U.S. patent application number 16/333770 was filed with the patent office on 2020-03-19 for bacteria.
The applicant listed for this patent is DUPONT NUTRITION BIOSCIENCES APS. Invention is credited to Sofia Forssten, Sampo Lahtinen, Harri Makivuokko, Jaana Matto, Janne Nikkila, Arthur Ouwehand, Heli Putaala.
Application Number | 20200085888 16/333770 |
Document ID | / |
Family ID | 56985467 |
Filed Date | 2020-03-19 |
United States Patent
Application |
20200085888 |
Kind Code |
A1 |
Putaala; Heli ; et
al. |
March 19, 2020 |
BACTERIA
Abstract
The present invention relates to bacteria and metabolites
thereof that are capable of binding to A, B and/or O blood type
antigens or which are acid and/or bile tolerant, their use in
probiotic compositions and food products, and methods for their
selection. The invention also relates to the use of said bacteria
and metabolites for the prevention and/or treatment of
gastrointestinal disorders.
Inventors: |
Putaala; Heli; (KANTVIK,
FI) ; Forssten; Sofia; (KANTVIK, FI) ;
Lahtinen; Sampo; (KANTVIK, FI) ; Ouwehand;
Arthur; (KANTVIK, FI) ; Matto; Jaana;
(HELSINKI, FI) ; Makivuokko; Harri; (HELSINKI,
FI) ; Nikkila; Janne; (HELSINKI, FI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DUPONT NUTRITION BIOSCIENCES APS |
Copenhagen K |
|
DK |
|
|
Family ID: |
56985467 |
Appl. No.: |
16/333770 |
Filed: |
September 12, 2017 |
PCT Filed: |
September 12, 2017 |
PCT NO: |
PCT/EP2017/072859 |
371 Date: |
March 15, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; A61K
35/745 20130101; A61P 1/12 20180101; C12R 1/23 20130101; A61K
35/741 20130101; A61K 35/747 20130101; A61K 35/745 20130101; A61K
2300/00 20130101; A61P 1/00 20180101; A61K 35/741 20130101; A23L
33/135 20160801; C12R 1/24 20130101; A61K 35/747 20130101; A23L
33/30 20160801; A61K 2300/00 20130101; C12R 1/225 20130101; A61P
1/14 20180101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/747 20060101
A61K035/747; A61K 35/745 20060101 A61K035/745; A61P 1/04 20060101
A61P001/04; A61P 1/14 20060101 A61P001/14; A23L 33/00 20060101
A23L033/00; A23L 33/135 20060101 A23L033/135; A61P 1/12 20060101
A61P001/12 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 16, 2016 |
EP |
16189291.4 |
Claims
1-15. (canceled)
16. A method of preventing or treating a gastrointestinal disorder
comprising administering to a subject in need thereof a
pharmaceutically effective amount of a bacteria or metabolite
thereof, wherein the bacteria are characterized as having: a) an
average A antigen adhesion of greater than 100% when compared to L.
crispatus LMG18199, b) an average B antigen adhesion of greater
than 100% when compared to L. crispatus LMG18199, c) an average O
antigen adhesion of greater than 100% when compared to L. crispatus
LMG18199, d) greater than -2.6 average acid tolerance represented
as a log change when measured at a pH of 2.5 for 1.5 hours as
defined in an acid resistance assay, and/or e) greater than 40%
average bile tolerance represented as growth in 0.9% dehydrated
fresh bile as a percentage of growth in MRS without bile as defined
in a bile resistance assay.
17. The method of claim 16, wherein the method comprises
administering to the subject a pharmaceutically effective amount of
the bacteria.
18. The method of claim 17, wherein the bacteria are characterized
as having an average A antigen adhesion of greater than 100% when
compared to L. crispatus LMG18199.
19. The method of claim 17, wherein the bacteria are characterized
as having an average B antigen adhesion of greater than 100% when
compared to L. crispatus LMG18199.
20. The method of claim 17, wherein the bacteria are characterized
as having an average O antigen adhesion of greater than 100% when
compared to L. crispatus LMG18199.
21. The method of claim 17, wherein the bacteria are characterized
as having greater than -2.6 average acid tolerance represented as a
log change when measured at a pH of 2.5 for 1.5 hours as defined in
an acid resistance assay.
22. The method of claim 17, wherein the bacteria are characterized
as having greater than 40% average bile tolerance represented as
growth in 0.9% dehydrated fresh bile as a percentage of growth in
MRS without bile as defined in a bile resistance assay.
23. The method of claim 17, wherein the method further comprises
administering to the subject Lactobacillus acidophilus NCFM,
Bifidobacterium lactis BL-04, Lactobacillus paracasei LPC37,
Bifidobacterium lactis HNO19 or Bifidobacterium lactis Bi-07.
24. The method of claim 17, wherein the bacteria comprise a mutant,
variant or progeny of DSM 32111, DSM 32108, DSM 32107, DSM 32098,
DSM 32104, DSM 32112, DSM 32109, DSM 32105, DSM 32110, DSM 32103,
DSM 32099, DSM 32106, DSM 32097, DSM 32114 or DSM 32115
characterized as having: a) an average A antigen adhesion of
greater than 100% when compared to L. crispatus LMG18199, b) an
average B antigen adhesion of greater than 100% when compared to L.
crispatus LMG18199, c) an average O antigen adhesion of greater
than 100% when compared to L. crispatus LMG18199, d) greater than
-2.6 average acid tolerance represented as a log change when
measured at a pH of 2.5 for 1.5 hours as defined in an acid
resistance assay, and/or e) greater than 40% average bile tolerance
represented as growth in 0.9% dehydrated fresh bile as a percentage
of growth in MRS without bile as defined in a bile resistance
assay.
25. The method of claim 17 comprising administering to the subject
a combination of two bacterial strains, wherein each of the
bacterial strains is characterized as having: a) an average A
antigen adhesion of greater than 100% when compared to L. crispatus
LMG18199, b) an average B antigen adhesion of greater than 100%
when compared to L. crispatus LMG18199, c) an average O antigen
adhesion of greater than 100% when compared to L. crispatus
LMG18199, d) greater than -2.6 average acid tolerance represented
as a log change when measured at a pH of 2.5 for 1.5 hours as
defined in an acid resistance assay, and/or e) greater than 40%
average bile tolerance represented as growth in 0.9% dehydrated
fresh bile as a percentage of growth in MRS without bile as defined
in a bile resistance assay.
26. The method of claim 16, wherein the gastrointestinal disorder
comprises inflammatory bowel disease, irritable bowel syndrome,
Chrone's disease, ulcerative colitis, constipation or diarrhea.
27. A method of preventing or treating a gastrointestinal disorder
comprising administering to a subject in need thereof a
pharmaceutically effective amount of bacteria or a metabolite
thereof, wherein the bacteria comprise DSM 32111, DSM 32108, DSM
32107, DSM 32098, DSM 32104, DSM 32112, DSM 32109, DSM 32105, DSM
32110, DSM 32103, DSM 32099, DSM 32106, DSM 32097, DSM 32114 or DSM
32115.
28. A method of claim 27, wherein the method comprises
administering to the subject a pharmaceutically effective amount of
DSM 32111, DSM 32108, DSM 32107, DSM 32098, DSM 32104, DSM 32112,
DSM 32109, DSM 32105, DSM 32110, DSM 32103, DSM 32099, DSM 32106,
DSM 32097, DSM 32114 or DSM 32115.
29. The method of claim 28, wherein the method further comprises
administering to the subject Lactobacillus acidophilus NCFM,
Bifidobacterium lactis BL-04, Lactobacillus paracasei LPC37,
Bifidobacterium lactis HNO19 or Bifidobacterium lactis Bi-07.
30. The method of claim 29, wherein the method comprises
administering to the subject DSM 32109, Lactobacillus acidophilus
NCFM, Bifidobacterium lactis BL-04, Lactobacillus paracasei LPC37,
Bifidobacterium lactis HNO19 and Bifidobacterium lactis Bi-07.
31. The method of claim 27, wherein the gastrointestinal disorder
comprises inflammatory bowel disease, irritable bowel syndrome,
Chrone's disease, ulcerative colitis, constipation or diarrhea.
32. A probiotic composition, wherein: the probiotic composition
comprises: bacteria or a metabolite thereof, and a carrier; and the
bacteria are characterized as having: a) an average A antigen
adhesion of greater than 100% when compared to L. crispatus
LMG18199, b) an average B antigen adhesion of greater than 100%
when compared to L. crispatus LMG18199, c) an average O antigen
adhesion of greater than 100% when compared to L. crispatus
LMG18199, d) greater than -2.6 average acid tolerance represented
as a log change when measured at a pH of 2.5 for 1.5 hours as
defined in an acid resistance assay, and/or e) greater than 40%
average bile tolerance represented as growth in 0.9% dehydrated
fresh bile as a percentage of growth in MRS without bile as defined
in a bile resistance assay.
33. The probiotic composition of claim 32 comprising bacteria
characterized as having: a) an average A antigen adhesion of
greater than 100% when compared to L. crispatus LMG18199, b) an
average B antigen adhesion of greater than 100% when compared to L.
crispatus LMG18199, c) an average O antigen adhesion of greater
than 100% when compared to L. crispatus LMG18199, d) greater than
-2.6 average acid tolerance represented as a log change when
measured at a pH of 2.5 for 1.5 hours as defined in an acid
resistance assay, and/or e) greater than 40% average bile tolerance
represented as growth in 0.9% dehydrated fresh bile as a percentage
of growth in MRS without bile as defined in a bile resistance
assay.
34. The probiotic composition of claim 33 further comprising
Lactobacillus acidophilus NCFM, Bifidobacterium lactis BL-04,
Lactobacillus paracasei LPC37, Bifidobacterium lactis HNO19 or
Bifidobacterium lactis Bi-07.
35. The probiotic composition of claim 33 comprising a combination
of two bacterial strains, wherein each of the bacterial strains is
characterized as having: a) an average A antigen adhesion of
greater than 100% when compared to L. crispatus LMG18199, b) an
average B antigen adhesion of greater than 100% when compared to L.
crispatus LMG18199, c) an average O antigen adhesion of greater
than 100% when compared to L. crispatus LMG18199, d) greater than
-2.6 average acid tolerance represented as a log change when
measured at a pH of 2.5 for 1.5 hours as defined in an acid
resistance assay, and/or e) greater than 40% average bile tolerance
represented as growth in 0.9% dehydrated fresh bile as a percentage
of growth in MRS without bile as defined in a bile resistance
assay.
36. The probiotic composition of claim 33 comprising a mutant,
variant or progeny of DSM 32111, DSM 32108, DSM 32107, DSM 32098,
DSM 32104, DSM 32112, DSM 32109, DSM 32105, DSM 32110, DSM 32103,
DSM 32099, DSM 32106, DSM 32097, DSM 32114 or DSM 32115
characterized as having: a) an average A antigen adhesion of
greater than 100% when compared to L. crispatus LMG18199, b) an
average B antigen adhesion of greater than 100% when compared to L.
crispatus LMG18199, c) an average O antigen adhesion of greater
than 100% when compared to L. crispatus LMG18199, d) greater than
-2.6 average acid tolerance represented as a log change when
measured at a pH of 2.5 for 1.5 hours as defined in an acid
resistance assay, and/or e) greater than 40% average bile tolerance
represented as growth in 0.9% dehydrated fresh bile as a percentage
of growth in MRS without bile as defined in a bile resistance
assay.
37. A probiotic composition, wherein: the probiotic composition
comprises: bacteria or a metabolite thereof, and a carrier; and the
bacteria comprise DSM 32111, DSM 32108, DSM 32107, DSM 32098, DSM
32104, DSM 32112, DSM 32109, DSM 32105, DSM 32110, DSM 32103, DSM
32099, DSM 32106, DSM 32097, DSM 32114 or DSM 32115.
38. The probiotic composition of claim 37 comprising DSM 32111, DSM
32108, DSM 32107, DSM 32098, DSM 32104, DSM 32112, DSM 32109, DSM
32105, DSM 32110, DSM 32103, DSM 32099, DSM 32106, DSM 32097, DSM
32114 or DSM 32115.
39. The probiotic composition of claim 38 further comprising
Lactobacillus acidophilus NCFM, Bifidobacterium lactis BL-04,
Lactobacillus paracasei LPC37, Bifidobacterium lactis HNO19 or
Bifidobacterium lactis Bi-07.
40. The probiotic composition of claim 39 comprising DSM 32109,
Lactobacillus acidophilus NCFM, Bifidobacterium lactis BL-04,
Lactobacillus paracasei LPC37, Bifidobacterium lactis HNO19 and
Bifidobacterium lactis Bi-07.
41. A food product, dietary supplement, medical food or
nutraceutical, wherein the food product, dietary supplement,
medical food or nutraceutical comprises the probiotic composition
of claim 32.
42. The food product, dietary supplement, medical food or
nutraceutical of claim 41 comprising a nutritionally acceptable
carrier, nutritionally acceptable diluent, nutritionally acceptable
excipient, nutritionally acceptable adjuvant or nutritionally
active ingredient in addition to the bacteria or metabolite.
43. The food product of claim 42 comprising a soft drink, fruit
juice, a beverage comprising whey protein, health tea, cocoa drink,
milk drink, lactic acid bacteria drink, yoghurt, cheese, ice cream,
water ice, confectionery, biscuit, cake, snack food, balanced food,
balanced drink, fruit filling, glaze, chocolate bakery filling,
cheese cake flavored filling, fruit flavored cake filling, cake
icing, doughnut icing, instant bakery filling cream, cookie
filling, ready-to-use bakery filling, reduced-calorie filling,
adult nutritional beverage, acidified soy/juice beverage,
aseptic/retorted chocolate drink, bar mix, beverage powder,
calcium-fortified milk or calcium-fortified coffee.
44. The food product of claim 42 comprising American cheese sauce,
anti-caking agent for grated or shredded cheese, chip dip, cream
cheese, dry-blended whip topping, fat-free sour cream, freeze/thaw
dairy whipping cream, freeze/thaw stable whipped topping, low fat
or light natural cheddar cheese, low fat Swiss style yoghurt,
aerated frozen dessert, hard pack ice cream, low-fat ice cream,
soft serve ice cream, barbecue sauce, cheese dip sauce, cottage
cheese dressing, dry mix Alfredo sauce, mixed cheese sauce or dry
mix tomato sauce.
45. A food product, dietary supplement, medical food or
nutraceutical wherein the food product, dietary supplement, medical
food or nutraceutical comprises the probiotic composition of claim
37.
46. A pharmaceutical composition, wherein the pharmaceutical
composition comprises the probiotic composition of claim 32.
47. The pharmaceutical composition of claim 46 comprising a
pharmaceutically acceptable carrier, pharmaceutically acceptable
diluent, pharmaceutically acceptable excipient, pharmaceutically
acceptable adjuvant or pharmaceutically active ingredient in
addition to the bacteria or metabolite.
48. A pharmaceutical composition, wherein the pharmaceutical
composition comprises the probiotic composition of claim 37.
49. A method of making the probiotic composition of claim 32
comprising combining the bacteria or metabolite with the
carrier.
50. A method of making the probiotic composition of claim 37
comprising combining the bacteria or metabolite with the
carrier.
51. A method of selecting bacteria or a metabolite thereof for
treating or preventing a gastrointestinal disorder, wherein the
method comprises: a) admixing test bacteria or a metabolite thereof
with an A antigen, B antigen, O antigen or a combination thereof;
b) admixing Lactobacillus reuteri RC-14 control bacteria with the A
antigen, B antigen, 0 antigen or combination thereof; c) incubating
said admixtures of step a) and b); d) comparing the affinity of
adhesion to said A antigen, B antigen, O antigen or combination
thereof of the test bacteria or metabolite thereof and
Lactobacillus reuteri RC-14; and e) selecting bacteria (or a
metabolite thereof) having a higher affinity of adhesion to said A
antigen, B antigen, O antigen or combination thereof than
Lactobacillus reuteri RC-14.
52. A method of selecting bacteria or a metabolite thereof for
treating or preventing a gastrointestinal disorder, wherein the
method comprises: assaying one or both of the following: the
average acid tolerance of the bacteria at a pH of 2.5 for 1.5
hours, and the average bile tolerance of the bacteria in 0.9% bile;
and selecting bacteria (or a metabolite thereof) having: greater
than -2.6 average acid tolerance represented as a log change when
measured at a pH of 2.5 for 1.5 hours, and/or greater than 40%
average bile tolerance represented as growth in 0.9% bile as a
percentage of growth in MRS without bile.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel bacteria and
metabolites thereof, their use in probiotic compositions and food
products, methods for selection of probiotic bacteria and methods
of personalising a probiotic composition or food product. The
invention also relates to the use of said bacteria, metabolites and
compositions for the prevention and/or treatment of
gastrointestinal disorders.
BACKGROUND
[0002] The human body is colonised with a myriad of microbes
representing over 1000 bacterial species. The composition and
density of the microbiota is specific for each body location. The
majority of the bacterial biomass resides in the gastrointestinal
tract (GIT), especially in the lumen of the large intestine, where
two populations are present, the lumen and mucosa-associated
populations that differ from each other. The microbiota has an
important role in human health. It contributes to the maturation of
the gut tissue, to host nutrition, pathogen resistance, epithelial
cell proliferation, host energy metabolism and immune response. An
altered composition and diversity of the GIT microbiota have been
associated with several diseases, such as inflammatory bowel
disease (IBD), irritable bowel syndrome (IBS), rheumatoid
arthritis, atopic eczema, asthma and type 1 diabetes.
[0003] The microbiota of adults is fairly stable over time and
unique within an individual. The similarity of the dominant
microbial population is higher in monozygotic twins compared to
unrelated subject suggesting the role of host genetic factors on
the microbiota composition. Some animal studies suggest that the
major histocompatibility complex is involved in the genetic
regulation of gut microbiota. However, little is known about which
genes or other factors determine or regulate the spectrum of
microbial composition.
[0004] The mucosal layer covering the gut epithelium has an
important role as the first layer of host defences, but it also
enables contacts between intestinal microbiota and the host. The
mucus is mainly composed of mucins, large glycoproteins containing
a protein core and attached oligosaccharides. Although the mucus
layer prevents the direct contact of the bacteria with the
epithelial cells in the colon, it provides adhesion sites for the
GIT bacteria and has thus an important role in bacterial
colonization. Besides adhesion sites, the secreted mucus provides
endogenous substrate for bacteria. The mucus may be a major
nutrient source in situations, where carbohydrates originating
elsewhere are limited.
[0005] Blood group antigens are attached to various components in
the red blood cell membrane, and the antigens expressed on the red
blood cell determine an individual's blood group. The main two
blood groups are called ABO (with blood types A, B, AB, and O) and
Rh (with Rh D-positive or Rh D-negative blood types). ABO blood
group antigens are expressed in the mucus of secretor type
individuals (roughly 80% of Western population). The expression of
the ABO antigens is site-specific. For example, in the GIT the
expression of fucosylated glycans including ABO blood group
antigens decreases towards the distal parts of the intestine. Some
microbes, such as Helicobacter pylori and some other pathogenic
bacteria and viruses, have been shown to use ABO blood group
antigens as adhesion receptors (Boren et al. 1993, Imberty and
Varrot 2008). ABO antigen binding ability has been reported also
for Lactobacillus spp., which tend to adhere in a strain-specific
manner (Uchida et al. 2006). Bifidobacteria and Bacteroides
thetaiotaomicron are, for example, also able to specifically
utilize blood group antigens, e.g. the glycan structures of ABO
antigens (Martens et al. 2008, Xiao et al. 2010). The ABO blood
group status of an individual also has an effect on the relative
proportions of the host microbiota (Makivuokko et al. 2012).
[0006] Many probiotic supplements and microbiota modulation
products currently available on the market are ineffective in
promoting the desired health effects for every individual and
effect commonly varies from person to person. Thus, there is a
continuous need for more specific or personally tailored products
that are able to mediate the health effects more efficiently.
SUMMARY OF THE INVENTION
[0007] The present invention is based on the inventors' surprising
discovery that certain bacterial strains showed enhanced in vitro
adhesion to one or more of the ABO blood group antigens. This
finding enables the selection of ABO blood group-specific bacteria
for use in probiotic compositions and for the treatment or
prevention of gastrointestinal disorders.
[0008] While not wishing to be bound by theory, it is thought that
as the glycan structures of the ABO blood groups are abundant in
the mucosa and serve as bacterial adhesion sites and nutrient
sources, stronger binding to one or more ABO antigens by a
probiotic bacterium will improve its colonization of the bacterium
in the gastrointestinal tract by enhancing its interaction with the
mucosa. The probiotic bacterium may also demonstrate enhanced
health effects by blocking the adhesion and invasion of certain
pathogenic microbes which also bind to ABO antigens. A probiotic
bacterium which adheres to an ABO antigen could also enhance the
probiotic responses by providing tighter and longer-lasting contact
between the host cells and probiotics. It could further provide the
host easier access to any beneficial metabolites produced by the
probiotics when probiotics are colonized tighter and more
long-lasting.
[0009] The inventors have further shown that certain probiotic
bacteria are more resistant to acid and/or bile. These properties
may be helpful to enable the bacterium or metabolite thereof to
survive the conditions of the stomach and gastrointestinal tract,
and are therefore advantageous properties for a probiotic
bacterium. Acid tolerance may also be beneficial if the strain is
fermented in a product such as yogurt.
[0010] Accordingly the present invention provides a bacterium or
metabolite thereof characterised by: [0011] a) an average A antigen
adhesion of more than 100% when compared to L. crispatus LMG18199;
and/or [0012] b) an average B antigen adhesion of more than 100%
when compared to L. crispatus LMG18199; and/or [0013] c) an average
O antigen adhesion of more than 100% when compared to L. crispatus
LMG18199; and/or [0014] d) more than -2. 6 average acid tolerance
represented as a log change when measured at pH 2.5 for 1.5 hours
as defined in an acid resistance assay; and/or [0015] e) more than
40% average bile tolerance represented as growth in 0.9% dehydrated
fresh bile as a percentage of growth in MRS without bile as defined
in a bile resistance assay.
[0016] The bacterium of the invention may be a bacterial strain
deposited as DSM 32111, DSM 32108, DSM 32107, DSM 32098, DSM 32104,
DSM 32112, DSM 32109, DSM 32105, DSM 32110, DSM 32103, DSM 32099,
DSM 32106, DSM 32097, DSM 32114, DSM 32115 or a mutant, a variant
and/or a progeny thereof.
[0017] According to another aspect of the present invention there
is provided a probiotic composition comprising a bacterium or
metabolite thereof according to the invention and a suitable
carrier.
[0018] The probiotic composition of the invention may comprise a
combination of 2, 3, 4, 5 or 6 bacterial strains or metabolites
thereof according to the invention, optionally in combination with
one or more further bacterial strains.
[0019] The probiotic composition may further comprise a prebiotic
component.
[0020] The present invention also provides a method of producing a
probiotic composition of the invention, the method comprising
combining the selected bacterium or metabolite thereof with a
suitable carrier.
[0021] The invention further provides a food product comprising a
probiotic composition according to the invention.
[0022] According to another aspect of the invention there is
provided a method of selecting a bacterium or metabolite thereof
comprising: [0023] a) admixing one or more test bacteria with an A
antigen, B antigen, O antigen or combinations thereof; [0024] b)
admixing one or more Lactobacillus reuteri RC-14 control bacteria
with A antigen, B antigen, O antigen or combinations thereof;
[0025] c) incubating said admixtures of step a) and b); [0026] d)
comparing the affinity of adhesion to said A antigen, B antigen, O
antigen or combinations thereof of the test bacteria and
Lactobacillus reuteri RC-14; and [0027] e) selecting bacteria
having a higher affinity of adhesion to said A antigen, B antigen,
O antigen or combinations thereof than Lactobacillus reuteri
RC-14.
[0028] In one embodiment, the admixtures are incubated in separate
vials in step (b).
[0029] In another embodiment, the method is a fluorescence based
method.
[0030] In a further embodiment, bacteria are selected which have:
[0031] a) an average A antigen adhesion intensity of fluorescence
of more than 549; [0032] b) an average B antigen adhesion intensity
of fluorescence of more than 519; and/or [0033] c) an average O
antigen adhesion intensity of fluorescence of more than 746.
[0034] The invention further provides a bacterium or metabolite
thereof selected by the method of the invention.
[0035] In a further aspect, the invention provides the use of a
bacterium or metabolite of the invention or selected by a method of
the invention, or a probiotic composition of the invention, for the
manufacture of a formulation for preventing and/or treating
gastrointestinal disorders.
[0036] The invention also provides a bacterium or metabolite of the
invention or selected by a method of the invention, or a probiotic
composition of the invention, for use in preventing and/or treating
gastrointestinal disorders.
[0037] In another aspect, the invention provides a method of
preventing and/or treating gastrointestinal disorders comprising
administering to a subject a bacterium or metabolite thereof
according to the invention or selected by a method of the
invention, or a probiotic composition of the invention, in a
pharmaceutically effective amount.
DETAILED DESCRIPTION
[0038] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale
& Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, NY (1991) provide one of skill with a general dictionary
of many of the terms used in this disclosure.
[0039] This disclosure is not limited by the exemplary methods and
materials disclosed herein, and any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of embodiments of this disclosure. Numeric ranges are
inclusive of the numbers defining the range.
[0040] The headings provided herein are not limitations of the
various aspects or embodiments of this disclosure which can be had
by reference to the specification as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0041] Other definitions of terms may appear throughout the
specification. Before the exemplary embodiments are described in
more detail, it is to be understood that this disclosure is not
limited to particular embodiments described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting, since the scope of the
present disclosure will be limited only by the appended claims.
[0042] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within this disclosure. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within this disclosure, subject to any specifically excluded limit
in the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in this disclosure.
[0043] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise.
[0044] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
such publications constitute prior art to the claims appended
hereto.
[0045] It will be understood that in the following, preferred
embodiments referred to in relation to one broad aspect of the
invention are equally applicable to each of the other broad aspects
of the present invention described herein. It will be further
understood that, unless the context dictates otherwise, the
preferred embodiments described herein may be combined.
[0046] The term "bacterium" or "bacterial" is used herein to refer
to any bacterial species, strains or combinations thereof, and is
not limited to strains currently accepted as probiotics. However,
bacterial strains used in the present invention are those that a
suitable for human and/or animal consumption. A skilled person will
be readily aware of specific species and or strains from within the
genera described herein which are used in the food and/or
agricultural industries and which are generally considered suitable
for human and/or animal consumption. Such bacterial strains are
typically non-pathogenic, and may be generally regarded as safe for
human use (e.g. GRAS).
[0047] The term "bacterium" is generally used to refer to whole
bacteria, for example whole viable bacteria.
[0048] Bacteria suitable for use in the present invention include,
but are not limited to, Bifidobacterium, Brevibacterium,
Propionibacterium, Lactococcus, Streptococcus, Lactobacillus,
Enterococcus, Pediococcus, Leuconostoc and/or Oenococcus.
[0049] In one embodiment the bacterium belongs to the genus
Lactobacillus. Suitable strains of Lactobacillus include L.
acidophilus, L. amylovorus, L. brevis, L. casei, L. crispatus, L.
fermentum, L. vaginalis, L. curvatis, L. de/brueckii bu/garicus, L.
gasseri, L. helveticus, L. jensenii, L. mucosae, L. paracasei, L.
plantarum, L. rhamnosus, L. silvarius and L. ruminis.
[0050] In a further aspect, the present invention provides the
novel bacterial strains DGCC11884, DGCC11864, DGCC11860, DGCC11873,
DGCC11852, DGCC11853, DGCC11865, DGCC1925, DGCC11866, DGCC5111,
DGCC11854, DGCC11858, DGCC11887, DGCC11862 and DGCC11881. These
strains have been deposited by DuPont Nutrition Biosciences ApS,
Langebrogade 1, P.O. Box 17, DK-1001 Copenhagen K, under the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the purposes of Patent Procedure at Leibniz
Institute DSMZ-German Collection of Microorganisms and Cell
Cultures, lnhoffenstr. 7 B, D-38124 Braunschweig on 29 Jul. 2015
under accession numbers DSM 32111, DSM 32108, DSM 32107, DSM 32098,
DSM 32104, DSM 32112, DSM 32109, DSM 32105, DSM 32110, DSM 32103,
DSM 32099, DSM 32106, DSM 32097, DSM 32114, DSM 32115.
[0051] We hereby confirm that the depositor has authorised the
applicant to refer to the deposited biological material in this
application and has given his unreserved and irrevocable consent to
the deposited material being made available to the public.
[0052] The invention further provides a mutant, a variant and/or a
progeny of the deposited bacterial strains.
[0053] As used herein, the term "mutant" refers to any
microorganism resulting from modification of the parent (i.e.
deposited) strain. For example, a mutant may be a microorganism
resulting from genetically modifying a deposited strain.
[0054] As used herein, the term "variant" refers to a naturally
occurring microorganism which is derived from the parent (i.e.
deposited strain). For example, a variant may be a microorganism
resulting from adaption to particular cell culture conditions.
[0055] As used herein, the term "progeny" means any microorganism
resulting from the reproduction or multiplication of any one of the
deposited strains. Therefore, "progeny" means any direct descendant
of any one of the deposited strains. As such, the progeny strain
may itself be identified as the same strain as the parent (i.e.
deposited) strain. It will be apparent to one skilled in the art
that due to the process of asexual reproduction, a progeny strain
will be genetically virtually identical to the parent strain.
Accordingly, in one embodiment, the progeny may be genetically
identical to the parent strain, and may be considered to be a
"clone" of the parent strain. Alternatively, the progeny may be
substantially genetically identical to the parent strain.
[0056] The mutant, variant or progeny may have at least 90, 95, 98,
99, 99.5 or 99.9% sequence identity over the entire length of the
bacterial genome with their parent strain. Furthermore, the mutant,
variant or progeny will retain the same phenotype as the deposited
parent strain, for example the mutant or variant may demonstrate
the same or equivalent level of in vitro adhesion to A, B and/or O
blood type antigen as the parent strain.
[0057] As used herein, the term "metabolite" refers to all
molecules produced or modified by the bacteria as a result of
bacterial metabolism during growth, survival, persistence, transit
or existence of bacteria during probiotic product manufacture and
storage and during gastrointestinal transit in a mammal. Examples
include all organic acids, inorganic acids, bases, proteins and
peptides, enzymes and co-enzymes, amino acids and nucleic acids,
carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids,
vitamins, all bioactive compounds, metabolites containing an
inorganic component, and all small molecules, for example nitrous
molecules or molecules containing a sulphurous acid.
[0058] A metabolite or metabolites are typically obtained from the
supernatant of a cell culture from which the bacterial cells have
been removed. In one embodiment, the cells may be grown in MRS
medium under anaerobic conditions for 6-24 hours at 37.degree. C.
According to a further embodiment, the bacterial cell culture may
be grown to a cell density of at least about OD.sub.600 0.5, 1.0,
1.5, 2.0, 2.5, or 3.0, for example from 1.5 to 2.5 OD.sub.600. The
cells may suitably be removed by centrifugation or by filtration.
It will be apparent that the supernatant may be used directly in
the formulations of the present invention, or that one or more of
the metabolites may be isolated from the supernatant by any
suitable means prior to use.
[0059] Suitable metabolites for use in the present invention
include, but are not limited to, metabolites from any of the above
mentioned bacteria.
[0060] According to the present invention, the bacterium or
metabolite thereof is characterised by its adhesion to a particular
ABO blood type antigen, with reference to a control strain. The
adhesion to the antigen may be measured by mixing the bacterium or
metabolite thereof with the antigen, and measuring adhesion
according to any suitable test or assay. In one embodiment, the
method is a fluorescence-based method. For example, the blood group
antigen may be biotinylated and combined with a sample of bacteria
to be tested, then transferred to streptavidin coated plates and
the attached bacteria detected with a suitable dye, such as a
fluorescent dye. Antigen may be bound to any immobilized matrix for
affinity binding assays and the binding of bacteria to it could be
detected using suitable antibodies, fluorometric or colorimetric
stains, or by labelling the bacteria with any labelling technique
such as radioactive labelling. It could be done the other way
around, by attaching bacterial components to immobilized matrix,
and by investigating if the blood group antigens can bind.
[0061] According to one aspect of the invention, the antigen
adhesion is compared to Lactobacillus crispatus LMG18199 (available
from BCCM/LMG collection). According to another aspect of the
invention, the antigen adhesion is compared to Lactobacillus
reuteri RC-14 (available from Christian Hansen A/S, Denmark).
[0062] According to another aspect of the present invention, the
bacterium or metabolite thereof is selected on the basis of its
resistance to bile and/or acid. These properties may be helpful to
enable the bacterium or metabolite thereof to survive the
conditions of the stomach and gastrointestinal tract, and are
therefore advantageous properties for a probiotic bacterium.
[0063] Any suitable bile resistance assay may be used to determine
average bile tolerance. In one embodiment, the bile resistance
assay comprises incubating strain cultures in culture medium (e.g.
MRS) containing a defined amount of dehydrated fresh bile (e.g.
oxgall, available under the brand name Difco.TM. from BD), such as
0.9% or 0.3% at 37.degree. C. for 24 hours in anaerobic conditions.
Growth is measured before and after incubation and the bile
tolerance results are expressed as % growth (OD) with bile in
comparison to growth without bile.
[0064] Any suitable acid resistance assay may be used to determine
average acid tolerance. In one embodiment, the acid tolerance assay
comprises incubating bacterial cells in suitable medium (e.g. PBS)
at neutral pH (i.e. pH 7.2) levels and at a comparator acidic pH
(such as pH 2.5 or pH 3.5) for at least 90 min at 37.degree. C. The
ten-fold dilution series are grown on MRS agar, and colonies are
counted after 48 h incubation in anaerobic conditions at 37.degree.
C. The results for acid tolerance are expressed as growth log
reduction of CFU after exposure to acid conditions in comparison to
neutral pH.
[0065] In a preferred embodiment, the bacterium or metabolite
thereof is characterised by both its adhesion to a particular ABO
blood type antigen, with reference to a control strain, and by its
acid and/or bile tolerance. Accordingly, the invention provides a
bacterium or metabolite thereof characterised by: [0066] a) an
average A, B and/or O antigen adhesion of more than 100% when
compared to L. crispatus LMG18199; and [0067] b) more than -2.6
average acid tolerance represented as a log change when measured at
pH 2.5 for 1.5 hours as defined in an acid resistance assay and/or
more than 40% average bile tolerance represented as growth in 0.9%
dehydrated fresh bile as a percentage of growth in MRS without bile
as defined in a bile resistance assay.
[0068] The invention further provides a probiotic composition
comprising one or more bacterial strains that have been selected
according to methods of the invention. The composition may comprise
a single strain, or a combination of 2, 3, 4, 5, 6, 7, 8, 9 or 10
strains of the invention. The probiotic composition may further
comprise additional strains, for example commercially available
probiotic bacterial strains.
[0069] According to one embodiment, the probiotic composition of
the invention further comprises one or more further bacterial
strains.
[0070] In some embodiments the further micro-organism may be a
bacterium from one or more of the following genera: Lactococcus,
Streptococcus, Pediococcus, Enterococcus, Leuconostoc,
Carnobacterium, Propionibacterium, Bifidobacterium, Lactobacillus,
Brevibacterium, and Vagococcus. In one preferred embodiment the at
least one further probiotic microorganism is selected from the
genera Lactobacillus, Streptococcus, Enterococcus, Bifidobacterium
and Saccharomyces.
[0071] In preferred embodiments, the further probiotic
microorganism is a bacterium preferably a probiotic lactic acid
bacterium and/or a probiotic Bifidobacterium. In one embodiment
preferably the further microorganism is from the genus
Lactobacillus or the genus Bifidobacterium or is a mixture thereof.
Suitably, the microorganism may be a strain from the species L.
acidophilus, L. curvatus, L. rhamnosus, L. casei, L. paracasei, L.
salivarius, B. lactis. B animalis, B. longum and/or B. bifidum. In
one embodiment, preferably the microorganism may be a strain from
the species L. acidophilus, L. curvatus, L. salivarius and/or B.
lactis.
[0072] The bifidobacterium may be any bifidobacterium having a
probiotic effect, typically strains belonging to the species
Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium
infantis, Bifidobacterium lactis, Bifidobacterium longum,
Bifidobacterium bifidum and Bifidobacterium adolescentis are
used.
[0073] The Lactobacillus bacterium may be any of the following:
Lactobacillus acidophilus, Lactobacillis amylovorus. Lactobacillus
bulgaricus, Lactobacillus casei, Lactobacillus delbrueckii,
Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus
helveticus, Lactobacillus johnsonii, Lactobacillus lactis,
Lactobacillus paracasei, Lactobacillus pentosaceus, Lactobacillus
plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, and
Lactobacillus salivarius,
[0074] According to one embodiment, the probiotic composition of
the invention further comprises one or more, such as a combination
of 2, 3, 4 or 5 of the following commercially available strains:
Lactobacillus acidophilus NCFM; Bifidobacterium lactis BL-04;
Lactobacillus paracasei LPC37; Bifidobacterium lactis HNO19, and/or
Bifidobacterium lactis Bi-07.
[0075] As used herein, the term "probiotic" refers to a live
microorganism which, when administered in adequate amounts, confers
a health benefit on the recipient. They are suitable for human
consumption and therefore are non-pathogenic and non-toxic. These
probiotic strains generally have the ability to survive the passage
through the upper part of the digestive tract. Probiotic bacteria
typically exercise their beneficial effect on health on the one
hand via ecological interactions with the resident flora in the
digestive tract, and on the other hand via their ability to
influence the immune system in a positive manner via the "GALT"
(gut-associated lymphoid tissue). Probiotic bacteria, when given in
a sufficient number, have the ability to progress live through the
intestine. However they do not cross the intestinal barrier and
their primary effects are therefore induced in the lumen and/or the
wall of the gastrointestinal tract. They then form part of the
resident flora during the administration period. This colonization
(or transient colonization) allows the probiotic bacteria to
exercise a beneficial effect, such as the repression of potentially
pathogenic micro-organisms present in the flora and interactions
with the immune system of the intestine.
[0076] While there are no lower or upper limits for probiotic use,
it has been suggested that at least 10.sup.6-10.sup.12, such as at
least 10.sup.6-10.sup.10, for example 10.sup.8-10.sup.9 cfu as a
daily dose may be effective to achieve the desired health effects
in a subject. Accordingly, the probiotic bacteria used in
accordance with the present invention may comprise from 10.sup.6 to
10.sup.12 CFU of bacteria/g of support, and more particularly from
10.sup.8 to 10.sup.12 CFU of bacteria/g of support, typically
10.sup.9 to 10.sup.12 CFU/g for the lyophilized form.
[0077] Suitably, the bacterium may be administered at a dosage of
from about 10.sup.6 to about 10.sup.12 CFU of microorganism/dose,
typically about 10.sup.8 to about 10.sup.12 CFU of
microorganism/dose. By the term "per dose" it is meant that this
amount of microorganism is provided to a subject either per day or
per intake, typically per day. For example, if the microorganism is
to be administered in a food product (for example in a
yoghurt)--then the yoghurt will typically contain from about
10.sup.8 to 10.sup.12 CFU of the microorganism. Alternatively,
however, this amount of microorganism may be split into multiple
administrations each consisting of a smaller amount of microbial
loading--so long as the overall amount of microorganism received by
the subject in any specific time (for instance each 24 hour period)
is from about 10.sup.6 to about 10.sup.12 CFU of microorganism,
such as about 10.sup.8 to about 10.sup.12 CFU of microorganism.
[0078] In accordance with the present invention an effective amount
of at least one strain of a microorganism may be at least 10.sup.6
CFU of microorganism/dose, for example from about 10.sup.6 to about
10.sup.12 CFU of microorganism/dose, such as about 10.sup.8 to
about 10.sup.12 CFU of microorganism/dose.
[0079] CFU stands for "colony-forming units". By `support` is meant
the food product, dietary supplement or the pharmaceutically
acceptable support or carrier.
[0080] While it is possible to administer the bacterium or
metabolite alone according to the present invention (i.e. without
any support, diluent or excipient), they are typically administered
on or in a carrier or support as part of a product, in particular
as a component of a food product, a dietary supplement or a
pharmaceutical formulation. These products typically contain
additional components well known to those skilled in the art.
[0081] Any product which can benefit from the composition may be
used in the present invention. These include but are not limited to
foods, particularly fruit conserves and dairy foods and dairy
food-derived products, and pharmaceutical products.
[0082] The bacterial composition of the present invention may be
formulated as a nutritional supplement. The bacterial composition
may be in the form of, for example, a capsule, tablet, powder or
emulsion.
[0083] A typical probiotic ingredient is a freeze-dried powder
containing, for example, 10.sup.10-10.sup.12 viable probiotic
bacterial cells per gram. The powder may further comprise a
suitable carrier, such as skim milk or sugars, typically
oligosaccharides such as sucrose or trehalose.
[0084] Alternatively, the bacterial composition may be encapsulated
using a carrier such as alginate, starch or xanthan. A typical
capsule preparation may contain approximately 10.degree.-10.sup.11
viable probiotic bacterial cells per capsule.
[0085] The probiotic composition of the present invention may
additionally contain one or more prebiotics. The term "prebiotic
component" as used herein refers to any compound, nutrient or
additional microorganism used to support or enhance a desired
probiotic health effect or to assist the growth and/or activity of
probiotic bacteria. Typically, prebiotics are carbohydrates (such
as oligosaccharides), but the definition does not preclude
non-carbohydrates. The most prevalent forms of prebiotics are
nutritionally classed as soluble fibre. To some extent, many forms
of dietary fibre exhibit some level of prebiotic effect.
[0086] In one embodiment, a prebiotic is a selectively fermented
ingredient that allows specific changes, both in the composition
and/or activity in the gastrointestinal microflora that confers
benefits upon host well-being and health.
[0087] Suitably, the prebiotic may be used according to the present
invention in an amount of 0.01 to 100 g/day, such as 0.1 to 50
g/day, or 0.5 to 20 g/day. In one embodiment, the prebiotic may be
used according to the present invention in an amount of 1 to 10
g/day, such as 2 to 9 g/day, or 3 to 8 g/day. In another
embodiment, the prebiotic may be used according to the present
invention in an amount of 5 to 50 g/day, such as 10 to 25
g/day.
[0088] Examples of dietary sources of prebiotics include soybeans,
inulin sources (such as Jerusalem artichoke, jicama, and chicory
root), raw oats, unrefined wheat, unrefined barley and yacon.
[0089] Examples of suitable prebiotics include alginate, xanthan,
pectin, locust bean gum (LBG), inulin, guar gum,
galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),
polydextrose (i.e. Litesse.RTM.), lactitol, lactosucrose, soybean
oligosaccharides, isomaltulose (Palatinose.TM.),
isomalto-oligosaccharides, gluco-oligosaccharides,
xylo-oligosaccharides, manno-oligosaccharides, beta-glucans,
cellobiose, raffinose, gentiobiose, melibiose, xylobiose,
cyclodextrins, isomaltose, trehalose, stachyose, panose, pullulan,
verbascose, galactomannans, and all forms of resistant starches. A
particularly preferred example of a prebiotic is polydextrose.
[0090] The probiotic composition of the present invention may be
used as, or in the preparation of, a food product. Herein, the term
"food" is used in a broad sense and covers food for humans as well
as food for animals (i.e. a feed). In one aspect, the food is for
human consumption.
[0091] The food may be in the form of a solution or as a
solid--depending on the use and/or the mode of application and/or
the mode of administration.
[0092] A typical probiotic food product may contain approximately
10.sup.9-10.sup.11 viable probiotic bacterial cells per daily dose.
The probiotic bacteria may be incorporated in the food product as a
probiotic ingredient, such as a freeze-dried powder, or may be
cultured in the product.
[0093] When used as or in the preparation of a food, such as
functional food, the composition of the present invention may be
used in conjunction with one or more of: a nutritionally acceptable
carrier, a nutritionally acceptable diluent, a nutritionally
acceptable excipient, a nutritionally acceptable adjuvant, and a
nutritionally active ingredient.
[0094] By way of example, the probiotic composition of the present
invention can be used as an ingredient in soft drinks, a fruit
juice or a beverage comprising whey protein, health teas, cocoa
drinks, milk drinks and lactic acid bacteria drinks, yoghurt and
drinking yoghurt, cheese, ice cream, water ices and desserts,
confectionery, biscuits cakes and cake mixes, snack foods, balanced
foods and drinks, fruit fillings, care glaze, chocolate bakery
filling, cheese cake flavoured filling, fruit flavoured cake
filling, cake and doughnut icing, instant bakery filling creams,
fillings for cookies, ready-to-use bakery filling, reduced calorie
filling, adult nutritional beverage, acidified soy/juice beverage,
aseptic/retorted chocolate drink, bar mixes, beverage powders,
calcium fortified soy/plain and chocolate milk, calcium fortified
coffee beverage.
[0095] The probiotic composition can further be used as an
ingredient in food products such as American cheese sauce,
anti-caking agent for grated & shredded cheese, chip dip, cream
cheese, dry blended whip topping fat free sour cream, freeze/thaw
dairy whipping cream, freeze/thaw stable whipped tipping, low fat
and light natural cheddar cheese, low fat Swiss style yoghurt,
aerated frozen desserts, hard pack ice cream, label friendly,
improved economics & indulgence of hard pack ice cream, low fat
ice cream: soft serve, barbecue sauce, cheese dip sauce, cottage
cheese dressing, dry mix Alfredo sauce, mix cheese sauce, dry mix
tomato sauce and others.
[0096] The term "dairy product" as used herein is meant to include
a medium comprising milk of animal and/or vegetable origin. As milk
of animal origin there can be mentioned cow's, sheep's, goat's or
buffalo's milk. As milk of vegetable origin there can be mentioned
any fermentable substance of vegetable origin which can be used
according to the invention, in particular originating from
soybeans, rice or cereals.
[0097] According to one particular aspect, the food product
employed according to the invention is a fermented milk or
humanized milk.
[0098] For certain aspects, the present invention may be used in
connection with yoghurt production, such as fermented yoghurt
drink, yoghurt, drinking yoghurt, cheese, fermented cream, milk
based desserts and others.
[0099] Suitably, the composition can be further used as an
ingredient in one or more of cheese applications, meat
applications, or applications comprising protective cultures.
[0100] The present invention also provides a method of preparing a
food or a food ingredient, the method comprising admixing the
composition according to the present invention with another food
ingredient.
[0101] Advantageously, the present invention relates to products
that have been contacted with the probiotic composition of the
present invention (and optionally with other
components/ingredients), wherein the composition is used in an
amount to be capable of improving the nutrition and/or health
benefits of the product.
[0102] As used herein the term "contacted" refers to the indirect
or direct application of the composition of the present invention
to the product. Examples of the application methods which may be
used, include, but are not limited to, treating the product in a
material comprising the composition, direct application by mixing
the composition with the product, spraying the composition onto the
product surface or dipping the product into a preparation of the
composition.
[0103] Where the product of the invention is a foodstuff, the
composition of the present invention is typically admixed with the
product. Alternatively, the composition may be included in the
emulsion or raw ingredients of a foodstuff. In a further
alternative, the composition may be applied as a seasoning, glaze,
colorant mixture, and the like.
[0104] For some applications, it is important that the composition
is made available on or to the surface of a product to be
affected/treated. This allows the composition to impart one or more
of the following favourable characteristics: nutrition and/or
health benefits.
[0105] The compositions of the present invention may be applied to
intersperse, coat and/or impregnate a product with a controlled
amount of a microorganism.
[0106] In one embodiment, the composition is used to ferment milk
or sucrose fortified milk or lactic media with sucrose and/or
maltose where the resulting media containing all components of the
composition--i.e. said microorganism according to the present
invention--can be added as an ingredient to yoghurt milk in
suitable concentrations--such as for example in concentrations in
the final product which offer a daily dose of 10.sup.6-10.sup.10
cfu. The microorganism according to the present invention may be
used before or after fermentation of the yoghurt.
[0107] Advantageously, where the product is a food product, the
probiotic bacteria should remain effective through the normal
"sell-by" or "expiration" date during which the food product is
offered for sale by the retailer. The effective time may usefully
extend past such dates until the end of the normal freshness period
when food spoilage becomes apparent. The desired lengths of time
and normal shelf life will vary from foodstuff to foodstuff and
those of ordinary skill in the art will recognise that shelf-life
times will vary upon the type of foodstuff, the size of the
foodstuff, storage temperatures, processing conditions, packaging
material and packaging equipment.
[0108] The bacterial composition of the present invention may be
used as, or may be added to, a functional food. As used herein, the
term "functional food" means food which is capable of providing not
only a nutritional effect and/or a taste, but is also capable of
delivering a further beneficial effect to consumer, such as an
additional health benefit.
[0109] Accordingly, functional foods are ordinary foods that have
components or ingredients (such as those described herein)
incorporated into them that impart to the food a specific
functional benefit, such as a medical or physiological benefit,
other than a purely nutritional effect.
[0110] Although there is no legal definition of a functional food,
they are generally foods marketed as having specific health
effects.
[0111] Some functional foods are nutraceuticals. As used herein,
the term "nutraceutical" means a food which is capable of providing
not only a nutritional effect and/or a taste satisfaction, but is
also capable of delivering a therapeutic (or other beneficial)
effect to the consumer. Nutraceuticals cross the traditional
dividing lines between foods and medicine.
[0112] According to a further aspect, the present invention relates
to the use of a bacterium or metabolite thereof which is
demonstrates binding to at least one ABO blood type antigen, or a
probiotic composition comprising one or more such bacteria, for the
prevention and/or treatment of gastrointestinal disorders.
[0113] As used herein, the term "gastrointestinal disorder"
includes any disease or disorder relating to the gastrointestinal
tract. The disorder may, for example, be one that is known to be
associated with an altered composition and diversity of the GIT
microbiota. The gastrointestinal disorder may be inflammatory bowel
disease (IBD), irritable bowel syndrome (IBS), Crohn's disease,
ulcerative colitis, constipation or diarrhoea.
[0114] When used as--or in the preparation of--a pharmaceutical,
the composition of the present invention may be used in conjunction
with one or more of: a pharmaceutically acceptable carrier, a
pharmaceutically acceptable diluent, a pharmaceutically acceptable
excipient, a pharmaceutically acceptable adjuvant, a
pharmaceutically active ingredient.
[0115] A pharmaceutically acceptable support may be for example a
support in the form of compressed tablets, tablets, capsules,
ointments, suppositories or drinkable solutions. Other suitable
forms are provided below.
[0116] The pharmaceutical may be in the form of a solution or as a
solid--depending on the use and/or the mode of application and/or
the mode of administration.
[0117] The bacterium or metabolite thereof or probiotic composition
may be used according to the present invention in any suitable
form--whether when alone or when present in a combination with
other components or ingredients. The bacteria used in the present
invention may be referred to herein as "the composition". Likewise,
combinations comprising the composition of the present invention
and other components and/or ingredients (i.e. ingredients--such as
food ingredients, functional food ingredients or pharmaceutical
ingredients) may be used in any suitable form.
[0118] The bacterium or metabolite thereof or probiotic composition
may be used according to the present invention in the form of solid
or liquid preparations or alternatives thereof. Examples of solid
preparations include, but are not limited to tablets, capsules,
dusts, granules and powders which may be wettable, spray-dried or
freeze-dried. Examples of liquid preparations include, but are not
limited to, aqueous, organic or aqueous-organic solutions,
suspensions and emulsions.
[0119] Suitable examples of forms include one or more of: tablets,
pills, capsules, ovules, solutions or suspensions, which may
contain flavouring or colouring agents, for immediate-, delayed-,
modified-, sustained-, pulsed- or controlled-release
applications.
[0120] By way of example, if the composition of the present
invention is used in a tablet form--such for use as a functional
ingredient--the tablets may also contain one or more of: excipients
such as microcrystalline cellulose, lactose, sodium citrate,
calcium carbonate, dibasic calcium phosphate and glycine;
disintegrants such as starch (such as corn, potato or tapioca
starch), sodium starch glycollate, croscarmellose sodium and
certain complex silicates; granulation binders such as
polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),
hydroxypropylcellulose (HPC), sucrose, gelatin and acacia;
lubricating agents such as magnesium stearate, stearic acid,
glyceryl behenate and talc may be included.
[0121] Examples of nutritionally acceptable carriers for use in
preparing the forms include, for example, water, salt solutions,
alcohol, silicone, waxes, petroleum jelly, vegetable oils,
polyethylene glycols, propylene glycol, liposomes, sugars, gelatin,
lactose, amylose, magnesium stearate, talc, surfactants, silicic
acid, viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, petroethral fatty acid esters,
hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
[0122] Suitable excipients for the forms include lactose, starch, a
cellulose, milk sugar or high molecular weight polyethylene
glycols.
[0123] For aqueous suspensions and/or elixirs, the composition of
the present invention may be combined with various sweetening or
flavouring agents, colouring matter or dyes, with emulsifying
and/or suspending agents and with diluents such as water, propylene
glycol and glycerin, and combinations thereof.
[0124] The forms may also include gelatin capsules, fibre capsules,
fibre tablets or fibre beverages.
[0125] It will be apparent to the skilled person that the
formulation should ideally be able to remain stable during transit
though the gastrointestinal tract; for example, it may be resistant
to proteolytic degradation, stable at acid pH and resistant to the
detergent effects of bile.
[0126] The invention further provides the following disclosure,
presented in the form of numbered paragraphs: [0127] 1. A bacterium
or metabolite thereof characterised by: [0128] a) an average A
antigen adhesion of more than 100% when compared to L. crispatus
LMG18199; and/or [0129] a) an average B antigen adhesion of more
than 100% when compared to L. crispatus LMG18199; and/or [0130] b)
an average O antigen adhesion of more than 100% when compared to L.
crispatus LMG18199. [0131] 2. A bacterium or metabolite thereof
according to paragraph 1 wherein said bacterium or metabolite
thereof is further characterised by: [0132] c) more than -2.6
average acid tolerance represented as a log change when measured at
pH 2.5 for 1.5 hours as defined in an acid resistance assay; and/or
[0133] d) more than 40% average bile tolerance represented as
growth in 0.9% dehydrated fresh bile as a percentage of growth in
MRS without bile as defined in a bile resistance assay. [0134] 3. A
bacterium according to paragraph 1 or 2 wherein said bacterium is
deposited as DSM 32111, DSM 32108, DSM 32107, DSM 32098, DSM 32104,
DSM 32112, DSM 32109, DSM 32105, DSM 32110, DSM 32103, DSM 32099,
DSM 32106, DSM 32097, DSM 32114, DSM 32115 or a mutant, variant
and/or progeny thereof. [0135] 4. A probiotic composition
comprising a bacterium or metabolite thereof according to any one
of paragraphs 1 to 3. [0136] 5. A probiotic composition according
to paragraph 4 further comprising a suitable carrier. [0137] 6. A
probiotic composition according to any one of paragraphs 4 or 5,
which comprises a combination of 2, 3, 4, 5 or 6 bacterial strains
or metabolites thereof as defined in any one of claims 1 to 3,
optionally in combination with one or more further bacterial
strains. [0138] 7. A probiotic composition according to any one of
paragraphs 4 to 6, wherein the composition further comprises one or
more further bacterial strains. [0139] 8. A probiotic composition
according to paragraph 7, wherein said one or more further
bacterial strains are selected from: Lactobacillus acidophilus
NCFM; Bifidobacterium lactis BL-04; Lactobacillus paracasei LPC37;
Bifidobacterium lactis HNO19, and/or Bifidobacterium lactis Bi-07.
[0140] 9. A probiotic composition according to paragraph 8
comprising DAVE034, Lactobacillus acidophilus NCFM; Bifidobacterium
lactis BL-04; Lactobacillus paracasei LPC37; Bifidobacterium lactis
HNO19 and Bifidobacterium lactis Bi-07. [0141] 10. A probiotic
composition according to any one of the paragraphs 4 to 9, wherein
the composition is personalised for an A, B, AB or O blood group
individual by comprising one or more bacteria which demonstrate
antigen adhesion of more than 100% when compared to L. crispatus
LMG18199 for the appropriate blood group antigen(s). [0142] 11. A
probiotic composition according to any one of paragraphs 4 to 10,
which further comprises at least one prebiotic component. [0143]
12. A method of producing a probiotic composition according to any
one of paragraphs 4 to 11, the method comprising combining the
selected bacterium or metabolite thereof with a suitable carrier.
[0144] 13. A food product, dietary supplement, medical food,
neutraceutical or pharmaceutical composition comprising a probiotic
composition according to any one of paragraphs 4 to 11 [0145] 14. A
method of selecting a bacterium or metabolite thereof comprising:
[0146] a) admixing one or more test bacteria with an A antigen, B
antigen, O antigen or combinations thereof; [0147] b) admixing one
or more Lactobacillus reuteri RC-14 control bacteria with A
antigen, [0148] B antigen, O antigen or combinations thereof;
[0149] c) incubating said admixtures of step a) and b); [0150] d)
comparing the affinity of adhesion to said A antigen, B antigen, O
antigen or combinations thereof of the test bacteria and
Lactobacillus reuteri RC-14; and [0151] e) selecting bacteria
having a higher affinity of adhesion to said A antigen, B antigen,
O antigen or combinations thereof than Lactobacillus reuteri RC-14.
[0152] 15. A method according to paragraph 14 wherein said
admixtures are incubated in separate vials in step (b). [0153] 16.
A method according to paragraph 14 or 15, wherein the method is a
fluorescence based method. [0154] 17. A method according to
paragraph 16, wherein bacteria are selected which have: [0155] i)
an average A antigen adhesion intensity of fluorescence of more
than 549;
[0156] ii) an average B antigen adhesion intensity of fluorescence
of more than 519; and/or [0157] iii) an average O antigen adhesion
intensity of fluorescence of more than 746. [0158] 18. A method
according to any one of paragraphs 14 to 17 wherein said method
further comprises: [0159] f) assaying the average acid tolerance of
the bacteria selected in step e) at pH 2.5 for 1.5 hours; and/or
[0160] g) assaying the average bile tolerance of the bacteria
selected in step e) in 0.9% bile; and [0161] h) selecting one or
more bacteria having more than -2.56 average acid tolerance
represented as a log change when measured at pH 2.5 for 1.5 hours
and/or more than 40.16% average bile tolerance represented as
growth in 0.9% bile as a percentage of growth in MRS without bile.
[0162] 19. A bacterium or metabolite thereof selected by the method
of any one of paragraphs 14 to 18. [0163] 20. A bacterium according
to paragraph 19 wherein said bacterium is deposited as DSM 32111,
DSM 32108, DSM 32107, DSM 32098, DSM 32104, DSM 32112, DSM 32109,
DSM 32105, DSM 32110, DSM 32103, DSM 32099, DSM 32106, DSM 32097,
DSM 32114, DSM 32115 or a mutant, variant and/or progeny thereof.
[0164] 21. A probiotic composition comprising a bacterium or
metabolite thereof according to any one of paragraphs 19 to 20.
[0165] 22. A probiotic composition according to paragraph 21
further comprising a suitable carrier. [0166] 23. A probiotic
composition according to any one of paragraphs 21 or 22, which
comprises a combination of 2, 3, 4, 5 or 6 bacterial strains or
metabolites thereof as defined in any one of the paragraphs 19 to
20, optionally in combination with one or more further bacterial
strains. [0167] 24. A probiotic composition according to any of
paragraphs 21 to 23, wherein the composition further comprises one
or more further bacterial strains. [0168] 25. A probiotic
composition according to paragraph 24, wherein said one or more
further bacterial strains are selected from: Lactobacillus
acidophilus NCFM; Bifidobacterium lactis BL-04; Lactobacillus
paracasei LPC37; Bifidobacterium lactis HNO19, and/or
Bifidobacterium lactis Bi-07. [0169] 26. A food product, dietary
supplement, medical food, neutraceutical or pharmaceutical
composition comprising a probiotic composition according to any one
of paragraphs 21 to 25. [0170] 27. Use of a bacterium or metabolite
according to any one of paragraphs 1-3 or 19-20 or selected by the
method of any one of paragraphs 14-18, or a probiotic composition
according to any one of paragraphs 4-11 or 21-25, for the
manufacture of a formulation for preventing and/or treating
gastrointestinal disorders. [0171] 28. A bacterium or metabolite
according to any one of paragraphs 1-3 or 19-20 or selected by the
method of any one of paragraphs 14-18, or a probiotic composition
according to any one of paragraphs 4-11 or 21-25, for use in
preventing and/or treating gastrointestinal disorders. [0172] 29. A
method of preventing and/or treating gastrointestinal disorders
comprising administering to a subject a bacterium or metabolite
thereof according to any one of paragraphs 1-3 or 19-20 or selected
by the method of any one of paragraphs 14-18, or a probiotic
composition according to any one of paragraphs 4-11 or 21-25, in a
pharmaceutically effective amount. [0173] 30. A method of
personalising a probiotic composition or food product for an
individual with an A, B, AB or O blood group by selecting for
inclusion in the probiotic composition or food product one or more
bacteria which demonstrate antigen adhesion of more than 100% when
compared to Lactobacillus reuteri RC-14 or L. crispatus LMG18199
for the appropriate blood group antigen(s). [0174] 31. A bacterium,
metabolite, probiotic composition, food product, method, bacterium
or metabolite for use, use or combinations thereof substantially as
described herein with reference to the drawings.
[0175] The invention will now be described, by way of example only,
with reference to the following Examples.
EXAMPLES
[0176] Materials & Methods
[0177] Probiotic Properties of the Strains
[0178] Acid tolerance of the strains was tested in pH 2.5 and pH
3.5 for 1.5 h and bile tolerance in 0.9% and 0.3% Oxgall (Difco)
bile concentrations for 24 h (Saarela et al. 2009). Briefly, the
strains were cultivated in duplicate in MRS broth in anaerobic
conditions at 37.degree. C. for 18 h. The pelleted cells were
washed twice with 10 ml PBS pH 7.2 and resuspended in 0.01 mol/L
PBS pH 7.2 so that the optical density (OD.sub.600) of the sample
was 1 (equals to 1.times.10.sup.8 CFU/ml). Acid tolerance was
tested by incubating cells in PBS pH 7.2. PBS pH 2.5 and PBS pH 3.5
for 90 min at 37.degree. C. The ten-fold dilution series were grown
on MRS agar, and colonies were counted after 48 h incubation in
anaerobic conditions at 37.degree. C. The results for acid
tolerance were expressed as growth log reduction of CFU after
exposure to pH 2.5 or 3.5 in comparison to pH 7.2. Bile tolerance
was tested by incubating 1:10 diluted strain cultures in MRS
containing 0.9% Oxgall, MRS containing 0.3% Oxgall and in plain MRS
at 37.degree. C. for 24 in anaerobic conditions. The growth was
measured before and after incubation as OD.sub.595 by Multiskan RC
(Labsystems). The bile tolerance results were expressed as % growth
(OD) in MRS with 0.9% or 0.3% Oxgall in comparison to growth (OD)
in MRS without bile. Additionally, acid and bile tolerance of the
strains was compared with that of Lactobacillus rhamnosus LGG (VTT
E-96666) strain. All the measurements were performed in duplicates
and repeated twice for most of the strains.
[0179] ABH Antigen Adhesion
[0180] The ABH adhesion capability was tested for all strains (H
antigen corresponding to blood group 0 phenotype). The tested blood
group antigens A, B and H (Elicityl) were suspended in PBS pH7.2
and biotinylated. The stains were cultivated in anaerobic
conditions in MRS plates at 37.degree. C. for 48-72 h. A single
colony was re-inoculated to 10 ml MRS broth and cultivated
overnight in anaerobic conditions at 37.degree. C. The cells were
washed twice with 10 ml PBS pH 7.2 and resuspended in PBS pH 7.2,
so that the OD (600 nm) of the sample was set to 1 (equals to
1.times.10.sup.8 cfu/ml). The 1 ml sample and 1 ml 10 .mu.g/ml
biotinylated antigen solution were mixed together and incubated in
slow shaking for 30 min at room temperature, and 100 .mu.l of the
mixture transferred in to the Delfia Streptavidin coated 96-well
plates (Wallac), which were washed twice with 200 .mu.l PBS pH7.2,
followed by wash with SuperBlock (Pierce) three times (2.times.200
.mu.l+1.times.100 .mu.l) and once with 200 .mu.l sterile water. For
streptavidin--biotin interaction, the plates were incubated for 30
min at room temperature on slow agitation before washing of the
well three times with 200 .mu.l sterile water. Between the washes
the plate was incubated for 5 min. To detect the attached bacteria,
200 .mu.l Syto9 dye (diluted 1:6) (Invitrogen) was added to the
well and incubated for 15 min in dark. The intensity of Syto-9
stain in well was measured with Wallac Viktor.sup.2 1420 multilabel
counter (Perkin Elmer). All the measurements were done in four
replicates and the results were repeated at least twice. The
adhesion of the strains was compared to Lactobacillus reuteri RC-14
(available from Christian Hansen A/S, Denmark).
Example 1
[0181] A number of different criteria were used to select bacteria
of the invention that would have advantageous properties for the
production of a probiotic composition.
[0182] The criteria used and the cut-off values selected are
indicated below in Table 2.
TABLE-US-00001 TABLE 2 Cut-off values Selected Control criteria:
strain Cut-off value Results Acid pH 2.5 L. Rhamnosus -2.56 Average
acid tolerance, log change in LGG pH 2.5 for 1.5 h Bile 0.9% L.
Rhamnosus 40.16% Average bile tolerance, growth in 0.9% LGG oxgall
as % of growth in MRS w/o bile A antigen L. crispatus 100% Average
A antigen adhesion, intensity adhesion LMG18199 of fluorescence B
antigen L. crispatus 100% Average B antigen adhesion, intensity
adhesion LMG18199 of fluorescence O antigen L. crispatus 100%
Average O antigen adhesion, intensity adhesion LMG18199 of
fluorescence
[0183] The assays were carried out as indicated above in the
Materials and Methods section. The results of these analyses are
presented below for a number of strains, including the control
strains L. rhamnosus LGG, RC-14, GR-1 and L. crispatus
LMG18199.
TABLE-US-00002 TABLE 3 Results of the acid tolerance studies at
pH2.5 for 1.5 hours. Acid Acid tolerance tolerance Other strain log
log Average acid DGCC code/ change in change in tolerance, log
Deposit strain commercial pH 2.5 for pH 2.5 for change in pH
number(s) number ID Identification 1.5 h 1.5 h 2.5 for 1.5 h DSM
32107 11860 LX11860 L. brevis -5.2 -5.2 DSM 32363 4022 LA0893 L.
acidophilus -5.7 -4.3 -5.0 PTA-4800 9868 LS-33 L. salivarius -4.6
-4.8 -4.7 DSM 32109 11865 LX11865 L. fermentum -4.3 -5.1 -4.7 DSM
32112 11853 LX11853 L. fermentum -5.3 -3.6 -4.5 DSM 32110 11866
LX11866 L. fermentum -4.5 -4.5 DSM 32108 11864 LX11864 L. brevis
-5.4 -3.5 -4.5 SD 5584 8656 M61/Ll-23 Lactococcus lactis -4.3 -4.1
-4.2 Ll-23 SD 5214 9912 LBr-35 L. brevis -4.1 -3.2 -3.7 DSM32099
11854 LX11854 L. mucosae -3.2 -3.5 -3.4 DSM32105 1925 1925 L.
fermentum -4.0 -2.5 -3.2 SD5209, 4715 LP0115 L. plantarum -3.1 -2.9
-3.0 PTA-4799, DSM 22266 SD 5589 4106 LB0064 L. delbrueckii -3.2
-2.4 -2.8 bulgaricus SD 5212 9353 La-11/La-14 L. acidophilus -4.2
-1.2 -2.7 L. Rhamnosus LGG -2.6 DSM32111 11884 LA11884 L.
acidophilus -1.8 -3.2 -2.5 DSM32114 11862 LX11862 L. rhamnosus -3.3
-1.8 -2.5 PTA-4798 4981 LQ0281/LPC- L. paracasei -2.6 -2.5 -2.5 37
DSM32098 11873 LX11873 L. acidophilus -2.3 -2.2 -2.3 DSM 22193,
9913 Lr-32 L. rhamnosus -2.1 -2.4 -2.2 SD 5217 DSM22091, 8698 NCFM
L. acidophilus -1.3 -3.2 -2.2 SD5221, PTA-4797 DSM 22876, 1460
1460/HN001 L. rhamnosus -2.2 -2.2 -2.2 SD 5675 DSM 32115 11881
LX11881 L. rhamnosus -1.5 -2.6 -2.0 DSM 32097 11887 LX11887 L.
paracasei -1.1 -1.6 -1.3 DSM 32103 5111 LG0179 L. gasseri -0.6 -1.2
-0.9 SD 5585 10687 LG10687/LG- L. gasseri 0.1 -0.1 0.0 36
TABLE-US-00003 TABLE 4 Results of the acid tolerance studies at
pH3.5 for 1.5 hours Average Acid Acid acid Other strain tolerance,
tolerance, tolerance, DGCC code/ log change log change log change
Deposit strain commercial in pH 3.5 in pH 3.5 in pH 3.5 for
number(s) number ID Identification for 1.5 h for 1.5 h 1.5 h
DSM22091, 8698 NCFM L. acidophilus -0.1 -0.1 -0.1 SD5221, PTA-4797
DSM 32363 4022 LA0893 L. acidophilus 0.0 0.3 0.1 SD 5212 9353
La-11/La-14 L. acidophilus -0.4 0.0 -0.2 SD 5214 9912 LBr-35 L.
brevis -0.5 0.1 -0.2 SD 5213 9864 Lc-11 L. casei -0.1 -0.1 -0.1 SD
5589 4106 LB0064 L. delbrueckii -0.1 0.0 -0.1 bulgaricus DSM 32104
11852 LX11852 L. fermentum -0.2 -0.2 DSM 32112 11853 LX11853 L.
fermentum -0.1 -0.1 DSM 32109 11865 LX11865 L. fermentum -0.3 -0.3
DSM 32110 11866 LX11866 L. fermentum -0.3 -0.3 SD 5585 10687
LG10687/LG- L. gasseri 0.0 0.1 0.1 36 PTA-4798 4981 LQ0281/LPC- L.
paracasei -0.1 -0.1 37 SD5209, 4715 LP0115 L. plantarum -0.1 -0.1
-0.1 PTA-4799, DSM 22266 DSM 32106 11858 LX11858 L. paracasei -0.1
-0.1 DSM 22876, 1460 1460/HN001 L. rhamnosus 0.0 0.0 0.0 SD 5675
DSM 22193, 9913 Lr-32 L. rhamnosus -0.1 0.0 -0.1 SD 5217 PTA-4800
9868 LS-33 L. salivarius 0.0 0.1 0.0 SD 5584 8656 M61/Ll-23
Lactococcus -0.2 -0.2 -0.2 lactis Ll-23 DSM 32111 11884 LA11884 L.
acidophilus -0.2 -0.2 DSM 32108 11864 LX11864 L. brevis -0.2 -0.2
DSM 32107 11860 LX11860 L. brevis -0.5 -0.5 DSM 32098 11873 LX11873
L. acidophilus 0.0 0.0 DSM 32105 1925 1925 L. fermentum 0.1 0.2 0.2
DSM 32103 5111 LG0179 L. gasseri -0.3 0.1 -0.1 DSM 32099 11854
LX11854 L. mucosae 0.0 -0.4 -0.2 DSM 32097 11887 LX11887 L.
paracasei 0.0 0.0 DSM 32114 11862 LX11862 L. rhamnosus -0.1 -0.1
DSM 32115 11881 LX11881 L. rhamnosus 0.7 0.7 L. Rhamnosus -0.1
LGG
TABLE-US-00004 TABLE 5 Results of the bile tolerance studies in
0.9% oxgall as % of growth in MRS without bile. Average bile
tolerance, growth in 0.9% oxgall DGCC Other strain Bile tolerance,
growth in as % of Deposit strain code/commercial 0.9% oxgall as %
of growth growth in number(s) number ID Identification in MRS w/o
bile MRS w/o bile SD 5589 4106 LB0064 L. delbrueckii 5% 2% 3%
bulgaricus DSM 32363 4022 LA0893 L. acidophilus 9% 5% 2% 5% SD 5584
8656 M61/Ll-23 Lactococcus 1% 11% 6% lactis Ll-23 DSM 32103 5111
LG0179 L. gasseri 19% 5% 12% SD 5585 10687 LG10687/LG- L. gasseri
15% 16% 15% 36 SD 5213 9864 Lc-11 L. casei 17% 15% 16% PTA-4800
9868 LS-33 L. salivarius 23% 21% 22% PTA-4798 4981 LQ0281/LPC- L.
paracasei 22% 23% 22% 37 DSM 22876, 1460 1460/HN001 L. rhamnosus
29% 35% 32% SD 5675 DSM 32097 11887 LX11887 L. paracasei 29% 40%
35% DSM 32106 11858 LX11858 L. paracasei 36% 36% DSM 22193, 9913
Lr-32 L. rhamnosus 36% 35% 36% SD 5217 DSM 32114 11862 LX11862 L.
rhamnosus 27% 44% 36% DSM 32115 11881 LX11881 L. rhamnosus 44% 34%
39% L. Rhamnosus 40% LGG DSM 32107 11860 LX11860 L. brevis 42% 42%
DSM 32109 11865 LX11865 L. fermentum 34% 51% 43% DSM 32105 1925
1925 L. fermentum 55% 50% 35% 46% DSM 32112 11853 LX11853 L.
fermentum 43% 50% 47% DSM 32104 11852 LX11852 L. fermentum 46% 50%
48% DSM 32108 11864 LX11864 L. brevis 47% 65% 56% SD 5214 9912
LBr-35 L. brevis 71% 77% 74% DSM 32111 11884 LA11884 L. acidophilus
104% 110% 107% SD 5212 9353 La-11/La-14 L. acidophilus 113% 106%
113% 111% DSM22091, 8698 NCFM L. acidophilus 115% 111% 116% 114%
SD5221, PTA-4797 DSM 32098 11873 LX11873 L. acidophilus 120% 119%
119% DSM 32099 11854 LX11854 L. mucosae 142% 163% 152%
TABLE-US-00005 TABLE 6 Results of the bile tolerance studies in
0.3% oxgall as % of growth in MRS without bile. Average bile
tolerance, growth in 0.3% oxgall Other strain as % of DGCC code/
Bile tolerance, growth in growth in Deposit strain commercial 0.3%
oxgall as % of MRS w/o number(s) number ID Identification growth in
MRS w/o bile bile DSM22091, 8698 NCFM L. acidophilus 93% 91% 85%
90% SD5221, PTA-4797 DSM 32363 4022 LA0893 L. acidophilus 2% 3% 2%
2% SD 5212 9353 La-11/La-14 L. acidophilus 92% 89% 85% 88% SD 5214
9912 LBr-35 L. brevis 92% 93% 92% SD 5213 9864 Lc-11 L. casei 30%
31% 30% SD 5589 4106 LB0064 L. delbrueckii 9% 5% 7% bulgaricus DSM
32104 11852 LX11852 L. fermentum 64% 64% DSM 32112 11853 LX11853 L.
fermentum 64% 64% DSM 32109 11865 LX11865 L. fermentum 64% 63% 63%
DSM 32110 11866 LX11866 L. fermentum 21% 21% SD 5585 10687
LG10687/LG- L. gasseri 33% 39% 36% 36 PTA-4798 4981 LQ0281/LPC- L.
paracasei 46% 43% 45% 37 SD5209, 4715 LP0115 L. plantarum 85% 90%
88% PTA-4799, DSM 22266 DSM 32106 11858 LX11858 L. paracasei 55%
55% DSM 22876, 1460 1460/HN001 L. rhamnosus 62% 63% 63% SD 5675 DSM
22193, 9913 Lr-32 L. rhamnosus 70% 66% 68% SD 5217 PTA-4800 9868
LS-33 L. salivarius 73% 68% 71% SD 5584 8656 M61/Ll-23 Lactococcus
2% 8% 5% lactis Ll-23 DSM 32111 11884 LA11884 L. acidophilus 89%
85% 87% DSM 32108 11864 LX11864 L. brevis 76% 63% 70% DSM 32107
11860 LX11860 L. brevis 72% 72% DSM 32098 11873 LX11873 L.
acidophilus 99% 98% 99% DSM 32105 1925 1925 L. fermentum 83% 77%
76% 79% DSM 32103 5111 LG0179 L. gasseri 60% 47% 53% DSM 32099
11854 LX11854 L. mucosae 167% 167% DSM 32097 11887 LX11887 L.
paracasei 39% 68% 54% DSM 32114 11862 LX11862 L. rhamnosus 61% 60%
60% DSM 32115 11881 LX11881 L. rhamnosus 58% 58% 58% L. Rhamnosus
62.66% LGG
TABLE-US-00006 TABLE 7 Growth in MRS as a control for Tables 5 and
6 above. Other strain Average DGCC code/ growth in Deposit strain
commercial Growth in MRS, OD MRS, OD number(s) number ID
Identification (A600) (A600) DSM22091, 8698 NCFM L. acidophilus 1.1
1.1 1.1 1.1 SD5221, PTA-4797 DSM 32363 4022 LA0893 L. acidophilus
1.2 1.1 1.2 1.2 SD 5212 9353 La-11/La-14 L. acidophilus 1.1 1.1 1.1
1.1 SD 5214 9912 LBr-35 L. brevis 0.9 1.0 1.0 SD 5213 9864 Lc-11 L.
casei 1.2 1.2 1.2 SD 5589 4106 LB0064 L. delbrueckii 1.1 1.1 1.1
bulgaricus DSM 32104 11852 LX11852 L. fermentum 1.0 0.9 0.9 DSM
32112 11853 LX11853 L. fermentum 1.0 0.9 0.9 DSM 32109 11865
LX11865 L. fermentum 1.0 0.9 0.9 DSM 32110 11866 LX11866 L.
fermentum 0.8 0.8 SD 5585 10687 LG10687/LG- L. gasseri 1.0 1.0 1.0
36 PTA-4798 4981 LQ0281/LPC- L. paracasei 1.2 1.1 1.1 37 SD5209,
4715 LP0115 L. plantarum 1.2 1.2 1.2 PTA-4799, DSM 22266 DSM 32106
11858 LX11858 L. paracasei 1.2 1.2 DSM 22876, 1460 1460/HN001 L.
rhamnosus 1.2 1.2 1.2 SD 5675 DSM 22193, 9913 Lr-32 L. rhamnosus
1.2 1.2 1.2 SD 5217 PTA-4800 9868 LS-33 L. salivarius 1.3 1.3 1.3
SD 5584 8656 M61/Ll-23 Lactococcus 1.0 1.1 1.1 lactis Ll-23 DSM
32111 11884 LA11884 L. acidophilus 1.1 1.1 1.1 DSM 32108 11864
LX11864 L. brevis 0.9 1.1 1.0 DSM 32107 11860 LX11860 L. brevis 0.9
0.9 DSM 32098 11873 LX11873 L. acidophilus 1.0 1.0 1.0 DSM 32105
1925 1925 L. fermentum 1.1 1.0 1.0 1.1 DSM 32103 5111 LG0179 L.
gasseri 1.1 1.0 1.0 DSM 32099 11854 LX11854 L. mucosae 0.8 0.5 0.6
DSM 32097 11887 LX11887 L. paracasei 1.0 1.0 1.0 DSM 32114 11862
LX11862 L. rhamnosus 1.2 1.2 1.2 DSM 32115 11881 LX11881 L.
rhamnosus 1.1 1.2 1.2 L. Rhamnosus 1.1 LGG
TABLE-US-00007 TABLE 8 Results from the A antigen adhesion studies.
DGCC Other strain Average A antigen adhesion, Deposit strain code/
A antigen adhesion, % of adhesion of L. crispatus number(s) number
commercial ID Identification % of adhesion of L. crispatus LMG
18199 LMG 18199 DSM22091, 8698 NCFM L. acidophilus 0% 5% 2% SD5221,
PTA-4797 DSM 32363 4022 LA0893 L. acidophilus 2% 3% 3% SD 5212 9353
La-11/La-14 L. acidophilus 0% 2% 1% SD 5214 9912 LBr-35 L. brevis
0% 1% 1% SD 5213 9864 Lc-11 L. casei -2% -1% -1% SD 5589 4106
LB0064 L. delbrueckii -1% 0% 0% bulgaricus DSM 32104 11852 LX11852
L. fermentum 133% 188% 112% 144% DSM 32112 11853 LX11853 L.
fermentum 144% 151% 137% 122% 126% 222% 150% DSM 32109 11865
LX11865 L. fermentum 116% 91% 120% 104% 137% 201% 128% DSM 32110
11866 LX11866 L. fermentum 102% 135% 105% 106% 112% SD 5585 10687
LG10687/LG-36 L. gasseri -1% 0% 0% PTA-4798 4981 LQ0281/LPC-37 L.
paracasei 0% 1% 1% SD5209, 4715 LP0115 L. plantarum 0% 0% 0%
PTA-4799, DSM 22266 DSM 32106 11858 LX11858 L. paracasei 73% 169%
121% DSM 22876, 1460 1460/HN001 L. rhamnosus 46% 46% SD 5675 DSM
22193, 9913 Lr-32 L. rhamnosus -2% 59% 28% SD 5217 PTA-4800 9868
LS-33 L. salivarius 0% 2% 1% SD 5584 8656 M61/Ll-23 Lactococcus
lactis 1% 3% 2% Ll-23 DSM 32111 11884 LA11884 L. acidophilus 0% 5%
111% 39% DSM 32108 11864 LX11864 L. brevis 31% 47% 57% 45% DSM
32107 11860 LX11860 L. brevis -1% 37% 18% DSM 32098 11873 LX11873
L. acidophilus 30% 30% DSM 32105 1925 1925 L. fermentum -1% 0% 0%
DSM 32103 5111 LG0179 L. gasseri -1% 5% 2% DSM 32099 11854 LX11854
L. mucosae 4% 17% 11% DSM 32097 11887 LX11887 L. paracasei 0% 3% 3%
172% 45% DSM 32114 11862 LX11862 L. rhamnosus 0% 1% 24% 36% 88% 30%
DSM 32115 11881 LX11881 L. rhamnosus 55% 55% L. crispatus 100%
LMG18199
TABLE-US-00008 TABLE 9 Results from the B antigen adhesion studies.
Average B antigen Deposit DGCC strain Other strain B antigen
adhesion, % of adhesion of adhesion, % of adhesion number(s) number
code/commercial ID Identification L. crispatus LMG 18199 of L.
crispatus LMG 18199 DSM22091, 8698 NCFM L. acidophilus 0% 4% 2%
SD5221, PTA-4797 DSM 32363 4022 LA0893 L. acidophilus 1% 1% SD 5212
9353 La-11/La-14 L. acidophilus 0% 2% 1% SD 5214 9912 LBr-35 L.
brevis 1% 1% 33% 12% SD 5213 9864 Lc-11 L. casei -1% 1% 0% SD 5589
4106 LB0064 L. delbrueckii -1% 0% 0% bulgaricus DSM 32104 11852
LX11852 L. fermentum 121% 109% 179% 114% 131% DSM 32112 11853
LX11853 L. fermentum 132% 113% 148% 203% 118% 143% DSM 32109 11865
LX11865 L. fermentum 80% 111% 113% 139% 131% 117% 115% DSM 32110
11866 LX11866 L. fermentum 127% 103% 98% 100% 107% SD 5585 10687
LG10687/LG-36 L. gasseri 1% 1% 1% PTA-4798 4981 LQ0281/LPC-37 L.
paracasei 1% 2% 2% SD5209, 4715 LP0115 L. plantarum -1% 1% 0%
PTA-4799, DSM 22266 DSM 32106 11858 LX11858 L. paracasei -1% 58%
29% DSM 22876, 1460 1460/HN001 L. rhamnosus -1% 3% 1% SD 5675 DSM
22193, 9913 Lr-32 L. rhamnosus 1% 63% 32% SD 5217 PTA-4800 9868
LS-33 L. salivarius 1% 1% 1% SD 5584 8656 M61/Ll-23 Lactococcus 1%
2% 1% lactis Ll-23 DSM 32111 11884 LA11884 L. acidophilus 1% 4% 18%
8% DSM 32108 11864 LX11864 L. brevis 21% 23% 22% DSM 32107 11860
LX11860 L. brevis 0% 27% 14% DSM 32098 11873 LX11873 L. acidophilus
30% 30% DSM 32105 1925 1925 L. fermentum -1% 13% 1% 4% DSM 32103
5111 LG0179 L. gasseri 0% 5% 3% DSM 32099 11854 LX11854 L. mucosae
3% 14% 9% DSM 32097 11887 LX11887 L. paracasei 4% 60% -1% 21% DSM
32114 11862 LX11862 L. rhamnosus -1% 1% 20% 18% 34% 15% DSM 32115
11881 LX11881 L. rhamnosus 50% 50% L. crispatus 100% LMG18199
TABLE-US-00009 TABLE 10 Results from the O antigen adhesion
studies. Average DGCC Other strain O antigen adhesion, Deposit
strain code/commercial O antigen adhesion, % of adhesion of % of
adhesion number(s) number ID Identification L. crispatus LMG 18199
of L. crispatus LMG 18199 DSM22091, 8698 NCFM L. acidophilus 0% 4%
2% SD5221, PTA-4797 DSM 32363 4022 LA0893 L. acidophilus 2% 3% 3%
SD 5212 9353 La-11/La-14 L. acidophilus 1% 2% 2% SD 5214 9912
LBr-35 L. brevis 1% 2% 2% SD 5213 9864 Lc-11 L. casei -1% 1% 0% SD
5589 4106 LB0064 L. delbrueckii 0% 2% 1% bulgaricus DSM 32104 11852
LX11852 L. fermentum 112% 100% 176% 110% 124% DSM 32112 11853
LX11853 L. fermentum 122% 106% 138% 121% 269% 151% DSM 32109 11865
LX11865 L. fermentum 77% 111% 114% 99% 110% 130% 107% DSM 32110
11866 LX11866 L. fermentum 123% 94% 101% 116% 108% SD 5585 10687
LG10687/LG-36 L. gasseri 1% 2% 2% PTA-4798 4981 LQ0281/LPC-37 L.
paracasei 1% 2% 2% SD5209, 4715 LP0115 L. plantarum 0% 3% 1%
PTA-4799, DSM 22266 DSM 32106 11858 LX11858 L. paracasei 0% 0% 0%
DSM 22876, 1460 1460/HN001 L. rhamnosus 0% 2% 1% SD 5675 DSM 22193,
9913 Lr-32 L. rhamnosus 0% 1% 1% SD 5217 PTA-4800 9868 LS-33 L.
salivarius 1% 3% 2% SD 5584 8656 M61/Ll-23 Lactococcus 3% 5% 4%
lactis Ll-23 DSM 32111 11884 LA11884 L. acidophilus 0% 1% 5% 2% DSM
32108 11864 LX11864 L. brevis 16% 18% 22% 19% DSM 32107 11860
LX11860 L. brevis 1% 10% 6% DSM 32098 11873 LX11873 L. acidophilus
26% 26% DSM 32105 1925 1925 L. fermentum 0% 1% 0% DSM 32103 5111
LG0179 L. gasseri 1% 5% 3% DSM 32099 11854 LX11854 L. mucosae 3%
13% 8% DSM 32114 11862 LX11862 L. rhamnosus 3% 7% 20% 30% 22% 16%
DSM 32115 11881 LX11881 L. rhamnosus 37% 37% L. crispatus 100%
LMG18199 No data was obtained for DSM 32097 in this assay.
[0184] Based on the data acquired above and the selection criteria
used a number of strains were selected according to their
suitability for use in a probiotic composition. Table 11 below
shows those strains that performed better than control strains
based on A, B and O antigen adhesion, and which strains showed
enhanced bile and/or acid tolerance. A, B and/or O adhesion of five
strains (DSM 32104, DSM 32112, DSM 32109, DSM 32110 and DSM 32106)
was equal or better than the adhesion of control strain L.
crispatus LMG18199, which adheres well to the A antigen (Uchida et
al. 2006).
TABLE-US-00010 TABLE 11 Strains grouped in accordance with their
properties based on criteria as defined in Table 2 Strains
performing Properties better than control strain(s) A, B and/or O
antigen adhesion DGCC11852 DSM 32104 in vitro DGCC11853 DSM 32112
DGCC11865 DSM 32109 DGCC11866 DSM 32110 DGCC11858 DSM 32106 Acid
and/or bile tolerance DGCC11884 DSM 32111 DGCC11864 DSM 32108
DGCC11860 DSM 32107 DGCC11873 DSM 32098 DGCC1925 DSM 32105 DGCC5111
DSM 32103 DGCC11854 DSM 32099 DGCC11887 DSM 32097 DGCC11862 DSM
32114 DGCC11881 DSM 32115 A, B and/or O antigen adhesion DGCC11852
DSM 32104 in vitro + acid and/or bile DGCC11853 DSM 32112
tolerance
Example 3 (Gastrointestinal Health--Pain)
[0185] Male rats weighing approximately 200-220 g are surgically
prepared for electromyography of the abdominal muscles in order to
measure the pain response to colonic distension later on. Rats are
then gavaged with each probiotic (daily dose of 10.sup.7, 10.sup.8
and/or 10.sup.9 CFU) or saline for 7-21 days before a colorectal
distension test. Rats are accustomed to polypropylene tunnels 3-5
days before the distension test. The balloon used for distention is
taken from an embolectomy probe and is inserted into the rectum at
1 cm from the anus and fixed at the basis of the tail. Increasing
levels of distensions are then induced with the probe connected to
a barostat. A functional ingredient desensitizes the colonic wall
and leads to an increased pain threshold or decreased contraction
of the abdominal muscles. Contractions corresponding to each
pressure level are measured and compared between groups.
Example 4 (Gastrointestinal Health--Inflammation)
[0186] Male rats weighing approximately 200-220 g are gavaged with
each probiotic or saline for 15 days before inducing
trinitrobenzene sulfonic acid (TNBS) colitis. TNBS is infused
intrarectally after an overnight fast at a maximum of 80 mg/kg in
0.3 ml 50% ethanol. Two to four days after TNBS infusion, rats are
subjected to the colonic distension test and sacrificed. At
sacrifice, tissue specimens, blood and GI contents are collected
for further analyses, such as one or more of the following:
[0187] Scoring of Inflammation
[0188] Fresh tissue specimens are visually scored by an experienced
scientist to determine the level of inflammation.
[0189] Expression of Inflammation Markers
[0190] Sections of colon are snap-frozen in liquid nitrogen. RNA is
extracted and converted to cDNA with a commercially available kit.
The expression of inflammation markers, such as IL-6, TNF-alpha,
IL-1beta, matrix metalloproteinases (MMPs) and their tissue
inhibitors, in colonic tissue is analyzed with RT-qPCR.
[0191] Colonic Histology
[0192] Histological samples are cut from tissues embedded in
paraffin, and are visualized under a microscope to enumerate
infiltrated immune cells.
[0193] Circulating Inflammation Markers
[0194] Blood is centrifuged to separate plasma. Inflammation
markers (such as IL-6, TNF-alpha, IL-1beta, hsCRP) are then
determined with commercially available ELISA kits.
[0195] Circulating Lipopolysaccharide
[0196] Blood is centrifuged to separate serum. Lipopolysaccharides
are analyzed from serum samples using a standard kit based on a
Limulus amebocyte extract (the LAL kit), which is commercially
available.
[0197] Gut Microbiota Analysis
[0198] Gut microbiota is analyzed from feces or caecum by
extracting DNA. Samples then undergo gut microbial sequencing.
Results are analyzed by a trained bioinformatician.
[0199] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Although the present invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in biochemistry and biotechnology or related fields
are intended to be within the scope of the following claims.
* * * * *