U.S. patent application number 14/765682 was filed with the patent office on 2015-12-31 for use of microorganisms for the prevention and treatment of intestinal diseases.
This patent application is currently assigned to LUDWIG STOCKER HOFPFISTEREI GMBH. The applicant listed for this patent is LUDWIG STOCKER HOFPFISTEREI GMBH, TECHNISCHE UNIVERSITAT MUNCHEN. Invention is credited to Andreas DUNKEL, Dirk HALLER, Thomas HOFMANN, Dagmar KRUGER, Jurgen MAYER, Michael SCHEMANN, Anna ZHENCHUK.
Application Number | 20150374763 14/765682 |
Document ID | / |
Family ID | 47630212 |
Filed Date | 2015-12-31 |
United States Patent
Application |
20150374763 |
Kind Code |
A1 |
MAYER; Jurgen ; et
al. |
December 31, 2015 |
USE OF MICROORGANISMS FOR THE PREVENTION AND TREATMENT OF
INTESTINAL DISEASES
Abstract
The invention relates to acetylcholine-producing microorganisms
for use in the prevention and/or treatment of intestinal diseases,
and/or reduction of risks of intestinal diseases, and/or
improvement of intestinal health as well as promoting healthy gut
flora. The acetylcholine-producing microorganisms may be provided
as a pharmaceutical dosage form or as additive to functional food
or food supplemental products. Also encompassed is a method for the
production of acetylcholine by use of Lactobacilli. Further the
invention refers to microbially produced acetylcholine for use in
the treatment and/or prevention of intestinal diseases.
Inventors: |
MAYER; Jurgen; (Munchen,
DE) ; HALLER; Dirk; (Freising, DE) ; ZHENCHUK;
Anna; (Freising-Weihenstephan, DE) ; HOFMANN;
Thomas; (Freising-Weihenstephan, DE) ; DUNKEL;
Andreas; (Freising-Weihenstephan, DE) ; SCHEMANN;
Michael; (Freising-Weihenstephan, DE) ; KRUGER;
Dagmar; (Freising-Weihenstephan, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LUDWIG STOCKER HOFPFISTEREI GMBH
TECHNISCHE UNIVERSITAT MUNCHEN |
Munchen
Munchen |
|
DE
DE |
|
|
Assignee: |
LUDWIG STOCKER HOFPFISTEREI
GMBH
Munchen
DE
|
Family ID: |
47630212 |
Appl. No.: |
14/765682 |
Filed: |
February 4, 2014 |
PCT Filed: |
February 4, 2014 |
PCT NO: |
PCT/EP2014/052129 |
371 Date: |
August 4, 2015 |
Current U.S.
Class: |
424/93.45 ;
435/128; 435/252.9 |
Current CPC
Class: |
A61K 2035/115 20130101;
C12P 13/001 20130101; A61K 38/00 20130101; A61P 1/10 20180101; A61K
38/1787 20130101; A61P 1/04 20180101; A61K 35/747 20130101; C12R
1/24 20130101; C12R 1/225 20130101; C12R 1/25 20130101; C12N 1/26
20130101; A23L 33/135 20160801; C12N 1/00 20130101; A61P 1/14
20180101 |
International
Class: |
A61K 35/747 20060101
A61K035/747; C12R 1/24 20060101 C12R001/24; C12R 1/25 20060101
C12R001/25; C12R 1/225 20060101 C12R001/225; C12P 13/00 20060101
C12P013/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 5, 2013 |
EP |
13153996.7 |
Claims
1. An acetylcholine-producing microorganism for medical use in the
prevention and/or treatment of intestinal diseases, and/or
reduction of risk of intestinal diseases.
2. The acetylcholine-producing microorganism for use according to
claim 1, for maintaining and/or promoting a healthy gut flora
and/or reducing the toxic effects of the digestive process and/or
stimulating the digestive system and/or improving intestinal
control.
3. The acetylcholine-producing microorganism for use according to
claim 1, wherein the intestinal disease is a functional intestinal
disorder and/or a disorder associated with the secretions of the
intestinal wall controlled by the enteric nervous system, in
particular functional constipation, functional diarrhea and/or
irritable bowel syndrome (IBS), more particular constipation
predominant IBS, alternating IBS or diarrhea predominant IBS.
4. The acetylcholine-producing microorganism for use according to
claim 1, wherein the intestinal disease is an inflammatory bowel
disease, in particular ulcerative colitis and/or Crohn's
disease.
5. The acetylcholine-producing microorganism for use according to
claim 1, wherein the microorganism produces .gtoreq.30 mg/kg
acetylcholine under suitable culture conditions.
6. The acetylcholine-producing microorganism for use according to
claim 1, wherein the microorganism is a bacterium.
7. The acetylcholine-producing microorganism for use according to
claim 1, wherein the microorganism is a Lactobacillus strain, in
particular a Lactobacillus sanfranciscensis strain, a Lactobacillus
rossiae strain, a Lactobacillus brevis strain or a Lactobacillus
plantarum strain, more particularly strains DSM 23090 or DSM
23093.
8. The acetylcholine-producing microorganism for use according to
claim 1, wherein the Lactobacillus is selected from any one of
strains DSM 26024, DSM 23090, DSM 23091, DSM 23200, DSM 23092, DSM
23093, DSM 23201, DSM 23174 and DSM 23121 or a strain cultivated
therefrom.
9. The acetylcholine-producing microorganism for use according to
claim 1 in a pharmaceutical dosage form, in a functional food or in
a functional beverage.
10. The acteylcholine-producing microorganism for use according to
claim 9, wherein the functional food is sourdough bread.
11. The acetylcholine-producing microorganism for use according to
claim 9, further comprising at least one additional bacterium for
maintaining and/or restoring a favorable gut flora.
12. The acetylcholine-producing microorganism for use according to
claim 9, further comprising at least one additional medicament for
the treatment of intestinal diseases.
13. A method for the production of acetylcholine by use of
lactobacilli, particularly by the use of lactobacilli selected from
Lactobacillus sanfranciscensis and Lactobacillus rossiae
strains.
14. A method for the treatment and/or prevention of intestinal
diseases, in a patient in need of such, comprising orally
administering microbially produced acetylcholine to said
patient.
15. Non-medical use of an acetylcholine-producing microorganism for
maintenance and/or improvement of intestinal health.
16. (canceled)
Description
[0001] The invention relates to acetylcholine-producing
microorganisms for use in the prevention and/or treatment of
intestinal diseases, and/or reduction of risks of intestinal
diseases, and/or improvement of intestinal health as well as
promoting healthy gut flora. The acetylcholine-producing
microorganisms may be provided as a pharmaceutical dosage form or
as additive to functional food or food supplemental products. Also
encompassed is a method for the production of acetylcholine by use
of Lactobacilli. Further the invention refers to microbially
produced acetylcholine for use in the treatment and/or prevention
of intestinal diseases.
[0002] A large number of patients suffer from gastrointestinal
disorders associated with the lower small bowel and/or large bowel.
These disorders include irritable bowel syndrome (IBS), or spastic
colon, idiopathic alterative colitis, mucous colitis, collagenous
colitis, Crohn's disease, inflammatory bowel disease in general,
microscopic colitis, antibiotic-associated colitis, idiopathic or
simple constipation, diverticular disease, and AIDS
enteropathy.
[0003] Irritable bowel syndrome is the most common of all
gastrointestinal disorders, affecting 11-14% of adults and
accounting for more than 50% of all patients with digestive
complaints. (G. Triadafilopoulos et al., Bowel Dysfunction in
fibromyalgia, Digestive Dis., Sci. 36 (1): 59-64 [1991]; W. G.
Thompson, Irritable Bowel Syndrom: Pathogenesis and Management,
Lancet, 341:1569-1572 [1993]). It is thought that only a minority
of people with IBS actually seek medical treatment. Patients with
IBS present with disparate symptoms, for example, abdominal pain,
predominantly related to defecation, alternating diarrhea and
constipation, abdominal distention, gas, and excessive mucus in the
stool. There are three groups of IBS: constipation-predominant IBS
(C-IBS), alternating IBS (A-IBS) and diarrhea-predominant IBS
(D-IBS). IBS is recognized as a chronic condition, which may have
profound effect on the patient's quality of life.
[0004] A number of possible causes for IBS have been proposed such
as fiber-poor Western diet, intestinal motility malfunction,
abdominal pain perception, abnormal psychology or behavior, or
psycho-physiological response to stress. However, none of those
causes has been fully accepted (W. G. Thompson [1993] supra).
[0005] Patients suffering from IBS appear to perceive normal
intestinal activity as painful. For example, IBS patients
experience pain at lower volumes of rectal distention than normal
or have lower than normal threshold for perceiving migrating motor
complex phase III activity (W. E. Whitehead et al., Tolerance for
Rectosigmoid Distention in Irritable Bowel Syndrom, Gasteroenterol.
98:1187-92 [1990]; J. E. Kellow et al., Enhanced Perception of
Physiological Intestinal Motility in the Irritable Bowel Syndrom,
Gasteroenterol. 101 (6):1621-24 [1991]).
[0006] Bowel motility in IBS patients differs from a normal
controlled response to various stimuli such as drugs, hormones,
food, and emotional stress (D. G. Wangel and D. J. Deller,
Intestinal Motility in Man, III: Mechanisms of Constipation and
Diarrhea with Particular Reference to the Irritable Bowel,
Gasteroenterol. 48: 69-84 [1965]; R. F. Harvey and A. E. Read,
Effect of Cholecystokinin and Colon Motility and on Symptoms of
Patients with Irritable Bowel Syndrome, Lancet i: 1-3 [1973]; R. M.
Valori et al., Effects on Different Types of Stress and "Prokinetic
drugs" on the Control of the Fasting Motor Complex in Humans,
Gasteroenterol. 90: 1890-900 [1986]).
[0007] Evans at al. and Govath and Farthing recognized that
irritable bowel syndrome is frequently associated with disordered
gastro-intestinal motility. (P. R. Evans et al., Gastroparesis and
Small Bowel Dysmotility in Irritable Bowel Syndrome, Dig. Dis. Sci.
42 (10): 2087-93[1997]; D. A. Gorard and M. J. Farthing, Intestinal
Motor Function in Irritable Bowel Syndrome, Dig. Dis. 12 (2):
72-84[1994]). Treatment directed to bowel dysmotility in IBS
includes the use of serotonine antagonists (D. P. Becker et al.,
Mesoazacyclic Aromatic Acid Amides And Esters as Serotonergic
Agents, U.S. Pat. No. 5,612,366; M. Ohta at al., Methods for
Treatment of Intestinal Diseases, U.S. Pat. No. 5,547,961) and
Choleocytokinin antagonists (Y. Sato et al., Benzodiazepine
derivatives, U.S. Pat. No. 4,970,207; H. Kitajima et al.,
Thienylazole Compound and Thienotriazolodiazepine Compound, U.S.
Pat. No. 5,760,032). Colonic motility index, altered myoelectrical
activity in the colon and small intestinal dysmotility, however,
have not proven to be reliable diagnostic tools because they are
not IBS-specific (W. G. Thomson[1993], supra).
[0008] Administration of probiotics for the treatment of IBS has
been attempted. For example, Allan at al. described the use of a
strain of Enterococcus faecium to alleviate symptoms. (W. D. Allan
at al., Probiotic Containing Enterococcus faecium strain NCIMB
40371 U.S. Pat. No. 5,728,380 and Probiotic, U.S. Pat. No.
5,589,168). Borody taught a method of treating irritable bowel
syndrome by at least partial removal of the intestinal microflora
by lavage and replacement with a new bacteria community introduced
by fecal inoculum from a disease-screened human donor or by a
composition comprising Bacterioids and Escherichia coli species.
(T. J. Borody, Treatment of Gastro-Intestinal Disorders with a
Fecal Composition of Bacterioids and E. coli, U.S. Pat. No.
5,443,826).
[0009] It is a contention of many scientists that the health and
well-being of people can be positively or negatively influenced by
the microorganisms which inhabit the gastrointestinal tract, and in
particular the large intestine. These microorganisms, through the
production of toxines, metabolic by-products and short-chain fatty
acids, and the like, affect the physiological condition of the
host.
[0010] The constitution and quantity of the gut microflora can be
influenced by conditions or stress induced by disease, life-style,
travel and other factors. If microorganisms which positively effect
health and well-being of the individual can be encouraged to
populate in the large bowel, this should improve the psychological
well-being of the host.
[0011] The introduction of beneficial microorganisms or probiotics
may be accomplished by ingestion of the organisms in drinks,
yogurts, capsules, and other forms allowing viable organisms to
arrive at the large bowel.
[0012] However, until now, no reliable method has been found or
developed to stimulate the enteric nervous system (ENS) which
regulates the intestinal bowel movements and secretory functions of
epithelial layer in a sufficient manner.
[0013] The problem of the present invention was therefore to
provide a method to intervene in the regulation of the intestinal
bowel movements and secretory functions of epithelial layer in a
sufficient manner to influence thereby the course of intestinal
diseases.
[0014] The inventors of the present invention have conducted
intensive studies and found as a result that intestinal function
can be modulated by acetylcholine-producing microorganisms. Thereby
a method for the targeted dual stimulation of motility and
secretion by acetylcholine in the intestine through selection and
specific administration of acetylcholine-producing microorganism,
particularly lactic acid bacteria is provided. This treatment
method is a promising alternative or addition to known therapies
for chronic IBS and other disorders associated with impaired
intestinal motility and secretion.
[0015] A first aspect of the present invention is therefore an
acetylcholine-producing microorganism for the use in the prevention
and/or treatment of intestinal diseases and/or reduction of risk of
developing intestinal diseases.
[0016] A further aspect of the present invention is the non-medical
use of an acetylcholine-producing microorganism for the maintenance
and/or improvement of intestinal health, particularly for the
improvement of intestinal health.
[0017] The acetylcholine-producing microorganism is a live organism
and preferably capable of propagating in the intestinal area.
[0018] The term "intestinal area" as used herein is intended to
include the small intestine and large intestine. Large intestine is
intended to include the colon and rectum, and in humans, is
intended to include the colon, rectum and caecum.
[0019] The term "reduction of risk of developing intestinal
diseases" as used herein means that an individual being treated
with the acetylcholine-producing microorganism of the present
invention exhibits a lower risk to develop an intestinal disease
caused by external stimuli or physiological processes compared to a
non-treated individual.
[0020] The term "maintenance and/or improvement of intestinal
health" as used herein means that an individual, upon treatment
with the acetylcholine-producing microorganism, exhibits a
different gut flora, which is beneficial for human or animal health
and reasonable for a maintenance and/or an improvement of the
digestion of said individual. The improved gut flora further may
lead to an increased resistance of the subject to develop an
intestinal disease by out-competing harmful bacteria and
stimulating the normal bowel movement.
[0021] The term "microorganism" as used herein comprises bacteria
and yeasts. The bacteria are preferably Lactobacillaceae such as
Lactobacillus strains, in particular Lactobacillus sanfranciscensis
strains, Lactobacillus rossiae strains, Lactobacillus lactis and
Lactobacillus plantarum (Stephenson et al., The production of
acetylcholine by a strain of Lactobacillus plantarum,
[0022] In certain embodiments, the microorganism is not a
Lactobacillus plantarum strain, particularly not Lactobacillus
plantarum strain 299v (DSM 9843), or Lactobacillus plantarum strain
(ATCC 10241). In certain embodiments, the microorganism is not a
Lactobacillus rhamnosus strain, particularly not Lactobacillus
rhamnosus GG.
[0023] The intestinal diseases encompass inflammatory bowel
diseases (IBD), such as ulcerative colitis, Crohn's disease,
collagenous colitis, lymphocytic colitis, ischaemic colitis,
Behcet's disease, indeterminate colitis, diversion colitis,
pouchitis or microscopic colitis and/or colon cancer and/or
diseases associated with microorganisms, such as candidiasis, small
intestinal bacterial overgrowth, acute or chronic bowel infections,
and/or diseases induced by sulphate-reducing bacteria, and/or
intestinal diverticular, and/or intestinal carcinoma, and/or
functional bowel disorders (FBD) such as irritable bowel syndrome,
and/or disorders associated with the secretions of the intestinal
wall controlled by the enteronervous system.
[0024] The term "Functional bowel disorder" (FBD) refers to
gastro-intestinal disorders which are chronic or semi-chronic and
which are associated with bowel pain, disturbed bowel function and
social disruption. Particular combinations and prevalence of
symptoms characterize in following seven FBD subgroups, which are
defined in accordance with the classification system known as the
"Rome criteria": 1) C1: constipation-predominant irritable bowel
syndrome; 2) C1: diarrhea-predominant irritable bowel syndrome; 3)
C3: Functional constipation; 4) C4: Functional diarrhea; 5) C2:
Functional abdominal bloating; 6) F3a: Pelvic Floor dyssynergia; 7)
F3b: internal anal sphincter dysfunction.
[0025] More specifically, the intestinal disease may be a
functional intestinal disorder and/or a disorder associated with
the secretions of the intestinal wall controlled by the enteric
nervous system, in particular functional constipation, functional
diarrhea and/or irritable bowel syndrome (IBS), such as particular
constipation-predominant IBS, alternating IBS or
diarrhea-predominant IBS.
[0026] The acetylcholine-producing microorganism of the present
invention is preferably useful for maintaining and/or promoting a
healthy gut flora and/or reducing the toxic effects of the
digestive process and/or stimulating the digestive system and/or
improving intestinal control. The promotion of a healthy gut flora
leads to an out-competing of the harmful bacteria in the
intestines, in particular the large intestine, and more
particularly the colon, and thereby reducing the toxic effect of
the digestive process, stimulating the digestive system and
improving bowel control.
[0027] The acetylcholine-producing microorganism of the present
invention is preferably useful for modulating the course of
inflammatory bowel diseases (IBD) in a beneficial way and relieving
the symptoms of IBD patients by inhibition of the secretion of
pro-inflammatory chemokine IP-10.
[0028] In another embodiment, the intestinal disease may be an
inflammatory bowel disease. The diseases are preferably ulcerative
colitis, Crohn's disease, collagenous colitis, lymphocytic colitis,
ischaemic colitis, Behcet's disease, indeterminate colitis,
diversion colitis and/or microscopic colitis. The diseases are more
preferably ulcerative colitis and/or Crohn's disease.
[0029] The microorganism of the present invention is capable of
producing acetylcholine. Preferably, the acetylcholine-producing
microorganism produces .gtoreq.20, 25, 30, 35 or 40 mg/kg
acetylcholine under suitable culture conditions as described, more
preferably .gtoreq.40 mg/kg and even more preferably .gtoreq.35
mg/kg. Any culture medium might be used for the culture of the
acetylcholine-producing microorganisms, which are suitable for
Lactobacilli-culture. Preferably, MRS-broth is used as a culture
medium. Preferably, the acetylcholine concentration is adjusted to
a 10.sup.6/ml bacteria count.
[0030] The acetylcholine-producing microorganism is preferably a
bacterium. More preferably, the bacterium is a Lactobacillaceae
such as a Lactobacillus strain, in particular a Lactobacillus
sanfranciscensis strain, Lactobacillus rossiae strain,
Lactobacillus brevis strain, or Lactobacillus plantarum strain,
which are generally available from the public catalogue of Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig,
Germany).
[0031] In a more preferred embodiment, the Lactobacillus strain is
selected from any one of strains DSM 26024, DSM 23090, DSM 23091,
DSM 23200, DSM 23092, DSM 23093, DSM 23201, DSM 23174 and DSM 23121
or a strain cultivated therefrom, more particularly strain DSM
23090 or DSM 23093 or a strain cultivated therefrom. These strains
have been deposited at the Leibnitz-Institut DSM--Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany),
according to the Budapest Treaty. The novel Lactobacillus strains
have the following accession numbers and deposition dates: DSM
23090 (2012-06-21), DSM 23091 (2012-06-21), DSM 23200 (2012-06-21),
DSM 23092 (2012-06-21), DSM 23093 (2012-06-21), DSM 23201
(2012-06-21), DSM 26024 (2012-06-04), DSM 23174 (2012-06-21), DSM
23121 (2012-06-21).
[0032] The term "strain cultivated therefrom" as used herein refers
to offspring strains derived by cultivation of the original
strain.
[0033] The acetylcholine-producing microorganism is preferably an
acetylcholine-secreting microorganism. The term
"acetylcholine-secreting microorganism" as used herein means that
the microorganism secrets acetylcholine into the culture
medium.
[0034] The present invention refers to the medical use of an
acetylcholine-producing microorganism. Preferably, the
microorganism is provided as a pharmaceutically acceptable dosage
form or in form of a nutrient, e. g. food or beverage.
[0035] In one embodiment, the acetylcholine-producing microorganism
is provided in a pharmaceutical composition. The
acetylcholine-producing microorganisms can also be provided as an
additive in a functional food or in a functional beverage. The
pharmaceutical composition, food or beverages, incorporating the
microorganism can be safely consumed and are especially recommended
for subjects perceived to be at risk or suffering from
gastro-intestinal dysfunction or organic disorders or diseases,
e.g. conditions or symptoms related to IBS, or IBD. They contain
the acetylcholine-producing microorganism preferably in an
effective amount to treat or prevent said disorders or
diseases.
[0036] As used herein, the term "an effective amount" refers to an
amount effective to achieve a desired therapeutic effect, such as
treating and/or preventing diseases, conditions and symptoms
related to IBS or IBD.
[0037] The effective amount of the acetylcholine-producing
microorganisms preferably comprises a dosage in the range of
10.sup.6 to 10.sup.12 cfu/dosage form (colony forming units/dosage
form), more preferably in the range of 10.sup.7 to
0.5.times.10.sup.12 cfu/dosage form, even more preferably in the
range of 10.sup.9 to 10.sup.11 cfu/dosage form. The dosage form may
be administered once or several times, e. g. 2, 3 or more times
daily.
[0038] The pharmaceutical composition may be in liquid or solid
form. The composition contains at least one of the
acetylcholine-producing microorganisms or a mixture thereof and
optionally a pharmaceutically acceptable carrier.
[0039] The amount of, e. g. microorganisms, incorporated into a
pharmaceutical composition may vary from about 0.1 to about 100% by
weight, preferably from about 2 to about 20% by weight, even more
preferably from about 4 to about 10% by weight, based on the total
weight of the composition.
[0040] The pharmaceutical composition of the present invention can
be made in the usual pharmaceutical forms known in literature, such
as for example tablets, coated tablets, capsules, packets,
solutions, suspensions, emulsions, suppositories, pellets, syrups,
vaginal suppositories, ointments, creams and so on. Preferably the
composition comprises an enteric coating. They can be prepared in
the usual manner by mixing the active ingredient with excipients
and/or carriers, optionally adding adjuvants and/or dispersing
agents. Should water be used as a diluent, also other organic
solvents can be used in the form of adjuvants. Adjuvants can be e.
g. water, non-toxic organic solvents such as paraffins, vegetable
oils (peanut oil or sesame oil), alcohols (e. g. ethanol,
glycerol), glycols (propylene glycol, polyethylene glycol). Solid
carriers can be e. g. natural mineral flours (kaolin, talc),
synthetic mineral flours (e. g. silicates), sugar (e. g. cane
sugar). Emulsifiers can be alkyl sulphonates or aryl sulphonates
and the like, dispersers e. g. lignin, methyl cellulose, starch and
polyvinyl pyrrolidine and lubricants e. g. magnesium is stearate,
talc, stearic acid, sodium lauryl sulphonate.
[0041] The composition may contain the acetylcholine-producing
microorganisms lyophilized, pulverized and powdered, optionally for
reconstitution in a pharmaceutically acceptable liquid carrier
administered to intestinal area, e. g. oral, rectal or
naso-duodenal. The administration takes place in the usual manner,
preferably by oral/rectal route. It may then be infused, dissolved
such as in saline, as an enema. As a powder, it can preferably be
provided in a palatable form for reconstitution for drinking. The
powder may also be reconstituted to be infused via naso-duodenal
infusion.
[0042] Pharmaceutical forms adapted to this end can contain, in
addition to usual excipients such as lactulose, dextrose, lactose,
other additives such as sodium citrate, calcium carbonate, calcium
dihydrogen phosphate, together with several additional substances
such as starch, gelatin and the like. In case of liquid forms
compatible coloring agents or flavoring substances may be
added.
[0043] Further components of the composition containing the
acetylcholine-producing microorganism may include an active agent,
e. g. glutamin/glutamate or precursors thereof, mannans,
galacturonic acid oligomers, herbal extracts such as Regulat.RTM.
(registered trademark of Dr Niedermayer Pharma) and Iberogast.RTM.
(registered trademark of Steigerwald Arzneimittelwerk GmbH),
chokeberry beer yeast, a drug useful for the treatment of
ulcerative colitis, such as sulphasalazine, 5-ASA agents,
corticosteroids, such as adrenal steroid, prednisone,
hydrocortisone, or budesonide, or drugs used against pain,
diarrhea, infection or IBS such as serotonine-4 receptor agonist,
e. g. tegaserod. The composition can be combined with other
adjuvants such as antacids to dampen bacterial inactivation in the
stomach. Acid secretion in the stomach could also be
pharmacologically suppressed using H2 antagonists or
omeprazole.
[0044] The composition of the invention may be provided in the form
of a kit for is separate sequential or simultaneous administration
in conjunction with such active agents as described herein above.
These active agents may conveniently be formulated together with
the composition of the invention in standard pharmaceutical dosage
forms, e. g. in combination with at least one pharmaceutically
acceptable carrier.
[0045] In another preferred embodiment, a composition containing
the acetylcholine-producing microorganism can contain at least one
additional microorganism, i.e. non-acetylcholine-producing
microorganism such as a bacterium for maintaining and/or restoring
favorable gut flora. The additional bacteria are preferably
pro-biotic bacteria.
[0046] In another embodiment of the invention, the
acetylcholine-producing microorganisms can preferably be provided
as a probiotic, preferably as an additive to functional food or to
functional beverages.
[0047] The amount of the microorganisms as a nutrient additive may
vary from about 0.0001 to about 20% by weight, preferably from
about 0.01 to about 10% by weight and even more preferably from
about 0.1 to about 5% by weight.
[0048] In another preferred embodiment the acetylcholine-producing
microorganisms can be applied to an edible material, preferably a
food--or feed product such as cereals, in particular oat flakes or
bread, a beverage or dairy products, in particular yogurt,
sauerkraut juice, plant extracts such as Regulat.RTM., fermented
beverages or Brottrunk.RTM.. The application to the edible material
may be achieved by coating said material with the
acetylcholine-producing microorganisms, preferably by spraying the
microorganisms onto the edible material. The
acetylcholine-producing microorganisms can also be applied by
injecting them into an edible material, such as bread, yogurt, or
cheese, preferably sour dough bread.
[0049] The term "functional food" as used herein is food, which, in
addition to its nutritional and sensory functions, has a positive
effect on the metabolism and, within a balanced nutrition,
contributes to an improvement of health, an increase in wellbeing
and/or a reduction of health risks. The functional food may be a
natural food or a food which has been altered by adding or cutting
off a component.
[0050] In a preferred embodiment, the functional food is a
fermented product, more preferably, the functional food is sour
dough or sour dough bread.
[0051] The sour dough bread of the present invention has the
advantage that it may not only contain the acetylcholine-producing
strains, but in addition also high contents of other acetylated
compounds such as N-acetyl-glycine, homoserine, canavanine, and the
like.
[0052] The term "functional beverage" as used herein is a beverage,
which in addition to its nutritional and sensory function, has a
positive effect on the metabolism and, within a balanced nutrition,
contributes to an improvement of health, an increase in wellbeing
and/or a reduction of health risks. It takes its effect within
normal food habits in an amount common for consumption.
[0053] The functional beverage may comprise additional components
such as herbs, vitamins, minerals, aminoacids, or additional other
food or vegetable ingredients to provide specific health benefits
that go beyond general nutrition. Alternatively, it may include
stimulants such as taurin, glucoronolactone, caffeine, B-vitamins,
guarana, ginseng, gingko biloba, L-carnitine, sugars, antioxidants,
yerba mate, creatine, milk thistle and the like. In a preferred
embodiment, it additionally contains a prebiotic suitable to be
digested by the acetylcholine-producing microorganisms of the
present invention.
[0054] A preferred functional beverage is a drinking yogurt, a
fermented grain beverage, alcohol-free beer, Brottrunk.RTM.,
fruitjuice-based beverages or beverages containing plant or herbal
extracts such as Iberogast.RTM..
[0055] The functional food or functional beverage can preferably
also be administered with at least one additional active agent for
the treatment of intestinal diseases. The additional active agent
can be selected from the group of medicaments as described
above.
[0056] In another embodiment the active agent may preferably be at
least one additional bacterium for maintaining and/or restoring a
favorable gut flora. The additional bacterium can be selected form
the group of probiotic bacteria.
[0057] Preferred pro-biotic bacteria can be selected from the group
comprising Strains from Lactobacillus and Bifidobacterium such as
Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus
casei, Lactobacillus lactis, Lactobacillus reuteri, Lactobacillus
rhamnosus and/or Bifidobacterium lactis.
[0058] Optionally, the composition can also contain a prebiotic. As
used herein, a "prebiotic composition" is an at least
non-digestible food ingredient that beneficially affects the host
by selectively stimulating the growth, activity or both of one of a
limited number of species of microorganisms already resident in the
colon. The prebiotic is preferably a non-digestible oligosaccharide
such as fructo-oligosaccharides, galacto-oligosaccharides,
lactolose, xylo-oligosaccharides, isomalto-oligosaccharides, soy
bean oligosaccharides, gentio-oligosaccharides,
gluco-oligosaccharides, fructans, lactosuccrose, short-chain
fructo-oligosaccharides, and mixtures thereof.
[0059] The present invention also provides a method for the
production of acetylcholine by use of Lactobacilli. Preferably, a
single Lactobacillus strain is used or a combination of
Lactobacillus strains or a composition of Lactobacillus strains.
The Lactobacillus strains used therefore are in particular
Lactobacillus sanfranciscensis strains, Lactobacillus rossiae
strains, Lactobacillus brevis strains, or Lactobacillus plantarum
strains, more particularly strain DSM 26024, DSM 23090, DSM 23091,
DSM 23200, DSM 23092, DSM 23093, DSM 23201, DSM 23174 and DSM 23121
or a strain cultivated therefrom, even more particularly strains
DSM 23090 or DSM 23093.
[0060] Another aspect of the present invention is microbially
produced acetylcholine for use in the treatment and/or prevention
of intestinal diseases as described above. The acetylcholine may be
produced preferably by the microorganisms of the invention. The
microorganisms are preferably Lactobacillaceae such as
Lactobacillus strains, in particular Lactobacillus sanfranciscensis
strains, Lactobacillus rossiae strains, Lactobacillus brevis
strains, or Lactobacillus plantarum strains. The intestinal
diseases encompass the intestinal diseases described above such as
inflammatory bowel diseases or functional bowel disorders.
Preferably, the disease is irritable bowel syndrome and/or
disorders associated with secretions of the intestinal wall
controlled by the enteronervous system. The microbially produced
acetylcholine may be administered orally, e.g. added to food and/or
feed products. The addition may occur during the manufacture of the
food and/or feed product by using the microorganisms of the
invention also for the fermentation of the food and/or feed
product. Such a fermented food product might be sour dough bread,
wherein the live microorganisms are killed during the baking phase
by heat, resulting in a sour dough bread containing acetylcholine
produced by the microorganisms. The content of the microbial
produced acetylcholine is in a range of about 5 to 1000 mg
acetylcholine/kg food or feed product, preferably in a range from
about 20 to 500 mg acetylcholine/kg feed product or food product,
more preferably in a range from about 40-200 mg acetylcholine/kg
feed product or food product. Preferably, the amount of
acetylcholine equates to the recommended daily dosage.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1: Metabolite profile in water extracts of sourdough,
sourdough bread and analog bread. A: Heatmap of absolute metabolite
concentrations in water extract from sourdough (SD), sourdough
bread (BR) and analog bread (AN) in triplicates. Water extracts
constitute 5-10% of total dough or bread is dry mass. B: Principal
component analysis (PCA) of metabolites reveals significant
differences in metabolite concentrations between sourdough (SD),
sourdough bread (BR) and analog bread (AN).
[0062] FIG. 2: Sourdough extracts effectively stimulate stomach
muscle motility by acting directly on muscarinic acetylcholine
receptor (mACHR). A: Muscle tone change induced by either
acetylcholine (ACH) (2.5 .mu.M) or extracts of sourdough (SD),
sourdough bread (BR) or analog bread (AN BR) at 0.02%. Increase in
tone was immediately observed upon addition of the stimulants
(arrow sign) except for extract of analog bread. B: Median (n>4)
change of muscle tone upon differential treatments. The pro-kinetic
effect of acetylcholine (ACH) and extracts of sourdough (SD),
sourdough bread (BR) or analog bread (AN BR) is completely
abolished by mACHR specific antagonist atropine. This indicates
that acetylcholine in extracts directly acts on mACHR.
[0063] FIG. 3: Tetrodotoxin (TTX) has no significant effect on the
stimulation of muscle contraction by sourdough (SD)-derived
acetylcholine (ACH). The figure shows the muscle tone change
stimulated by either acetylcholine (2.5 .mu.M) or 0.02% extracts of
sourdough (SD), sourdough bread (BR) or analog bread (AN BR), and
the effect of TTX pre-treatment. TTX did not exhibit significant
effect on the muscle contraction induced by sourdough ACH
suggesting stimulation is induced by direct action on muscle mACHR
without neuronal mediation.
[0064] FIG. 4: Sourdough and sourdough bread extracts stimulate
chloride ions secretion when applied from either serosal or mucosal
side of intestinal mucosa. A: Ussing chamber set-up with mucosa
piece separating the two chambers. The lower set of electrodes
measure transepithelial voltage (V.sub.TE) and the lateral set--the
short circuit current (I.sub.SC). The secretion is measured
estimated by the change in I.sub.SC necessary to maintain V.sub.TE
at 0 mV. B: Representative I.sub.SC traces stimulated by
acetylcholine (ACH), sourdough (SD), sourdough bread (SD BR) or
analog bread (AN BR) extracts. Area under the curve is calculated
using an integral (.rho.A*s/cm.sup.2) where blue shows response to
extracts and red-striped shows response to electric field
stimulation (EFS). Figure C shows median (n>4) value of change
in I.sub.SC upon treatment on either mucosal side (MUC) or serosal
side (SER) of guinea pig colonic mucosa. Acetylcholine (ACH) and
sourdough (SD) as well as sourdough bread (SD BR) extracts clearly
stimulate secretion when applied to either side of mucosa while
analog brad extract has no effect. Showing that acetylcholine in
sourdough and sourdough bread is responsible for the
stimulation.
[0065] FIG. 5: Atropine completely abrogates chloride ion secretion
stimulated with sourdough (SD) and sourdough bread (SD BR)
extracts. Shown is the mean (n>4) value of change in short
circuit current (I.sub.SC) over time upon acetylcholine (ACH) (10
.mu.M) and extract (0.1%) application on either mucosal side (A) or
serosal side (B) of guinea pig colonic mucosa with and without
atropine pretreatment (1 .mu.M). Atropine completely abrogated
secretion stimulation suggesting the role of mACHR in the secretory
effect of sourdough extracts.
[0066] FIG. 6: Atropine completely abrogates secretion stimulation
by ACH and extracts of sourdough (SD), sourdough bread (BR) or
analog bread (AN BR) when applied serosally but not mucosally.
Shown is the mean (n>4) value of change in short circuit current
(I.sub.SC) over time subsequent to ACH and extract treatment on the
mucosal side with and without atropine pre-treatment (1 .mu.M).
Atropine was added on either serosal or mucosal side. Atropine
completely abrogated secretion when applied on serosal but not
mucosal side.
[0067] FIG. 7: Metabolic profile of sourdough lactic acid bacteria.
A: Heatmap of absolute metabolite values in MRS broth after 24 hour
lactic acid bacteria inoculation (mean of three experiments). B:
PCA plot of metabolites indicates sharp distinction between tested
sourdough bacteria and L. paracasei (LC). C: Acetylcholine (ACH)
plays the strongest role in the separation observed in PCA plot
since L. paracasei does not produce any Acetylcholine.
[0068] FIG. 8: Acetylcholine concentration in MRS media upon 24
hours of incubation with lactic acid bacteria. A: Acetylcholine
concentration in MRS broth upon 24 hour inoculation with
0.25.times.10.sup.7 of bacteria/mL. B: Acetylcholine (ACH)
concentration adjusted to 10.sup.6/mL bacteria count in MRS.
Concentration was determined using LC-MS/MS by comparing the peak
area to the area of solutions with known concentration of
acetylcholine.
[0069] FIG. 9: Concentrated conditioned media of sourdough lactic
acid bacteria significantly inhibits the secretion of interferon
inducible protein 10 (IP-10) by tumor necrosis factor
(TNF)-activated intestinal epithelial cells (IEC). The
concentration of IP-10 in the culture media of Mode-k cells as
measured by ELISA is shown. The cells were incubated for 24 hours
with concentrated conditioned media (cCM) (black bar) and cCM plus
10 ng/mL TNF (grey bar). L. paracasei (L.p.) expresses Lactocepin
PrtP that is capable of efficiently degrading IP-10 and as expected
has the highest inhibitory activity. The cCM of sourdough lactic
acid bacteria significantly inhibit IP-10 secretion but to a lesser
extent. L. sanfranciscensis strains DSM 23174 and DSM 23200 are the
most efficient in inhibiting IP-10 secretion next to L.
paracasei.
[0070] FIG. 10: Formaldehyde-fixed sourdough lactic acid bacteria
significantly inhibit the secretion of interferon inducible protein
(IP-10) by TNF-activated intestinal epithelial cells. The
concentration of IP-10 in the culture media of Mode-k cells as
measured by ELISA is shown. The cells where incubated for 24 hours
with 20 MOI of fixed lactic acid bacteria (black bar) and 20 MOI of
fixed lactic acid bacteria plus 10 ng/mL tumor necrosis factor
(TNF) (grey bar). Fixed L. paracasei (L.p.) has, similar to fixed
L. sanfranciscensis strains DSM 23090 and DSM 23092, the highest
inhibitory activity on the IP-10 secretion by TNF-activated
intestinal epithelial cells (grey bar) as compared to TNF-activated
control.
[0071] Further, the invention shall be explained in more detail by
the following examples.
EXAMPLES
1) Methods and Materials
1.1) Sourdough and Bread
[0072] Sourdough (Vollsauer) was prepared by traditional
propagation of type I sourdough rye starter containing the
Lactobacilli strains DSM 26024, DSM 23090, DSM 23091, DSM 23200,
DSM 23092, DSM 23093, DSM 23201, DSM 23174 and DSM 23121. The
composition of sourdough and sourdough bread is: 71% rye flour, 25%
wheat flour, 1.8% salt and 2% bread crumbs (pH 4.5, acidity 9-10).
The dough was baked at 298.degree. C. for 1.5 hours. Analog bread
was identical to sourdough bread with substitution of sourdough
starter with 2.5% sodium bicarbonate, 0.13% acetic acid and 1.2%
lactic acid.
1.2) Metabolite Analysis
[0073] Water extracts (<10 kDa) of sourdough, sourdough bread
and analog bread as well as MRS growth media of lactic acid
bacteria were subjected to LC-MS/MS analysis for metabolite
quantification. MRS media was filtered with 10 kDa Vivaspin 500
filters (Sartorius Stedim biotech, Goettingen, Germany) before
analysis.
[0074] Samples were measured using:
[0075] Dionex Ultra High Performance Liquid Chromatography
UltiMate.RTM. 3000 (Dionex, Idstein, Germany) [0076]
Pump--HPG-3400SD [0077] Degasser--SRD-3400 [0078]
Autosampler--WPS--3000TSL [0079] Column oven--TCC-3000SD [0080] API
4000 QTRAP, Linear Ion Trap Quadrupole Mass Spectrometer (AB Sciex,
Darmstadt, Germany): [0081] Ionization type--electrospray
ionization (ESI) [0082] Instrument control--Analyst software
(AbSciex, Darmstadt, Germany) [0083] Stationary phase: TSKgel
Amide-80 3 .mu.m (150.times.2 mm, Tosoh Bioscience, Stuttgart,
Germany) [0084] Stationary phase temperature: 40.degree. C.
TABLE-US-00001 [0084] Mobile phase: eluent A: acetonitrile/5 mM/L
ammonium acetate in water (95 + 5) eluent B: 5 mM/L ammonium
acetate in water (95 + 5) gradient: 0 min 90% A 10% B 5 min 90% A
10% B 10 min 80% A 20% B 15 min 50% A 50% B 18 min 0% A 100% B 21
min 0% A 100% B 24 min 90% A 10% B 30 min 90% A 10% B flow: 200
.mu.l/min
[0085] The Chromatograms were analysed with Multiquant 2.0 (AB
Sciex, Darmstadt, Germany) and concentrations in the samples were
calculated according to the spectra of standards.
1.3) Extraction
[0086] Sourdough, sourdough bread and analog bread were
freeze-dried and grinded into powder. 100 g of powdered bread or
sourdough was solubilized in 500 mL distilled water and extracted
for 3 hours at 50.degree. C. with constant stirring. The suspension
was centrifuged at 9000 rpm for 20 min. The supernatant was
collected and kept at 4.degree. C. The pellet was re-suspended
again in 500 mL distilled water and 3 hour extraction repeated.
After centrifugation the pellet was again re-suspended in 500 mL
distilled water and extracted overnight. Supernatants after three
extraction steps (total volume of approx. 1.5 L) were pooled
together and step-wise filtered using Vivaflow 200 cassettes of 0.2
.mu.m, 100 kDa and 10 kDa exclusion thresholds (Sartorius,
Goettingen, Germany). The filtrates of the 100 kDa and 10 kDa
exclusion thresholds were freeze dried and re-suspended in
distilled water to 25% for in vitro assays. 10 g of <10 kDa
fraction was extracted from 100 g freeze-dried bread and
sourdough.
1.4) Endotoxin Measurement and Clean-Up
[0087] Endotoxin concentrations measurement in water extracts from
sourdough, sourdough bread and analog bread were determined using
Limulus Amebocyte Lysate (LAL) Chromogenic Endpoint Assay (Hycult
biotech, Uden, Netherlands). The assay was performed according to
the manufacturer's instructions. Endotoxin contamination in water
extracts from sourdough, sourdough bread and analog bread was
removed using Detoxi-Gel.TM. Endotoxin Removing Columns (Thermo
Scientific, Rockford, USA), containing a resin with immobilized
polymyxin B to bind and remove pyrogens from solution. The removal
of endotoxin was performed according to the manufacturer's
instructions.
1.5) ELISA
[0088] Interferon inducible protein (IP-10) (murine/human) and
(murine) concentrations in cell culture supernatants were
determined using the appropriate ELISA kits (R&D Europe,
Abington, England) according to the manufacturer's instructions.
The ELISA was performed using Nunc MaxiSorp.RTM. flat-bottom 96
well plates (Greiner Bio-One GmbH, Frickenhausen, Germany). Briefly
96-well plates were coated with the appropriate capture antibody
overnight at RT. Plates were washed 3 times with phosphate buffered
saline (PBS), blocked with 1% bovine serum albumin in PBS and
incubated with cell culture supernatants for 1.5 h at RT. Plates
were washed and incubated with the appropriate detection antibody
for 1.5 h at RT. Plates were washed and incubated with a detection
enzyme. Plates were washed and incubated with a substrate solution.
Protein concentration was determined by photometrical analysis of
the reaction of substrate and detection enzyme.
1.6) Bacterial Culture
[0089] L. sanfranciscensis strains (DSM 23090, DSM 23091, DSM
23092, DSM 23093, DSM 23174, DSM 23200, DSM 23201) and L. rossiae
(DSM 26024) isolated from sourdough, L. sanfranciscensis type
strain DSM 20451 (DSMZ GmbH, Braunschweig, Germany), L. plantarum
FUA 3038 and L. brevis 3113 (provided by Prof. Ganzle from
University of Alberta, Canada), L. paracasei VSL#3 (provided by Dr.
DeSimone, L'Aquila, Italy) were grown at 30.degree. C. in MRS broth
(pH 5.4) containing freshly added 0.15% L-cystein under anaerobic
conditions using Anaerogen packages (Anaerogen, Basingstoke, Oxoid,
UK). Fixed bacteria (5% formaldehyde, 4 hours, 4.degree. C.) were
washed three times with sterile PBS before use. Concentrated
conditioned media (CM) were generated by transferring bacteria
(5.times.10.sup.7 cfu/ml) from anovernight culture to DMEM (1%
glutamine, 20 mM HEPES) and anerobical cultivation overnight at
30.degree. C. Bacteria and bacterial supernatant (CM) were
separated after centrifugation (4500 g, 10 min, RT). CM was
adjusted to pH 7.4, filter sterilized (0.22 .mu.m), and
concentrated (100.times.) using Vivacell filter systems with an
exclusion size of 100 kDa (Satorius Stedim Biotech, Goettingen,
Germany). Concentrated conditioned media was diluted to 1.times. in
the cell culture stimulation experiments. Agar plates were obtained
by adding 1.5% of agar to the above described respective
medium.
1.7) Motility
[0090] Motility measurements were performed with corpus circular
muscle preparations from Dunkin Hardley guinea pigs (Sulzfeld and
Harlan Winkelmann GmbH, Borchen, Germany). Contractile force of the
muscle was measured using force transducer in organ bath using
LabChart 5 software (ADInstruments, Spechbach, Germany). Briefly,
stomach muscle tissue was dissected from mucosa layer in
continuously perfused ice-cold preparation Krebs solution (pH 7.4)
(MgCl.sub.2.times.6H.sub.2O 1.2 mM, CaCl.sub.2.times.2H.sub.2O 2.5
mM, NaH.sub.2PO.sub.4 1.2 mM, NaCl 117 mM, NaHCO.sub.3 25 mM,
C.sub.6H.sub.12O.sub.6 11 mM, KCl 4.7 mM). A 1.5 cm.sup.2 piece of
corpus circular muscle was cut out and mounted from both ends with
polyamide thread between two electrodes into organ bath in 20 mL
experimental Krebs solution (identical to preparation Krebs except
for NaHCO.sub.3 20 mM) at 37.degree. C. and aerated continuously
with Carbogen (95% O.sub.2 and 5% CO.sub.2). After an equilibration
period of 45 min muscle is preparations were stimulated by
electrical field stimulation (EFS) to test vitality. The change in
contractile force during EFS as well experimental treatment was
measured by the force transducer. The time lapse between any
treatments was always 20 min.
1.8) Ussing Chamber
[0091] The ion movement across intestinal epithelia was measured
with Ussing chamber technique (Easy mount chambers, Physiologic
instruments, San Diego, USA) and LabChart 5 software
(ADInstruments, Spechbach, Germany). Briefly segments, of the
distal colon of Dunkin Hardley guinea pigs (Sulzfeld and Harlan
Winkelmann GmbH, Borchen, Germany) were dissected, the muscle
layers removed and mucosa/submucosa preparations were mounted into
slider with a recording area 0.5 cm.sup.2. Apical and basolateral
sides were bathed separately in 5 mL Krebs solution. During
experimental procedures, the bath was maintained at 37.degree. C.
and aerated continuously with Carbogen (95% 0.sub.2 and 5%
CO.sub.2). After an equilibration period of 45 min tissue was
electrically stimulated (Parameters: stimulus strength 6V, duration
10 sec, frequency 10 Hz, single pulse duration 0.5 ms) to assess
tissue vitality. For assessment of active ion transport spontaneous
occurring transepithelial voltage (V.sub.TE) formed by passive ion
transport across the tissue was set to 0 mV by applying short
circuit current (I.sub.SC). When the active chloride ion secretion
is induced an increase in I.sub.SC is observed necessary to keep
V.sub.TE at 0 mV. The change in I.sub.SC is equivalent to the
current generated by the anions secretion or cation absorption.
Transepithelial resistance (TER=V.sub.TE/I.sub.SC.times.1000/2) of
tissue was measured at the beginning and at the end of each
experiment to assess the tissue integrity.
1.9) Statistical Analysis
[0092] Data are expressed as mean values.+-.standard deviation
(SD). All statistical computations were performed using Statistical
programming platform R comparing treatment vs. corresponding
control group were analyzed using unpaired t-tests. Data comparing
several treatments vs. corresponding control group were analyzed
using One-Way ANOVA followed by an appropriate multiple comparison
procedure. If data was not normally distributed or comprised
discontinuous data, non-parametrical tests (Mann-Whitney/Rank sum
test, ANOVA on ranks) were used. Differences were considered
significant if p-values were <0.05 (*) or <0.01 (**).
Principal component analysis (PCA) is described in Pearson, K.; On
Lines and Planes of Closest Fit to Systems of Points in Space,
Philosophical Magazine (1901), 2 (11), 559-572 and Theodoridis, G.,
Gika, H. G., Wilson, I. D.; LC-MS-based methodology for global
metabolite profiling in metabonomics/metabolomics, TrAC Trends in
Analytical Chemistry (2008), 27 (3), 251-260.
2.) Results
[0093] 2.1) LC-MS/MS Analysis of Metabolites in Extracts from
Sourdough, Sourdough Bread and Analog Bread
[0094] To compare the effect of fermentation on sourdough and
sourdough bread water soluble extracts (<10 kDa, triplicates) of
raw sourdough, sourdough bread and analog bread prepared from three
different batches were subjected to LC-MS/MS analysis. The
concentration of metabolites in extracts was determined by
comparison to the standard solution with known concentration of
metabolites.
[0095] Principal component analysis (PCA) demonstrated significant
differences in metabolites isolated from sourdough, sourdough bread
and analog bread (FIG. 1). Raw sourdough has significantly higher
amounts of free amino acids reflecting proteolitic acitivity of
endogenous flour enzymes as well as lactic acid bacteria proteases.
Before baking fresh unfermented flour is added to the sourdough
explaining why there are no differences in the free amino acid
content between analog and sourdough bread. Acetylcholine is a
metabolite that is consistently present in sourdough and sourdough
bread but not in analog bread (Table 1). Fermentation by sourdough
bacteria is the only difference between sourdough bread and analog
bread, suggesting acetylcholine is produced by microorganisms
present in sourdough.
TABLE-US-00002 TABLE 1 Sourdough and sourdough bread contain high
amounts of acetylcholine of with 38.6 mg/kg in dry bread.
Concentration was determined using LC-MS/MS by comparing the peak
area to the area of solutions with standard concentrations of
acetylcholine. ACH concentration ACH concentration in bread Water
extracts in water extracts or sourdough, dry mass Sourdough, 10 kDa
1819 .+-. 717 .mu.M 26.5 .+-. 10.4 mg/kg Sourdough bread, 10 2644
.+-. 273 .mu.M 38.6 .+-. 4.03 mg/kg kDa Analog bread, 10 kDa 44.5
.+-. 1.0 .mu.M 0.64 .+-. 0.03 mg/kg
2.2) Sourdough-Derived Acetylcholine Triggers Muscle Contraction In
Vitro
[0096] Acetylcholine (ACH) is a neurotransmitter that is
responsible for the activation of motility in the gastro-intestinal
tract by stimulating either muscarinic (mACHR) or nicotinic ACH
receptors (nACHR) on the muscle cells. To determine if the
sourdough-derived acetylcholine mimicks this activity, isolated
corpus muscle of guinea pig was stimulated with extracts of
sourdough, sourdough bread and analog bread and the contraction
stimulation was measured. Both sourdough and sourdough extracts but
not analog bread extract induced muscle contractions similar to
acetylcholine at equivalent concentration (FIG. 2). Atropine,
mACHR-specific antagonist, was used to determine whether extracts
stimulate contraction by activating muscarinic or nicotinic ACHR.
Pre-treatment of muscle strips with atropine completely abrogated
stimulation by ACH as well as by sourdough and sourdough bread
extracts indicating that sourdough-derived acetylcholine is acting
via mACHR.
[0097] Furthermore to clarify whether sourdough-derived ACH acts
through the activation of neurons that consequently stimulate
muscle cells, muscle preparations were pre-treated with
tetrodotoxin (TTX). TTX blocks action potential generated by
neurons that abrogates downstream signalling. TTX pre-treatment had
no significant effect on muscle contraction induced by
acetylcholine and extracts suggesting that both directly activate
mACHR on muscle cells (FIG. 3). Motility is one of the crucial
functions of the GI tract. Agonists and antagonists of serotonin
(5-hydroxytryptamine) receptors are a common treatment option for
modulating motility and consequently bowel movements of IBS
patients (Camilleri, M. and V. Andresen, Current and novel
therapeutic options for irritable bowel syndrome management. Dig
Liver Dis, 2009. 41(12): p. 854-62). These observations show that
external application of ACH at local sites could also be utilised
to modulate ENS-mediated motility.
2.3) Sourdough-Derived Acetylcholine Stimulates Secretion by the
Intestinal Mucosa from the Luminal Side
[0098] Acetylcholine (ACH), released by the enteric neurons to the
serosal side of the intestinal wall, stimulates secretion of
chloride ions by the mucosa subsequently driving the passive
transport of water into the lumen. This effect is transient due to
rapid degradation of acetylcholine by acetylcholine esterase. The
effect of sourdough-derived ACH on the intestinal secretory
function was tested in guinea pig colon. Extracts of sourdough,
sourdough bread, analog bread as well as (pure) ACH were applied on
either luminal (mucosal) or serosal side of intestinal
mucosa/submucosa preparations from guinea pig distal colon. The
experiment was performed in Ussing chamber and the change in
short-circuit current (I.sub.SC) was measured. In this system,
passive flow of ions across a tissue or epithelial cell layer is
eliminated by balancing electrical, osmotic, hydrostatic and
chemical gradients across the preparation, such that only active
ion transport is measured. In the Ussing chamber, electrodes are
placed close to each side of the tissue to allow detection of the
spontaneous potential difference (PD) across the epithelium,
generated as a consequence of active ion transport (Hirota, C. L.
and McKay D. M., Cholinergic regulation of epithelial ion transport
in the mammalian intestine, Br. J. Pharmacol., 2006, 149(5):p.
463-79). Surprisingly an increase in I.sub.SC could be observed
when the preparations were stimulated from both mucosal and serosal
side by ACH as well as ACH-containing extracts (analog bread
extract had no effect) (FIG. 4), showing that acetylcholine in
sourdough and sourdough bread is suitable for the stimulation.
[0099] The tissue was pre-treated with atropine and the effect of
extracts on secretion measured again (FIG. 5). Atropine completely
abolished the response corroborating the role of mACHR in the
pro-secretory effect of sourdough extracts.
[0100] Earlier research provides evidence of the expression of ACHR
in intestinal epithelial cells on the basolateral side but not on
the apical side of the cell layer (Hirota, C. L. and McKay D. M.,
Cholinergic regulation of epithelial ion transport in the mammalian
intestine, Br. J. Pharmacol., 2006, 149(5):p. 463-79). Therefore it
is highly probable that ACH applied from apical side crosses the
cell layer and stimulates the receptors on the basolateral side.
This hypothesis was verified by the fact that when ACH is applied
apically, i.e. on the mucosal or luminal side, the secretory effect
was significantly inhibited when atropine was applied to the
serosal but not to the mucosal side (FIG. 6). Atropine applied on
the serosal side blocked all mACHR on basolateral side abrogating
ACH activity. However, when atropine is applied on the mucosal side
this results into smaller amounts reaching basolateral side and
thus only partially inhibiting ACH activity. The observation that
atropine can cross epithelial layer and block basolateral mACHR was
confirmed by the fact that when ACH was applied serosally and
atropine mucosally secretion was inhibited (data not shown).
[0101] These observations show that external application of ACH at
local sites could also be utilised to modulate ENS-mediated fluid
secretion. This is especially important, since fluid secretion into
the intestine is hypothesized to provide the ideal environment for
enzymatic digestion and to facilitate the passage of stool through
the intestinal tract. Furthermore recent studies suggest that acute
and locally targeted water secretion serves as a protective measure
against epithelial damage at points of particular mechanical stress
(Barrett, K. E. and S. J. Keely, Chloride secretion by the
intestinal epithelium: molecular basis and regulatory aspects. Annu
Rev Physiol, 2000. 62: p. 535-72 and Sidhu, M. and H. J. Cooke,
Role for 5-HT and ACh in submucosal reflexes mediating colonic
secretion. Am J Physiol, 1995. 269(3 Pt 1): p. G346-51).
2.4) LC-MS/MS Analysis Revealed Presence of Acetylcholine in Growth
Media of Sourdough Lactic Acid Bacteria
[0102] Seven strains of L. sanfranciscensis (DSM 23090-DSM 23201)
and L. rossiae (DSM 26024) isolated from sourdough, L. plantarum
FUA 3038 and L. brevis 3113 isolated from another sourdoughs, and
L. paracasei (VSL#3) were grown for 24 hours in MRS media. The
growth media was collected, filtered and analyzed using
LC-MS/MS.
[0103] PCA analysis shows significant difference in the metabolites
profiles of all sourdough isolated bacteria compared to L.
paracasei. The separation is due in major part to the impact of
acetylcholine present in sourdough bacteria growth media but not in
L. paracasei media (FIG. 7). The highest ACH producer over 24 hours
is L. brevis 3113, which additionally has the highest growth rate
(FIG. 8A). However when the concentration is adjusted to a
bacterial number, e.g. 10.sup.6/ml, in the broth, the most ACH per
bacterial cell is produced by L. sanfranciscensis strains DSM 23090
and DSM 23093 (FIG. 8B).
2.5) Sourdough Lactic Acid Bacteria Inhibit Secretion of Chemokine
IP-10 by TNF-Activated Intestinal Epithelial Cells
[0104] Lactocepin PrtP, a serine protease expressed in L. paracasei
(VSL#3), selectively degrades pro-inflammatory chemokine
interferone inducible protein 10 (IP-10). To investigate if PrtP is
also present in the Lactobacilli of the present invention, the
total bacterial DNA was isolated from eight sourdough strains: L.
sanfranciscensis (DSM 23090, DSM 23091, DSM 23092, DSM 23093, DSM
23174, DSM 23200, DSM 23201) and L. rossiae (DSM 26024) as well as
L. paracasei as positive control. DNA was amplified using
Lactocepin PrtP specific primers and visualized on agarose gel. No
detectable amounts of lactocepin PrtP gene were present in
sourdough-isolated lactic acid bacteria. The eight sourdough lactic
acid bacteria strains and L. paracasei were tested for their
effects on the secretion of pro-inflammatory chemokine IP-10 by
unstimulated and TNF-activated Mode-K cells. Interestingly, both
conditioned media (FIG. 9) and fixed lactic acid bacteria (FIG. 10)
demonstrated IP-10 inhibitory activity despite the fact that no
Lactocepin PrtP gene was detected. This suggests that there are
both secreted and cell surface-bound factors produced by sourdough
lactic acid bacteria suitable to inhibit secretion of
pro-inflammatory chemokine IP-10. This result provide a basis for
potential treatment of IBD and relief of their symptoms, since
IP-10 has been implicated in re-enforcing intestinal inflammation
in IBD patients.
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