U.S. patent application number 13/847064 was filed with the patent office on 2014-02-06 for prebiotic oligosaccharides.
This patent application is currently assigned to DSM Food Specialties USA Inc.. The applicant listed for this patent is DSM Food Specialties USA Inc., The Regents of the University of California. Invention is credited to Mariana Barboza, Samara Freeman, J. Bruce German, William Robert King, Carlito B. Lebrilla, David Mills.
Application Number | 20140037785 13/847064 |
Document ID | / |
Family ID | 42728831 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140037785 |
Kind Code |
A1 |
Barboza; Mariana ; et
al. |
February 6, 2014 |
PREBIOTIC OLIGOSACCHARIDES
Abstract
The present invention provides galacto-oligosaccharide
compositions that preferentially stimulate growth of specific
Bifidobacterium species and subspecies.
Inventors: |
Barboza; Mariana; (Longmont,
CO) ; German; J. Bruce; (Davis, CA) ;
Lebrilla; Carlito B.; (Davis, CA) ; Mills; David;
(Davis, CA) ; Freeman; Samara; (San Francisco,
CA) ; King; William Robert; (Walnut Creek,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California;
DSM Food Specialties USA Inc.; |
|
|
US
US |
|
|
Assignee: |
DSM Food Specialties USA
Inc.
Parsippany
NJ
The Regents of the University of California
Oakland
CA
|
Family ID: |
42728831 |
Appl. No.: |
13/847064 |
Filed: |
March 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12722813 |
Mar 12, 2010 |
8425930 |
|
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13847064 |
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61160088 |
Mar 13, 2009 |
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Current U.S.
Class: |
426/2 ; 426/64;
426/71 |
Current CPC
Class: |
A61P 35/00 20180101;
A23L 7/00 20160801; A61K 31/702 20130101; A61P 1/14 20180101; A61K
9/0095 20130101; C12Y 302/01108 20130101; A23L 29/30 20160801; A61P
1/10 20180101; A61P 1/04 20180101; A61K 38/47 20130101; A61K 35/745
20130101; A61P 1/00 20180101; A23L 33/125 20160801; A23L 5/00
20160801; A61K 2035/115 20130101; A23V 2200/3204 20130101; A23V
2200/32 20130101; A23L 33/135 20160801; A23V 2250/28 20130101; A61P
1/12 20180101; A61P 1/06 20180101; A23V 2200/3202 20130101; A23L
33/40 20160801; A23V 2002/00 20130101; A23V 2002/00 20130101; A23L
33/21 20160801 |
Class at
Publication: |
426/2 ; 426/71;
426/64 |
International
Class: |
A23L 1/308 20060101
A23L001/308 |
Claims
1. A composition comprising galacto-oligosaccharides, wherein at
least 45% of the galacto-oligosaccharides by weight are tetra or
penta galacto-oligosaccharides or wherein at least 25% of the
galacto-oligosaccharides by weight are tetra
galacto-oligosaccharides.
2. The composition of claim 1, wherein the composition has less
than 20% by weight of dimeric galacto-oligosaccharides, based on
weight of the total oligosaccharides.
3. The composition of claim 1, wherein the composition has less
than 10% by weight of dimeric galacto-oligosaccharides, based on
weight of the total oligosaccharides.
4. The composition of claim 1, wherein the composition has less
than 5% by weight of monomeric sugars based on total sugar and
oligosaccharide solids.
5. The composition of claim 1, wherein the composition has less
than 5% by weight of lactose, based on weight of the total
oligosaccharides.
6. The composition of claim 1, wherein the composition comprises a
lactase enzyme.
7. The composition of claim 1, wherein the composition is a food
product or dietary supplement product.
8. The composition of claim 1, wherein the food product is selected
from the group consisting of an infant formula, a follow-on
formula, and a toddler beverage.
9. The composition of claim 1, wherein less than 10% of the
galacto-oligosaccharides by weight have a degree of polymerization
of 6 or greater.
10. The composition of claim 1, wherein less than 10% of the
galacto-oligosaccharides by weight are trimeric
galacto-oligosaccharides.
11. The composition of claim 1, wherein more than 30% of the
galacto-oligosaccharides by weight are trimeric
galacto-oligosaccharides.
12. The composition of claim 1, prepared by a method comprising the
step of treating a mixed galacto-oligosaccharide solution (GOS) to
reduce monomeric, dimeric and/or trimeric sugars.
13. The composition of claim 12, wherein the monomeric, dimeric
and/or trimeric sugars are removed by size exclusion or
enzymatically, or by selective microbial consumption of particular
sugars or oligosaccharides.
14. The composition of claim 1, further comprising Bifidobacterium
breve or Bifidobacterium longum bv. infantis.
15. A method for stimulating beneficial Bifidobacterium microflora
in an animal, the method comprising administering a sufficient
amount of the composition of claim 1 to the animal to stimulate
colonization of the gut of the animal by at least one beneficial
Bifidobacterium strain.
16. The method of claim 15, wherein the strain is a strain of
Bifidobacterium breve or Bifidobacterium longum bv. infantis.
17. The method of claim 15, wherein the animal is a human.
18. The method of claim 15, wherein the animal is a non-human
mammal.
19. The method of claim 17, wherein the human is less than 5 years
old.
20. The method of claim 17, wherein the human is over 50 years
old.
21. The method of claim 17, wherein the human has a condition
selected from the group consisting of inflammatory bowel syndrome,
constipation, diarrhea, colitis, Crohn's disease, colon cancer,
functional bowel disorder, irritable bowel syndrome, and excess
sulfate reducing bacteria.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/722,813 filed Mar. 12, 2010, which application claims
benefit of priority to U.S. Provisional Patent Application No.
61/160,088 filed Mar. 13, 2009, both of which are incorporated by
reference for all purposes.
BACKGROUND OF THE INVENTION
[0002] Galacto-oligosaccharides (GOS) are non-digestible
carbohydrates and versatile food ingredients that possess prebiotic
properties (Angus, F., Smart, S. and Shortt, C. 2005. In Probiotic
Dairy Products ed. Tamine, A. pp. 120-137. Oxford: Blackwell
Publishing). In addition, many other health benefits have been
reported for these oligosaccharides including: improvement of
defecation, stimulation of mineral absorption, elimination of
ammonium, colon cancer prevention, as well as protection against
certain pathogenic bacteria infections (Hopkins, M. J. and
Macfarlane, G. T. 2003 Appl Environ Microbiol 69, 1920-1927; Shoaf,
K., G. L. Mulvey, G. D. Armstrong, and R. W. Hutkins. 2006 Infect
Immun 74:6920-8; Macfarlane, G. T., Steed H., Macfarlane S. 2008
Journal of Applied Microbiology 104, 305-44).
[0003] The human gastrointestinal tract (GIT) hosts a large
bacterial population of 500-1000 different phylotypes that reside
in the colon (Ninonuevo, M. R., et al. 2007 Anal Biochem
361,15-23). Among them, Bifidobacterial species are the predominant
microbial in the infant GIT, exerting beneficial effects to their
host such us immuno-stimulation, human pathogen inhibition, vitamin
production, and anticarcinogenic activity, among others (Harmsen,
H. J., et al. 2000 J Pediatr Gastroenterol Nutr 30:61-7; Casci, T.,
et al. 2007 Human Gut microflora in Health and Disease: Focus on
Prebiotics. In Functional food and Biotechnology. Ed Taylor and
Francis. pp 401-434). Due to these beneficial health effects,
Bifidobacteria are considered probiotics and have being
increasingly used in functional foods and pharmaceutical products
(Stanton, C., et al. 2003. Challenges facing development of
probiotics-containing functional foods. In Handbook Fermented
Functional Foods, Functional Foods and Nutraceutical Series. CC
Press, Boca Raton, Fla. pp 27-58).
[0004] The physicochemical characteristics of GOS have enabled them
to be incorporated as prebiotic food ingredients in a variety of
designed foods (Sako, T., et al. 1999 Int Dairy J 9, 69-80). GOS
are of particular interest in confectionary acidic beverage and
fermented milk formulations as they possess increased thermal
stability in acidic environments compared to FOS (Watanuki, M., et
al. 1996 Ann Report Yakult Central Inst Microbiol Res 16, 1-12).
Thus, in the past decade, GOS have also had an increasing
application in human food products, including dairy products, sugar
replacements and other diet supplements as well as infant formula
(Macfarlane, G. T., Steed H., Macfarlane S. 2008 Journal of Applied
Microbiology 104, 305-44).
[0005] Galacto-oligosaccharides are naturally occurring in human
milk, however, commercial GOS preparations are produced by
enzymatic treatment of lactose with .beta.-galactosidases from
different sources such as fungi, yeast and/or bacteria, yielding a
mixture of oligomers with varied chain lengths (Angus, F., supra).
Thus, the basic structure of GOS includes a lactose core at the
reducing end which is elongated typically with up to six galactose
residues. GOS structural diversity dependents on the enzyme used in
the trans-galactosylation reaction, and the experimental conditions
such as pH and temperature (Dumortier, V., et al. 1990. Carbohydr
Res 201:115-23.).
[0006] Despite the amount of research claiming GOS bifidogenic
effect, the vast majority of studies used commercially available
preparations of GOS, containing high concentrations of
monosaccharide (i.e. galactose and glucose) and the disaccharide
lactose, all remaining reagents of the trans-galactosylation
reaction. Notably, in the majority of reported cases,
monosaccharides are the preferred substrates for microorganism when
available in a mixed carbon source (Saier, M. H. Jr. 1996. Res.
Microbiol, 147, 439-587; Bruckner, R. and Titgemeyer, F. 2002 FEMS
Microbiology Letters 209, 141-48).
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides compositions for stimulating
growth of particular Bifidobateria. In some embodiments, the
compositions comprise galacto-oligosaccharides, wherein at least
45% of the galacto-oligosaccharides by weight are tetra or penta
galacto-oligosaccharides or wherein at least 25% of the
galacto-oligosaccharides by weight are tetra
galacto-oligosaccharides. In some embodiments, the compositions
comprise galacto-oligosaccharides, wherein at least 30%, 40%, 50%,
60%, 75%, or 80% of the galacto-oligosaccharides by weight are
tetra or penta galacto-oligosaccharides.
[0008] In some embodiments, the composition has less than 20% by
weight of dimeric galacto-oligosaccharides based on weight of the
total oligosaccharides. In some embodiments, the composition has
less than 10% by weight of dimeric galacto-oligosaccharides based
on weight of the total oligosaccharides.
[0009] In some embodiments, the composition has less than 5% by
weight of monomeric sugars based on total sugar and oligosaccharide
solids.
[0010] In some embodiments, the composition has less than 5% by
weight of lactose , based on weight of the total
oligosaccharides.
[0011] In some embodiments, the composition comprises a lactase
enzyme (e.g., an encapsulated lactase that is degraded when
ingested).
[0012] In some embodiments, the composition has less than 20%
(e.g., less than 10%) by weight of dimeric
galacto-oligosaccharides, and/or less than 5% by weight of
monomeric galacto-oligosaccharides and/or less than 5% lactose.
[0013] In some embodiments, the composition is a food product or
dietary supplement product.
[0014] In some embodiments, the food product is selected from the
group consisting of an infant formula, a follow-on formula, and a
toddler beverage.
[0015] In some embodiments, less than 10% of the
galacto-oligosaccharides by weight have a degree of polymerization
of 6 or greater.
[0016] In some embodiments, less than 10% of the
galacto-oligosaccharides by weight are trimeric
galacto-oligosaccharides.
[0017] In some embodiments,more than 30% of the
galacto-oligosaccharides by weight are trimeric
galacto-oligosaccharides.
[0018] In some embodiments, the compositions are prepared by a
method comprising the step of treating a mixed
galacto-oligosaccharide solution (GOS) to reduce monomeric, dimeric
and/or trimeric sugars. In some embodiments, the monomeric, dimeric
and/or trimeric sugars are removed by size exclusion or
enzymatically, or by selective microbial consumption of particular
sugars or oligosaccharides.
[0019] In some embodiments, the composition further comprises
Bifidobacterium breve or Bifidobacterium longum bv. infantis.
[0020] The present invention also provides methods for stimulating
beneficial Bifidobacterium microflora in an animal. In some
embodiments, the method comprises administering a sufficient amount
of the compositions described above or elsewhere herein to the
animal to stimulate colonization of the gut of the animal by at
least one beneficial Bifidobacterium strain.
[0021] In some embodiments, the strain is a strain of
Bifidobacterium breve or Bifidobacterium longum bv. infantis.
[0022] In some embodiments, the animal is a human. In some
embodiments, the animal is a non-human mammal.
[0023] In some embodiments, the human is less than 5 years old. In
some embodiments ,the human is over 50 years old. In some
embodiments, the human has a condition selected from the group
consisting of inflammatory bowel syndrome, constipation, diarrhea,
colitis, Crohn's disease, colon cancer, functional bowel disorder,
irritable bowel syndrome, and excess sulfate reducing bacteria.
[0024] Other aspects of the invention will be evident from the
remaining text.
Definitions
[0025] The "degree of polymerization" or "DP" of a
galacto-oligosaccharide refers to the total number of sugar monomer
units that are part of a particular oligosaccharide. For example, a
tetra galacto-oligosaccharide has a DP of 4, having 3 galactose
moieties and one glucose moiety.
[0026] The term "Bifidobacteria" and its synonyms refer to a genus
of anaerobic bacteria having beneficial properties for humans.
Bifidobacteria is one of the major strains of bacteria that make up
the gut flora, the bacteria that reside in the gastrointestinal
tract and have health benefits for their hosts. See, e.g., Guarner
F and Malagelada J R. Lancet (2003) 361, 512-519, for a further
description of Bifidobacteria in the normal gut flora.
[0027] A "prebiotic" or "prebiotic nutrient" is generally a
non-digestible food ingredient that beneficially affects a host
when ingested by selectively stimulating the growth and/or the
activity of one or a limited number of bacteria in the
gastrointestinal tract. As used herein, the term "prebiotic" refers
to the above described non-digestible food ingredients in their
non-naturally occurring states, e.g., after purification, chemical
or enzymatic synthesis as opposed to, for instance, in whole human
milk.
[0028] A "probiotic" refers to live microorganisms that when
administered in adequate amounts confer a health benefit on the
host.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1. Positive MALDI-FTICR ion spectra of syrup GOS. Major
peaks correspond to sodium coordinated ions showing the degree of
polymerization of GOS. Minor signals observed at 18 mass units less
could correspond to B-type fragments.
[0030] FIGS. 2A-2E. Positive MALDI-FTICR ion spectrum of
GOS-Bio-Gel P-2 fractions. Fractions are (ml) 45 (FIG. 2A), 56
(FIG. 2B), 67 (FIG. 2C), 74 (FIG. 2D), and 82 (FIG. 2E). Signals
with m/z values 527, 689, 851, 1013, 1175, 1337, 1449, 1662, 1824,
1966, 2148, 2310, and 2473 represent sodium coordinated
galacto-oligosaccharides with a DP ranging from 3 to 15.
[0031] FIGS. 3A-3C. IRMPD MALDI-FTICR spectra of GOS. FIGS. 3A, 3B
and 3C correspond to galactooligosaccharides with DP 5, 4 and 3,
respectively. Fragments ions corresponding to glycosidic-bond
cleavages (Hex) and cross-ring cleavages (60, 90 and 120) were
obtained.
[0032] FIG. 4. Positive MALDI-FTICR spectra of pGOS with selected
DP used in bifidobacterial fermentation experiments.
[0033] FIGS. 5A-5D. Growth of B. adolescentis, B. breve, B. longum
bv. Infantis, and B. longum bv. longum on modified MRS containing:
0.5% (5A), 1% (5B), 1.5% (5C) and 2% (5D) (w:v) of pGOS.
[0034] FIGS. 6A-6D. Positive MALDI-FTICR MS ion spectum of
remaining pGOS purified from supernatants of bifidobacterial
culture growth on mMRS containing 0.5% pGOS. FIG. 6A)
Bifidobacterium adolescentis, FIG. 6B) B. breve, FIG. 6C) B. longum
bv. Infantis, and FIG. 6D) B. longum bv. longum.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0035] Galacto-oligosaccharides are carbohydrates that possess
prebiotic properties and that are non-digestible by humans. The
present invention is based in part on the discovery that particular
Bifidobacterium species or subspecies consume
galacto-oligosaccharide polymers having a specific degree of
polymerization (DP) but do not significantly consume other DPs. In
view of these results, the invention provides for
galacto-oligosaccharide compositions specifically designed to
preferentially stimulate growth of specific Bifidobacterium species
or subspecies in humans or other animals relative to other enteric
bacteria.
II. Galacto-oligosaccharide Compositions
[0036] The galacto-oligosaccharide compositions of the invention
can comprise the galacto-oligosaccharides themselves as well as
optionally other components as desired for a particular use. The
galacto-oligosaccharide compositions are synthetic (e.g., are
generated by purified enzymatic reactions or as part of a
human-directed fermentation process), and in some embodiments are
purified. As discussed in more detail below, the
galacto-oligosaccharides can be combined with various ingredients
to manufacture food stuffs and food supplements including, for
example, infant formulas. The compositions can further optionally
comprise beneficial bacteria, notably particular Bifidobacterium
species or subspecies.
A. Galacto-oligosaccharides
[0037] Galacto-oligosaccharides refer to straight or branched
polymers of galactose. Generally, galacto-oligosaccharides are made
up solely of galactose units with the exception that the terminal
sugar is glucose. Galacto-oligosaccharides can therefore be
represented by the formula Gal-(Gal).sub.n-Glc, where Gal is a
galactose residue, Glc is a glucose residue, and n is an integer of
zero or greater.
[0038] The present invention provides for GOS compositions that are
enriched for particular DPs that can be used to preferentially
stimulate growth of specific Bifidobacteria. For example, the
following summarizes some of the findings of the inventors: [0039]
1. Infant-borne Bifidobacteria (e.g., B. breve and B. longum bv
infantis) growth can be preferentially stimulated (e.g., relative
to other enteric bacteria including other Bifidobacteria) using GOS
that is enriched for DP 4-5 galacto-oligosaccharides. [0040] 2.
Adult-borne Bifidobacteria (B. longum bv longum) growth can be
preferentially stimulated using GOS that is enriched for DP 6-8
galacto-oligosaccharides. [0041] 3. B. longum bv infantis and B.
adolescentis species growth can be preferentially stimulated using
GOS that is enriched for DP 3 galacto-oligosaccharides.
i. Galacto-oligosaccharides that Enrich Bifidobacteria infantis or
breve
[0042] As noted above and in the Example, galacto-oligosaccharides
of DP 4-5 are consumed by Bifidobacteria typically found in
infants, e.g., Bifidobacteria infantis or breve. Accordingly, in
some embodiments, the compositions of the present invention
comprise galacto-oligosaccharides, wherein at least 20%, 25%, 30%,
35%, 40%, 45%, or 50% of the galacto-oligosaccharides by weight are
tetra galacto-oligosaccharides and/or optionally at least 20%, 25%,
30%, 35%, 40%, 45%, or 50% of the galacto-oligosaccharides by
weight are penta galacto-oligosaccharides. All composition
percentages as provided herein, unless indicated otherwise, are
determined by mass spectrometry (e.g., MALDI-FTICR as described in
the Examples). In some embodiments, the compositions of the present
invention comprise galacto-oligosaccharides, wherein at least 30%,
40%, 50%, 60%, 70%, 80%, or 90% of the galacto-oligosaccharides by
weight are DP4-5 galacto-oligosaccharides. These embodiments are
useful, for example, for enriching for Bifidobacteria infantis or
breve. In some embodiments, the compositions have less than 10% or
less than 5% of monomeric sugars (e.g., galactose) and/or less than
10% or less than 5% of lactose and/or optionally less than 10% or
less than 5% of dimeric galacto-oligosaccharides. In some
embodiments, the compositions also have less than 10% or less than
5% of trimeric (DP3) galacto-oligosaccharides. As used herein, a
percentage of a particular DP refers to the amount by weight of the
particular DP relative to the weight of total sugars (including
galactose monomers) in the composition.
[0043] Alternatively, in some embodiments, compositions are
enriched for DP 3-6, i.e., including trimeric,
galacto-oligosaccharides. In some embodiments, at least 30%, 40%,
50%, 60%, 70%, 80%, or 90% of the sugars in the composition are
galacto-oligosaccharides having a DP of 3-6. Such embodiments will
optionally have less than 10% or less than 5% of monomeric sugars
(e.g., galactose) and optionally less than 10% or less than 5% of
dimeric galacto-oligosaccharides.
[0044] Any of the compositions of the invention, including but not
limited to infant or follow-on formula, can include supplements of
lactose as well as other sugars or vitamins as well as other
components, including but not limited to, Bifidobacteria species
and subspecies as described herein.
[0045] Any of the above-described compositions can also be selected
to have low or no galacto-oligosaccharides of DP 6 or above. Thus,
in some embodiments, the compositions have less than 10% or less
than 5% of DP 6+ galacto-oligosaccharides.
[0046] The present invention also provides for compositions
comprising galacto-oligosaccharides wherein
galacto-oligosaccharides having DP 4-5 are enriched (e.g., are at
least 5%, 10%, 15%, 20%, 30%, 40% more than) compared to the amount
by weight of DP 4-5 in a mixed galacto-oligosaccharide solution. "A
mixed galacto-oligosaccharide solution" refers to a mix of
galacto-oligosaccharides having different DPs, e.g., as is produced
using a .beta.-galactosidase in a transgalactosylation reaction
(e.g., as described in Japanese Patent JP105109 or U.S. Pat. No.
4,957,860). Exemplary mixed galacto-oligosaccharide solutions
include, e.g., Vivinal.TM. GOS (available from Friesland Foods
Domo, The Netherlands). In some embodiments, the enriched
compositions of the invention have less than 10% or less than 5% of
sugar monomers (e.g., galactose) and optionally less than 10% or
less than 5% of dimeric galacto-oligosaccharides. In some
embodiments, the enriched compositions of the invention also have
less than 10% or less than 5% of trimeric (DP3)
galacto-oligosaccharides.
ii. Galacto-oligosaccharides that Enrich Bifidobacteria longum
[0047] As noted above and in the Example, galacto-oligosaccharides
of DP 6-8 are consumed by Bifidobacteria typically found in adults,
e.g., Bifidobacteria longum. Accordingly, in some embodiments, the
compositions of the present invention comprise
galacto-oligosaccharides, wherein at least 30%, 40%, 50%, 60%, 70%,
80%, or 90% of the galacto-oligosaccharides by weight are DP 6-8
galacto-oligosaccharides. In some embodiments, the compositions
have less than 10% or less than 5% of monomeric sugars (e.g.,
galactose) and optionally less than 10% or less than 5% of dimeric
galacto-oligosaccharides. In some embodiments, the compositions
also have less than 10% or less than 5% of galacto-oligosaccharides
with a DP of 3, 4, and/or 5. Any of the compositions of the
invention can include supplements of lactose as well as other
sugars or vitamins as other components, including but not limited
to, Bifidobacteria species and subspecies as described herein.
[0048] The present invention also provides for compositions
comprising galacto-oligosaccharides wherein
galacto-oligosaccharides having DP 6-8 are enriched (e.g., are at
least 5%, 10%, 15%, 20%, 30%, 40% more than) compared to the amount
by weight of DP 6-8 in mixed galacto-oligosaccharide solutions,
e.g., such as described above or as in Vivinal.TM. GOS. In some
embodiments, the compositions have less than 10% or less than 5% of
monomeric sugars (e.g., galactose) and optionally less than 10% or
less than 5% of dimeric galacto-oligosaccharides. In some
embodiments, the compositions also have less than 10% or less than
5% of DP 3, 4, 5, and/or 6 galacto-oligosaccharides.
iii. Additional galacto-oligosaccharides that Enrich B. longum bv
infantis and B. adolescentis species
[0049] As noted above and in the Example, galacto-oligosaccharides
of DP 3 are consumed by B. longum bv infantis and B. adolescentis
species. Accordingly, in some embodiments, the compositions of the
present invention comprise galacto-oligosaccharides, wherein at
least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the
galacto-oligosaccharides by weight are DP 3
galacto-oligosaccharides. In some embodiments, the compositions
have less than 10% or less than 5% of sugar monomers (e.g.,
galactose) and optionally less than 10% or less than 5% of dimeric
galacto-oligosaccharides. In some embodiments, the compositions
also have less than 10% or less than 5% of DP 4 or greater
galacto-oligosaccharides. Any of the compositions of the invention
can include supplements of lactose as well as other sugars or
vitamins as other components, including but not limited to,
Bifidobacteria species and subspecies as described herein.
[0050] The present invention also provides for compositions
comprising galacto-oligosaccharides wherein
galacto-oligosaccharides having DP 3 are enriched (e.g., are at
least 5%, 10%, 15%, 20%, 30%, 40% more than) compared to the amount
by weight of DP 3 in mixed galacto-oligosaccharide solutions such
as described above or as in Vivinal.TM. GOS. In some embodiments,
the compositions have less than 10% or less than 5% of monomeric
sugars (e.g., galactose) and optionally less than 10% or less than
5% of dimeric galacto-oligosaccharides.
iv. Methods of Making the galacto-oligosaccharide Compositions of
the Invention
[0051] In some embodiments, galacto-oligosaccharides are produced
as mixtures (known in the art as "GOS") of oligosaccharides having
different degrees of polymerization (i.e., "DP" or the number of
monomeric units in the polymer). For example, in some embodiments,
galacto-oligosaccharides are synthesized enzymatically from
monomeric or dimeric sugars. Galacto-oligosaccharides can be
produced, for example, from lactose syrup using the
transgalactosylase activity of the enzyme .beta.-galactosidase
(Crittenden, (1999) Probiotics: A Critical Review. Tannock, G.(ed)
Horizon Scientific Press, Wymondham, pp. 141-156). Other general
GOS production methods include, e.g., production of
galacto-oligosaccharide by treating lactose with beta-galactosidase
derived from Bacillus circulans (see, e.g., Japanese Patent
JP105109 and production by the reaction between lactose and
beta-galactosidase from Aspergillus oryzae (see, e.g., U.S. Pat.
No. 4,957,860). See also, e.g., Ito et al., Microbial Ecology in
Health and Disease, 3, 285-292 (1990). A related method utilizes
the .beta.-galactosidase of Bifidobacterium bifidum NCIMB 41171 to
synthesize prebiotic galacto-oligosaccharides (see, Tzortzis et
al., Appl. Micro. and Biotech. (2005), 68:412-416). Commercial GOS
products are also available that generally and generally include a
wide spectrum of different-sized galacto-oligosaccharides.
[0052] Thus, to generate the specific purified
galactooligosaccharides of the present invention (e.g., lacking, or
being enriched for, sugars of a particular size), in some
embodiments, the compositions of the present invention can be
generated by obtaining a GOS mixture containing a variety of
different-sized galacto-oligosaccharides and then reducing the
proportion of galacto-oligosaccharides having a DP that is not
desired. For example, in some embodiments, galacto-oligosaccharides
having a DP of 1, 1-2, 1-3, etc. can be reduced, for example, by
size exclusion technology, enzymatic degradation, selective
microbial consumption or a combination thereof. An example of
selective microbial consumption is the use of Kluyveromyces lactis
or other Kluyveromyces species to selectively consume DP2 sugars,
for example.
[0053] Alternatively, or optionally in addition, enzymatic methods
can be used to synthesize the galacto-oligosaccharides of the
present invention. In general, any oligosaccharide biosynthetic
enzyme or catabolic enzyme (with the reaction running in reverse)
that converts a substrate into any of the target DP of the
galacto-oligosaccharide(or their intermediates) may be used in the
practice of this invention. For example, prebiotic
galacto-oligosaccharides have been synthesized from lactose using
the .beta.-galactosidase from L. reuteri (see, Splechtna et al., J.
Agricultural and Food Chemistry (2006), 54: 4999-5006). The
reaction employed is known as transgalactosylation, whereby the
enzyme .beta.-galactosidase hydrolyzes lactose, and, instead of
transferring the galactose unit to the hydroxyl group of water, the
enzyme transfers galactose to another carbohydrate to result in
oligosaccharides with a higher degree of polymerization (Vandamme
and Soetaert, FEMS Microbiol. Rev. (1995), 16:163-186). The
transgalactosylation reaction can proceed intermolecularly or
intramolecularly. Intramolecular or direct galactosyl transfer to
D-glucose yields regioisomers of lactose. Through intermolecular
transgalactosylation di-, tri-, and tetra saccharides and
eventually higher oligosaccharides specific to Bifidobacteria can
produced and subsequently purified as desired.
[0054] Optionally, the galacto-oligosaccharide compositions of the
invention can be made by contacting a first solution comprising
lactose with a lactase (e.g., a transferase type of lactase) to
convert at least part of the lactose into oligosaccharides,
resulting in a second solution of oligosaccharides and lactose,
contacting the second solution with a lactase (e.g., a hydrolytic
type of lactase), and optionally separating monomeric or other
sugars (e.g., lactose, dimeric sugars) from the solution. In some
embodiments, the galacto-oligosaccharide composition will comprise
lactose and the composition is formulated to comprise one or more
lactase (e.g., an encapsulated lactase that is degraded following
ingestion, thereby allowing for relase of the lactase and digestion
of the lactose).
[0055] In some embodiments, the process for the preparation of the
claimed galactose-oligosaccharides compositions can comprise the
following steps:
[0056] 1. Incubation of a lactose containing solution under proper
conditions with a .beta.-galactosidase preparation. The
.beta.-galactosidase preparation can be characterized by containing
(optionally only) enzymes that have high transgalactosidase
activity (transferase type lactases such as provided by the
.beta.-galactosidases derived from Aspergillus oryzae, Bacillus
circulans, Streptococcus thermophilus and Lactobacillus
bulgaricus). The .beta.-galactosidase preparation may also consist
of a mixture of such .beta.-galactosidases. Reaction conditions can
be optimized for the .beta.-galactosidase enzyme preparation. In
some embodiments, the reaction is allowed to proceed until no
significant additional formation of oligosaccharides is
observed.
[0057] 2. Addition of a .beta.-galactosidase preparation that shows
high hydrolytic activity (a hydrolytic type lactase) such as
lactases derived from Kluyveromyces lactis, Kluyveromyces fragilis
or Aspergilus niger. Reaction conditions can be optimized for the
.beta.-galactosidase enzyme preparation. In some embodiments, the
reaction is allowed to proceed until lactose levels are at least
lower than 5% of total sugars.
[0058] The reaction mixture can then optionally be further
processed as desired, including steps like heat-inactivation of the
enzymes, ultra-filtration to remove enzymes and nano-filtration to
reduce mono sugar concentrations. The final preparations may be
stored as a stabilized liquid or alternatively it may be dried.
Methods for stabilization and drying are known to the expert in the
art. In some embodiments, the second step in the process does not
lead to a reduction in concentration of galacto-oligosaccharides
but instead leads to an increase of yield of these components.
[0059] A detailed process for the preparation of improved
oligosaccharide compositions is provided below: An aqueous solution
containing lactose (e.g., 50-400 g/L) is prepared. At this stage,
cofactors like metal ions (e.g. Mg.sup.2+, Mn.sup.2+, Zn.sup.2+,
Na.sup.+, K.sup.+, etc) may be added to improve enzyme stability in
the process. The production method consists of three main steps. In
step 1, most of the galacto-oligosaccharides are produced. In step
2, lactose levels are reduced below 5% of total sugars and
oligosaccharide production is further increased. In step 3,
monomeric sugars are optionally removed from the oligosaccharide
composition and the remaining solution is further processed into a
stabilized liquid; alternatively, it may be dried using methods
known to the expert in the field.
[0060] In step 1 of the process, the solution is treated with a
transferase type .beta.-galactosidase. To this purpose transferase
type acid lactases may be used, and the lactose containing solution
is in this case adjusted preferably to a pH between 2.5 and 5.5,
using hydrochloric acid, acetic acid or any other suitable acid.
Alternatively, buffer solutions such as 50 mM Na-acetate buffer or
any other suitable buffer may be used to set the pH. After pH
adjustment, acid lactase derived from Aspergillus oryzae (Tolerase,
DSM, The Netherlands), is added to an end concentration of
preferably 1,000-10,000 ALU per liter. Other suitable examples
include but are not limited to a .beta.-galactosidase derived from
Bacillus circulans or Lactobacillus reuteri. "ALU" refers to Acid
Lactase Units, which is defined as the amount of enzyme required to
release one micromole of o-nitrophenol from
o-nitrophenyl-.beta.-D-galactopyranoside in one minute under the
defined conditions (pH=4.5, T=37.00 C).
[0061] Instead of Tolerase, any suitable other transferase type
acid lactase may be added, or a combination of suitable transferase
type acid lactases may be used. The reaction mixture can optionally
be heated to any suitable temperature preferably between 30.degree.
C. and 60.degree. C. The optimal temperature depends on the
specific lactase or combination of lactases used. In some
embodiments, the reaction mixture is kept at this optimal
temperature for, e.g., 2-48 hours, but alternatively temperature
gradients may be applied during this period. Optionally, a
transferase type acid lactase may be added to the reaction mixture
during this period to improve formation of oligosaccharides. A
transferase type neutral lactase, like the lactase from Bacillus
circulans, may also be used in the first step of the process
instead of an acid lactase or combination of acid lactases. In that
case, the pH of the concentrated lactose solution is adjusted to
any suitable pH between preferably pH 5.0 and 8.0 using HCl, acetic
acid, or any suitable acid, NaOH, ammonium hydroxide or any
suitable base or buffer, after that the reaction is allowed to
proceed as described for the acid lactases. The use of a
combination of transferase type neutral lactases or the addition of
a neutral lactase during step 1 is optional. After this first step,
the reaction mixture is optionally cooled to any suitable
temperature, and when required the pH is adjusted to the pH that is
most suitable for step 2 of the process.
[0062] In step 2 of the process, a hydrolytic type lactase is used.
For example, a hydrolytic type neutral lactase such as derived from
Kluyveromyces lactis (Maxilact, DSM, The Netherlands) is used at a
concentration preferably between 1,000 and 10,000 NLU per liter.
"NLU" refers to Neutral Lactase Units, which is defined as the
amount of enzyme that will form 1.30 umol ortho-nitro-phenol from
the synthetic substrate ortho-nitro-phenol-galacto-pyranoside under
the test conditions (pH=6.5, T=37.00 C). Other suitable examples
include, but are not limited to, a hydrolytic type neutral lactase
derived from Aspergillus niger or Streptococcus thermophilus.
[0063] In some embodiments, the reaction is allowed to proceed for
2-48 hours, e.g., at temperatures between 10 and 60.degree. C.
Alternatively, temperature gradients may be used during the
incubation. Reaction conditions are optimized for lactose
hydrolysis. The reaction is allowed to proceed until lactose
concentration is below 5% of total sugars. In step 2, combinations
of hydrolytic type neutral lactases may be used. Hydrolytic type
neutral lactases may be added during the incubation of step 2 to
help to reduce lactose levels. A hydrolytic type acid lactase may
also be used in step 2 instead of the hydrolytic type neutral
lactase. In that case the pH of the solution is adjusted to any
suitable pH, including but not limited to, between 2.5 and 5.5,
using hydrochloric acid, acetic acid or any other suitable acid.
Alternatively, buffers like 50 mM Na-acetate buffer or any other
suitable buffer may be used to set the pH. Suitable lactases may be
derived from e.g. Aspergillus niger and may be added to
concentrations of preferably 1,000-10,000 ALU/L and the reaction is
allowed to proceed, e.g., between 2-48 hours at temperatures
between, e.g., 20 and 60.degree. C. Instead of a single hydrolytic
type acid lactase, combinations of hydrolytic type acid lactases
may be used in this step. It is an option to add an additional
lactase during the incubation in this second step. The reaction
conditions are optimized to obtain lactose hydrolysis until final
lactose concentration is below 5% of total sugars and without
significant degrading of formed previously oligosaccharides. At the
end of step 2, the temperature may be raised to inactivate
enzymes.
[0064] In step 3, the solution containing galacto-oligosaccharides
is optionally further processed to remove enzymes and mono sugars.
Enzymes may be removed by ultra filtration;
[0065] suitable filters are well known to the person skilled in the
art. The resulting mono sugars (primarily glucose and galactose)
may subsequently be removed by nanofltration. Suitable filters and
filtration conditions are known to the person skilled in the art,
and have been described in literature as described previously in
this text. The resulting oligosaccharide composition is than
essentially free from enzymes and monomeric sugars and can be
further processed into a stabilized liquid or can be dried using
methods known to the person skilled in the art to obtain e.g. a
powder or granulate products.
[0066] The enzymes used in a method of the invention can be used
either in the free form without restriction of movement in the
reaction mixture or alternatively can be immobilized on a suitable
carrier. Immobilization can be obtained by covalent coupling of the
enzyme to a carrier substrate or by physical entrapment of the
enzyme in e.g. a gel matrix. Methods to immobilize enzymes are
known to the expert in the field; recent reviews have appeared on
this topic (see e.g. Mateo et al 2007, Enz. Micr. Technol. 40,
1451-1463). Enzymes may also be cross-linked to form large
aggregates that can easily be separated from the reaction mature by
filtration (see for review e.g. Margolin et al, 2001, Angew. Chem.
Int. Ed. 40, 2204-2222).
[0067] Alternatively, conventional chemical methods may be used for
the de novo organic synthesis of or conversion of pre-existing
oligosaccharides into the galacto-oligosaccharides having DPs of
the present invention. See, e.g., March's Advanced Organic
Chemistry: Reactions, Mechanisms, and Structure, 5th Edition.
[0068] B. Prebiotic and Probiotic Formulations
[0069] The galacto-oligosaccharides compositions of the present
invention can be administered as a prebiotic formulation (i.e.,
without bacteria) or as a probiotic formulation (i.e., with
desirable bacteria such as bifidobacteria as described herein). In
general, any food or beverage that can be consumed by human infants
or adults or animals may be used to make formulations containing
the prebiotic and probiotic compositions of the present invention.
Exemplary foods include those with a semi-liquid consistency to
allow easy and uniform dispersal of the prebiotic and probiotic
compositions of the invention. However, other consistencies (e.g.,
powders, liquids, etc.) can also be used without limitation.
Accordingly, such food items include, without limitation,
dairy-based products such as cheese, cottage cheese, yogurt, and
ice cream. Processed fruits and vegetables, including those
targeted for infants/toddlers, such as apple sauce or strained peas
and carrots, are also suitable for use in combination with the
galacto-oligosaccharides of the present invention. Both infant
cereals such as rice- or oat-based cereals and adult cereals such
as Musilix are also be suitable for use in combination with the
galacto-oligosaccharides of the present invention. In addition to
foods targeted for human consumption, animal feeds may also be
supplemented with the prebiotic and probiotic compositions of the
invention.
[0070] Alternatively, the prebiotic and probiotic compositions of
the invention may be used to supplement a beverage. Examples of
such beverages include, without limitation, infant formula,
follow-on formula, toddler's beverage, milk, fermented milk, fruit
juice, fruit-based drinks, and sports drinks. Many infant and
toddler formulas are known in the art and are commercially
available, including, for example, Carnation Good Start (Nestle
Nutrition Division; Glendale, Calif.) and Nutrish A/B produced by
Mayfield Dairy Farms (Athens, Tenn.). Other examples of infant or
baby formula include those disclosed in U.S. Pat. No. 5,902,617.
Other beneficial formulations of the compositions of the present
invention include the supplementation of animal milks, such as
cow's milk.
[0071] Alternatively, the prebiotic and probiotic compositions of
the present invention can be formulated into pills or tablets or
encapsulated in capsules, such as gelatin capsules. Tablet forms
can optionally include, for example, one or more of lactose,
sucrose, mannitol, sorbitol, calcium phosphates, corn starch,
potato starch, microcrystalline cellulose, gelatin, colloidal
silicon dioxide, talc, magnesium stearate, stearic acid, and other
excipients, colorants, fillers, binders, diluents, buffering
agents, moistening agents, preservatives, flavoring agents, dyes,
disintegrating agents, and pharmaceutically compatible carriers.
Lozenge or candy forms can comprise the compositions in a flavor,
e.g., sucrose, as well as pastilles comprising the compositions in
an inert base, such as gelatin and glycerin or sucrose and acacia
emulsions, gels, and the like containing, in addition to the active
ingredient, carriers known in the art. The inventive prebiotic or
probiotic formulations may also contain conventional food
supplement fillers and extenders such as, for example, rice
flour.
[0072] In some embodiments, the prebiotic or probiotic composition
will further comprise a non-human protein, non-human lipid,
non-human carbohydrate, or other non-human component. For example,
in some embodiments, the compositions of the invention comprise a
bovine (or other non-human) milk protein, a soy protein, a rice
protein, betalactoglobulin, whey, soybean oil or starch.
[0073] The dosages of the prebiotic and probiotic compositions of
the present invention will be varied depending upon the
requirements of the individual and will take into account factors
such as age (infant versus adult), weight, and reasons for loss of
beneficial gut bacteria (e.g., antibiotic therapy, chemotherapy,
disease, or age). The amount administered to an individual, in the
context of the present invention should be sufficient to establish
colonization of the gut with beneficial bacteria over time. The
size of the dose also will be determined by the existence, nature,
and extent of any adverse side-effects that may accompany the
administration of a prebiotic or probiotic composition of the
present invention. In some embodiments, the dosage range will be
effective as a food supplement and for reestablishing beneficial
bacteria in the intestinal tract. In some embodiments, the dosage
of a galacto-oligosaccharide composition of the present invention
ranges from about 1 micrograms/L to about 25 grams/L of
galacto-oligosaccharides. In some embodiments, the dosage of a
galacto-oligosaccharide composition of the present invention is
about 100 micrograms/L to about 15 grams/L of
galacto-oligosaccharides. In some embodiments, the dosage of a
galacto-oligosaccharide composition of the present invention is 1
gram/L to 10 grams/L of galacto-oligosaccharides. Exemplary
Bifidobacteria dosages include, but are not limited to, 10.sup.4 to
10.sup.12 colony forming units (CFU) per dose. A further
advantageous range is 10.sup.6 to 10.sup.10 CFU.
[0074] The prebiotic or probiotic formulations of the invention can
be administered to any individual in need thereof. In some
embodiments, the individual is an infant or toddler. For example,
in some embodiments, the individual is less than, e.g., 3 months, 6
moths, 9 months, one year, two years or three years old. In some
embodiments, the individual is an adult. For example, in some
embodiments, the individual is over 50, 55, 60, 65, 70, or 75 years
old. In some embodiments, the individual is immuno-deficient (e.g.,
the individual has AIDS or is taking chemotherapy).
[0075] Exemplary Bifidobacteria that can be included in the
pro-biotic compositions of the invention include, but are not
limited to, B. longum bv infantis, B. longum bv longum, B. breve,
and B. adolescentis. The Bifidobacterium used will depend in part
on the target consumer.
[0076] For example, in some embodiments, B. longum bv infantis is
administered with the galacto-oligosaccharide compositions of the
invention to an infant or young child (e.g., under 5 years old). In
some embodiments, B. longum bv infantis is included in, or in
conjunction with, an infant formula or follow-on formula. In some
of these embodiments, the galacto-oligosaccharide compositions of
the invention are enriched for DP 4-5 galacto-oligosaccharides,
optionally having less than 5% by weight of dimeric and trimeric
galacto-oligosaccharides. In some embodiments, the compositions are
administered to an adult or an elderly person. In some embodiments,
the person is at least 50, 60, 70, or 80 years old.
[0077] It will be appreciated that it may be advantageous for some
applications to include other Bifidogenic factors in the
formulations of the present invention. Such additional components
may include, but are not limited to, fructoligosaccharides such as
Raftilose (Rhone-Poulenc, Cranbury, N.J.), inulin (Imperial Holly
Corp., Sugar Land, Tex.), and Nutraflora (Golden Technologies,
Westminister, Colo.), as well as lactose, xylooligosaccharides,
soyoligosaccharides, lactulose/lactitol, among others. In some
applications, other beneficial bacteria, such as Lactobacillus, can
be included in the formulations.
[0078] In some embodiments, the compositions of the invention are
administered to a human or animal in need thereof. For example, in
some embodiments, the compositions of the invention are
administered to a person or animal having at least one condition
selected from the group consisting of inflammatory bowel syndrome,
constipation, diarrhea, colitis, Crohn's disease, colon cancer,
functional bowel disorder (FBD), irritable bowel syndrome (IBS),
excess sulfate reducing bacteria, inflammatory bowel disease (IBD),
and ulcerative colitis. Irritable bowel syndrome (IBS) is
characterized by abdominal pain and discomfort, bloating, and
altered bowel function, constipation and/or diarrhea. There are
three groups of IBS: Constipation predominant IBS (C-IBS),
Alternating IBS (A-IBS) and Diarrhea predominant IBS (D-IBS). The
compositions of the invention are useful, e.g., for repressing or
prolonging the remission periods on Ulcerative patients. The
compositions of the invention can be administered to treat or
prevent any form of Functional Bowel Disorder (FBD), and in
particular Irritable Bowel Syndrome (IBS), such as Constipation
predominant IBS (C-IBS), Alternating IBS (A-IBS) and Diarrhea
predominant IBS (D-IBS); functional constipation and functional
diarrhea. FBD is a general term for a range of gastrointestinal
disorders which are chronic or semi-chronic and which are
associated with bowel pain, disturbed bowel function and social
disruption.
[0079] In another embodiment of the invention, the compositions of
the invention are administered to those in need stimulation of the
immune system and/or for promotion of resistance to bacterial or
yeast infections, e.g., Candidiasis or diseases induced by sulfate
reducing bacteria.
EXAMPLES
[0080] The following examples are offered to illustrate, but not to
limit the claimed invention. Those of skill in the art will readily
recognize a variety of noncritical parameters that could be changed
or modified to yield essentially similar results.
Example 1
[0081] We have previously developed analytical methods employing
high mass accuracy and high resolution Fourier Transform Ion
Cyclotron (FTICR) mass spectrometry to characterize bacterial
consumption of human milk oligosaccharides (HMOs) and
fructo-oligosaccharides (FOS) (Ninonuevo, M. R. et al., Anal
Biochem, 361:15-23 (2007); LoCascio, R. G. et al., J Agric Food
Chem, 55:8914-9 (2007); Seipert, R. R. et al., Anal Chem, 80:159-65
(2008)). MALDI-FTICR was shown to be a sensitive and robust
analytical method with high-performance capabilities, allowing
rapid and unambiguous assignments of oligosaccharide signals.
[0082] In the present study, the oligosaccharide composition in GOS
syrup preparations was investigated by MALDI-FTICR. Moreover,
disaccharide- and monosaccharide-free fractions of GOS (termed
pGOS) were prepared by size-exclusion chromatography and used in
bacterial fermentation experiments. Four major bifidobacterial
species, Bifidobacterium adolescentis, B. breve, B. longum subsp.
Infantis, and B. longum subsp. longum, present in infants and adult
intestinal microbiota were assayed and pGOS consumption profiles
were obtained by MALDI-FTICR mass spectrometry.
Material and Methods
[0083] Bacterial strains. Bifidobacterium adolescentis ATCC 15703,
B. breve ATCC 15700 and B. longum subsp. infantis ATCC 15697 were
obtained from the American type Culture Collection (Manassas, Va.).
B. longum subsp. longum DJO10A was a gift from D. O'Sullivan,
University of Minnesota.
[0084] Galacto-oligosaccharides purification.
Galacto-oligosaccharides purification. The purified GOS mixture
(termed pGOS) was obtained by purification from Vivinal.TM. GOS
(Dorno Friesland Food, location?). Sugars with degree of
polymerization (DP) less than 2 (including lactose, glucose and
galactose) were removed by Bio-Gel P-2 gel size-exclusion
chromatography (110.times.2.6 cm with a 200/400 mesh, Bio-Rad) at
room temperature using water as the eluent and a flow rate was 0.16
ml/min. One mL fractions were collected and analyzed by MALDI-FTICR
MS. Fractions containing oligosaccharides with a DP>=3 were
pooled for bacterial fermentation experiments. Thin layer
chromatography was performed to confirm lactose-free pGOS obtained
in a solvent mixture of acetonitrile/water (8:2 v/v). The plate was
developed twice at room temperature, dried and visualized using
0.3% (w/v) N-(1-naphthyl)-ethylenediamine and 5% (v/v)
H.sub.2SO.sub.4 in methanol, followed by heating at 110.degree. C.
for 10 min (Lee H Y, M. J. et al., Journal of Molecular Catalysis
B: Enzymatic, 26:293-305 (2003)).
[0085] Bacterial fermentations. Bifidobacteria cultures were
initially propagated on a semi-synthetic MRS medium supplemented
with 1% L-cysteine and 1.5% (w/v) lactose as a carbon source.
Cultures were then inoculated at 1% into a modified MRS medium
supplemented with 1% L-cysteine, containing 0.5, 1, 1.5 or 2% (w/v)
of pGOS as a sole carbon source. Growth studies were carried out in
a 96 well-plate (clear, non-treated, polysterene 96 well-plate from
Nunc), containing 100 .mu.l of media/well and each well was covered
with 40 .mu.l of mineral oil. Incubations were carried out at
37.degree. C. and cell growth was measured by assessing optical
density (OD) at 600 nm with an automated PowerWave microplate
spectrophotometer (BioTek Instruments, Inc.), placed inside of an
anaerobic chamber (Coy Laboratory Products, Grass Lake, Mich.).
Each fermentation experiment was performed in triplicates, and
controls consisted of inoculated medium lacking pGOS and
un-inoculated medium containing pGOS.
[0086] pGOS purification after fermentation. After cell growth, the
residual pGOS was recovered and purified from supernatant cultures.
Samples (100 .mu.L) were collected 72 hours post-inoculation,
centrifuged at 4000.times.g for 10 min. The resulting supernatant,
were transferred into new tubes, heated at 95.degree. C. for 5 min,
sterile-filtered with Millex-GV (0.22 .mu.m, Millipore, Mass.), and
stored at -80.degree. C. Oligosaccharides were then purified from
the supernatant using microcolumns containing 100 .mu.L Dowex 50
W.times.8 H.sup.+ form (Supelco, Bellefonte, Pa.) (bottom) and 100
.mu.L of C18 resins (taken from disposable C18 cartridge (Waters,
Milford, Mass.) (top). Resins were packed into empty columns
(MicroBio-Spin columns, Bio-Rad, Hercules, Calif.) with nano-pure
water. Supernatants samples were applied and pGOS was eluted with
0.3 mL water, dried down in vacuum and stored at -80.degree. C.
Samples were then reconstituted in deionized water to initial
concentration before MS analyses.
[0087] MALDI-FTICR MS analysis. All mass analyses were carried out
with a ProMALDI-FT-ICR MS instrument with an external MALDI source,
a 355 nm pulsed Nd:YAG laser, a hexapole accumulation cell, a
quadrupole ion guide, and a 7.0-T superconducting magnet
(Varian/IonSpec, Lake Forest, Calif.). Tandem MS was performed by
IRMPD and a CO2 laser (10.6 im, 20-W maximum power, Parallax,
Waltham, Mass.) was added to the instrument in order to provide IR
photons for these experiments. DHB (0.4 M in acetonitrile:water
(50% v/v)) and 0.10 mM NaCl, were used as matrix and dopant,
respectively; samples were spotted onto a 100-well stainless steel
sample plate (Applied Biosystems, Foster City, Calif.), according
to the "thin layer" method. Samples were analyzed in the positive
ion mode, with external accumulation of ions in the hexapole; ions
were then transferred to the ICR cell via the ion guide for
excitation and detection. In tandem, IRMPD experiments select
precursor ions were isolated in the ICR cell and irradiated with
photons for 500 ms.
Results
[0088] MALDI-FTICR analysis of GOS syrup. To determine the degree
of polymerization (DP) of galacto-oligosaccharides in GOS syrup
preparations, samples were diluted and analyzed by MALDI-FTICR mass
spectrometry. Both glucose and galactose, monomer components of
GOS, have an exact residue mass of 162.0528 Da. Exact mass
measurement was used to identify the DP of GOS, and the
quasimolecular ions were assigned with less than 5 ppm difference
between theoretical and calculated mass. Positive ion mode
MALDI-FTICR spectrum obtained showed that GOS syrup contains
oligosaccharides with DPs ranging from 2 to 11 (FIG. 1). In
addition, when GOS syrup preparations where fractionated in a size
exclusion chromatography column, MALDI-FTICR analysis of Bio-Gel
P-2 excluded fractions showed that GOS mixtures contain oligomers
with a DP up to 15 (FIG. 2a). Tandem mass spectrometry is usually
required to verify composition and elucidate structures; thus,
select oligosaccharide ions were interrogated using infrared
multiphoton dissociation (IRMPD) tandem MS method. The IRMP mass
spectra of GOS with DP 5, 4 and 3 are shown in FIG. 3 (A, B and C).
Fragment ions with shifted masses of 162 toward lower masses were
observed, corresponding to glycosidic-bonds cleavages and loss of
galactose residues. IRMPD tandem MS analysis also yield fragment
ions shifted in 60, 90 and 120 mass units from the parental ion
corresponding to cross-ring cleavages fragments.
[0089] GOS purification. To better understand the GOS bifidogenic
effect, GOS syrup was fractionated and purified from
monosaccharides (glucose and galactose) and disaccharides
(including lactose and GOS with DP 2) by size-exclusion
chromatography. Fractions were collected and analyzed by
MALDI-FTICR, displaying DP of oligomers eluted in each fraction
(FIGS. 2a-e). Di- and mono- saccharide-free fractions were
confirmed by TLC (data not shown) and pooled according to the
desired DP. MALDI-FTICR mass spectrum of purified GOS (pGOS)
preparations obtained indicated that the DP ranging from 3 to 8
(FIG. 4).
[0090] Rapid-throughput screen of pGOS bifidogenic effect:
microscale fermentations coupled to MALDI-FTICR MS analysis. The
concept that prebiotics can selectively modulate gastrointestinal
microbiota fermentation to influence physiological processes, which
are known biomarkers of potential illness and health, has been an
important development in nutritional research and food product
innovation. However, the lack of analytical methods available to
perform comparative analysis of bacterial prebiotics consumption
has limited this field. Thus, a fast-throughput method to screen
and compare the prebiotic effect of pGOS was developed, coupling
bifidobacterial microscale fermentations and pGOS consumption
profiling using MALDI-FTICR MS.
[0091] pGOS microscale fermentations. Microscale fermentations were
performed anaerobically in a 96 well-plate format. The ability to
grow on pGOS preparations as the sole carbon source was tested at
varying substrate concentrations: 0.5%, 1%, 1.5% and 2%. Four
Bifidobacterium phylotypes were used in the present work:
Bifidobacterium breve and B. longum subsp. infantis, both common
infant-associated microbiota, and B. adolescentis and B. longum
subsp. Longum, which are typically referred to as "adult-type"
bifidobacteria (Mitsuoka, T., Bifid Micro, 3:11-28 (1984); Ventura,
M. et al., FEMS Microbiol Ecol, 36:113-121 (2001)).
[0092] Growth curves obtained (FIGS. 4A-D) showed that all
bifidobacteria assayed were able to utilize and grow on pGOS at the
four concentrations tested further confirming GOS bifidogenic
properties. Interestingly, a differential pGOS growth phenotype was
observed among the various assayed bifidobacteria. pGOS strongly
stimulated the growth of B. longum subsp. infantis, reaching the
highest cell density at all four pGOS concentrations tested
(OD.sub.600nm 1.2). On the other end, pGOS showed a moderate effect
on B. longum subsp. longum cultures, producing the lowest endpoint
biomass while growing on 0.5% pGOS (max. OD.sub.600nm 0.4), with a
slight increase in cell mass observed at higher pGOS concentrations
(max. OD.sub.600nm 0.5-0.7). An intermediate growth profile was
displayed by B. adolescentis and B. breve with a maximum density
occurring at OD.sub.600nm.about.0.7 at all pGOS concentrations.
[0093] pGOS consumption determined by MALDI-FTICR MS. With the aim
to further understand the prebiotic effect of pGOS, a methodology
to determine consumption profiles after bifidobacterial
fermentation was developed. pGOS remaining in culture supernatants
were recovered 72 hours post-inoculation, purified, and analyzed
using MALDI-FTICR MS. Positive MALDI-FTICR MS ion spectra of
remaining pGOS purified from supernatants of bifidobacterial
culture containing 0.5% pGOS are shown in FIGS. 5A-D. A comparative
analysis of the mass spectra obtained clearly show a differential
fermentative capacity among the bifidobacteria assayed, signaling
substrate preferences in the utilization of pGOS.
[0094] B. breve and B. longum subsp. infantis showed to be the most
efficient in pGOS consumption (FIGS. 5b and c). Although slightly
different, signals with m/z values 689, 851, 1013, 1175, and 1337
were strongly reduced in both samples, indicating pGOS consumption
with DP range from 4 to 8. Remarkably, signal with m/z value 689,
corresponding to tetra-saccharides, were almost absent following
fermentation by B. longum subsp. infantis, demonstrating the
preferential consumption of pGOS with DP 4. Unlike B. breve, B.
longum subsp. infantis also showed an important signal reduction
corresponding to oligosaccharides with DP 3. Similarly, B.
adolescentis showed a significant decrease in signal with m/z value
527, indicating consumption of GOS with DP 3. Although signals
corresponding to longer oligosaccharides were not greatly altered,
some consumption of oligosaccharides with DP 4 and 5 were evident
(FIG. 5a).
[0095] Contrastingly, B. longum subsp. longum did not consume GOS
with DP 4 and 5 either, but showed a complete reduction of GOS
masses corresponding to DP 6, 7, and 8. Unlike the other strains
tested, signals corresponding to trisaccharides were not altered,
indicating that pGOS with DP 3 were not consumed by B. longum
subsp. longum.
[0096] Genomics of bifidobacterial GOS utilization. The
availability of complete genome sequences have enabled various
metabolic reconstruction approaches to understand and often predict
phenotypes of fermentative bacteria (Schell, M. A. et al., Proc
Natl Acad Sci USA, 99:14422-7 (2002); Azcarate-Peril, M. et al.,
Appl Environ Microbiol, 74:4610-25 (2008); Sela, D. A. et al., The
Complete Genome Sequence of Bifidobacterium longum subsp. infantis
Reveals Adaptations for Milk Utilization within the Infant
Microbiome (Submitted, 2008)).
[0097] Bifidobacteria have adapted to the utilization of a diverse
range of host-indigestible oligosaccharides encountered in the
lower bowel. Accordingly, GOS oligomers are degraded to galactose
and glucose by bifidobacterial enzymes to generate energy and
substrates for anabolic reactions. The requisite catabolic reaction
in GOS utilization is .beta.-galactosidase activity (EC 3.2.1.23)
exerted on terminal .beta.-galactosyl linkages which are found in
industrially produced or naturally occurring GOS. In general,
bifidobacterial .beta.-galactosidases are classified into glycosyl
hydrolase (GH) family 42 and GH family 2, along with a few
exceptions. In addition, several .beta.-galactosidases are fused to
other glycosidic domains.
[0098] Accordingly, the genome sequence of B. adolescentis ATCC
15703, B. longum subsp. infantis ATCC15697 and B. longum subsp.
longum NCC2705 contains 10, 7, and 3 sequences, respectively, that
have been assigned a .beta.-galactosidase functionality (FIG. 6,
Table 1). All 20 enzymes are predicted to be intracellular or are
secreted by unknown or non-classical pathways as they lack
transmembrane helices or signal peptides. Conversely, one B.
bifidum .beta.-galactosidase isozyme, termed BIF3, possesses a
signal peptide and is likely secreted to the extracellular surface,
where it is believed to be active in GOS utilization (5). While an
exact homolog of BIF3 is not evident in the ATCC15703, NCC2705 or
ATCC15697 genomes, a B. longum subsp. infantis .beta.-galactosidase
(Blon.sub.--2334; GH 2) with 25% identity is located in a gene
cluster dedicated to human milk oligosaccharide (HMO) utilization.
Homologs of Blon.sub.--2334 are present in two copies: B.
adolescentis ATCC15703 (BAD.sub.--1605 and BAD.sub.--1582) and B.
longum subsp. longum NCC2705 (BL.sub.--0978) whose genomes do not
contain the same complement HMO-related genes found in B. longum
subsp. infantis. Interestingly, these .beta.-galactosidases have
been previously isolated and characterized from B. infantis HL96
(termed .beta.-gall) as possessing high transgalactosylation
activity (3). The presence of this large .beta.-galactosidase (1023
a.a.) in the B. longum subsp. infantis HMO cluster, as well as high
in vitro transgalactosylation activity, offers a link to
oligosaccharide metabolism which may enable bifidobacteria to
cleave terminal galactosyl residues from GOS and HMO.
[0099] In addition to .beta.-galactosidases, an endogalactanase (EC
3.2.1.89) from B. longum subsp. longum NCC2705 (BL.sub.--0257;
GH53) was experimentally determined to release
galactotrisaccharides from hydrolysis of .beta.1-4 and .beta.1-3
linkages in GOS. This extracellular enzyme likely acts
progressively on GOS molecules with trimeric products imported
across the cell membrane. B. longum subsp. longum preference for
GOS with DP.gtoreq.6 suggests that this endogalactanase is coupled
to intracellular transport. The in vitro specificity of
purified
TABLE-US-00001 TABLE I Beta-Galactosidases of the Sequenced
Bifidobacteria protein TM Locus length (aa) signalP helices COG
PFAM GH notes B. longum subsp. infantis ATCC15697 Blon_2334 1023 no
no COG3250 02837, 00703, 00703, 02929 2 unique region, but gene is
similar to adol and longum Blon_1905 423 no no COG2723 00232 1
potential beta-glucosidase Blon_0268 606 no no COG3250 00703, 02836
2 unique to infantis Blon_0346 674 no no COG1874 08532 42 unique to
infantis, posseses trimerization domain Blon_2016 691 no no COG1874
02449, 01373, 08532, 08533 42/35 experimental evidence .beta.(1-4)
(Hinz, et. al, 2004) Blon_2416 706 no no COG1874 02449,08532, 42/14
Blon_2123 720 no no COG1874 02449, 01373, 08532, 42/5 experimental
evidence .beta.(1-4) (Hinz, et. al, 2004) B. longum subsp. longum
NCC2705 BL_0259 710 no no COG1874 02449, 01373, 08532, 08533 42
bgaB BL_0978 1023 no no COG3250 02837, 00703, 02836, 02929 2 lacZ
BL_1168 691 no no COG1874 02449, 01373, 08532, 08533 42/14 bga B.
longum adolescentis ATCC15703 BAD_1605 1023 no no COG3250 02837,
00703, 02836, 02929 2 lacZ BAD_1582 1049 no no COG3250 02837,
00703, 02836, 02929 2 lacZ BAD_1534 788 no no COG3250 02837, 00703,
02836, 2 lacZ BAD_0435 328 no no COG1874 02449, 08532, 08533 42
BAD_1287 391 no no COG2723 00232 1 potential beta-glucosidase
BAD_0156 423 no no COG2723 00232, 02449 1/42 potential
beta-glucosidase BAD_1211 688 no no COG1874 02449, 08532 42
BAD_1603 692 no no COG1874 02449, 01373, 08532, 08533 42/14
BAD_1401 711 no no COG1874 02449, 01373, 08532, 42/14 BAD_1402 751
no no COG1874 01301 35
BL.sub.--0257 towards DP.gtoreq.5 GOS is somewhat consistent with
this coupling. The existence of a transporter possessing affinity
for galactotrisaccharides to the exclusion of trimeric GOS is
strongly supported by the DP3 GOS fraction remaining unaltered
following fermentation by B. longum subsp. longum. Accordingly, the
endogalactanase appears in a gene cluster with a potential
oligosaccharide transporter (BL.sub.--0260-BL.sub.--0264), as well
as a .beta.-galactosidase (BL.sub.--0259) and a lad family
regulatory protein (BL.sub.--0257) (FIG. 7). The expression of this
.beta.-galactosidase and components of the ABC transporter has been
recently demonstrated to be upregulated while growing on GOS
(Gonzalez, R. et al., Appl Environ Microbiol, 74:4686-94 (2008)).
This specific response to GOS provides further evidence that this
locus is a primary contributor to GOS metabolism in B. longum
subsp. longum. A homolog of this endogalactanase is absent from the
B. adolescentis genome although the putative GOS operon remains
intact along with a duplication of the cluster's
.beta.-galactosidase (BAD.sub.--01566 and BAD.sub.--01567) (FIG.
7). Interestingly, B. longum subsp. infantis possesses a truncated
endogalactanase gene (Blon.sub.--0440), which lacks the majority of
its catalytic domain and is located next to a degraded
.beta.-galactosidase remnant with a complete absence of a proximal
sugar transporter (FIG. 7). It appears that these genes became
expendable subsequent to the evolutionary divergence of subsp.
infantis and longum. This is consistent with the general remodeling
of the subsp. infantis catabolic potential towards host-derived
glycans at the expense of plant sugars such as type I
arabinogalactans on which this cluster is active on.
[0100] Clearly, the genetics underlying bifidobacterial GOS
utilization is diverse and is reflected in their varied consumption
glycoprofiles. It is currently unclear if these differential
phenotypes are attributable to specific isozymes, unexpected
disparity in enzyme localization, variation in signal transduction
and regulatory circuits, or other physiological parameters.
Likewise, it is possible that specific transporters may facilitate
efficient GOS utilization as the ATCC15697 genome encodes twice as
many copies of family 1 solute binding proteins (potentially
oligosaccharide binding) as the other two fully sequenced
bifidobacteria.
Discussion
[0101] The MALDI-FTICR analysis of GOS clearly demonstrated that
oligosaccharides longer than previously described (DP>8) are
present in the examined GOS mixtures. These GOS with higher DP did
not agree with the manufacturer's claim and is likely due to the
superior sensitivity of FT-ICR mass spectrometry over HPLC and NMR
techniques previously used for GOS analysis (Dumortier, V. et al.,
Carbohydr Res, 201:115-23 (1990); Kimura, K. et al., Carbohydr Res,
270:33-42 (1995); Van Laere, K. M. et al., Appl Environ Microbiol,
66:1379-84 (2000)). In general, the efficacy of prebiotics toward
promoting human health has been strongly related to their chemical
structure (Casci, T. et al., In Functional food and Biotechnology,
pp. 401-434, Ed Taylor and Francis (2007)). It is known that GOS
structures are highly variable and dependent on the enzyme and
conditions used during their synthesis process; thus
oligosaccharides with the same DP can contain up to eight isomeric
structures (Dumortier, V. et al., Carbohydr Res, 201:115-23 (1990);
Kimura, K. et al., Carbohydr Res, 270:33-42 (1995); Yanahira, S. et
al., Biosci Biotechnol Biochem, 59:1021-6 (1995)). (TANDEM) Select
oligosaccharide ions were interrogated using infrared multiphoton
dissociation (IRMPD) tandem MS method.
[0102] All together, these variations observed in bacterial growth
reflect that pGOS selectively stimulates the development of
specific bifidobacterial phylotypes in a differential manner.
Collectively, MALDI-FTICR mass spectrometry analysis of remaining
sugars after fermentation experiments accurately demonstrated
species-specific bifidobacterial preferences on pGOS utilization
with certain DP. Two predominant species encountered in the infant
GIT, B. breve and B. longum subsp. infantis, were more effective in
utilizing a diverse range of pGOS masses hinting at a potential
adaptive advantage within the infant intestinal environment, where
human milk has provided GOS over evolutionary time.
[0103] Previous studies on carbohydrate utilization by
bifidobacteria have found that individual strains possess specific
substrate preferences towards monosaccharide mixtures containing
glucose, mannose, galactose, arabinose, and xylose (Macfarlane, G.
T. et al., Journal of Applied Microbiology, 104:305-44 (2008)). In
addition, preferences for different prebiotic substrates, including
galacto-oligosaccharides, have been largely described in
comparative growth and/or fecal enrichment approaches (Sako, T. et
al., Int Dairy J, 9:69-80 (1999); Rabiu, B. A. et al., Appl Environ
Microbiol, 67:2526-30 (2001); Perez-Conesa, D. et al., Journal of
Food Science, 70:6, M279-85 (2005); Perez-Conesa, D. et al.,
Journal of Food Science, 71:1, M7-11 (2006); Vernazza, C. L. et
al., J Appl Microbiol, 100:846-53 (2006); Depeint, F. et al., Am J
Clin Nutr, 87:785-91 (2008)). So far, GOS consumption with specific
DP has only been determined in B. adolescentis cultures using
HPAEC-PAD (Van Laere, K. M. et al., Appl Environ Microbiol,
66:1379-84 (2000)). However, the relative concentration of
oligomers could not be accurately determined due to the significant
variation of the response factor of the detector (PAD) toward
oligosaccharides with higher DP.
Conclusions
[0104] This work demonstrates, for the first time, the genuine
bifidogenic effect of purified galacto-oligosaccharides with DP
from 3 to 8, in pure in vitro cultures of the major bifidobacterial
species present in the infant and adult GIT. Our results
demonstrate that pGOS selectively stimulates the different
bifidobacterial phylotypes.
[0105] In addition, a high-throughput analytical method was
developed to compare pGOS consumption after Bifidobacteria
fermentation. Selectivity was also demonstrated, highlighting pGOS'
potential for the rational design and development of functional
food, which can target the enrichment of select bifidobacterial
phylotypes.
[0106] Our results show that MALDI-FTICR is a useful tool for
comprehensive profiling of oligosaccharide species within GOS
mixtures and enhances the speed to rapidly investigate the
prebiotic effect of GOS, can be easily applied to other
oligosaccharides, non-digestible carbohydrates or any other
polymeric system.
[0107] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *