U.S. patent application number 12/992862 was filed with the patent office on 2011-11-10 for isolated lactobacillus plantarum strain inducia dsm 21379 as probiotic that enhances natural immunity and food products and medicinal preparations comprising it.
Invention is credited to Pirje Hutt, Kalle Kilk, Marika Mikelsaar, Imbi Smidt, Epp Songisepp, Jelena Stsepetove, Kai Truusalu, Mihkel Zilmer.
Application Number | 20110274789 12/992862 |
Document ID | / |
Family ID | 40957913 |
Filed Date | 2011-11-10 |
United States Patent
Application |
20110274789 |
Kind Code |
A1 |
Mikelsaar; Marika ; et
al. |
November 10, 2011 |
ISOLATED LACTOBACILLUS PLANTARUM STRAIN INDUCIA DSM 21379 AS
PROBIOTIC THAT ENHANCES NATURAL IMMUNITY AND FOOD PRODUCTS AND
MEDICINAL PREPARATIONS COMPRISING IT
Abstract
The present invention relates to a microorganism Lactobacillus
plantarum strain Inducia DSM 21379 as probiotic, its functional
properties, food product (e.g. cheese) and composition comprising
said microorganism and use of said microorganism for production of
medicine for enhancing cellular immunity. L. plantarum Inducia
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 barrier function, increases number of
immunocytes in blood and induces cytokine production for
enhancement of organisms' natural defense.
Inventors: |
Mikelsaar; Marika; (Tartu,
EE) ; Songisepp; Epp; (Tartu, EE) ; Smidt;
Imbi; (Tartumaa, EE) ; Stsepetove; Jelena;
(Tartu, EE) ; Hutt; Pirje; (Tartu, EE) ;
Zilmer; Mihkel; (Tartu, EE) ; Truusalu; Kai;
(Tartu, EE) ; Kilk; Kalle; (Tartu, EE) |
Family ID: |
40957913 |
Appl. No.: |
12/992862 |
Filed: |
May 12, 2009 |
PCT Filed: |
May 12, 2009 |
PCT NO: |
PCT/EE09/00006 |
371 Date: |
July 26, 2011 |
Current U.S.
Class: |
426/61 ;
435/252.9 |
Current CPC
Class: |
A23C 19/054 20130101;
A23C 19/062 20130101; A23C 9/1234 20130101; A23K 10/18 20160501;
A61K 35/747 20130101; A61K 2035/115 20130101; C12R 1/25 20130101;
A23V 2002/00 20130101; A61P 1/12 20180101; A61P 3/06 20180101; A23L
33/135 20160801; A61P 31/04 20180101; A23Y 2220/67 20130101; A23C
19/061 20130101; C12N 1/20 20130101; A61K 31/047 20130101; A23V
2002/00 20130101; A23V 2200/00 20130101; A23V 2200/3204 20130101;
A23V 2200/324 20130101; A23V 2200/326 20130101; A23V 2200/3262
20130101; A23V 2250/6422 20130101 |
Class at
Publication: |
426/61 ;
435/252.9 |
International
Class: |
C12N 1/20 20060101
C12N001/20; A23C 19/00 20060101 A23C019/00; A23C 9/152 20060101
A23C009/152; A23C 9/123 20060101 A23C009/123 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2008 |
EE |
P200800027 |
Claims
1. An isolated probiotic microorganism strain Lactobacillus
plantarum Inducia DSM 21379 that produces polyamines from ornithine
and glutamate and nitric mono-oxide (NO) from arginine.
2. The probiotic microorganism strain of claim 1 in freeze-dried
form.
3. The isolated probiotic microorganism of claim 1, wherein said
microorganism possesses antioxidative activity.
4. The isolated probiotic microorganism of claim 1, wherein said
microorganism is capable of inducing adaptively proinflammatory
cytokines.
5. The isolated probiotic microorganism of claim 1, wherein said
microorganism is capable of enhancing the natural defense potential
of human organism.
6. The isolated probiotic microorganism of claim 1, wherein said
microorganism is capable of improving the intestinal barrier
function and increasing cellular immunity of the intestinal mucosa
and blood.
7. A composition for enhancing the natural defense potential of a
human organism and cellular immunity comprising the probiotic
microorganism of claim 1.
8. The composition of claim 7, which is selected from the group
consisting a pharmaceutical composition and a food composition.
9. A food product comprising the probiotic microorganism of claim
1.
10. The food product of claim 9, wherein the food product is a
dairy product.
11. The food product of claim 10, wherein the food product is a
fermented milk product.
12. The food product of claim 11, wherein the food product is
cheese.
Description
TECHNICAL FIELD
[0001] 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.
BACKGROUND ART
[0002] During the past decades lactobacilli have widely been used
as probiotics in functional foods. Food can be regarded as
functional, if beyond adequate nutritional components it contains
some natural additives (pre- or probiotics), which beneficially
affect one or more target functions in the body, either improving
the state of health and well-being and/or reducing disease risk.
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.
[0003] 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.sup.st century. Clin Inf. Dis., 2007; 45: S85-S88).
[0004] The cause of all of mentioned diseases is the decrease of
the capability of several physiological functions of the organism
(Timiras, P. S. Physiology of aging: standards for age-related
functional competence In: Comprehensive Human Physiology. Greger, R
(edt)/Windhorst, U (eds) Springer Verlag, 1996; pp 2391-2405).
Ageing is regarded as a decrease of stress-resistance and weakened
immune response. First of all, this affects the counts of defence
cells (leucocytes, macrophages, lymphocytes, trombocytes)
circulating in human body and the decrease of the activity of
phagocytes as well as the decrease of the barrier function of
mucosa (Hebuterne X., Gut changes attributed to ageing: effects on
intestinal microflora. Curr. Opin. Clin. Nutr Metab Care, 2003; 6
(1):49-54; Fang H., Intestinal microbiota of the elderly. In:
Gastrointestinal Microbiology: The Intestinal Microbiota of Elderly
Edited by A. Ouwehand and E. Vaughan 2006; pp 75-91).
[0005] 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). Butyrate, produced by eubacteria
and clostridia is essential for enhancement of barrier function of
the mucosa, likewise the effect of fiber-rich diet on colonic
microflora, which has been described in several studies (O'Keefe S
J D. Nutrition and colonic health: the critical role of the
microbiota. There is strong evidence for the ability of diet to
influence the colonic microbiota and cancer risk as measured by
epithelial proliferation rate. Current Opin Gastoenterol. 2008; 24:
51-58).
[0006] For enhancement of mucosal barrier besides short chain fatty
acids also polyamines are essential. Polyamines are linear
aliphatic compound, in which amino acids are situated along the
structure. Putrescine, spermidine and spermine belong to polyamines
(Larque, 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.
[0007] Besides, arginine serves as the source of nitricmono-oxide
(NO); 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).
[0008] The physiological impact of polyamines is targeted to cell
growth and differentiation, regulation of immune cells and
inflammatory response, polyamines possess also anti-allergic,
protein synthesis-stimulating, nucleic acid structure stabilizing
and enzyme activity controlling effect. Polyamines reduce the
development of ulcers in intestine and improve the intestinal
mucosa, thus reducing its permeability for macromolecules.
Polyamines possess the ability to induce apoptosis, avoiding the
hyperproliferation of epithelium and the development of primary
cancer cells (Moinard C, Cynober L, De Bandt J P Polyamines:
metabolism and implications in human disease. Clin Nutr., 2005; 24:
184-197). Polyamines are produced either endogenously or they are
obtained actively from food. Only 20% of consumed putrescine is
absorbed from intestine into bloodstream, accompanied by the raise
of acetylated putrescine, while majority is metabolized by liver
and enterocytes (Milovic V, Odera G, Murphy G M, Dowling R H
Jejunal putrescine absorption and the
pharmakokinetic/biotransformation of ingested putrescine in humans.
Gut, 1997; 41: A63). It is important especially for elderly people,
whose endogenous polyamine-synthesizing ability is lowered, to gain
polyamines with food. Abundance of putrescine, spermidine and
spermine can be found in acetylated or oxidized form in urine
(Seiler N Catabolism of polyamines. Amino Acids 2004; 26: 217-233).
Putrescine is considered toxic in concentrations of 2000 mg/kg;
however, putrescine and cadavereine can also intensify the toxic
effect of tyramine and histamine. Therefore, >300 mg/kg per food
is considered the total toxicity of polyamines (Larque, M.,
Sabater-Molina, S. Zamora E. Biological significance of dietary
polyamines, Nutrition 2007; 23(1): 87-95).
[0009] In the case of the damage of epithelial cells, 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). On the other
hand, strains of Lactobacillus acidophilus utilize putrescine and
reduce odour of faces (WO 2008/019887, BASF AG). Japanese patent
application JP2006191808 (Toyota Motor Corporation) discloses
putrescine production technology with enterobacteria. Probi AB
Estonian patent EE03597 discloses pharmaceutical composition that
contains L. plantarum strains 299 and 299v together with arginine
for prevention translocation of intestinal microbes during liver
injury. These strains of L. plantarum resemble over 70% (REA) to L.
plantarum strain 299 and they are able to colonise human intestinal
mucosa in vivo. In this patent no information is available
concerning the end products of arginine utilization (putrescine,
cadaverine, tyramine, enhancement of NO or antioxidativity) by L.
plantarum, which are responsible for aforementioned effect. In
close relationship with previous patent another Probi AB patent
EE04097 discloses the strains L. plantarum 299v and 299, which can
colonize the mucosa due to mannose-specific adhesins. This patent
also does not disclose the polyamines or NO production ability of
mentioned L. plantarum strains.
[0010] However, due to administration of high amounts of arginine
daily (3.4 g/daily), significant raise of NO is observed in
comparison with lower doses (3.2 g/daily) (Jablakad A. Checinski
P., Krauss H., Mickerbe M. Astcef J. The influence of two different
doses of L-arginine oral supplementation on nitric oxide (N=9)
concentration and total antioxidant status (TAS) in atherosclerotic
patients. Clinical Research, 2004). It has been demonstrated, that
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, Malmstrom 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). It has been demonstrated, that NO protects
mucosa for damages and excessive permeability, arising after
reperfusion (Payne D, Kubes P. Nitric oxide donors reduce the rise
in reperfusion-induced intestinal mucosal permeability. Am J.
Physiol. 1993; 265 (1 Pt 1):G189-G195).
[0011] USA patent application US20060078595 (Friesland Brands B.V.)
discloses method to avoid the excessive permeability of the
intestinal bather in newborns by glutamate and its precursors as
well as by polyamines spermidine, spermine, putrescine in the case
of different syndromes (malnutrition, allergy, sepsis,
translocation of microbes, endotoxemia, viral diarrhoea).
Lactobacillus Reuteri (BIOGAIA) served as glutamate source.
[0012] Only a few probiotics are available for elderly population.
The counts of intestinal lactobacilli were higher in the regularly
probiotics consuming elderly in comparison with these who did not
consume probiotics (Soovares, P., Salusaar L, Kolk H, Sepp E,
Kullisaar T, Vihalemm T, Zilmer M, Mikelsaar M. Kas soole
laktobatsillide hulk ja soomisharjumused mojutavad ateroskleroosi
riski eakatel isikutel? Eesti Arst, 2007; 9: 704). 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. No data is presented which
components of immune system were stimulated, however from the
publication it turned out that the best stimulators of 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).
[0013] Polyamine spermidine has inflammation-lowering property. It
has been demonstrated that spermidine, when added to human
monocytes stimulated with lipopolysaccharides, inhibits effectively
the synthesis of TNF, IL-1, IL-6 and other proinflammatory
cytokines (Zang M, Caragine T. Wong H et al. Spermine inhibits
proinflammatory cytokine synthesis in human mononuclear cells: a
counter regulatory mechanism, that restrains the immune response.
J. Exp. Med., 1997, 185: 1759-1768). Matsumoto with co-authors
described the suppression of proinflammatory cytokine synthesis
(Matsumoto M, Ohisshi H, Benno Y Impact of LKM512 yoghurt on
improvement of intestinal environment of the elderly. FEMS immunol.
Medical Microbiol, 2001; 31:181-186). Excessive suppression of
proinflammatory cytokine synthesis could be harmful, as it lowers
activation of lymphocytes and natural killers acting against cancer
cells. 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.
Besides, polyamines improve the apoptosis of epithelial cells in
order to avoid hyperproliferation, an essential prerequisite to
colon cancer development (Hamilton-Miller J M T, Probiotics and
prebiotics in the elderly. Postgrad. Med., 2004; 80:447-451; Gill H
S, Darragh A J, Cross M. Optimizing immunity and gut function in
the elderly. J. Nutr. Health Aging 2001, 5: 60-91). Due to the
aforementioned effects the consumption of polyamines-rich diets by
elderly is contradictory. At the same time it is evident, that
different lactic acid bacteria incl. lactobacilli species and
strains differ by their ability to induce pro- and
anti-inflammatory cytokines and non-specific cellular immune
response. Up to now, no lactobacillus species/strain has been
described, which would be able to produce physiologically relevant
amounts of polyamines, which could be detected in urine after the
consumption of this particular strain comprising composition and
which promote simultaneously the adaptive activation of immunocytes
due to interleukin IL-6.
[0014] Proinflammatory cytokine IL-6 synthesis has been described
after 24 h of stimulation with different strains of Bifidobacterium
animalis and Lactobacillus rhamnosus (Miettinen M., Vuopio-Varkila
J, Varkila K. Synthesis of human tumour necrosis factor alpha,
interleukin-6 and interleukin-10 is induced by lactic acid
bacteria. Infection and Immunity, 1996, 64:5403-5408). It is
important to observe inflammation markers like counts of leucocytes
(WBC) and amount of CRP in sera on the induction of IL-6
(Kiecolt-Glaser J K, Preacher K J, MacCallum R C et al. Chronic
stress and age-related increases in the proinflammatory cytokine
IL-6, PNAS, 2003; 100:9090-9095) to avoid the overproduction of
IL-6. Aforementioned is associated with cardio-vascular diseases,
arthritis, type II diabetes, cancer, periodontal diseases, cachexy
and decrease of organisms functions (Rose-John S., J. Scheller, G.
Elson, and S. A. Jones. Interleukin-6 biology is coordinated by
membrane-bound and soluble receptors: role in inflammation and
cancer J. Leukoc. Biol., Aug. 1, 2006; 80 (2): 227-236).
[0015] 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. Balows A., Trupes H. S., Drowkin M.,
Schleifer K-H., 1992; 2: 1535-1594; Xanthopoulos, V., Hatzikamari,
M., Adamidis, T., Tsakalidou, E., Tzanteakis, N.,
Litopoulou-Tzanteaki, E. Heterogeneity of Lactobcillus plantrum
isolates from Feta cheese througout ripening. J. Appl. Microbiology
2000, 88: 1056-1064). Lactobacillus plantarum is able to reorganize
its metabolism according to environmental conditions. Probiotic
Lactobacillus plantarum is available in probiotic foods as well as
in food supplements (e.g. Lactobacillus plantarum 299v DSM 9843,
Probi AB, Sweden, Skanemejeriers' ProViva probiotic brand in Sweden
or as one of the components in bacterial composition VSL#3 (VSL
Pharmaceuticals, Inc. USA). WO2007/108764 (Probac AB) discloses the
action mechanisms of Lactobacillus plantarum strains, which are
able to enhance immunotolerance in the case on autoimmune coeliac
disease. 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.
[0016] Cheese as a probiotic carrier has several controversial
aspects. Incorporation lactobacilli of human origin into a food
product different from other milk-based products and having a long
ripening period could be complicated (Gardiner, G, Ross, R. P,
Collins, J. H, Fitzgerald, G, Stanton. C. Development of a
probiotic cheddar cheese comprising human derived Lactobacillus
paracasei strains, Appl. Environ. Microbiol., 1998, 64, 6:
2192-2199; Madkor, S., A., Tong, P. S., El Soda, M. Ripening of
Cheddar cheese with attenutaed adjunct cultures of Lactobacilli. J.
Dairy Sci. 1999; 1684-1691). At the same time the fat and
protein-rich cheese matrix protects a probiotic microbial strain
throughout the passage of gastrointestinal tract better than other
milk products (yoghurt, kefir).
[0017] 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). 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.
[0018] 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. Various non-starter
lactobacilli have been described (Lactobacillus paracasei,
Lactobacillus curvatus), which are able to gain energy for
proliferation from ornithine (ornithine is released from milk
casein arginine) after depletion of carbohydrates (Laht T.-M., Kask
S., Elias P., Adamberg K., Paalme T. Role of arginine in the
development of secondary microflora in Swiss-type cheese. Int.
Dairy Journal, 2002, 12: 831-840).
[0019] Thus, till now no lactobacillus species/strain have been
described, the culture of which produces NO and additionally
physiologically relevant amounts of polyamines in food product,
whereas the latter could be detectable in urine after the
consumption of this strain comprising food product (cheese) or
composition and that is able to regulate through polyamines the
apoptosis of intestinal epithelium and increase the count of the
mucosal lymphatic follicles and blood monocytes, regulating the
condition of mucosa by NO and antioxidative compounds and to
enhance the activation of immune cells particularly due to the
activation of macrophages by central interleukine.
DISCLOSURE OF THE INVENTION
[0020] 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.
[0021] The Lactobacillus plantarum strain DSM 21379, which is the
object of the investigation, was isolated from a faecal sample of a
healthy child during a comparative study of the microflora of
Estonian and Swedish children. The microorganism strain
Lactobacillus plantarum Inducia DSM 21379 was isolated by seeding
the dilutions of the faeces of a healthy one-year old Estonian
child (10.sup.-2-10.sup.4 in phosphate buffer with 0.04% thioglycol
acid; pH 7.2). The dilutions were seeded on freshly prepared MRS
agar medium and cultivated at 37.degree. C. microaerobically. The
strain, which is the object of the investigation, was isolated
according to the characteristic morphology of colonies and cell to
Lactobacillus sp. A provisional and more precise identification
followed as described next.
[0022] The fact that the microbial strain originates from the
intestinal tract of a healthy child proves its GRAS (generally
recognized as safe) status i.e. that this strain of microorganism
is harmless for human organism and is suitable for oral
application.
[0023] Cultural--morphological characteristics were detected after
cultivation on MRS agar and in MRS broth (OXOID). Cells of
Lactobacillus plantarum Inducia DSM 21379 are Gram-positive,
non-spore-forming rods of regular shape, occurring singly, in pairs
or in short chains.
Physiological-Biochemical Characteristics
[0024] 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.degree. C. in microaerobic conditions
(atmosphere CO.sub.2/O.sub.2/N.sub.2: 10/5/85) are round, 2-2.5 mm
of diameter, sooth, entire, convex and white.
[0025] The optimal growth temperature is 37.degree. C.; it
multiplies also at 15.degree. C. and 45.degree. C. The optimal pH
of the growth environment is 6.5.
[0026] Lactobacillus plantarum strain Inducia DSM 21379 is catalase
and oxidase negative, facultatively heterofermentative, no gas is
produced from glucose and no arginine hydrolysis.
[0027] Strain Lactobacillus plantarum Inducia DSM 21379 was
identified on the basis of biochemical activity with API 50CHL
System (bioMerieux, 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
[0028] 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, .alpha. methyl-D-mannoside, .alpha.
methyl-D-glucoside, N acetyl-glucosamine, amygdalin, arbutine,
esculine, salitsin, cellobiose, maltose, lactose, melibiose,
sachharose, trehalose, melezitose, .beta.-gentiobiose, D-turanose,
gluconate. Weak reaction for starch.
[0029] Negative reaction for L-arabinose, D-raffinose glycerol,
erythrol, D-arabinose, D-xylose, L-xylose, adonitol, .beta.
methyl-xyloside, L-sorbose, rhamnose, dulcitol, inositol, inulin,
glycogen, xylitol, D-lyxsose, D-tagatose, D-fucose, L-fucose,
D-arabitol, L-arabitol, 2 keto-gluconate, 5 keto-gluconate.
[0030] According to API ZYM test-kit (bioMerieux, France)
Lactobacillus plantarum Inducia DSM 21379 possesses leucine
arylamidase, valine arylamidase, acid phosphatase,
naphthol-AS-BI-phosphohydrolase, .alpha.-glucosidase,
.beta.-glucosidase activities and acetoin activity. Weak reaction
for valine arylamidase, naphthol-AS-BI-phosphohydrolase,
.beta.-galactosidase, cystine arylamidase, esterase (C4), esterase
(C8), N-acetyl-.beta.-glucosaminidase was detected.
[0031] The molecular identification of Lactobacillus plantarum
Tensia DSM 21379 was confirmed by Internal-Transcribed Spacer
Polymerase Chain Reaction (ITS-PCR). The banding pattern of the
isolates was visually compared with that of the reference strain
Lactobacillus plantarum ATCC 14917 (FIG. 1).
[0032] Method. Strain identification was confirmed by (ITS-PCR)
Internal-Transcribed Spacer Polymerase Chain Reaction in comparison
with the reference strain Lactobacillus plantarum ATCC 14917.
[0033] The DNA extraction from Lactobacillus isolates was performed
using lysozyme (Serva, Sweden; 20 mg/ml), mutanolysin (Sigma; 0.5
mg/ml) and proteinase K solutions (Fermentas, Lithuania; 14.6
mg/ml).
[0034] The DNA amplification was performed in a reaction volume of
50 .mu.l containing 1.times.Taq polymerase buffer (Fermentas,
Lithuania), 1.5 U Taq polymerase (Fermentas), 0.5 .mu.M of each
primer (16S-1500F and 23S-32R; DNA Technology AS), 200 .mu.M
deoxynucleoside triphosphates (Amersham Pharmacia Biotech,
Germany), 2 mM MgCl.sub.2 and 2 .mu.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.times.TBE [Tris(Hydroxymethyl)aminomethane-borate/disodium
ethylendiamine tetraacetate] buffer. The banding patterns were
visualized and visually compared with reference stain L. plantarum
ATCC 14917.
[0035] Lactobacillus plantarum Inducia was deposited in Deutsche
Sammlung von Mikroorganismen and Zellkulturen GmbH under the
registration number DSM 21379 at 16.04.2008.
Pulsfield-Gel-Electrophoresis (PFGE) Profile
[0036] Method. For pulsfield-gel-electrophoresis (PFGE) procedure
the lactobacillus strains were grown in MRS broth at 37.degree. C.
for 24 h. The cells were washed in SE buffer (75 mM NaCl, 25 mM
EDTA, pH=7.4), density of the cell suspension was adjusted to 1.5
(OD.sub.600). 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.degree. C. The
banding patterns were visualized in UV light illuminator (FIG.
2).
Antibiotic Resistance
[0037] Method. 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.
TABLE-US-00001 TABLE 1 Antibiotic resistance of Lactobacillus
plantarum Inducia DSM 21379 MIC* MIC* (.mu.g/ml) (.mu.g/ml) L.
plantarum Control strain EUC antibiotic resis- Inducia L. plantarum
tance MIC* breakpoint Antibiotic DSM 21379 DSM 21380 values
(.mu.g/ml) Ampicillin 0.19 0.25 4 Gentamicin 1 1.5 64 Streptomycin
6 16 64 Erytromycin 0.25 0.19 4 Clindamycin 0.016 0.032 2
Tetracycline 6 8 32 Chloramphenicol 2 2 8 Cipro/ofloxacin 32 32 4
*minimal inhibitory concentration
[0038] In vitro study of antibiotic susceptibility showed that
Lactobacillus plantarum Inducia DSM 21379 did not express
antibiotic resistance against most important antimicrobial
preparations. A somewhat higher MIC for ciprofloxacin was detected,
revealed in normal range of wild strains described previously
(Vankerckhoven V, Huys G, Vancanneyt M, Vael C, Klare I, Romond
M-B, Entenza J M, Moreillon P, D. Wind R, Knol J, Wiertz E, Pot B.,
Vaughan E. E, Kahlmeter G, Goossens H. Biosafety assessment of
probiotics used for human consumption: recommendations from the
EU-PROSAFE project. Trends in Food Science & Technology 2008;
19: 102e114). Therefore, no horizontal transfer of antibiotic
resistance genes of Lactobacillus plantarum Inducia DSM 21379
during the application of the strain as a probiotic could be
predicted.
Antimicrobial Activity
[0039] Lactobacillus plantarum Inducia DSM 21379 expresses in vitro
on MRS agar medium antagonistic activity against several enteric
pathogens (Table 2).
TABLE-US-00002 TABLE 2 Lactobacillus plantarum Inducia DSM 21379
antimicrobial activity against pathogens and non-starter
lactobacilli on modified MRS agar medium (pathogen growth
inhibition zone, mm) Pathogen Growth inhibition zone (mm)
Non-starter lactobacilli (NSLAB) 2.67 .+-. 3.4 Listeria
monocytogenes 22.7 .+-. 2.4 Yersinia enterocolitica 11.2 .+-. 2.7
Salmonella enteritidis 22.1 .+-. 1.9 S. typhimurium 20.8 .+-. 2.8
Shigella sonnei 24.0 .+-. 0.1 Escherichia coli 23.0 .+-. 1.4
Enterobacter sakazakii 18.1 .+-. 1.8 Campylobacter jejuni 12.0 .+-.
7.6
[0040] Lactobacillus plantarum Inducia DSM 21379 antimicrobial
activity in vitro in streak-line procedure (antimicrobial effect of
metabolites) was highest against E. coli, followed by growth
inhibition of Salmonella sp., Shigella and Listeria. The lowest
antimicrobial activity was detected against other lactobacilli
(NSLAB).
Functional Properties
The Profile of Metabolites
[0041] Method. The profile of Lactobacillus plantarum Inducia DSM
21379 metabolites was determined by gas chromatographic method
(Hewlett-Packard 6890) after incubation in microaerobic milieu for
24 h and 48 h (Table 2). L. plantarum strain was grown on MRS agar
for 48 h microaerobically (10% CO.sub.2). A suspension (McFarland
4.0 turbidity standard, 10.sup.9CFU/ml) of lactobacillus culture
was prepared in 0.9% NaCl solution. 1.0 ml of this suspension was
transferred to 9.0 ml of MRS broth. The metabolite concentration
(mmol/l) was detected using the capillary column HP--INNOWax (15
m.times.0.25 mm; 0.15 .mu.m). The column temperature program was
60.degree. C. 1 min, 20.degree. C./min 120.degree. C. 10 min;
detector (FID) 350.degree. C.
[0042] Electrochemical measurements of H.sub.2O.sub.2 were carried
out with 24 h old intact cells in 500 .mu.l of MRS broth with
Apollo 4000 free radical analyzer (WPI, Berlin, Germany) and
electrodes of type ISO-HPO2 and ISO-NOP.
[0043] ISO-HPO2 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. H.sub.2O.sub.2 concentration was calculated
according to the standard curves correlation with the strength of
the electrodes signal.
TABLE-US-00003 TABLE 3 Acetic acid, lactic acid and succinic acid
concentration (mmol/l) of Lactobacillus plantarum DSM 21379 Inducia
in MRS broth at microaerobic cultivation for 24 and 48 h and
H.sub.2O.sub.2 concentration (.mu.g/ml) of intact cells
H.sub.2O.sub.2 Acetic acid Lactic acid Succinic acid (.mu.g/ml)
(mmol/l) (mmol/l) (mmol/l) intact cells 24 h 48 h 24 h 48 h 24 h 48
h 24 h L. plantarum 2.1 2.4 133.3 186.6 0.6 0.6 288.9 .+-. 175.8
Inducia DSM 21379 Control 1.4 1.7 112.2 129.2 0.6 0.6 196.4 .+-.
128.8 strain L. plantarum DSM 21380
[0044] Lactobacillus plantarum Inducia DSM 21379 produces
substantial amounts, of acetic acid, lactic acid and H.sub.2O.sub.2
in comparison with the control and which are essential for the
trophic of the colon mucosa.
Total Antioxidative Activity of Lactobacillus plantarum Inducia DSM
21379
[0045] Method. For the detection of TAA and TAS of the microbial
cells, the strain L. plantarum Tensia was incubated in MRS broth
(Oxoid, U.K.) for 24 h at 37.degree. C. Microbial cells were
harvested by centrifugation (1500 RPM, during 10 min) at 4.degree.
C. and the pellet was washed with isotonic saline (4.degree. C.)
and suspended in 1.15% KCl (Sigma, USA). The density of the
suspension was OD.sub.600 of 1.1.times.10.sup.9 bacterial cells
ml.sup.-1). Total antioxidative activity (TAA) was assessed by
using the linolenic acid test (LA-test). (Kullisaar, T, Songisepp,
Mikelsaar M, Zilmer, K, Vihalemm, T, Zilmer, M. Antioxidant
probiotic fermented milk decreases oxidative stress-mediated
atherogenicity in human. British J of Nutrition 2003, 90, 2,
449-456) and total antioxidative status (TAS) was measured by
commercial kit (TAS, Randox Laboratories Ltd.; UK).
TABLE-US-00004 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
Production of Nitrogen Mono-Oxide (NO)
[0046] Method. Nitrogen mono-oxide production measurements were
carried out with 24 h old intact cells in 500.mu. 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.
TABLE-US-00005 TABLE 5 NO concentration (.mu.M) produced by
Lactobacillus plantarum DSM 21379 Strain number NO concentrations
(.mu.M) L. plantarum Inducia DSM 21379 2.7 .+-. 1.2 L. plantarum
DSM 21380 2.6 .+-. 0.8 L. coprophilus 2.1 .+-. 1.1 L. plantarum 2.1
.+-. 0.9 L. paracasei ssp paracasei strain no 1 1.3 .+-. 0.8 L.
paracasei ssp paracasei strain no 2 1.8 .+-. 0.9 L. paracasei ssp
paracasei strain no 3 2.8 .+-. 1.6 L. buchneri 2.0 .+-. 1.1
[0047] Lactobacillus plantarum Inducia DSM 21379 was the best NO
producer among the tested heterofermentative lactobacilli.
In Vitro Polyamines Production of Lactobacillus plantarum Inducia
DSM 21379
[0048] Method. Microbial strains were suspended in physiological
saline according to McFarlandi turbidity standard (10.sup.9 CFU/ml)
and 0.5 ml of each strain suspension was seeded into
decarboxylation medium (a 4.5 ml) and incubated at 37.degree. 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)).
[0049] For detection of BA 200 .mu.l of medium was derivatized for
GC analyze by modified method of Nakovich (Nakovich, L. Analysis of
biogenic amines by GC/FID and GC/MS. Thesis, Va. polytechnic
institute, USA. 2003).
[0050] GC analysis were carried out by gas chromatograph HP 6890
Series GC System, with capillary colonna HP-5 19091J-413 (30
m.times.0.32 mm; 0.25 .mu.m) with 160.degree. C. 1 min, 20.degree.
C./min 280.degree. C. 15 min; and detector (FID) 300.degree. C.
TABLE-US-00006 TABLE 6 Production of polyamines in vitro in the
decarboxylation medium (Arena, M. E. and Manca de Nadra, M. C.
Biogenic amine production by Lactobacillus. J Appl Microbiol, 2001;
90; 158-162) Polyamines (.mu.g/ml) and biogenic amines Arginine
Glutamine Lysine Ornithine Histidine Putres- Cadav- Putres- Cadav-
Putres- Cadav- Putres- Cadav- Cadav- Sample cine erine cine erine
cine erine cine erine erine L. plantarum 0 0.4 1.2 0.5 0 0.4 1.9 0
0 Inducia DSM 21379 L. plantarum 0 0 0 0 0 0.3 0.5 0.6 0 DSM
2137980
[0051] Lactobacillus plantarum strain Inducia DSM 21379 was able to
produce cadaverine from arginine, putrescine both from glutamine as
well as from ornithine. But the control strain was able to produce
putrescine in low amounts only from ornithine (FIG. 3). Traces of
cadaverine were detected both in control strain as well as in
Lactobacillus plantarum Inducia DSM 21379. No histamine production
was detected (Table 6).
DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1. Molecular identification of the strain by
ITS-PCR
[0053] 1. L. plantarum Inducia DSM 21379
[0054] 2. L. plantarum DSM 21380
[0055] 3. L. plantarum CRL 972 (ATCC 14917)
[0056] M-100 bp marker (Fermentas)
[0057] FIG. 2. L. plantarum Inducia DSM 21379 molecular
fingerprints in comparison with L. plantarum control strain (Pulse
field-gel-electrophoresis profile, PFGE).
[0058] 1. Lambda Ladder PFG Marker (New England Bio Labs Inc.)
[0059] 2. L. plantarum DSM 21380 (control)
[0060] 3. L. plantarum DSM 21379
[0061] FIG. 3. Production of polyamines by L. plantarum DSM 21379
in vitro in the decarboxylation medium (Arena, M. E. and Manca de
Nadra, M. C. Biogenic amine production by Lactobacillus. J Appl
Microbiol, 2001; 90; 158-162) (a) from ornithine (b) from
glutamine
[0062] 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)
[0063] FIG. 5. Lactobacillus species by Pearson UPMAG
clusteranalyses in L. plantarum Inducia DSM 21379 group.
[0064] Subjects no 3, 6, 9, 11, 14, 15, 19 and 20.
[0065] Four samples: at the recruitment (1), after consumption of
probiotic cheese (2), after wash-out period (3) and after
consumption of control cheese (4).
[0066] FIG. 6. IL-6 and blood monocytes
[0067] Positive correlation between IL-6 and blood monocytes in
last (IV) sample--r=0.653, p=0.029, n=11. The linear regression
model confirmed the correlation (R.sup.2=0.405, R.sup.2 adj=0.338,
=0.035).
DESCRIPTION OF THE EMBODIMENTS
Example 1
Obtaining Optimal Viable Counts of Lactobacillus plantarum Inducia
DSM 21379 in Food Product
[0068] Test with Estonia Cheese
[0069] Method. Microorganism Lactobacillus plantarum Inducia DSM
21379 was added to the cheese milk of Dairy Cooperative E-Piim,
(inoculation dose 3.times.10.sup.8 CFU/vat) and the milk was
renneted (25 min). The curds were cut (25 min), heated (34.degree.
C. 15 min), dried (25 min), pressed, drained (1 h), salted in brine
(12.degree. C.; 20% NaCl; pH 4.7) 20 h, drained and dried (8 h),
backed into plastic and ripened at 12.degree. C. for at least 4
weeks.
TABLE-US-00007 TABLE 7 Dynamics of microflora in Lactobacillus
plantarum Inducia DSM 21379 comprising cheese. L. plantarum Inducia
DSM 21379 comprising cheese Control cheese Sample Day 14 Day 21 Day
30 Day 14 Day 21 Day 30 total counts 8 .times. 10.sup.8 4 .times.
10.sup.8 10.sup.10 2 .times. 10.sup.8 3 .times. 10.sup.8 2 .times.
10.sup.8 Lactobacillus 5 .times. 10.sup.8 2 .times. 10.sup.8
10.sup.10 10.sup.5 -- 3 .times. 10.sup.3 sp L. plantarum 5 .times.
10.sup.8 2 .times. 10.sup.8 10.sup.10 -- -- 3 .times. 10.sup.3
cocci 3 .times. 10.sup.8 2 .times. 10.sup.8 10.sup.9 2 .times.
10.sup.8 3 .times. 10.sup.8 10.sup.8 spore-forming -- -- 10.sup.3
-- 10.sup.7 10.sup.8 microbes L. casei -- -- -- 10.sup.5 . --
Example 2
Production of Polyamines in Food Product by Lactobacillus plantarum
Inducia DSM 21379
[0070] Method. Cheese samples were extracted (20 ml 50% methanol
solution was added to 10 g of cheese and incubated at 45.degree. C.
for 1 h, cooled to 30.degree. C. and centrifuged) and 200 .mu.l of
upper layer was derivatized for GC analyze by modified method of
Nakovich (Nakovich, L. 2003 Analysis of biogenic amines by GC/FID
and GC/MS) in Department of Microbiology of the University of
Tartu.
[0071] GC analysis were carried out by gas chromatograph HP 6890
Series GC System, with capillary colonna HP-5 19091J-413 (30
m.times.0.32 mm; 0.25 .mu.m). The column temperature program
160.degree. C. 1 min, 20.degree. C./min 280.degree. C. 15 min; and
detector (FID) 350.degree. C. (FIG. 4).
[0072] The production of putrescine and tyramine was related to
that: in different lots in comparison with control-cheese
putrescine content increased 3-11 times and tyramine content
accordingly 2-5 times.
[0073] 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.
TABLE-US-00008 TABLE 8 Biogenic amines and polyamines in
Lactobacillus plantarum Inducia DSM 21379 comprising test-cheeses
from industrial test-trials Viable count of L. Viable counts of
strain plantarum incorporated into cheese Amines (mg/kg) Tensia
(CFU/g) at day 3-4 after Tyra- Putres- Cadav- DSM 21379 Sample
preparation. mine cine erine in ripe L. plantarum Inducia 3 .times.
10.sup.8 4.83 20.28 0 2 .times. 10.sup.7 Inducia DSM 21379, 1.
Batch Control cheese -- 2.31 1.82 0 -- 1. Batch L. plantarum
Inducia 3.3 .times. 10.sup.7 13.57 24.67 0 2 .times. 10.sup.6 DSM
21379, 2. Batch Control cheese -- 2.63 6.64 0 -- 2. Batch
[0074] 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 (Larque,
M., Sabater-Molina, S. Zamora E. Biological significance of dietary
polyamines. Nutrition 2007; 23(1): 87-95).
[0075] By consumption of 100 g Lactobacillus plantarum Inducia DSM
21379 comprising cheese the subject gets ca 3 mg of putrescine.
Thus, in the case of person of 70 kg the concentration is up to 50
.mu.g per kg, which does not express toxic effect.
Example 3
Enhancement of Defence Capability of Intestinal Mucosa with
Lactobacillus plantarum Inducia DSM 21379 Comprising Food
[0076] In the experimental model with NIH mice 3 groups of mice
consumed different cheeses during 30 days (control cheese with no
additives, Lactobacillus plantarum strain Inducia DSM 21379
2.times.10.sup.8 cfu/g comprising cheese).
[0077] Cheese was administered to mice at night (normal awake time
for mice) 4.4 g/per mouse, daily ad libitum regular commercial
diet. Consumed amount of cheese was 3.5-4.2 g/per night.
[0078] Mice stayed in good condition, no changes in skin, fur and
digestion was detected. Cheese administration caused increase of
body weight: body weight at the beginning of the trial was
22.9-29.8 g and at the end of the trial a weight gain was up to
2-6.1 g. The mice were sacrificed by cervical dislocation at day
30. No translocation of lactobacilli or other bacteria of the
microbiota to blood or organs was detected at the autopsy.
TABLE-US-00009 TABLE 9 Total counts of lactobatsilli in faeces,
ileum and colon Sample Control group Test group (cheese (cheese
without L. comprising L. plantarum plantarum Inducia DSM Inducia
DSM 21379 was Faecal samples 21379 was administered) administered)
Day 0 6.7 7.6 Day 10 8.0 8.3 Day 15 7.0 8.0 ileum 3.0-7.1/5.95*
6.3-7.7/6.95* colon 4.4-7.3/6.65* 6.9-7.8/7.45* *Student t-test p =
0.001
[0079] With seeding from ileum and colon from the mice administered
with Lactobacillus plantarum Inducia DSM 21379 total counts of
lactobacilli were found to be increased significantly both in ileum
and colon.
[0080] 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.
[0081] No morphological shifts were found in organs (liver and
spleen), which proves the safety of Lactobacillus plantarum Inducia
DSM 21379.
[0082] In the ileum the status of intestinal mucosa, count of
goblet and Paneth cells and also the formation of follicles and
diffuse multiple deposits by lymphocytes was evaluated. In the
colon, the attention was paid to the numerously goblet cells
containing surface epithelium and to the characteristics of
lymphatic tissue in connective tissue of mucosa.
TABLE-US-00010 TABLE 10 Patomorphological evaluation of mice organs
after administration of Lactobacillus plantarum Inducia DSM 21379
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
[0083] Administration of 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.
Example 4
Examination of Clinical Blood Indices of Healthy Volunteers
Consumed Lactobacillus plantarum DSM 21379 Comprising Food and the
Increase of Lactobacillus plantarum Percentage in the Intestinal
Microbiota
[0084] 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.
[0085] The study group consisted of 12 healthy volunteers, both
male and female (M/F 4/8) aged 20-48 years. For exclusion criteria
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.times.10.sup.7 CFU/g.
[0086] Before consumption the test cheese was incubated with
Lactobacillus plantarum Inducia DSM 21379 for 30 days at 12.degree.
C. Regular Estonian cheese of Edam type without Lactobacillus
plantarum Inducia DSM 21379 served as a control.
[0087] 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.
Results
1) Safety
[0088] No discomfort, abdominal pain or other negative symptoms
were reported by trial participants. After cheese trial with
volunteers, the values of systemic inflammation markers (U-CRP,
ultrasensitive CRP, and leucocytes) were not changed and were
within the normal range.
[0089] The consumption of probiotic Lactobacillus plantarum Inducia
DSM 21379 cheese did not cause changes in WBC counts (leucogram)
(Table 11). No change was also detected in the values of essential
allergy marker IgE in comparison with pre-trial period. Consumption
of Lactobacillus plantarum Inducia DSM 21379 comprising cheese had
no negative impact on organisms' kidney and liver function nor
affected according parameters (ASAT, ALAT albumine, blood sera
creatinine).
[0090] Thus in healthy subjects the consumption of Lactobacillus
plantarum DSM 21379 comprising cheese does not cause systemic
inflammation, allergic sensibilisation or causes harm to essential
organs.
Intestinal Microbiota
[0091] Method. 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)
(Heilig 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). Subsequently, the PCR product was
separated by DGGE electrophoresis in 30-60% acrylamide containing
gel with Dcode.TM. System technique. (Bio-Rad, Hercules, Calif.).
Gels were analyzed by BioNumerics 2.5 (Applied Maths, Belgium)
software according to Peasoni correlation (Fromin, N.; Hamelin, J.;
Tarnawski, S.; Roesti, D.; Jourdain-Miserez, K.; Forestier, N.;
Teyssier-Cuvelle, S.; Gillet, F.; Aragno, M.; Rossi, P. Statistical
analysis of denaturing gel electrophoresis (DGE) fingerprinting
patterns Environmental Microbiology 2002; 4 (11), 634-643) (FIG.
5).
Results
[0092] Consumption of Lactobacillus plantarum Inducia DSM 21379
comprising cheese changed lactobacilli pattern profile in feces in
5 persons of 12. These changes remained stabile in 3 persons of 5
even 2 weeks after completing the trial.
[0093] Thus, consumption of Lactobacillus plantarum Inducia DSM
21379 comprising cheese affects the composition of human intestinal
lactoflora.
3) Humoral and Cellular Parameters of Blood Sera
[0094] It appeared that Lactobacillus plantarum Inducia DSM 21379
comprising cheese induces the raise of cytokine IL-6, in
volunteers, which indicates the stimulation of cellular immunity.
This finding was confirmed by the increase of blood monocytes at
the end of the trial (p=0.015), whereas indices of blood cells were
within the normal range. Positive correlation after completing the
cheese consumption appeared between IL-6 and blood monocytes
(r=0.653, p=0.029, n=11). The linear regression model confirmed the
correlation (R.sup.2=0.405, R.sup.2 adj=0.338, =0.035) (FIG.
6).
[0095] Enhancement of parameters of cellular immunity is in
accordance with results on animal model described above; where the
administration of Lactobacillus plantarum Inducia DSM 21379 induced
significantly Peyer's patches i.e. lymph follicles in the
intestine. In these follicles interaction of the components of the
immune system occurs. Proinflammatory cytokines incl. induction of
IL-6 play important role in activation of TH1 type lymphocytes
against bacteria through which macrophages i.e. blood monocytes are
retroactive activated. Thereupon blood monocytes produce also
IL-6.
[0096] The increase of the share of cellular immunity is
explainable also by the suppression of humoral response e.g.
somewhat decreased production of antibodies (IgA, IgG), which
however remained within the normal range.
TABLE-US-00011 TABLE 11 Immunological parameters of blood after
consumption of probiotic Lactobacillus plantarum Inducia DSM 21379
comprising cheese At the End of the P Normal range Immunity
parameters recruitment trial values and units Ultrasensitive CRP
1.9 .+-. 1.6 3.3 .+-. 2.7 1.0 <5 mg/L Leukocyte counts .times.
10.sup.9 6.3 .+-. 1.7 6.9 .+-. 1.6 0.116 4-10 .times. 10.sup.9/L
Lymphocytes 2.3 .+-. 0.5 2.2 .+-. 0.4 0.878 0.8-2.9 .times.
10.sup.9/L Monocytes 0.55 .+-. 0.17 0.64 .+-. 0.15 0.032 0.15-0.75
.times. 10.sup.9/L Cytokine IL-6 2.7 .+-. 1.0 3.8 .+-. 1.7 0.020
<3.4 ng/L IgA antibodies 2.5 .+-. 0.9 2.3 .+-. 0.8 0.009 0.7-4.0
g/L IgM antibodies 1.3 .+-. 0.5 1.3 .+-. 0.5 0.776 0.4-2.3 g/L IgG
antibodies 12.9 .+-. 3.2 12.4 .+-. 3.0 0.017 7.0-16 g/L IgE
antibodies 19.6 .+-. 21.2 21.4 .+-. 25.9 0.232 <85 kU/L
4) The Urine Metabolites
[0097] For the evaluation of the content biogenic amines before the
consumption of the probiotic and the efficiency of the
stabilization period, the morning urine and gas chromatography
method were used.
[0098] Method: urine samples were derivatized with
propylchlorophormate for GC analyze by modified method of Uglandi
(Ugland H G; Krough M, Rasmussen K E: Aqueous alkylchloroformate
derivatization and solid-phase microextraction: determination of
amphetamines in urine by capillary gas chromatography. J
Chromatography B Biomed Sci Appl 1997; 701:29-38)
[0099] GC analysis were carried out by gas chromatograph HP 6890
Series GC System (Hewlett Packard, Avondale, Pa., USA), with
capillar colonne HP-5 19091J-433 (30 m.times.0.25 mm; 0.25 .mu.m)
The column temperature program 150.degree. C. 1 min, 20.degree.
C./min 280.degree. C. for 5 min; and detector (FID) 250.degree. C.
The biogenic amines concentration was calculated according to
nmol/mol creatinine.
TABLE-US-00012 TABLE 12 Polyamines and biogenic amines content in
the morning urine (nmol/mol creatinine) of probiotic cheese
consumers Control cheese (without Probiotic cheese comprising L.
Lactobacillus plantarum plantarum Inducia DSM 21379 Inducia DSM
21379) BL1 PRO BL2 PL P values mean .+-. stdev mean .+-. stdev mean
.+-. stdev mean .+-. stdev paired t-test range range range range
BL1 vs PRO/ (median) (median) (median) (median) BL2 vs PL Put 0.064
.+-. 0.072 0.082 .+-. 0.058 0.043 .+-. 0.044 0.044 .+-. 0.060
0.432/0.432 0-0.191 0-0.191 0-0.126 0-0.216 (0.030) (0.077) (0.033)
(0.031) acPut 0.606 .+-. 0.559 1.087 .+-. 1.451 0.796 .+-. 0.689
0.635 .+-. 0.291 0.021/0.850 0.151-2.104 0.307-5.049 0.068-2.167
0.154-1.219 (0.435) (0.447) (0.600) (0.594) DAP 0.079 .+-. 0.092
0.059 .+-. 0.089 0.056 .+-. 0.089 0.117 .+-. 0.142 0.411/0.195
0-0.249 0-0.216 0-0.253 0-0.418 (0.055) (0) (0) (0.055) acSpd 0.251
.+-. 0.227 0.384 .+-. 0.198 0.354 .+-. 0.210 0.425 .+-. 0.260
0.089/0.464 0-0.813 0.043-0.686 0.085-0.668 0.065-0.831 (0.232)
(0.384) (0.304) (0.396) Cad 0.066 .+-. 0.123 0.069 .+-. 0.162 0.067
.+-. 0.093 0.044 .+-. 0.085 1.0/0.540 0-0.364 0-0.569 0-0.293
0-0.228 (0) (0) (0.016) (0) His 0.231 .+-. 0.226 0.387 .+-. 0.524
0.211 .+-. 0.364 0.478 .+-. 0.684 0.910/0.250 0-0.595 0-1.401
0-1.229 0-2.093 (0.156) (0.122) (0) (0.235) Tyr 0.153 .+-. 0.161
0.101 .+-. 0.132 0.093 .+-. 0.096 0.212 .+-. 0.285 0.167/0.149]
0-0.476 0-0.427 0-0.257 0-1.035 (0.102) (0.050) (0.093) (0.179)
Put--putrescine, acPut--N-acetylputrescine,
DAP--1.3-diaminopropane, acSpd--N 8-acetylspermidine,
Cad--cadaverine; His--histamine, Tyr--tyramine
[0100] The content of polyamines (putrescine, acetylputrescine and
acetylspermidine) in urine increased after consumption of L.
plantarum Inducia DSM 21379 comprising probiotic cheese. A
significant correlation (R=0.383 p<0.01, n=48) appeared between
the contents of acetylputrescine and acetylspermidine in the urine
of volunteers.
[0101] At the same time the content of all polyamines in
putrescine, acetylspermidine and acetylspermine as well as biogenic
amines remained within the normal range in the urine.
[0102] 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.
[0103] 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.
[0104] 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.sub.2O.sub.2 is essential for the organisms' defence against
foreign cells (microbes, cancer cells).
[0105] 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.
[0106] Consumption of Lactobacillus plantarum Inducia DSM 21379
cheese regulates the amount and activity of blood monocytes through
IL-6 which finding together with lymph follicles (increase of
carriers of cellular immunity) demonstrated in experimental animals
improves the barrier function of intestinal mucosa and supports
organisms' immunological defence functions.
Sequence CWU 1
1
2126DNAUnknownprimer_bind(503425) (503404)Designation
Uni-515-GC-rev, location the sequence measured against the positive
control Lactobacillus plantarum WCFS1 1atcgtattac cgcggctgct ggcagc
26221DNAArtificial sequenceprimer_bind(2181565)
(2181551)Designation Lab-159-f, location the sequence measured
against the positive control Lactobacillus plantarum WCFS1
2ggaaacagag tgctaatacc g 21
* * * * *