US20110274789A1 - Isolated lactobacillus plantarum strain inducia dsm 21379 as probiotic that enhances natural immunity and food products and medicinal preparations comprising it - Google Patents

Isolated lactobacillus plantarum strain inducia dsm 21379 as probiotic that enhances natural immunity and food products and medicinal preparations comprising it Download PDF

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US20110274789A1
US20110274789A1 US12/992,862 US99286209A US2011274789A1 US 20110274789 A1 US20110274789 A1 US 20110274789A1 US 99286209 A US99286209 A US 99286209A US 2011274789 A1 US2011274789 A1 US 2011274789A1
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lactobacillus plantarum
cheese
dsm
microorganism
probiotic
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Marika Mikelsaar
Epp Songisepp
Imbi Smidt
Jelena Stsepetove
Pirje Hutt
Mihkel Zilmer
Kai Truusalu
Kalle Kilk
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Tervisliku Piima Biotehnologiate Arenduskeskus OU
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Assigned to TERVISLIKU PIIMA BIOTEHNOLOOGIATE ARENDUSKESKUS OU (BIO-COMPETENCE CENTRE OF HEALTHY DAIRY PRODUCTS) reassignment TERVISLIKU PIIMA BIOTEHNOLOOGIATE ARENDUSKESKUS OU (BIO-COMPETENCE CENTRE OF HEALTHY DAIRY PRODUCTS) ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HUTT, PIRJE, KILK, KALLE, MIKELSAAR, MARIKA, SMIDT, IMBI, STSEPETOVA, JELENA, TRUUSALU, KAI, ZILMER, MIHKEL, SONGISEPP, EPP
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    • A23C19/05—Treating milk before coagulation; Separating whey from curd
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    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
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    • A23V2400/00—Lactic or propionic acid bacteria
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    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the present invention relates to the field of biotechnology and application in food industry and medicine.
  • the present invention relates to a microorganism Lactobacillus plantarum strain Inducia DSM 21379 as probiotic, which produces polyamines, nitric mono-oxide and antioxidative compounds, improves the intestinal barrier function, intestinal mucosa and blood cellular immunity and induces pro-inflammatory cytokines.
  • the invention relates to its use in the composition of a functional food, e.g. in milk products and in a pharmaceutical composition enhancing natural immunity of organism through improvement of the intestinal barrier function and enhancement of intestinal mucosa and blood cellular immunity.
  • probiotics are live microorganisms, which, when administered in adequate scientifically proven amounts, confer a health benefit on the host. Commonly probiotics are used for creating functional foods. Probiotic products may be conventional foods (yoghurt, quark, curds) or dietary supplements (freeze-dried microbial cultures). Probiotics are often used for enhancement of organisms' defence capability.
  • Enhancement of organisms' natural immunity has become essential in connection with the ageing of population and diseases connected with immunodeficiency (HIV infection, tissue transplantation induced immunosuppression). Infections, disturbances in metabolism and tumorigenic processes are observed more frequently in elderly (Hebuterne X., Gut changes attributed to ageing: effects on intestinal microflora. Curr. Opin. Clin. Nutr Metab Care, 2003; 6 (1):49-54; Wenzel, R. P., Health care—associated infections. Major issues in early years of 21 st century. Clin Inf. Dis., 2007; 45: S85-S88).
  • the permeability of the intestinal mucosa frequently increases on the background of chronic inflammation.
  • the mentioned condition is a basis for the generalization of intestinal infections due to autoallergic reactions and microbial translocation.
  • Microbial metabolites play essential role in the integrity of mucosa, e.g. short chain fatty acids (SCFA), produced by lactic acid bacteria in the colon in the case of fiber-rich (substances of plant origin) diet (Roy C C, Kien C L, Bouthillier L., Levy E. Short chain fatty acids: ready for prime time? Nutr. Clin. Pract., 2006; 21:351-366).
  • SCFA short chain fatty acids
  • Polyamines are linear aliphatic compound, in which amino acids are situated along the structure. Putrescine, spermidine and spermine belong to polyamines (Larqué, M., Sabater-Molina, S. Zamora E. Biological significance of dietary polyamines. Nutrition 2007; 23(1): 87-95). Polyamines are produced by decarboxylation from amino acids ornithine and arginine. Putrescine is produced straight from ornithine; arginine is primarily converted into agmantine which is then converted into putrescine (Halaris A, Plietz, Agmatine: metabolic pathway and spectrum of activity in brain. CNS Drugs, 2007; 21: 885-90). Diamine oxidase metabolizes putrescine into gamma-butyric acid, which is an essential molecule in neurons metabolism.
  • NO nitricmono-oxide
  • the system NOS—NO could possess hyperemia-dependent protection mechanism against stress-induced damage of intestinal mucosa (Brzozowski T., Brzozowski, Konturek P., C., Sliwowski Z., Drozdowicz D., Burnat G., Pajdo R., Pawlik M., Bielanski W., Kato I., Kuwahara A., Konturek S. J. and Pawlik W. W. Gastroprotective action of orexin-A against stress-induced gastric damage, is mediated by endogenous prostaglandins, sensory afferent neuropeptides and nitric oxide. Regul Pept 2008 in press).
  • the production of polyamines by the intestinal microflora is considered one of the compensatory mechanisms for modification of immune response and apoptosis regulation.
  • Putrescine is produced by several anaerobes, Escherichia coli and lactobacilli. Lactobacilli comprise majority of microflora of the proximal colon (Marteau, P., Pochart, P., Dore, J., Bera-Maillet, C., Bernalier, A. and Corthier, G. Comparative study of bacterial groups within the cecal and fecal microbiota. Appl. Env. Microbiol. 2001; 67, 4939-4942).
  • Lactobacilli produce polyamines through decarboxylation of amino acids, particularly at the high pH of the intestinal content (Lonvaud-Funel A, Biogenic amines in wine: role of lactic acid bacteria. FEMS Microbiol. Letters, 2001, 199: 9-13).
  • strains of Lactobacillus acidophilus utilize putrescine and reduce odour of faces (WO 2008/019887, BASF AG).
  • Japanese patent application JP2006191808 discloses putrescine production technology with enterobacteria.
  • Probi AB Estonian patent EE03597 discloses pharmaceutical composition that contains L.
  • Lactobacillus rhamnosus GG could enhance NO production in the epithelial cells of the intestine or by proinflammatory cytokines and it has been indicated, that some beneficial effects of Lactobacillus rhamnosus GG could be due to the production of NO by macrophages and epithelial cells (Korhonen K, Reijonen T M, Remes K, Malmström K, Klaukka T, Korppi M. Reasons for and costs of hospitalization for paediatric asthma: A prospective 1-year follow-up in a population-based setting. Pediatr Allergy Immunol 2001; 12:331-338).
  • Probiotics for elderly are usually targeted for the reduction of chronic inflammations by using immune suppressing and IL-10 inducing bacteria (bifidobacteria, Lactobacillus acidophilus ) e.g. Valio Ltd.
  • Estonian patent application EE200300351 discloses composition of bacteria ( L. rhamnosus G G, L. rhamnosus DSM LC705 7061, Propionibacterium freudenreichii ssp. Shermani DSM7067 and Bifidobacterium infantist DSM13642) together with galacto-oliogosaccharides (GOS) for improvement of general health status and stimulation of immune system.
  • OOS galacto-oliogosaccharides
  • TNF-alfa and IL-6 were Lactobacillus GG and another L. rhamnosus strain E509 (Miettinen M., Vupio-Varkila J, Varkila K. Production of TNF-alpha and interleukin ⁇ 6 and interleukin ⁇ 10 is induced by lactic acid bacteria. Infection & Immunity, 1996, 64: 5403-5405).
  • Bifidobacterium lactis LKM512 comprising yoghurt administration to elderly decreased the glucoprotein haptoglobuline caused inflammatory acute phase response due to IL-1, IL-6 and TNF-alfa, but the probiotic administration was also accompanied by decrease of mutagenicity of the intestinal epithelial cells.
  • Lactobacillus plantarum is a widely spread representative of the genus Lactobacillus .
  • Aforementioned lactobacillus species is present on fermented plants (sauerkraut, pickles, and silage), fermented dairy/meat products (cheese, salami) as well as in human gastrointestinal tract (Bottazzi, V. Other fermented dairy products. Food and feed production with microorganisms. Biotechnology. Verlag Chemie, Weinhein, Ed. Reed G. 1983, 5: 315-364; Hammes, W. P., Weiss, N., Holzapfel, W. The genera Lactobacillus and Carnobacterium . The Procaryots. Springer—Werlag, Heidelberg, New York, Eds.
  • Korean patent applications KR20020072913 and KR20050080630 discloses a mix of lactobacilli, where belongs also Lactobacillus plantarum , at least one of its cytoplasm fraction or the strain itself is able to inhibit cell lines of stomach and colon cancer.
  • Antimicrobial and antioxidative probiotic cheese has been produced by using Lactobacillus fermentum ME-3 (DSM 14241) (Estonian patent EE04580, Russian patent RU2284354, U.S. Pat. No. 6,190,879).
  • DSM 14241 Lactobacillus fermentum ME-3
  • U.S. Pat. No. 6,190,879 discloses microorganism from the genus Streptomyces for production of transglutaminase and a method for incorporation putrescine into casein during cheese production.
  • European patent EP1064857B1 (Snow Brand Milk Products Co Ltd., 2004) discloses methods for production substances incl. putrescine with lactobacilli, bifidobacteria and propionibacteria from Gouda cheese milk ultrafiltration. These methods are either polymerization reactions for incorporating putrescine into casein or vice versa—purification of these compounds by ultrafiltration, that have already been produced into milk, methods are different from this one described in present invention, where the putrescine, that has been produced by lactobacilli into cheese milk is still present in cheese after 30 days of ripening.
  • the present invention relates to a novel isolated microorganism strain Lactobacillus plantarum Inducia DSM 21379 as probiotic, its functional properties, food product (e.g. cheese) and composition comprising the said microorganism and application of the said microorganism for production of medicine for enhancing cellular immunity.
  • L. plantarum Inducia DSM 21379 produces polyamines from ornithine and glutamate, nitric mono-oxide (NO) from arginine, possesses antioxidative activity and with produced polyamines, nitric mono-oxide and antioxidative compounds improves the intestinal bather function, increases the number of immunocytes in blood and induces cytokine synthesis for enhancement of organisms' natural defence.
  • the MRS broth was suitable for cultivation of the strain during 24-48 h in microaerobic environment, after which homogenous turbid growth occurred in the broth.
  • Colonies on MRS agar plates after 48 h of growth at 37° C. in microaerobic conditions are round, 2-2.5 mm of diameter, sooth, entire, convex and white.
  • the optimal growth temperature is 37° C.; it multiplies also at 15° C. and 45° C.
  • the optimal pH of the growth environment is 6.5.
  • Lactobacillus plantarum strain Inducia DSM 21379 is catalase and oxidase negative, facultatively heterofermentative, no gas is produced from glucose and no arginine hydrolysis.
  • Strain Lactobacillus plantarum Inducia DSM 21379 was identified on the basis of biochemical activity with API 50CHL System (bioMérieux, France) kit as Lactobacillus plantarum (Coincidence with the type strain: excellent, ID %-99.9, T index ⁇ 0.81) and molecular identification by ITS-PCR (Internal-Transcribed Spacer Polymerase Chain Reaction).
  • the comparison with the reference strain Lactobacillus plantarum ATCC 14917 confirmed the preliminary identification by API 50CHL
  • Carbohydrate utilization profile of Lactobacillus plantarum Inducia DSM 21379 according to API CHL 50 is following. Positive reaction for: ribose, galactose, D-glycose, D-fructose, D-mannose, mannitol, sorbitol, ⁇ methyl-D-mannoside, ⁇ methyl-D-glucoside, N acetyl-glucosamine, amygdalin, arbutine, esculine, salitsin, cellobiose, maltose, lactose, melibiose, sachharose, trehalose, melezitose, ⁇ -gentiobiose, D-turanose, gluconate. Weak reaction for starch.
  • Lactobacillus plantarum Inducia DSM 21379 possesses leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, ⁇ -glucosidase, ⁇ -glucosidase activities and acetoin activity.
  • Lactobacillus isolates were prepared using lysozyme (Serva, Sweden; 20 mg/ml), mutanolysin (Sigma; 0.5 mg/ml) and proteinase K solutions (Fermentas, Lithuania; 14.6 mg/ml).
  • the DNA amplification was performed in a reaction volume of 50 ⁇ l containing 1 ⁇ Taq polymerase buffer (Fermentas, Lithuania), 1.5 U Taq polymerase (Fermentas), 0.5 ⁇ M of each primer (16S-1500F and 23S-32R; DNA Technology AS), 200 ⁇ M deoxynucleoside triphosphates (Amersham Pharmacia Biotech, Germany), 2 mM MgCl 2 and 2 ⁇ l of extracted DNA. Subsequently, the PCR product was restricted using a Taq I restriction enzyme (Fermentas Lithuania).
  • DNA fragments were separated by electrophoresis (1.5 h, 100 V) on a 2% agarose gel in 1 ⁇ TBE [Tris(Hydroxymethyl)aminomethane-borate/disodium ethylendiamine tetraacetate] buffer. The banding patterns were visualized and visually compared with reference stain L. plantarum ATCC 14917.
  • Lactobacillus plantarum Inducia was deposited in Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH under the registration number DSM 21379 at 16.04.2008.
  • PFGE pulsfield-gel-electrophoresis
  • the DNA extraction from Lactobacillus isolates was performed in EC buffer (50 mM EDTA (pH 8.5), 0.5% Na-laurylsarcosine, 0.2% Na-deocycholate 2 mg/ml lysozyme, 10 U mutanolysin), followed by 1 mg/ml proteinase K solution (100 mM EDTA-1% sarcosyl-0.2% deoxycholate, pH 8.0) containing buffer. Lysed probes were washed in TE buffer and cut to 2 mm, extracted overnight with enzyme 50 U Not I (Bio-Rad). The electrophoresis was carried out in CHEF-DR II (Bio-Rad) for 22 h at 14° C. The banding patterns were visualized in UV light illuminator ( FIG. 2 ).
  • Lactobacillus plantarum Inducia DSM 21379 was tested using antibiotic strips of the E-test (AB Biodisk, Solna). The MIC (minimal inhibitory concentration) was determined according to European Commission (EUC) suggested epidemiological break-points.
  • EUC European Commission
  • Electrochemical measurements of H 2 O 2 were carried out with 24 h old intact cells in 500 ⁇ l of MRS broth with Apollo 4000 free radical analyzer (WPI, Berlin, Germany) and electrodes of type ISO-HPO2 and ISO-NOP.
  • Lactobacillus plantarum Inducia DSM 21379 produces substantial amounts, of acetic acid, lactic acid and H 2 O 2 in comparison with the control and which are essential for the trophic of the colon mucosa.
  • TAA and TAS of the microbial cells were incubated in MRS broth (Oxoid, U.K.) for 24 h at 37° C. Microbial cells were harvested by centrifugation (1500 RPM, during 10 min) at 4° C. and the pellet was washed with isotonic saline (4° C.) and suspended in 1.15% KCl (Sigma, USA). The density of the suspension was OD 600 of 1.1 ⁇ 10 9 bacterial cells ml ⁇ 1 ). Total antioxidative activity (TAA) was assessed by using the linolenic acid test (LA-test).
  • TABLE 4 Total antioxidative activity (TAA) and total antioxidative status (TAS) of Lactobacillus plantarum Inducia DSM 21379 Strain TAA (%) TAS (mmol/l) L. plantarum Inducia DSM 21379 26 ⁇ 1.2 0.13 ⁇ 0.04 L. plantarum DSM 21380 22 ⁇ 5 0.05 ⁇ 0.02
  • Nitrogen mono-oxide production measurements were carried out with 24 h old intact cells in 500 ⁇ of MRS broth with Apollo 4000 free radical analyzer and electrodes (WPI, Berlin, Germany).
  • ISO-NOP electrode signals were registered during 5-7 minutes. Mean signal strength was calculated. Each experimental point was measured in 4 independent parallels and each parallel was measured twice. NO concentration was calculated according to the standard curves correlation with the strength of the electrodes signal.
  • Lactobacillus plantarum Inducia DSM 21379 was the best NO producer among the tested heterofermentative lactobacilli.
  • Microbial strains were suspended in physiological saline according to McFarlandi turbidity standard (10 9 CFU/ml) and 0.5 ml of each strain suspension was seeded into decarboxylation medium (a 4.5 ml) and incubated at 37° C. for 4 days (Bover-Cid and Holzapfel W. H. Improved screening procedure for biogenic amine production by lactic acid bacteria. Int J food Microbiol 1999; 53, (1); 33-41(9)).
  • FIG. 2 L. plantarum Inducia DSM 21379 molecular fingerprints in comparison with L. plantarum control strain (Pulse field-gel-electrophoresis profile, PFGE).
  • FIG. 4 Production of polyamines in cheese by Lactobacillus plantarum DSM 21379 (detected according to the method of Nakovich, L. Analysis of biogenic amines by GC/FID and GC/MS. Thesis, Va. Polytechnic Institute, USA. 2003)
  • FIG. 5 Lactobacillus species by Pearson UPMAG cluster analyses in L. plantarum Inducia DSM 21379 group.
  • FIG. 6 IL-6 and blood monocytes
  • Microorganism Lactobacillus plantarum Inducia DSM 21379 was added to the cheese milk of Dairy Cooperative E-Piim, (inoculation dose 3 ⁇ 10 8 CFU/vat) and the milk was renneted (25 min). The curds were cut (25 min), heated (34° C. 15 min), dried (25 min), pressed, drained (1 h), salted in brine (12° C.; 20% NaCl; pH 4.7) 20 h, drained and dried (8 h), backed into plastic and ripened at 12° C. for at least 4 weeks.
  • putrescine and tyramine were related to that: in different lots in comparison with control-cheese putrescine content increased 3-11 times and tyramine content accordingly 2-5 times.
  • Lactobacillus plantarum Inducia DSM 21379 belongs to the facultatively heterofermentative group of lactobacilli and therefore the content of tyramine in cheeses was significantly lower than that of strains of OHEL group. On the other hand, the content of putrescine was higher.
  • Permitted concentration of tyramine in food e.g. in cheese is 200 mg/kg (Karovicova and Kohajdova. Biogenic amines in food. Chem pap. 2005; 59 (1); 70-79; Larque, M., Sabater-Molina, S. Zamora E. Biological significance of dietary polyamines. Nutrition 2007; 23(1): 87-95).
  • Tyramine is considered toxic in concentrations of 1000-8000 mg/kg.
  • Putrescine is considered toxic if detected in organism in concentration of 2000 mg/kg per body weight and total toxicity of polyamines is >300 mg/kg per food product (Larqué, M., Sabater-Molina, S. Zamora E. Biological significance of dietary polyamines. Nutrition 2007; 23(1): 87-95).
  • Lactobacillus plantarum Inducia DSM 21379 comprising cheese the subject gets ca 3 mg of putrescine.
  • the concentration is up to 50 ⁇ g per kg, which does not express toxic effect.
  • Tissue samples from the liver, spleen, ileum and colon were fixed in formalin and embedded in paraffin.
  • Microtome-cut tissue samples were stained with hematoxyline-eosine.
  • mice organs after administration of Lactobacillus plantarum Inducia comprising cheese.
  • Mice group Liver Spleen Ileum Colon L. plantarum Inducia Hyperaemia ii Lymph Lymph DSM 21379 comprising 6/10* follicles follicles cheese consumed 6/10* 8/10* animals Control cheese Hyperaemia Hyperaemia Lymph Lymph consumed animals 3/10* 1/10 follicles follicles 3/10* 4/10* *p ⁇ 0.05
  • Lactobacillus plantarum Inducia DSM 21379 comprising cheese during 1 month enhanced liver hyperemia and raised significantly lymphatic follicles (immunocytes) of mice ileum and colon in comparison with control mice. These results refer to enhancement of the defence capability of intestinal mucosa and liver functions.
  • the clinical trial with healthy volunteers evaluated the impact of putrescine, NO and antioxidative compounds producing Lactobacillus plantarum Inducia DSM 21379 comprising food consumption on (Estonian cheese) 1) safety for the consumer; 2) humoral and cell immunity parameters of blood; 3) effect on intestinal microbiota; 4) and urine metabolites, to detect possible health-promoting effects.
  • the study group consisted of 12 healthy volunteers, both male and female (M/F 4/8) aged 20-48 years.
  • diabetes glucose and glycohemoglobin HbA1c from blood sera were detected.
  • the trial was randomized double-blind cross-over study. Trial started with 3-week consumption of test cheese. Volunteers consumed the test cheese for 3 weeks. After a 2-week washout period, volunteers were crossed over to another 3 weeks of control cheese administration. Lactobacillus plantarum Inducia DSM 21379 content in 30 old test cheeses was 6 ⁇ 10 7 CFU/g.
  • test cheese Before consumption the test cheese was incubated with Lactobacillus plantarum Inducia DSM 21379 for 30 days at 12° C. Regular Estonian cheese of Edam type without Lactobacillus plantarum Inducia DSM 21379 served as a control.
  • the trial was a randomized blinded cross-over placebo controlled trial. Trial started with 3-week consumption of test cheese, followed by 2 week washout period, after which the control cheese was consumed for 3 weeks in a dose 50 g/day.
  • Lactobacillus plantarum DSM 21379 comprising cheese does not cause systemic inflammation, allergic sensibilisation or causes harm to essential organs.
  • Microbial DNA was isolated from cheese by QIAamp DNA Mini Kit (QIAGEN) and amplified with primers Uni-515-GC-rev (ATCGTATTACCGCGGCTGCTGGCA-GC), Lab-159-f (GGAAACAGA/GTGCTAATACCG) (Hilor H G, Zoetendal E G, Vaughan E E, Marteau P, Akkermans, A D L, de Vos W M, /et al./Molecular diversity of / Lactobacillus /ssp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA./Appl Envir Microbiol/2002; 68: 114-123).
  • Lactobacillus plantarum Inducia DSM 21379 comprising cheese affects the composition of human intestinal lactoflora.
  • Lactobacillus plantarum strain Inducia DSM 21379 is able to produce putrescine in vitro as well as in cheese. Consumption of probiotic Lactobacillus plantarum Inducia DSM 21379 cheese elevated the content of acetylputrescine in urine of trial participants.
  • Acetylputrescine represents a detoxified compound, elevated content of which proves putrescine production by Lactobacillus plantarum Inducia DSM 21379 in gastrointestinal tract of volunteers or absorption and metabolism of additional amounts of putrescine, consumed with cheese. On the other hand, this indicates the successful adaptational reaction of organism to deal with superfluous amounts of putrescine by excreting it with urine in acetylated form.
  • the immunostimulative effect of putrescine produced by Lactobacillus plantarum Inducia DSM 21379 was confirmed by the correlation between blood cytokine IL-6 and the quantity of macrophages (monocytes), which in this case could be considered activated macrophages.
  • the finding mentioned together with H 2 O 2 is essential for the organisms' defence against foreign cells (microbes, cancer cells).
  • Physiological doses of putrescine occurring in the gut due to Lactobacillus plantarum Inducia DSM 21379 could theoretically enhance the regeneration of the epithelium of intestinal mucosa and apoptosis of old cells, thus avoiding the hyperproliferation of epithelium. These mechanisms ensure the barrier function of intestinal mucosa and protects against penetration of allergens.

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US11419893B2 (en) 2013-11-26 2022-08-23 Central Biomedia, Inc. Method of producing a designer blood product, method of using a designer blood product, and diet for selectively enhancing blood profile
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US9730969B2 (en) 2015-11-06 2017-08-15 Mead Johnson Nutrition Company Nutritional compositions for promoting gut barrier function and ameliorating visceral pain
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Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MIKELSAAR, MARIKA;SONGISEPP, EPP;SMIDT, IMBI;AND OTHERS;SIGNING DATES FROM 20111221 TO 20111222;REEL/FRAME:027526/0789

STCB Information on status: application discontinuation

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