a significant reduction (−5.37; p <0.001) of oxidative stress (OSI) from baseline to week 8 in the probiotic group. (Tables 6a and 6b). The change was also significant for placebo (p = 0.04). No effect was recorded in the placebo group.

  Throughout the study, a stable significant intergroup reduction in total peroxide count (TPX) was recorded in the probiotics (ie, probiotics containing L. plantarum Inducia) group (Tables 7a and 7b). No effect was recorded in the placebo group. No significant change in total antioxidant capacity was detected (Tables 8a and 8b).

Conclusion. According to human intervention studies, L. A composition comprising Plantarum Inducia DSM 21379 has a cardioprotective effect through the reduction of LDL-cholesterol in the blood, while at the same time protecting the human body from oxidative damage and oxLDL, OSI and oxidative stress markers Strengthens antioxidant activity by lowering TPX.
II. C. L. as an antimicrobial agent by preventing germination of difficile spores and proliferation of vegetative cells. Use of Plantarum Inducia strain DSM21379

Example 4
C. in an anaerobic environment in vitro. L. difficile reference strain for vegetative cells Antagonism of Plantarum Inducia DSM 21379 The aim of the study was to investigate C. aeruginosa in anaerobic environment. L. difficile reference strain It was to evaluate the antagonist activity of Plantarum Inducia DSM 21379.

  Method. The strains tested are Plantarum Inducia DSM 21379 and C.I. Difficile VPI 10463 (ATCC 43255), M13042 highly toxic strain.

Strains were planted on solid media and incubated in an anaerobic environment. A suspension of bacteria in saline was prepared according to MacFarland standard 3 (10 ⁸ CFU / ml). A suspension (0.2 ml) in 200 ml BHI medium was planted to reach a concentration of 10 ⁵ CFU / ml.

At various time scales, MRS and LAB160 media were seeded with bacteria for growth (0.1 ml). Plates were incubated under anaerobic and microaerobic conditions. Growth was checked after 2-5 days and results were expressed as log ₁₀ CFU / ml.

  L. Plantar Inducia DSM 21379 according to C.I. Complete suppression of difficile vegetative cells was found after co-culture in BHI medium and was further inoculated into MRS and CD LAB180 selective medium. Plantarum Inducia DSM 21379 showed a maximum value in BHI medium at 24 hours. 48 hours later, Plantum Inducia DSM 21379 is grown in a single culture (6.3 log CFU / g) or C.I. When combined with the difficile strain (7.3 and 6.0 log CFU / g), it was equally moderately suppressed (Table 9).

(Example 5)
Effect of Lactobacillus plantarum Inducia DSM 21379 Supernatant on Clostridium difficile reference and vegetative cells of clinical strains in vitro The purpose of the following in vitro experiments is the reference and clinical C.I. L. difficile strain It was to determine the antimicrobial action of Plantarum Inducia DSM 21379 vegetative cells. L. The distinction between the suppressive action of the plantarum Inducia DSM 21379 natural (acidic) supernatant and neutralized supernatant (inhibitor, eg peptide) is due to two mechanisms: the effect of organic acids or the presence of bacteriocin To help identify.
Materials Co., Ltd. (Table 10)
L. Plantarum Inducia DSM 21379
Clostridium difficile reference strains VPI10463 and M13042
Clostridium difficile clinical strain (11 strains)
Method. By microtiter plate (MTP) assay, reference and clinical C.I. L. difficile strain The antimicrobial activity of the supernatant of plantarum Inducia DSM 21379 was determined.

L. Plantarum Inducia DSM 21379 was maintained at −80 ° C. in a microtube on glass beads and activated in MRS liquid medium containing 0.15% agar in a short time under microaerobic conditions (10% CO ₂ ). The mixture was incubated at 37 ° C. for 24 hours. Overnight L. Plantarum Inducia DSM 21379 cultures were used to inoculate BHI liquid medium at 1% v / v and incubated for 24 hours in microaerobic conditions. L. To detect the antimicrobial activity of Plantarum Inducia DSM 21379, extracellular cell-free supernatant (CFS) was collected by centrifugation from a BHI liquid medium culture after 24 hours. The pH of the cell-free supernatant was measured and divided in half. One half was left acidic, the other half was neutralized with 6N NaOH to pH 6.0, and both supernatants were filter sterilized.

C. The difficile strain is maintained on glass beads in a microtube at −80 ° C. in an anaerobic environment (anaerobic glove box, gas is 90% N: 5% CO ₂ : 5% H ₂ ) It was activated in a short period of time over 24 hours with blood-supplemented anaerobic agar (FAA). Overnight C.I. The difficile culture was used for a suspension having a density according to MacFarland 3.0. L. To evaluate the antimicrobial activity of plantarum Inducia DSM 21379, 20 μl C.I. Difficile cell suspension was prepared in 180 μl: (1) BHI liquid medium (as a control), (2) cell-free BHI supernatant, (3) cell-free neutralized BHI supernatant, (4) diluted none Cell supernatant (1: 1 with sterile BHI liquid medium) and (5) diluted and neutralized cell-free supernatant (1: 1 with sterile BHI liquid medium) were added.

C. The growth of difficile (change in optical density value) was measured at OD _{620 nm} after 48 hours using an MTP reader and the growth rate was calculated.

L. using the Kruskal-Wallis test. The inhibitory activity of plantarum Inducia DSM 21379 was tested and C.I. The growth density (OD _{620 nm} ) of the difficile control was determined by comparing the C. difficile control with C. Compared with the data on the growth density of difficile. Statistical analysis of the data was performed using a program provided by the PAST Statistics Web.

  Clinical strain: C.I. Difficile CDE, CDP1-9; reference strain: C.I. Difficile VPI 10463 (ATCC 43255) and C.I. Vegetative cells of difficile M13042 (a pandemic strain from Canada belonging to ribotype 027).

  Therefore, L. Plantarum Inducia DSM 21379 is a strain of antimicrobials that is still active after acid production in natural products (decreasing pH) and, to a lesser extent, neutralization of the supernatant (neutralized product). For both sex protein-like substances, C.I. Has antimicrobial activity against difficile (Table 10).

(Example 6)
L. in a medium containing xylitol. Plantar Inducia DSM 21379 Growth and pH Value Testing The purpose of the study was that xylitol was C.I. L. for difficile It was to find out whether it would affect the antagonist activity of Plantarum Inducia DSM 21379.

Inducia (10 ⁵ CFU / ml) was incubated in MRS medium with glucose replaced with 5% xylitol or used without sugar in a microaerobic and anaerobic environment. The number of lactobacilli was recorded as CFU per ml of medium. We have described L. in vitro. Whether Plantarum Inducia DSM 21379 uses xylitol for growth in microaerobic and anaerobic conditions was tested at 2, 6, 24, and 48 hours.

  Using a medium supplied with or without xylitol at 24 hours, L. There was no difference in the growth of Plantarum Inducia DSM 21379, growing best in 5% xylitol medium at 24 hours in the microaerobic environment, but 2 logs lower than the glucose-containing medium in the anaerobic environment (Table 11).

  From this experiment, L. Plantar Inducia DSM 21379 proved not to metabolize xylitol effectively in MRS medium. L. in a modified MRS medium containing 5% xylitol. The pH change after growth of plantarum Inducia DSM 21379 was tested.

The lowest pH value due to organic acid production was seen in glucose-containing control medium incubated in an anaerobic environment. In xylitol-containing media, there was still a pH drop from pH 6.2 to pH 5.0 under both microaerobic and anaerobic conditions, but nevertheless of the glucose-containing control media from pH 7.5 to pH = 3.2. It was substantially different from pH (FIG. 1).
L. Use of Xylitol in the Metabolism of Plantarum Inducia DSM 21379 The purpose of this study was to investigate L. It was to measure the use of xylitol by Plantarum Industia DSM 21379.

  Method: L. in MRS medium containing 5% xylitol in a microaerobic and anaerobic environment for 2 to 120 hours. Duplicate experiments were performed with plantarum Inducia DSM 21379 culture. Xylitol was detected using mass spectrometry QTRAP3200 (Applied Biosystems, USA). Samples were centrifuged at 10000 g for 3 minutes, diluted 100-fold, and 50 μl was mixed with 50 μl internal standard (5 mM D4-succinic acid in acetonitrile, 50 μl). 5 μl of the diluent was injected into the mass spectrometer using 50% acetonitrile / water eluent. Substances were identified by multiple reaction monitoring (MRM) ion pairs 151/101 (xylitol) and 121/77 (internal standard). The concentration was calculated from a calibration curve prepared with a solution having a known concentration of commercially available xylitol (Sigma-Aldrich, Germany).

  According to repeated experiments, Inducia used xylitol in a very low amount since the sugar content was maintained at a change of basal value ± max 0-0.6 mM (Table 12).

Therefore, the present inventors have used L.L. Plantarum Inducia has been confirmed to metabolize xylitol in very low amounts.
C. in an environment containing xylitol and ampicillin. Growth of difficile reference strains The purpose of this study was to develop elaborate C. It was to simulate the gastrointestinal environment similar to the difficile infection model in vitro.

  In vitro in Brainhart medium (BHI). Plantarum Inducia DSM 21379 and C.I. The effect of xylitol and ampicillin on the growth of difficile reference strains (VPI 10463 and highly virulent ribotype 027 strain M13042) was tested.

  L. Plantarum Inducia DSM 21379 and two C.I. The difficile reference strain increased CFU numbers by approximately 2 to 4 logs after 48 hours in control BHI medium. C. No changes were observed after xylitol and ampicillin were allowed to act on the difficile strain (Table 13).

  L. Plantarum Inducia strain DSM21379 was inhibited by approximately 2 logs under both 5% xylitol and 0.75 μl / ml ampicillin. Thus, survival of L plantarum Inducia was observed in the gastrointestinal microenvironment administered antibiotic ampicillin and up to 5% xylitol.

(Example 7)
L. by co-culture with different concentrations of xylitol and ampicillin. Plantarum Inducia DSM 21379 and C.I. Growth of the difficile reference strain The purpose of this study was to investigate the growth of L. cerevisiae by co-culture with different concentrations of xylitol and ampicillin. Plantarum Inducia DSM 21379 and C.I. It was to evaluate the growth of the difficile reference strain.

  BHI medium supplemented with 5% xylitol and 0.75 μl / ml ampicillin was treated with L. in an anaerobic environment (workstation Concept 400, UK) for 24 and 48 hours. Plantarum Inducia DSM 21379 and C.I. Application to co-culture of difficile reference strain.

Single L. pneumoniae in BHI medium containing 5% ampicillin and xylitol. Plantarum Inducia DSM 21379, C.I. Viability after co-culture of difficile VPI and M reference strain was tested (1, 2a, 2b). Then, L. 10-fold serial dilutions in MRS liquid medium to determine plantarum Inducia number in a CO ₂ environment; In the case of difficile, it was cultured in LAB160 medium in an anaerobic environment.

  In vitro in BHI medium containing xylitol and ampicillin. Plantarum Inducia DSM 21379 (3) and C.I. During co-culture of the difficile reference strain (4, 5) (simulating a hamster model), Growth of vegetative cells of the difficile strain was suppressed. Complete suppression can be detected in VPI strains, C.I. In the case of difficile strain M, a 5 log substantial reduction (5.0 to 2.0 log CFU / ml) was detected.

  Therefore, from in vitro experiments simulating the gastrointestinal environment of the experimental hamster model after antibiotic treatment, Plantarum Inducia DSM 21379 is used alone (Table 5) or in combination with xylitol. Either complete or high inhibition of difficile growth was shown (Table 14).

(Example 8)
The combination of Lactobacillus plantarum Inducia DSM 21379 and xylitol inhibits germination of Clostridium difficile reference strain VPI spores.

  The purpose of this experiment is Prevent germination of Clostridium difficile when plantarum Inducia DSM 21379 is preincubated in culture medium It was to evaluate the ability of Plantarum Inducia DSM 21379. The effects of xylitol and taurocholate on the germination of Clostridium difficile spores were examined. Taurocholate is a natural substance in the small intestine that promotes germination of Clostridium difficile spores.

  L. Plantarum Inducia DSM 21379 was incubated with Brain Heart Infusion (BHI) liquid medium containing various drugs in anaerobic conditions for 24 hours. L. After incubation of plantarum Inducia DSM 21379, Clostridium difficile spores were added to the mixture and BHI liquid medium was concentrated at a ratio of 1:10.

  Experimental and control mixtures were incubated for up to 48 hours in anaerobic conditions. After 24 and 48 hours, the mixture was planted on Fastiodious anaerobic agar and human Rogosa Sharpe agar at the start of the experiment. L. The number of plantarum Inducia DSM 21379 and Clostridium difficile was calculated.

  In a medium containing xylitol or a combination of xylitol and taurodeoxycholic acid. Plantarum Inducia DSM 21379 preincubation inhibits the germination of Clostridium difficile (Table 15). L. Plantarum Inducia DSM 21379 was not affected by the presence of xylitol and / or taurodeoxycholic acid. These results support the results of animal experiments.

Example 9
Animal Experiments The purpose of this study is C.I. L. to infection caused by Clostridium difficile reference strain VPI10463 in the intestine of hamsters challenged with difficile spores. It was to evaluate the effects of Plantarum Inducia DSM 21379.

  Syrian Hamster (Mesocritus auratus) provides a well-characterized model of Clostridium difficile infection. The microbiota of the hamster colon treated with antibiotics is destroyed and then C.I. When exposed to S. difficile spores, animals will be C.I. It develops difficile-associated diarrhea (CDAD) and the mortality rate is 100%.

We follow L. in accordance with our in vitro results. By administering Plantarum Inducia DSM 21379, C.I. We tested whether the mortality of hamsters attacked with difficile can be reduced. First, the inventors have determined that L.I. at a concentration of 10 ¹⁰ cfu / ml with / without 1 ml of 20% xylitol for 5 consecutive days before administration of ampicillin (30 mg / kg). Plantarum Inducia DSM 21379 was given daily to hamsters by gavage. L. at the above concentrations. Administration of plantarum Inducia DSM 21379 was continued during the entire experiment. On the seventh day, C.I. Ten to thirty spores of difficile VPI 104631 were infected. In the next 5 days, the health status of the hamster is followed, and in the case of symptoms showing CDAD: wet tail, C.I. A difficile A / B toxin test was performed (X / pect Remel test). C. A group of hamsters infected with the difficile VPI strain was used as a control group.

  Therefore, C.I. Before attacking with difficile spores, Maximum survival was found when the combination of Plantarum Inducia DSM 21379 and xylitol solution was given in advance (Table 16). In this case, all surviving toxin tests were negative in viable hamsters (7/9), ie C.I. Sprouting of difficile spores was suppressed. When xylitol alone was applied, 4 of 5 surviving animals tested negative for toxins and only 1 surviving animal tested positive for toxins.

  Thus, xylitol and L. The combination with Plantarum Inducia DSM 21379 is capable of producing toxins. It functions by suppressing spore germination of difficile into vegetative cells.

  The inventors have described C.I. even after intervention of Inducia and xylitol. Whether C. difficile growth is indicated, and C.I. Whether difficile A and B toxins were found (Table 17) was tested. L. In living hamsters pre-given with the combination of plantarum Inducia DSM 21379 and xylitol, C.I. No difficile vegetative cells were found in the jejunum and ileum in 6 out of 7 cases (only 1 case showed C. difficile growth). Anaerobic bacteria replaced by native lactobacilli in the jejunum and ileum and L. Since both the numbers of plantarum Inducia DSM 21379 were high, ampicillin administration, C.I. Attack with difficile spores, and It is shown that the gut microbiota was reconstituted and stabilized after use of treatment with plantarum Inducia DSM 21379 and xylitol.

  The survival rate of hamsters is It was 22% higher for Plantarum Inducia DSM 21379 (Table 16). This was also evidenced by the absence of toxins in the intestinal contents of the large intestine (Table 17).

L. Plantarum Inducia DSM 21379 growth was high in the jejunum (range 0-6.0, median 4.0 CFUlog ₁₀ / g, ileum 6.0-7.6, median 6.7) . L. Plantarum Inducia DSM 21379 was acting by its acid production according to its SCFA profile.
C. Morphological evaluation of hamsters infected with difficile Since organ damage by toxins is a major virulence modulator in difficile infection, a typical CDAD infection was modeled in a hamster model.

  In our study, C.I. S. difficile VPI 10463 has both toxins A and B, which are also present in the liver and small intestine (FIGS. 2 and 3).

  C. In hamsters that survived difficile infection and had not been examined for extensive mucosal damage during pathological dissection, there was no severe infiltration with pseudomembrane and polymorphonuclear cells (FIG. 4a).

  C. a few hours before death. The characteristic morphological findings of hamsters damaged by difficile infection were inflammation due to infiltration of polymorphonuclear cells in the mucosa and the presence of pseudomembrane.

  Severe enterocolitis caused infiltration of the gastrointestinal mucosa, liver and spleen by erythrocytes and polymorphonuclear leukocytes and the animals died. In the organ, hyperemia occurred (Figs. 5a-f).

Claims (15)

  Used in the prevention of cholesterol metabolism disorders and ongoing cardiovascular disease by lowering LDL-cholesterol levels in the blood, and at the same time of oxidized low density lipoprotein (ox-LDL), and the degree of oxidative stress (OSI) A composition comprising the probiotic microorganism, Lactobacillus plantarum Inducia strain DSM21379, for use in enhancing the antioxidant activity of the human body by reducing levels.   A probiotic microorganism, lactate for use in reducing the risk of Clostridium difficile-associated diarrhea (CDAD) by preventing germination of Clostridium difficile spores and inhibiting the growth of Clostridium difficile vegetative cells A composition comprising Bacillus plantarum Inducia strain DSM21379.   The composition of claim 2 further comprising xylitol.   The composition according to claim 1 or 2, selected from the group consisting of a pharmaceutical composition, a food composition, and an animal composition.   A food comprising the composition according to claim 1 or 2.   The food according to claim 5, wherein the food is a dairy product.   The food according to claim 6, wherein the food is a fermented dairy product.   The food according to claim 7, wherein the food is cheese.   The food according to claim 7, wherein the food is yogurt.   A food supplement comprising the composition according to claim 1, 2 or 3.   A pharmaceutical or animal product comprising the composition according to claim 1, 2 or 3   Used as a cholesterol-lowering agent in the prevention of cholesterol metabolism disorders and ongoing cardiovascular disease by lowering LDL-cholesterol levels in the blood; Plantarum Inducia DSM 21379.   Used as an antioxidant in enhancing the antioxidant activity of the human body by reducing the level of oxidized low density lipoprotein (ox-LDL), L. Plantarum Inducia DSM 21379.   In order to reduce the risk of Clostridium difficile-associated diarrhea (CDAD) from endogenous and / or exogenous origin, to prevent germination of Clostridium difficile spores and to inhibit the growth of Clostridium difficile vegetative cells, Lactobacillus plantarum Inducia DSM 21379 used as an antimicrobial agent.   15. Lactobacillus plantarum Inducia DSM 21379 according to claim 14, for use as an antimicrobial agent together with xylitol.