U.S. patent application number 11/569239 was filed with the patent office on 2010-03-18 for synergism of gos and polyfructose.
This patent application is currently assigned to N.V. Nutricia. Invention is credited to Gelske SPEELMANS.
Application Number | 20100069320 11/569239 |
Document ID | / |
Family ID | 34928228 |
Filed Date | 2010-03-18 |
United States Patent
Application |
20100069320 |
Kind Code |
A1 |
SPEELMANS; Gelske |
March 18, 2010 |
SYNERGISM OF GOS AND POLYFRUCTOSE
Abstract
The present invention relates to the filed of prebiotics.
Provided are uses for compositions comprising synergistically
effective amounts of polyfructose and galactooligosaccharides
(GOS).
Inventors: |
SPEELMANS; Gelske;
(Wageningen, NL) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
N.V. Nutricia
|
Family ID: |
34928228 |
Appl. No.: |
11/569239 |
Filed: |
May 17, 2005 |
PCT Filed: |
May 17, 2005 |
PCT NO: |
PCT/NL2005/000372 |
371 Date: |
January 16, 2008 |
Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A23L 5/00 20160801; A61P
1/10 20180101; A61P 17/00 20180101; A23L 33/40 20160801; A23V
2002/00 20130101; A23L 29/244 20160801; A61P 1/06 20180101; A23L
29/30 20160801; A61P 1/14 20180101; A61K 31/01 20130101; A61P 21/02
20180101; A61K 31/702 20130101; A23L 33/22 20160801; A61P 37/08
20180101; A23L 33/125 20160801; A61P 1/00 20180101; A61P 3/02
20180101; A23L 33/10 20160801; A23L 7/00 20160801; A61P 29/00
20180101; A61K 31/733 20130101; A61P 1/04 20180101; A23V 2200/318
20130101; A61K 31/715 20130101; A61K 31/01 20130101; A61K 2300/00
20130101; A61K 31/702 20130101; A61K 2300/00 20130101; A61K 31/715
20130101; A61K 2300/00 20130101; A61K 31/733 20130101; A61K 2300/00
20130101; A23V 2002/00 20130101; A23V 2250/28 20130101; A23V
2250/5046 20130101; A23V 2200/3202 20130101; A23V 2002/00 20130101;
A23V 2250/28 20130101; A23V 2250/5062 20130101; A23V 2200/3202
20130101; A23V 2002/00 20130101; A23V 2200/304 20130101; A23V
2250/5062 20130101; A23V 2250/28 20130101; A23V 2002/00 20130101;
A23V 2200/318 20130101; A23V 2250/5062 20130101; A23V 2250/28
20130101 |
Class at
Publication: |
514/54 |
International
Class: |
A61K 31/715 20060101
A61K031/715; A61P 1/00 20060101 A61P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 17, 2004 |
EP |
04076479.7 |
Claims
1-15. (canceled)
16. A method of (a) increasing intestinal and/or faecal short chain
fatty acid levels, (b) increasing intestinal and/or faecal acetate
levels relative to total short chain fatty acids, (c) increasing
intestinal lactate levels, (d) decreasing intestinal and/or faecal
pH, (e) decreasing total intestinal and/or faecal butyrate,
isobutyrate, valerate and/or isovalerate relative to total short
chain fatty acids, (f) decreasing intestinal gas production, and/or
(g) promoting the production of short chain fatty acids at the
beginning, middle or end of the colon, without having a significant
effect on the total and/or relative number of intestinal
bifidobacteria, the method comprising administering to a mammal a
composition comprising galacto-oligosaccharides and
polyfructose.
17. The method according to claim 16, wherein the ratio of
galacto-oligosaccharide:polyfructose is 3:97 to 97:3.
18. The method according to claim 16, wherein the ratio of
galacto-oligosaccharide:polyfructose is 5:95 to 95:5.
19. The method according to claim 16, wherein the ratio of
galacto-oligosaccharide:polyfructose is 90:10 to 45:55.
20. The method according to claim 16, wherein the
galacto-oligosaccharide is transgalacto-oligosaccharide.
21. The method according to claim 16, wherein the polyfructose is
inulin.
22. The method according to claim 21, wherein the inulin has an
average degree of polymerisation of 20 or above.
23. The method according to claim 16, wherein the
galacto-oligosaccharide is transgalacto-oligosaccharide and the
polyfructose is inulin.
24. The method according to claim 16, wherein the faecal pH,
intestinal pH, or both is decreased to below pH 6.
25. The method according to claim 24, wherein the faecal pH,
intestinal pH, or both is decreased to below pH 5.75.
26. The method according to claim 16, wherein the concentration of
intestinal lactate, faecal lactate, or both is increased to above
about 10 mmol/kg faeces.
27. The method according to claim 16, wherein the concentration of
intestinal acetate, faecal acetate, or both is increased to above
about 85% of the total SCFA.
28. The method according to claim 16, wherein the sum of intestinal
and/or faecal butyrate, isobutyrate, valerate and isovalerate is
decreased to below 7% of total SCFA.
29. The method according to claim 16, wherein the composition is
suitable for oral administration to an infant.
30. A method of relaxing contractions of the colon comprising
administering to a mammal a composition comprising
galacto-oligosaccharides and polyfructose.
31. A method of increasing the intestinal barrier functioning
and/or mucus production of the large intestine comprising
administering to a mammal a composition comprising
galacto-oligosaccharides and polyfructose.
32. The method of preventing or treating a condition selected from
abdominal bloating, gas formation, abdominal pain, flatulence,
allergy, eczema, atopic diseases, irritable bowel syndrome and
inflammatory bowel disease, the method comprising administering to
a mammal a composition comprising galacto-oligosaccharides and
polyfructose.
33. The method according to claim 32, wherein the ratio of
galacto-oligosaccharide:polyfructose is 3:97 to 97:3.
34. The method according to claim 32, wherein the ratio of
galacto-oligosaccharide:polyfructose is 5:95 to 95:5.
35. The method according to claim 32, wherein the ratio of
galacto-oligosaccharide:polyfructose is 90:10 to 45:55.
36. The method according to claim 32, wherein the
galacto-oligosaccharide is transgalacto-oligosaccharide.
37. The method according to claim 32, wherein the polyfructose is
inulin.
38. The method according to claim 32, wherein the
galacto-oligosaccharide is transgalacto-oligosaccharide and the
polyfructose is inulin.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of human health
and nutrition. It provides novel synergistic mixtures of prebiotic
carbohydrates, especially mixtures of galacto-oligosaccharides
(GOS, for example TOS) and polyfructose (for example inulin), as
well as nutritional compositions comprising these. The nutritional
compositions have beneficial effects when fed to bottle fed or
partially bottle fed infants and also have health improving effects
when ingested by adults having intestinal problems, such as
Inflammatory Bowel Disease (IBD) or Irritable Bowel Syndrome
(IBS).
BACKGROUND OF THE INVENTION
[0002] The microflora of the human large intestine (typically
divided into caecum, colon and rectum) plays a crucial role in both
human nutrition and health. The bacterial composition is influenced
and can be modulated by dietary intake. Carbohydrates which have
passed through the stomach and small intestine are metabolised by
the bacteria and as a major end-product of metabolism short-chain
fatty acids (SCFA), such as acetate, propionate, butyrate and
valerate, are formed, which are subsequently released into the
blood. Other end products of bacterial fermentation include for
example lactate and succinate. The total amounts and compositions
(relative amounts) of these end products in turn have a profound
effect on bacterial growth, pH, exclusion of pathogenic species,
etc. A method to beneficially influence the microbial flora and
human health is the administration of prebiotics. "Prebiotics" were
defined as "non-digestible food ingredients that beneficially
affect the host by selectively stimulating the growth and/or
activity of one or a limited number of bacteria in the colon, and
thus improve the hosts health (Gibson and Roberfroid 1995, J. Nutr.
125, 1401-1412). The criteria which a compound must fulfil in order
to be classified as a prebiotic are: 1) it must not be hydrolysed
or absorbed in the upper part of the gastrointestinal tract
(stomach, small intestine), 2) it must be selectively fermented by
one or more potentially beneficial bacteria in the colon, 3) it
must alter the colonic microbiota towards a healthier composition
and 4) it must preferably induce effects which are beneficial to
the health of the subject. Commonly used prebiotics are so-called
non-digestible carbohydrates (or "soluble dietary fibres"), which
pass undigested through the upper part of the gastrointestinal
tract into the large intestine. These include for example
fructooligosaccharides (FOS), oligofructose, inulin and
transgalacto-oligosaccharides (TOS). It should be noted that in the
literature, there is often inconsistency between the use of various
terms, such as fructooligosaccharide, inulin, oligofructose and
inulo-oligosaccharides, and the same term may be used for different
compounds or compositions.
[0003] Well known beneficial bacteria, which are stimulated by the
uptake of prebiotics, are lactic acid bacteria, such as
Lactobacilli and Bifidobacteria, and health benefits have been
ascribed to be due to this stimulatory effect. For example,
beneficial effects of inulin and inulin-type fructans, such as
oligofructose, on intestinal function have been described (Jenkins
et al. 1999, J. Nutrition 129, 1431S-1433S) and this effect is
thought to be due to a bifidogenic effect, which refers to a
selective growth stimulating effect on total bifidobacteria,
measured either in vivo through bifidobacterial counts of the
faeces or in vitro (see e.g. Roberfroid, Am J Clin Nutr 2001,
73(suppl), 406S-409S).
[0004] Human milk appears also to have a bifidogenic effect, as the
dominant bacteria which become established in breast-fed infants
are bifidobacteria. In contrast, bacterial colonisation of milk
formula fed infants is not dominated by bifidobacteria and is more
diverse in bacterial species (Harmsen et al. 2000, J. Pediatr.
Gastroenterol. Nutr. 30, 61-67). It is thought that
oligosaccharides found in the human milk are responsible for the
bifidogenic or prebiotic effect and efforts have been made to
modify infant formula in such a way that it resembles human milk as
closely as possible and especially that it has the same or a very
similar prebiotic effect as human milk. This has been done by
adding prebiotics to infant milk formula (Boehm et al. Acta
Paediatr. Suppl. 2003, 441, 64-67 and Moro et al. 2002, J Pediatr
Gastroenterol Nutr 34, 291-295). For example, supplementation of
bovine milk formula with an oligosaccharide mixture comprising
trans-galactooligosaccharides (TOS) and inulinHP has been described
to increase the faecal count of bifidobacteria in bottle fed
infants (Boehm et al. 2002, Arch Dis Child 86, F178-F181).
[0005] Also, supplementation of infant milk formula with TOS and
inulin has been described to have a bifidogenic effect, and to
decrease faecal pH (Boehm et al. 2003 supra; Moro et al. 2003, Acta
Paediatr. Suppl. 441:77-79; Marini et al. 2003, Acta Paediatr.
Suppl. 441:80-81; Boehm et al. 2002, Arch. Dis. Child Fetal.
Neonata. Ed. 86:F178-F181; Moro et al. 2002, J. Pediatr.
Gastroenterol. Nutr. 34:291-295; Schmelzle et al. 2003, J. Pediatr.
Gastroenterol. Nutr. 36:343-51).
[0006] Upon fermentation of prebiotics by lactic acid bacteria
organic acids are produced and the pH is lowered. Lactobacilli
produce either lactate or lactate and acetate (a Short Chain Fatty
Acid; SCFA). The lactate can be in the L- or D-form.
Bifidobacteria, on the other hand, produce L-lactate and acetate,
but no D-lactate. Bifidobacteria (and other lactic acid bacteria)
usually do not lead to the production of gases, such as H.sub.2 and
CH.sub.4. They also do not produce other SCFA, such as propionate,
butyrate, isobutyrate, valerate and isovalerate. The presence of
SCFA, such as isobutyrate and isovalerate, are indicative of the
fermentation of carbohydrates by other bacterial species, such as
Clostridia and Bacteroides or Enterobacteriaceae, or are indicative
for the fermentation of proteins (of which the Bifidobacteria have
a poor capability). Also, other intestinal bacteria are capable of
producing acetate or lactate, such as Propionibacteria, Enterococci
and Pediococci.
[0007] Previously it has been reported that the intake of certain
prebiotic carbohydrates increase the (relative) amounts of
Bifidobacteria and/or Lactobacilli. Concomitantly an increased
formation of SCFA and decrease of pH has been observed. But also
the formation of gases, which results in unwanted symptoms such as
flatulence and abdominal pain, has been reported when introducing
prebiotics, such as inulin or GOS, into the diet. Furthermore, not
only acetate but also increases in, for example, butyrate have been
reported, which is undesirable. A range of undesirable effects have
therefore been described, resulting from the consumption of
nutrition supplemented with certain prebiotics.
[0008] In the colon and faeces of breast fed infants the
predominant SCFA found is acetate. Furthermore high concentrations
of lactate are found. These (relative) amounts are higher than
those observed in adults (where concentrations of lactate are
generally negligible) or in standard milk formula fed infants.
Subsequently, concentrations of propionate and especially butyrate
are much lower in the colon and faeces of breast fed infants than
in adults and even lower than in infants fed with standard infant
milk formula. The pH of the faeces is lowest in breast fed
children. As mentioned above, it is desirable to provide nutrient
compositions, especially milk formula compositions, which, when
consumed, result in an intestinal microflora closely resembling
that of breast fed infants.
[0009] Many health effects of SCFA and lactate and a low colon pH
have been described. The lowered pH and the presence of organic
acids have been described to have an anti-pathogenic effect, and
provide an advantage to acid tolerant bacteria such as the lactic
acid bacteria (including Bifidobacteria). Also effects of SCFA on
the intestinal wall have been described. SCFA are an energy source
of colonocytes and thereby aid to the intestinal barrier integrity.
SCFA are also involved in effects on peristalsis, bile acid
metabolism, water absorption and cell differentiation (see e.g.
EP1105002). SCFA are known to stimulate the production of mucus and
are involved in mineral absorption and mucus production.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present inventors surprisingly found that GOS
(especially TOS) and polyfructose (especially inulin), when
administered together, provide a number of beneficial effects which
cannot be explained by an increase in the number of intestinal
bifidobacteria and which are, therefore, not due to the bifidogenic
effect described for TOS or inulinHP in the prior art (e.g. in
Boehm et al. 2002, supra). In particular, it was unexpectedly found
that, following administration of GOS and polyfructose mixtures,
there was an increase in the total amounts of SCFA formed, an
increase in the relative amounts of acetate and lactate and a
decrease in faecal pH of infants, whereby these effects were not
correlated with an increased (relative) amount of Bifidobacteria.
Beside a beneficial SCFA amount and profile and an increased amount
of lactate, a decrease in gas formation was observed. Further,
lactate itself was found to have a hitherto unknown beneficial
effect on the colon, as it was found to increase the secretion of
prostaglandin E1 and prostaglandin E2. This effect was previously
only reported for SCFA, especially acetate (Willemsen et al. 2003,
Gut 52, 1442-1447). Compositions comprising lactate, and
compositions comprising prebiotics which stimulate lactate
production, may therefore be used to stimulate mucin production and
support the mucosal barrier integrity (mucoprotection). Lactate was
further found to decrease spontaneous contractions and tension in
colonic muscles, resulting in relief of cramp and pain.
[0011] The prebiotic compositions are therefore novel, in that they
do not, or do not significantly, increase the number of
Bifidobacteria, while they do result in an increase in total SCFA
amounts, beneficial changes in SCFA profile (increases in acetate,
decreases in butyrate and propionate), increase in lactic acid (and
resulting beneficial contraction and tension reducing effects and
muco-stimulatory effect), a decrease in faecal pH, a decrease in
formation of gases, and more gradual formation of SCFA (including
formation in the distal part of the colon), i.e. overall an optimal
pattern of fermentation products (more L-lactate, less butyrate
etc.). All these changes make the colonic environment more resemble
that of breast fed babies. In one embodiment of the invention the
use of mixtures of GOS and polyfructose in effective amounts for
the preparation of compositions which lead to a colonic environment
essentially similar to that of breast fed infants is, therefore,
provided herein.
[0012] The beneficial effects found were significantly more
pronounced upon co-administration than when either GOS or
polyfructose (e.g. TOS or inulin) alone was administered,
indicating that GOS and polyfructose act synergistically. In the
prior art no studies were carried out wherein the individual
effects of TOS and inulin administration were directly compared to
the co-administration of these two types of compounds and clearly
such independent individual studies cannot be compared to one
another and are unsuitable to identify synergistic effects.
[0013] This novel synergistic interaction of GOS and polyfructose
(e.g. TOS and inulin), and the in vivo effects of co-administration
thereof, leads to new uses of compositions comprising both GOS and
polyfructose in suitable (synergistic) amounts, such as treatment
or prevention of colic and/or abdominal cramps, abdominal bloating,
flatulence, abdominal pain, constipation, IBS, IBD, allergy and/or
increased muco-protection of the intestine. In addition, a
composition comprising lactate (D-lactate and/or L-lactate,
preferably L-lactate) may be used to treat or prevent one or more
of these symptoms or disorders.
[0014] Thus, unexpectedly, fermentation of the specific mixtures of
prebiotics (GOS and polyfructose) by the intestinal flora results
in an enhanced and kinetically advantageous formation of SCFA and
lactate and an improved pattern of metabolic end products. As a
consequence many beneficial effects result, such as effects on
mucus production, anti-inflammatory effects, effects on pain
perception and effects on abdominal cramps/colic (both via
relaxation of the colon and decrease of spontaneous contractions).
The nutrition compositions comprising these prebiotic mixtures can
also be used for adults having intestinal problems such as IBD or
IBS or a decreased intestinal barrier integrity due to
malnutrition.
DEFINITIONS
[0015] "Polysaccharides" refers to carbohydrate chains of
monosaccharide units with a chain length of at least 10 units. In
contrast, "oligosaccharides" have a chain length of less than 10
units.
[0016] "Degree of polymerisation" or "DP" refers to the total
number of saccharide units in an oligo- or polysaccharide chain.
The "average DP" refers to the average DP of oligosaccharides or
polysaccharide chains in a composition, without taking possible
mono- or disaccharides into account (which are preferably removed
if present). The average DP of a composition is used to distinguish
between compositions. In addition the % saccharide units, such as
the % glucose and % fructose units, in a composition are
distinguishing.
[0017] "Polyfructose" or "polyfructan" or "fructopolysaccharide"
refers to a polysaccharide carbohydrate comprising a chain of
.beta. linked fructose units with a degree of polymerisation of 10
or more and comprises, for example, inulin (e.g. inulin HP), levan
and/or a "mixed type of polyfructan" (see below).
[0018] "Inulin" or "non-hydrolysed inulin" or "inulin HP" is used
herein to refer to glucose-terminated fructose chains with the
majority of chains (at least 90%, preferably at least 95%) having a
degree of polymerisation (DP) of 10 or more. Inulin can thus be
described as GF.sub.n, wherein G represents a glucosyl unit, F
represents a fructosyl unit and n is the number of fructosyl units
linked to each other, n being 9 or more. The G/F ratio is about 0.1
to 0. A small part of the inulin molecules, however, may have no
terminal glucose unit. The average DP is preferably at least 15,
more preferably 20 or more, such as 20, 21, 22, 23, 25, 30, 40, 60,
70, 100, 150 or more herein. In inulin the fructose units are
linked with a .beta.(2.fwdarw.41) linkage. A suitable inulin is for
example the commercially available as Raftiline.RTM.HP (Orafti)
with an average DP>23.
[0019] "Hydrolised inulin" or "oligofructose" refers to mixtures of
glucose- and fructose-terminated fructose chains, with a DP below
10. Thus, hydrolysed inulin can be described as a mixture of
GF.sub.n chains and F.sub.n chains (wherein G is a glucosyl unit, F
is fructosyl unit and n=1-8). Hydrolysed inulin is an (enzymatic or
acidic) hydrolysis product or partial hydrolysis product of inulin,
resulting from cleavage of .beta.(1.fwdarw.2) fructosyl-fructose
linkages. The term hydrolysed inulin also encompasses synthetically
made or recombinantly made inulin which have the same structural
makeup.
[0020] "Levan" or "levulan" or "levulin" or "levulosan" refers to a
polysaccharide consisting of polyfructose in which the fructose
units are linked with .beta.(2.fwdarw.6) linkages. A starting
glucose moiety can be present, but this is not necessary. The
degree of polymerisation is above 10.
[0021] In "mixed type polyfructans" the fructose units are linked
with .beta.(2.fwdarw.1) and .beta.(2.fwdarw.6) linkages. Mixed type
polyfructans are branched and have a DP>10.
[0022] "GOS" or "galactooligosaccharides", or
"trans-galactooligosaccharides" or "TOS" refers to oligosaccharides
composed of galactose units, with a DP of 10 or less and an average
DP of 2, 3, 4, 5 or 6. A glucose unit may be present at the
reducing end of the chain. Preferably the GOS contains at least 2/3
galactose units. Most preferred are trans-galactooligosaccharides
(TOS) with 1341-4) glycosidic bonds. Such a GOS is for example that
found in Vivinal.RTM.GOS (commercially available from Borculo Domo
Ingredients, Zwolle, Netherlands), comprising
trans-galactooligosaccharides with .beta.-(1-4) glycosidic bonds
and .beta.-(1.fwdarw.6) glycosidic bonds.
[0023] "SCFA" or "short chain fatty acids" refers to fatty acids,
with a carbon chain lengths of up to C6, produced as an end-product
of bacterial intestinal fermentation, such as acetate (C2),
propionate (C3), butyrate and isobutyrate (C4), valerate and
isovalerate (C5) and others The expression "iC4-5" refers to the
sum of isobutyrate, valerate and isovalerate.
[0024] A "synergistically effective amount" refers to an amount of
GOS and polyfructose (for example TOS and inulin) which, when
co-administered, confers one (or more) specific physiological
effects (as described elsewhere herein), whereby the total effect
of co-administration is significantly larger than the sum of the
effect of individual administration of GOS or polyfructose. For
example, if the effect of administration of GOS alone is X and the
effect of administration of polyfructose alone is Y, then the
effect of co-administration of GOS and polyfructose is larger than
X+Y and for example the effect of co-administration of 1/2 the
concentration of GOS plus 1/2 the concentration of polyfructose has
an effect larger than 1/2 X+1/2Y, etc.
[0025] "Co-administration" of two or more substances refers to the
administration of these substances to one individual, either in one
composition or in separate compositions (kit of parts; as a
combined composition) which are administered at the same time
(simultaneously) or within a short time-span (separate or
sequential use, e.g. within minutes or hours).
[0026] "Enteral" refers herein to the delivery directly into the
gastrointestinal tract of a subject (e.g. orally or via a tube,
catheter or stoma).
[0027] The term "comprising" is to be interpreted as specifying the
presence of the stated parts, steps or components, but does not
exclude the presence of one or more additional parts, steps or
components.
[0028] "Infant" refers herein to humans aged 0-36 months,
preferably 0-18 months, or more preferably 0-12 months.
[0029] "Breast fed infants" refers to infants exclusively fed with
human breast milk. "Non- or partially breast fed infants" are
infants not exclusively fed on human breast milk. This includes
infants fed with at least one bottle (about 80 ml) of formula milk
per day.
[0030] "Percentage" or "average" generally refers to percentages of
averages by weight, unless otherwise specified or unless it is
clear that another basis is meant.
[0031] In one embodiment the present invention provides a number of
novel uses of compositions comprising both GOS and polyfructose in
suitable (synergistically effective) amounts.
Uses and Methods According to the Invention
[0032] In the various uses described herein, a subject is
preferably a human subject, more preferably an infant, such as a
new born infant up to a 12 months old infant. Especially bottle fed
or partially bottle fed infants are referred to. Alternatively, a
subject may also be a child, teenager or adult. In particular
intestinal problems, such as but not limited to Inflammatory Bowel
Disease (IBD), Irritable Bowel Syndrome (IBS), flatulence,
abdominal cramps, colic, abdominal bloating, abdominal pain, etc.,
which may also result in fatigue, depression or moodiness, may be
prevented and/or treated using compositions according to the
invention. Also, mucosal production may be enhanced and allergies
resulting from a suboptimal functioning of the intestine may be
prevented or treated. Also the intestinal barrier function may be
improved in patients with an impaired barrier function as a result
from malnutrition, surgery, chemotherapy etc.
[0033] Compositions comprising both GOS and polyfructose, such as
compositions comprising TOS and inulin, may in one embodiment be
used to induce the production of SCFA in the large intestine of a
subject and to significantly increase the (intestinal and/or
faecal) total amount of SCFA produced following administration
and/or to significantly modify the relative amounts of (intestinal
and/or faecal) SCFA produced, in particular to increase the
(intestinal and/or faecal) relative amount of acetate out of total
SCFA, and/or to significantly increase the amount (absolute and
relative) of intestinal lactate produced, and/or to significantly
reduce or prevent intestinal gas formation, and/or to extend the
SCFA production to the distal part of the colon, and/or to
significantly reduce the pH in the large intestine and faecal pH.
Also, the sum of intestinal and/or faecal butyrate, isobutyrate,
valerate and/or isovalerate relative to the total SCFA may be
decreased. These effects are achieved following administration,
without having a significant effect on the total and/or relative
number of intestinal bifidobacteria. Compositions for the treatment
or prophylaxis of any diseases or disorders or discomforts
associated with or caused by one or more of these intestinal
effects are provided herein.
[0034] The present invention provides the use of
galacto-oligsaccharides and polyfructose for the manufacture of a
composition for the treatment or prevention of abdominal bloating,
gas formation, abdominal pain and/or flatulence. In a further
embodiment the present invention provides the use of
galactooligosaccharides and polyfructose for the manufacture of a
composition for the treatment or prevention of allergy, eczema or
atopic diseases. In a further embodiment the present invention
provides the use of galacto-oligosaccharides and polyfructose for
the manufacture of a composition for the treatment and/or
prevention of colics and/or for the relaxation of contractions of
the colon, preferably the tonic and/or phasic contraction. In
another embodiment the present invention provides the use
galacto-oligosaccharides and polyfructose for the manufacture of a
composition for the treatment or prevention of irritable bowel
syndrome or inflammatory bowel disease. In an even further
embodiment the present invention provides the use of
galacto-oligosaccharides and polyfructose for the manufacture of a
composition for the increase of intestinal barrier functioning
and/or mucus production in the large intestine.
[0035] Unless specified otherwise, a "significant increase" (of for
example SCFA, acetate or lactate) refers herein to an increase of
at least 5%, preferably at least 10%, more preferably at least 20%,
or more, compared to the amount produced when either GOS or
polyfructose (e.g. inulin) alone are administered. Similarly,
unless specified otherwise a "significant reduction" (of for
example gas formation) refers to a reduction of at least 5%,
preferably at least 10%, more preferably at least 20%, 25%, 50% or
more (e.g. 70%, 80%, 90%, 100%) compared to the amount produced
when polyfructose (e.g. inulin) alone is administered.
[0036] An increase in the total amount of SCFA produced as
fermentation product of GOS and polyfructose compositions can also
be measured as a significant decrease in pH of faecal samples of
subjects, reflecting an in vivo decrease in pH in the large
intestine. In infants it was found that faecal pH decreased to
about pH 5.8 or less, such as pH 5.6, 5.5 or 5.4 or 5.2 following
co-administration of TOS and polyfructose (e.g. inulin), while the
pH of standard formula fed infants was around or above pH 6, such
as about pH 5.9, 6.8 or even 7.0. A significant decrease in pH
refers therefore to a decrease in pH by at least 15% or 0.9 unit
compared to infants fed a standard formula.
[0037] Another beneficial effect of co-administration of GOS and
polyfructose in suitable amounts is that the composition of SCFA is
significantly different than when either GOS or polyfructose are
administered. Especially the relative amount of butyrate is
significantly reduced, while the relative amount of acetate is
significantly increased. The compositions may, thus, be used to
alter not only total SCFA quantities, but also relative SCFA
proportions. In infants co-administered with TOS and polyfructose
(e.g. inulin), the relative SCFA levels resembled more the levels
found in breast fed infants, with acetate:propionate:butyrate
ratios generally about 80-85%:10-15%:1-5%, while in infants fed on
standard milk formula ratios were generally around
69-74%:16-19%:5-6% (as shown in the examples). Thus, especially
relative amounts of acetate are increased and relative amounts of
propionate and butyrate are decreased by the compositions according
to the invention. The co-administration of GOS and polyfructose
results in acetate:propionate:butyrate proportions resembling that
of breast-fed infants much more closely than that of formula fed
infants (which produce high levels of butyrate and propionate and
relatively low levels of acetate). The effect on relative SCFA
proportions can be measured by methods known in the art, such as
gas chromatography of faecal samples at various time points after
administration, either using in vivo studies or an in vitro
fermentation systems as e.g. described in the Examples. A
"significantly modified SCFA composition" or a "significantly
increased amount of acetate" refers to the amount of acetate (% of
total SCFA) being at least about 4%, 5%, 10%, 15%, or more, higher
than when no GOS or polyfructose are administered or when GOS or
polyfructose are administered individually. Preferably the relative
amounts of propionate and/or butyrate are lower than when no GOS or
polyfructose are administered or when GOS or polyfructose are
administered individually. In one embodiment, the composition
according to the invention is suitable for increasing intestinal
and/or faecal acetate to above about 85%, such as 86%, 87%, 88%,
90% or more, of the total SCFA.
[0038] Yet a further beneficial effect observed when
co-administering GOS and polyfructose is a significant decrease in
branched SCFA, compared to the proportion of branched SCFA found
when only GOS or only polyfructose are administered. A "significant
decrease in branched SCFA", as used herein, refers to a decrease at
least 70% compared to the concentration found in infants not fed
prebiotics or resulting in a faecal proportion of less than 1.5% of
total SCFA. The proportion of branched SCFA relative to the total
SCFA can be measured by dividing the sum of branched SCFAs, i.e.
isobutyrate, plus isovalerate plus valerate, by the sum of total
SCFA, i.e. acetate, plus propionate, plus butyrate, plus
isobutyrate, plus isovalerate, plus valerate, etc.
[0039] Reducing the proportion of branched SCFA is beneficial to
the health of the subject, as branches SCFA are damaging. This
indicates less protein degradation, which is unwanted because
protein fermentation results in an increase of pH and in the
formation of damaging agents such as H.sub.2S.
[0040] A significant decrease of butyrate relates to a decrease by
at least 50% compared to the concentration found in infants not fed
prebiotics or resulting in a faecal proportion of less than 4% of
total SCFA. In one embodiment the composition according to the
invention is suitable for decreasing the sum of intestinal (and/or
faecal) butyrate, isobutyrate, valerate and isovalerate is below 7%
of total SCFA, such as 6.5%, 6%, 5%, 4% or less of total SCFA.
[0041] In another embodiment, compositions comprising GOS and
polyfructose are suitable to increase the length of fermentation
within the colon. In particular, bacterial fermentation will still
be active in the most distal parts of the colon following
co-administration, as indicated by SCFA production in the distal
part of the colon. No or only very little SCFA is produced in the
distal part of the colon following administration of compositions
comprising only GOS or only polyfructose. In addition, fast
fermentation at the beginning of the colon is seen following
co-administration, which is especially important for
anti-pathogenic effects and is also observed in breast fed infants.
Overall, this indicates the compositions are suitable for
establishing and/or maintaining a relatively even fermentation
pattern throughout the colon of a subject and to extend
fermentation and SCFA production to the distal end of the colon.
SCFA, and most likely also other fermentation products, especially
lactate, are therefore produced throughout the colon, in the
beginning, middle and end of the colon.
[0042] In a further embodiment, co-administration of GOS and
polyfructose in synergistic amounts may be used for significantly
increasing lactate production, as can be determined again by
analysing faecal samples of test subjects and control subjects,
which received compositions comprising either GOS alone or
polyfructose alone or equivalent base compositions without
prebiotics. A "significant increase in lactate" refers to at least
5%, 10%, 20% or even 50% or more lactate being produced in subjects
being co-administered compositions comprising GOS and polyfructose,
compared to subjects not administered GOS or polyfructose. For
example, infants fed on standard milk formula supplemented with 6
g/l of TOS/inulin mixtures (90%:10%) produced at 16 weeks about 16%
lactate (as % of total acids), while standard formula fed infants
produced about 0.6% and breast fed infants produced about 35%
lactate. Although not matching the amount of lactate produced by
breast fed infants, the supplementation of the standard milk
formula with TOS and inulin mixtures led to a significant increase
in lactate production. As shown in the Examples, lactate was found
to have unexpected beneficial effects. Similarly to acetate it
increased the prostaglandin E1 and prostaglandin E2 production and
resulted in enhanced epithelial mucin expression. The intestinal
mucosa plays an important role in human health, as it serves as a
barrier to infectious pathogens, allergens and carcinogens.
Compositions which positively influence the development and/or
integrity of the mucosa may therefore be used to aid the build up
of a healthy mucosa in new born infants and to aid the
establishment of a healthy mucosa in bottle-fed or partially
bottle-fed infants and in subjects suffering from symptoms or
diseases resulting from or being associated with a not properly
functioning intestinal mucosa. For example, intestinal problems,
such as Inflammatory Bowel Disease (IBD, for example colitis or
Crohn's disease) or Irritable Bowel Syndrome (IBS) may be treated
or prevented, or pathogen attachment and/or entry through the
mucosa may be reduced or other immunological problems, such as the
development of asthma, may be prevented or reduced. Also the loss
of intestinal barrier integrity as a result from malnutrition,
surgery, chemotherapy etc. can be prevented or reduced.
[0043] Furthermore, spontaneous contractions and abdominal tension
was significantly reduced in a dose dependent manner following D-
or L-lactate administration. The compositions according to the
invention may thus be used to increase intestinal lactate levels
and thereby treat or prevent symptoms such as abdominal pain,
colic, abdominal contractions, abdominal tension and the like.
[0044] As lactate itself was found to have previously unknown
effects, it is also an object of the invention to provide
compositions comprising lactate in suitable amounts, which may be
used to treat or prevent the above described symptoms and
disorders. In one embodiment compositions comprises only lactate in
suitable amounts, while in another embodiment the GOS an
polyfructose compositions described herein further comprises
lactate to further increase intestinal lactate levels. One such
composition according to the invention comprises TOS and inulin in
synergistically effective amounts and further comprises lactate in
suitable amounts. In one embodiment compositions are provided which
are suitable for increasing intestinal and/or faecal lactate to
above about 10 mmol/kg faeces.
[0045] It is understood that when referring to the analysis of
faecal samples this is an indirect measure of the actual in vivo
effect. Likewise, in vitro assays may be carried out, whereby
bacterial populations are grown in vitro and suitable amounts of
compositions are added to the cultures. In particular, the in vitro
fermentation system described in the Examples may be used.
[0046] Co-administration of compositions comprising GOS and
polyfructose can therefore be used to achieve one or more of the
following physiological effects: [0047] a significant increase in
total SCFA [0048] a significant decrease in pH [0049] a
significantly modified SCFA composition [0050] a significant
decrease in branched SCFA [0051] a significant decrease in gas
production per mol SCFA produced [0052] a longer and more even
fermentation, including fermentation even in the most distal parts
of the colon, and/or [0053] a high initial formation of SCFA and
for lactic acid formation in the most proximal part of the colon
[0054] promoting the production of SCFA at the beginning, middle
and end of the colon [0055] a significant increase in lactic acid
(absolute and/or relative) and/or [0056] an increase in the
relative amount of L-lactate as a ratio of total lactic acid
formed.
[0057] In a preferred embodiment at least 2, 3, 4, 5, 6, 7, 8, 9 or
all these effects are significantly modified when using
compositions comprising both GOS and polyfructose, compared to when
administering compositions comprising only GOS or polyfructose, or
neither GOS or polyfructose. In particular, at least 2, 3, 4, 5, 6,
7, 8, 9 or all these effects are significantly larger following
co-administration of a (synergistically effective amount) of GOS
and polyfructose than the sum of the effect(s) of administration of
GOS and polyfructose individually.
[0058] The synergistic compositions according to the invention are,
therefore, particularly suited to build up and/or maintain a
healthy microflora within an infant's large intestine following
administration of a synergistically effective amount. In a
preferred embodiment an infant is fed solely on a base composition,
such as milk formula, supplemented with a synergistically effective
amount of GOS and polyfructose within the first weeks after birth.
A preferred composition is, therefore, a dietary composition for
infants. In another embodiment an infant is only partly fed on a
composition according to the invention. In one embodiment the
composition is used to treat or prevent symptoms selected from one
or more of: gas formation, flatulence, colitis, abdominal bloating,
abdominal cramps, abdominal pain, IBS, IBD, allergy and/or as a
mucoprotectant. GOS and polyfructose may, thus, be used for the
preparation of a composition or a combined composition for
simultaneous, separate or sequential use in treatment of
flatulence, excessive gas formation, colitis, abdominal bloating,
abdominal cramps, abdominal pain, IBS, IBD, allergy, decreased
intestinal barrier functioning and/or as a mucoprotectant. The
subject may in this case be any subject, ranging from infant,
child, teenager to adult. Clearly, the base compositions to which
GOS and polyfructose are added in synergistically effective
amounts, will vary depending on the age of the subject, the mode of
administration and on the main symptoms to be treated or prevented.
For infants for example the base composition is preferably a liquid
or powder form infant or follow-on formula, while for adults a
nutrient supplement composition (liquid, semi-liquid or solid) or
tube feeding may be more suitable.
[0059] In a preferred embodiment GOS and polyfructose are
co-administered in a synergistically effective amount suitable to
significantly increase the total amount of intestinal SCFA formed
and/or to significantly improve intestinal SCFA composition
(especially to significantly increase the percentage of acetate
relative to the total amount of SCFA) and/or to significantly
increase lactate production and/or to and to lower intestinal pH
and/or to significantly reduce intestinal gas formation.
[0060] Use of compositions comprising a synergistically effective
amount of GOS and polyfructose for the treatment and/or prevention
of colic and/or abdominal cramps, abdominal bloating and/or
flatulence, abdominal pain, IBS, IBD and/or allergy is provided.
Use of such compositions for increasing mucoprotection and
strengthening the intestinal barrier is also provided. It is
understood that not necessarily one composition is referred to, but
that GOS and polyfructose may be present in separate compositions,
which provides a synergistically effective amount when
co-administered.
[0061] Through these changes in SCFA levels, profile and intestinal
pH, the compositions are able to cause one or more of the following
downstream beneficial effects: [0062] a) The intestinal
permeability at the site of SCFA production is decreased. This is
important for preventing disease and maintaining health, especially
to prevent allergies from developing. The finding that
co-administration of GOS and polyfructose causes more even
fermentation and extends fermentation to the distal part of the
colon is important in this respect, as the permeability of the
intestine, including the distal parts of the colon, can be evenly
reduced and the intestine can thereby be evenly maintained in a
healthy state. [0063] b) The motility or peristaltic movement of
the intestine is enhanced, which reduces or prevents constipation,
a common problem observed in formula fed infants. [0064] c)
Decreasing the amount of spontaneous contractions and the colonic
muscle tension resulting in less cramps and less abdominal pain.
[0065] d) Calcium-ion absorption is increased, which is important
for bone mineralisation and bone development of the subject,
especially if the subject is an infant [0066] e) Secondary symptoms
of reduced health such as fatigue, depression and mood fluctuation
may be reduced, [0067] f) Mucus production of the intestinal mucosa
is significantly enhanced, which provides protection against
pathogen attachment and colonization. In particular, it was found
that lactate levels correlated positively with mucus production.
The compositions according to the invention may thus be used to
stimulate mucus production. [0068] g) Bile metabolism may be
modified to levels and/or patterns as observed in breast fed
infants. [0069] h) Growth of intestinal pathogenic microorganisms
is inhibited along the entire colon.
[0070] A composition comprising a synergistically effective amount
of GOS and polyfructose may therefore be used to either maintain or
establish (e.g. in a newborn, premature or mature born baby or in a
non- or partially breast fed baby, or other subjects, such as
adults, one or more of the above physiological effects and maintain
or improve the health of the subject by ensuring or establishing a
healthy large intestinal environment and optimal activity of the
large intestine.
[0071] In particular, incidence, duration and/or severity of
diseases, disorders or symptoms such as colic and/or abdominal
cramps, abdominal bloating and/or flatulence, abdominal pain, IBS,
IBD and/or allergy, intestinal pathogen infection (e.g. bacteria,
viruses, fungi), diarrhoea, eczema, allergy induced asthma and
other atopic diseases, and/or constipation can be reduced,
abolished or prevented by administration of a composition according
to the invention. The compositions according to the invention may
also be used to relax contractions of the colon, preferably the
phasic and/or tonic contractions.
Compositions According to the Invention
[0072] Compositions suitable for the uses described above comprise
both polyfructose and GOS in synergistically effective amounts. The
compositions comprise GOS/polyfructose in ratios ranging from 3/97
to 97/3, preferably 5/95 to 95/5, more preferably 90/10 to 45/55.
All individual ratios between these end-points are encompassed
herein, such as 10/90, 20/80, 30/70, 40/60, 50/50, 55/45, 60/40,
70/30, 80/20, 85/15, 90/10, etc.
[0073] The dosages required and the GOS/polyfructose ratio for
achieving the (optimal) synergistic effect may vary, depending on
the type of composition and the method and frequency of
administration. A few examples are however given below. A skilled
person may easily determine what dosages of each component is
required to achieve the best physiological effect and what
GOS/polyfructose ratio is the most suitable. For example, if the
main aim is to enhance the total level of SCFA following oral
administration of a composition comprising GOS and polyfructose, a
skilled person will make various compositions (comprising GOS plus
polyfructose, GOS alone, polyfructose alone) and compare their
efficiency in inducing high SCFA levels using either in vivo or in
vitro tests as known in the art. It is understood that when
referring to "daily dose" or `dosage per day`, this does not imply
that the dosage must be administered to the subject at one
time.
[0074] A synergistically effective infant formula may, for example,
consist of a base composition (e.g. standard infant formula)
comprising about 4 g/l, 5 g/l or more of a mixture of GOS and
polyfructose, whereby the ratio's of GOS to polyfructose may vary
as described elsewhere herein (e.g. 90% GOS:10% polyfructose).
[0075] Polyfructose may, for example, be levan and/or inulin.
Polyfructose, such as levan or inulin, for use in the compositions
may be either extracted from natural sources (plants or bacteria)
or may be made by de novo synthesis or by recombinant DNA
technologies as known in the art. Extraction, size separation and
purification methods of inulin have been described, for example in
De Leenheer (1996), U.S. Pat. No. 6,569,488 and U.S. Pat. No.
5,968,365. Recombinant production methods have been for example
described in U.S. Pat. No. 6,559,356. Synthesis generally involves
the use of sucrose molecules and enzymes with fructosyl transferase
activity. Inulin suitable for use in the compositions is also
already commercially available, e.g. Raftiline.RTM.HP, Orafti.
[0076] Plant inulins generally have a much lower degree of
polymerisation than bacterial inulins (up to 150, compared to up to
100,000 in bacteria). Plant sources include dicotyledenous species,
such as Compositae. Examples of species which produce relatively
large amounts of inulin, mainly in their roots, bulbs or tubers,
are chicory, asparagus, dahlia, Jerusalem artichoke, garlic, and
others (see Kaur and Gupta, J. Biosci. 2002, 27, 703-714). As the
inulin should preferably not comprise oligofructose, hydrolysis
should be avoided or oligofructose and/or mono- or disaccharides
present should be removed prior to use.
[0077] Especially preferred is polyfructose with a PD of at least
10, 15, 20, 50, 70, 100, 120, 130, 150, 200, 300, 500 or more.
Polyfructoses, such as levans, may be hydrolysed to avoid viscosity
problems associated with too long chains, as long as a PD of 10 or
more is retained.
[0078] Levans may also be obtained from natural sources such as
plants (e.g. monocotyledons) yeast, fungi, bacteria, or made
chemically or using recombinant DNA technology.
[0079] GOS may be obtained from natural sources, such as plants
(e.g. chicory, Soya) or bacteria, or may be made synthetically or
by recombinant DNA technology as known in the art. GOS may be
.beta.-galacto-oligosaccharide or .alpha.-galacto-oligosaccharide
or a mixture of both. In particular, galactose residues are linked
by .beta.(1.fwdarw.4) and .beta.(1.fwdarw.6) glycosidic bonds
(trans GOS or TOS). GOS suitable for use in the compositions is
also already commercially available, e.g. Vivinal.RTM.GOS, Borculo
Domo Ingredients, Zwolle, The Netherlands. GOS may also be derived
from lactose, by treatment of lactose enzymatically with
.beta.-galactosidase or by hydrolysis from polyglucan. In a
preferred embodiment TOS is used. In one embodiment the preferred
polyfructose is inulin HP, so that the combination of TOS and
inulin HP are also a preferred embodiment herein.
[0080] Other compositions provided are compositions comprising
lactate in suitable amounts, in particular for the uses described
above. Suitable amounts of lactate may vary, and may for example
range from 1 to 30 grams per day.
[0081] Compositions according to the invention may be either food
compositions, food supplement compositions or pharmaceutical
compositions. Apart from polyfructose and GOS (and/or lactate) they
may comprise additional ingredients. For food compositions or food
supplement compositions these should be food grade and
physiologically acceptable. "Food" refers to liquid, solid or
semi-solid dietetic compositions, especially total food
compositions (food-replacement), which do not require additional
nutrient intake or food supplement compositions. Food supplement
compositions do not completely replace nutrient intake by other
means. Food and food supplement compositions are in a preferred
embodiment baby food or food supplements, food or food supplements
for prematurely born babies, infant food, toddler food, etc., which
are preferably administered enterally, preferably orally several
times daily. The food or food supplement compositions are
particularly suited for non- or partially breast fed infants. Also,
the composition may be beneficially administered to infants in
their adaptation period to solid food or infants changing from
breast to bottle feeding. The composition may also be part of a
human milk fortifier supplement.
[0082] In one embodiment the composition is an infant or follow-on
formula as e.g. known in the art, especially as described in the
European Commission directive 91/321/EEC and the amendments
thereof, but may be modified to comprise an effective amount of
polyfructose and GOS. The infant or follow-on formula may be based
on milk (cows milk, goats milk, etc.), infant milk formula (IMF) or
soy for lactose intolerant infants or may contain amino acids as a
nitrogen source for infants having problems regarding allergy or
absorption. Commercially available infant or follow-on formulae
comprise, thus, a milk protein or soy protein base, fat, vitamins,
digestible carbohydrates and minerals in recommended daily amounts
and may be powders, liquid concentrates or ready-to-feed
compositions.
[0083] Administration of the modified infant or follow-on formula
results in a large intestinal environment which resembles that of
breast-fed infants, as can be determined by analyses of faecal pH,
bacterial composition, SCFA production and profiles, gas
production, etc. When the composition is a drink, preferably the
volume (comprising the daily effective dose) consumed or
administered on a daily basis is in the range of about 100 to 1500
ml, more preferably about 450 to 1000 ml per day. When the formula
is a solid preferably the amount (comprising the daily effective
dose) consumed or administered on a daily basis is in the range of
about 15 to 220 g/day, preferably about 70 to 150 g/day of formula
powder.
[0084] A daily effective dose of GOS and polyfructose ranges from
about 1 to 30 g/day, preferably from about 2 to 10 g/day for
infants and preferably for adults from about 5 to 20 g/day.
[0085] Food or food supplement compositions according to the
invention may additionally comprise other active ingredients, such
as vitamins (A, B1, B2, B3, B5, B12, C, D, E, K, etc.), probiotics
(e.g. bifidobacteria, lactobacilli, etc.), other prebiotics,
fibres, lactoferrin, immunoglobulins, nucleotides, and the like.
Nutrients such as proteins, lipids and other carbohydrates (e.g.
digestible carbohydrates, non-digestible carbohydrates, soluble or
insoluble carbohydrates) may be present in various amounts. Typical
in-soluble non-digestible carbohydrates present in infant nutrition
are soy polysaccharides, resistant starch, cellulose and
hemicellulose. Typical soluble and digestible carbohydrates for use
in infant nutrition are for example maltodextrin, lactose, maltose,
glucose, fructose, sucrose and other mono- or disaccharides or
mixtures thereof. The composition may also comprise other inactive
ingredients and carriers, such as e.g. glucose, lactose, sucrose,
mannitol, starch, cellulose or cellulose derivatives, magnesium
stearate, stearic acid, sodium saccharin, talcum, magnesium
carbonate and the like. The compositions may also comprise water,
electrolytes, essential and non-essential amino acids, trace
elements, minerals, fibre, sweeteners, flavorings, colorants,
emulsifiers and stabilisers (such as soy lecithin, citric acid,
esters of mono- or di-glycerides), preservatives, binders,
fragrances, and the like.
[0086] Lipids suitable for the compositions, especially for infant
food or food supplements, are milk fats, plant lipids, such as
canola oil, safflower oil, sunflower oil, olive oil, marine oils,
etc. or fractions or mixtures thereof comprising suitable fatty
acids (polyunsaturated and/or saturated).
[0087] Proteins suitable for the compositions especially for infant
food or food supplements, include casein, whey, condensed skimmed
milk, soy, beef, collagen, corn and other plant proteins or
hydrolysed proteins, free amino acids, etc. Preferably proteins
comprised in infant food or food supplement compositions are
extensively hydrolysed and/or partially hydrolysed to reduce the
risk of allergies. The infant food compositions according to the
invention preferably comprise all vitamins and minerals essential
in the daily or weekly diet in nutritionally significant amounts,
such as minimal recommended daily amounts.
[0088] The food or food supplement composition may also in one
embodiment be made on the basis of (i.e. starting from or
comprising) a food base. It may, for example, based on, or
comprises, a dairy product, such as a fermented dairy product,
including but not limited to milk, yoghurt, a yoghurt-based drink
or buttermilk. Such compositions may be prepared in a manner known
per se, e.g. by adding an effective amount of polyfructose and GOS
or TOS to a suitable food or food base. Other food bases suitable
may be plant bases, meat bases and the like.
[0089] The food or food supplement composition according to the
invention may be used either as a treatment and/or
prophylactically. This is to say that they may be either
administered after gastrointestinal problems or diseases have been
diagnosed in a subject or, alternatively, prior to the occurrence
of symptoms (for example to high risk patients, likely to develop
gastrointestinal problems). For example, if symptoms associated
with the disease or suboptimal functioning if the large intestine,
such as constipation, flatulence, allergies, colics, abdominal
pain, abdominal cramps, IBD, IBS are observed, administration of
the composition will aid in re-establishing a healthy large
intestinal environment or prevent the development of such
symptoms.
[0090] In one embodiment the compositions are administered
prophylactically, to support the development of a healthy
microflora and/or healthy large intestinal environment. A "healthy
large intestinal environment" refers to a normal intestinal
physiology and activity, especially normal absorption of nutrient,
water, resistance of the mucosa to pathogen attachment,
colonisation and infection, etc. as can be determined by faecal
analysis and gas production. Especially, an optimal physiology and
activity is referred to. Any suboptimal functioning of the large
intestine results in symptoms as described and can be determined by
faecal analysis and/or gas production. Intestinal diseases or
disorders resulting from a suboptimal functioning are included
herein.
[0091] Pharmaceutical compositions for the treatment or prophylaxis
of intestinal disorders or symptoms of suboptimal intestinal
functioning, such as IBD, colitis, IBS and/or increased barrier
integrity may comprise additional biologically active ingredients,
such as drugs, biologically active proteins or peptides,
probiotics, and others. The embodiments described for food or food
supplement compositions apply also to pharmaceutical
compositions.
[0092] The compositions according to the invention may be in any
dosage form, such as liquid, solid, semi-solid, tablets, drinks,
powders, etc., depending on the method of administration.
Administration to a subject is preferably oral, although for some
uses rectal or tube feeding (with a tube directly entering the
stomach, duodenum, or small intestine or large intestine) may be
suitable.
[0093] It is a further embodiment of the invention to provide a
method for the manufacture of a composition according to the
invention by adding a synergistically effective amount of GOS (or
TOS) and polyfructose to a suitable composition base, as described
above.
[0094] The following non-limiting Examples describe the synergikic
effect of GOS and polyfructose. Unless stated otherwise, the
practice of the invention will employ standard conventional methods
of molecular biology, pharmacology, immunology, virology,
microbiology or biochemistry. Such techniques are described in
Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual,
Third Edition, Cold Spring Harbor Laboratory Press, NY, in Volumes
1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular
Biology, Current Protocols, USA and Remington's Pharmaceutical
Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed.
(1985), Microbiology: A Laboratory Manual (6th Edition) by James
Cappuccino, Laboratory Methods in Food Microbiology (3.sup.rd
edition) by W. Harrigan (Author) Academic Press, all incorporated
herein by reference.
FIGURE LEGENDS
[0095] FIG. 1
[0096] Formation of acetate, propionate and butyrate after 48 h in
vitro fermentation of TOS, Inulin, a mixture of TOS/Inulin 9/1 and
a mixture of oligofructose and inulin 1/1 by fresh faeces obtained
from babies.
[0097] FIG. 2
[0098] Relative amounts of acetate, propionate and butyrate formed
after 48 h in vitro fermentation of TOS, Inulin, a mixture of
TOS/Inulin 9/1 and a mixture of oligofructose and inulin 1/1 by
fresh faeces obtained from babies. The sum of acetate plus
propionate plus butyrate formed was set at 100% for each of the
fibre tested.
[0099] FIG. 3
[0100] Formation of gas after 48 h in vitro fermentation of TOS,
Inulin, a mixture of TOS/Inulin 9/1 and a mixture of oligofructose
and inulin 1/1 by fresh faeces obtained from babies. The amount of
gas formed (ml was related to the amount of total SCFA formed (in
mmol per g fibre)
[0101] FIG. 4
[0102] Effects of mixtures of SCFA (acteate/propionate/butyrate in
a ratio of 90/5/5 on a molar basis) and of L-lactate.
[0103] FIG. 4A: Effect of 0, 0.1, 0.5, 1 and 4 mM of the SCFA
mixture or L-lactate on muc-2 expression in a CCD18/T84 co-culture
(n=6). Error bars show the SEM. The increase at 1 and 4 mM SCFA
mixture and 0.5, 1 and 4 mM L-lactate is statistically
significant.
[0104] FIG. 4B: Effect of 0, 50, 100, 250 and 500 .mu.M SCFA
mixture or L-lactate on the spontaneous PGE1 and PGE2 response in
CCD18 cells (n=7). Error bars show the SEM. The increase of PGE1 at
100, 250 and 500 .mu.M SCFA mixture and 100 .mu.M L-lactate is
statistically significant. The increase of PGE2 at 100, 250 and 500
.mu.M SCFA mixture and 250 .mu.M L-lactate is statistically
significant (P<0.05)
[0105] FIG. 5
[0106] Effects of sodium acetate and sodium L-lactate on the
spontaneous contraction in the distal and proximal part of the
colon. The blanc (set to a tension of 1 g) is 0%. The tension after
addition of 40 mM KCl was set to 100%.
EXAMPLES
Example 1
In Vitro Fermentation Studies Show Synergistic Effects on
Fermentation Patterns
1.1 Materials and Methods
Microorganisms
[0107] Microorganisms were obtained from fresh faeces from bottle
fed babies. Fresh faecal material from babies ranging 1 to 4 month
of age was pooled and put into preservative medium within 2 h.
Compositions/Substrate
[0108] As substrate either prebiotics (TOS; TOS (from VivinalGOS,
Borculo Domo Ingredients, The Netherlands) and inulin (raftilinHP
from Orafti, Belgium) mixture in a 9/1 (w/w) ratio; inulin;
oligofructose and inulin mixture in a 1/1 (w/w) ratio, or none
(blanc) was used.
Media
[0109] McBain & MacFarlane medium: Buffered peptone water 3.0
g/l, yeast extract 2.5 g/l. mucin (brush borders) 0.8 g/l, tryptone
3.0 g/l, L-Cysteine-HCl 0.4 g/l, bile salts 0.05 g/l,
K.sub.2HPO.sub.4.3H.sub.2O 2.6 g/l, NaHCO.sub.3 0.2 g/l, NaCl 4.5
g/l, MgSO.sub.4.7H.sub.2O 0.5 g/l, CaCl.sub.2 0.228 g/l,
FeSO.sub.4.7H.sub.2O 0.005 g/l. 500 ml Scott bottles were filled
with the medium and sterilised for 15 minutes at 121.degree. C.
[0110] Buffered medium: K.sub.2HPO.sub.4.3H.sub.2O 2.6 g/l,
NaHCO.sub.3 0.2 g/l, NaCl 4.5 g/l, MgSO.sub.4.7H.sub.2O, 0.5 g/l,
CaCl.sub.2 0.228 g/l, FeSO.sub.4.7H.sub.2O 0.005 g/l. pH was
adjusted to 6.3.+-.0.1 with K.sub.2HPO.sub.4 or NaHCO.sub.3. 500 ml
Scott bottles were filled with the medium and sterilised for 15
minutes at 121.degree. C.
[0111] Preservative medium: Buffered peptone 20.0 g/l,
L-Cysteine-HCl 0.5 g/l, Sodium thioglycollate 0.5 g/l, resazurine
tablet 1 per litre. pH was adjusted to 6.7.+-.0.1 with 1 M NaOH or
HCl. The medium was boiled in microwave. 30 ml serum bottles were
filled with 25 ml medium and sterilised for 15 minutes at
121.degree. C.
[0112] The fresh faeces were mixed with the preservative medium.
Fresh faeces can be preserved in this form for several hours at
4.degree. C.
[0113] Faecal suspension: The preserved solution of faeces was
centrifuged at 13,000 rpm for 15 minutes. The supernatant was
removed and the faeces was mixed with the McBain & Mac Farlane
medium in a weight ratio of 1:5.
Fermentation
[0114] 3.0 ml of the faecal suspension were combined with 85 mg
glucose or prebiotic or with no addition (blanc) in a bottle and
mix thoroughly. A t=0 sample was withdrawn (0.5 ml). 2.5 ml of the
resulting suspension was brought in a dialysis tube in a 60 ml
bottle filled with 60 ml of the buffered medium. The bottle was
closed well and incubated at 37.degree. C. Samples were taken from
the dialysis tube (0.2 ml) or from the dialysis buffer (1.0 ml)
with a hypodermic syringe after 3, 24, and 48 hours and immediately
put it on ice to stop fermentation.
Determination of the Short Chain Fatty Acids and Lactate
[0115] See Example 2. Values were corrected for blanc.
Gas Determination
[0116] At t=3, t=24 and t=48 the gas pressure in the head space of
the 60 ml bottle was measured by a gas pressure meter
(Druckmessumformer, Econtronic, Germany) by stinging a hypodermic 6
ml syringe through the rubber cap of the bottle and withdrawal of
gas from the headspace by this syringe until the gas pressure was 0
bar. The volume in the syringe was the volume of gas formed. Values
were corrected for blanc.
1.2 Results
[0117] In vitro fermentation was carried out using the following
samples:
1.) 85 mg TOS (from VivinalGOS, Borculo Domo Ingredients, The
Netherlands) 2.) 85 mg inulin (RaftilinHP, from Orafti, Belgium)
3.) 85 mg TOS/inulin with a ratio of TOS/inulin of 9/1 (w/w) and
4.) 85 mg OF (raftiloseP95, from Orafti, Belgium)/inulin
(raftilinHP) in a ratio of OF/inulin of 1/1 (w/w).
Total Amount of SCFA Produced
[0118] Results are shown in FIG. 1. FIG. 1 shows that the mixture
of TOS/Inulin resulted in a significantly higher amount of SCFA per
g fibre than the single components, but also higher than the
mixture of oligofructose (OF) and inulin. Also tested were 85 mg
TOS/inuline in a ratio of 1/1 (data not shown), which also provided
a synergistic effect.
L- and D-Lactate production
[0119] L- and D-lactate could only be determined at t=3. Table 1
shows the metabolic end products formed at that time point.
TABLE-US-00001 TABLE 1 metabolic end-products (mmol/g fibre) formed
after 3 hours in vitro fermentation Acetate Propionate Butyrate
L-lactate D-lactate TOS 0.23 0 0 0.14 0.03 Inulin 0 0 0 0.00 0.00
TOS/Inulin 0.40 0 0 0.17 0.04 OF/Inulin 0.30 0 0 0.04 0.02
[0120] Again a synergistic higher formation of lactate is observed
for the mixture TOS/Inulin compared to the single components TOS
and Inulin. Compared to the mixture with OF and Inulin the
percentage lactate (based on total acids) and ratio L-/D-lactate is
higher in the TOS/Inulin mixture.
Relative Amounts of SCFA
[0121] FIG. 2 shows the pattern of fermentation products after 48
h. The mixture of TOS/inulin shows a significantly higher
percentage of acetate than the single components, which is also
higher than for the mixture of oligofructose (OF) and Inulin.
Faeces of breast fed babies show a high percentage of acetate, so
the mixture of TOS/Inulin results in a pattern of fermentation
products which most resembles that of breast fed babies.
Gas Formation
[0122] Results are shown in FIG. 3. Regarding the formation of gas,
TOS and the mixture TOS/Inulin form the lowest amount of gas per
mmol SCFA formed. Inulin and the mixture of OF/inulin show a much
higher amount of gas formation. Per mmol SCFA formed the amount of
gas is lowest in the TOS/inulin mixture.
Kinetics of SCFA Formation
[0123] Table 2 shows the kinetics of SCFA formation. The
combination of TOS/inulin still shows a high SCFA formation between
24 and 48 h, indicating that in the distal part of the colon still
SCFA is formed and having a beneficial effect on the colon
permeability, mucus formation and anti-pathogenic effects etc. Also
in the first 3 h the highest amount of SCFA is formed, as is the
case with human milk oligosaccharides (data not shown). A fast
fermentation at the beginning of the colon is of importance because
of the antipathogenic effects.
TABLE-US-00002 TABLE 2 kinetics of SCFA formation (mmol) SCFA
(blanc corrected) Time interval (hours) Prebiotics 0-3 hrs 3-24 hrs
24-48 hrs TOS 0.23 3.85 0.13 TOS/inulin HP 0.40 4.49 0.24 Inulin HP
0.00 3.05 0.05 OF/Inulin HP 0.11 4.26 0.00 OF = oligofructose
Example 2
Clinical Study with TOS/Inulin: a Relative Increase of Acetate and
Relative Decrease of Butyrate is not Correlated with an Increase of
Bifidobacteria. TOS and Inulin have a Synergistic Effect
2.1 Materials and Methods
[0124] 63 pregnant woman who had decided to breast-feed and 57 who
chose not to, were recruited during their last trimester of
pregnancy. Infants with normal birth weight, no congenital
abnormality, congenital disease or gastrointestinal disease were
enrolled within 3 days after delivery. The study was approved by
the ethical committee of the Medical Center, St. Radboud, Nijmegen,
The Netherlands. Written informed consent was obtained from the
parents before enrolment in the study.
[0125] Infants of mothers who had decided not to breast-feed, were
randomly and double blindly allocated to one of two formula groups
(OSF, SF). The standard formula group (SF; n=19 received a regular,
non-supplemented infant formula (Nutrilon I, Nutricia, The
Netherlands). The main compositional data of the standard formula
at standard dilution of 131 g/l are given in Table 3. The prebiotic
formula group (OSF; n=19) received the same standard infant formula
supplemented with a mixture of 6 g/l transgalacto-oligosaccharides
(TOS; Vivinal GOS, Borculo Domo Ingredients, Zwolle, The
Netherlands) and inulin (PF; RaftilineHP, Orafti active food
ingredient, Tienen, Belgium). The mixture comprised 90% TOS and 10%
inulin (polyfructose). The study formulas were fed ad libitum
during the study period. Mothers who decided to breast feed were
stimulated to continue breast feeding during the course of the
study and were supported by a lactation consultant when needed. At
termination of breast feeding their infants received one of two
formulae. Compliance was assessed by counting the number of unused
formula tins during each visit and comparing the amount of consumed
formula with the recorded food intake.
TABLE-US-00003 TABLE 3 Composition of the standard formula per
litre Energy kcal 670 Protein g 14 Casein/whey ratio 40/60 Fat g 35
Total carbohydrates g 75 Lactose g 75 Minerals Calcium mg 540
Phosphorus mg 270 Magnesium mg 50 Sodium mg 190 Potassium mg 680
Chloride mg 430 Iron mg 5 Zinc mg 5
Questionnaires
[0126] Demographic, clinical anthropometrical data of the mother
are collected prior to delivery. Information on delivery was
obtained from the mothers at day 5 after delivery. Information of
the infants' food intake, formula tolerance, stool characteristics,
health and anthropometrics was obtained from questionnaires at
postnatal day 5, 10, 28 and once every 4 weeks thereafter until the
end of the study.
Faecal Samples
[0127] Parents were asked to take faecal samples from their
infants, at postnatal day 5, 10, 28 and once every 4 weeks
thereafter. The samples were taken from the diaper, as soon as
possible after defecation, collected in faeces containers (Greiner
Labortechnik, the Netherlands) and stored immediately at
-20.degree. C. by the parent and transported in a portable freezer
to the laboratory by the investigators.
Preparation of Faecal Samples:
[0128] For the determination of SCFA, 1 gram of the samples was
thawed in ice water diluted 10.times. in MilliQ and homogenised for
10 minutes using a stomacher (IUL Instruments, Barcelona, Spain).
350 .mu.l homogenised faeces was mixed with 200 .mu.l 5% (v/v)
formic acid, 100 .mu.l 1.25 g/l 2-ethylbutyric acid (Sigma-Aldrich,
Zwijndrecht, The Netherlands) and 350 .mu.l 1 MilliQ. The samples
were centrifuged for 5 minutes at 14,000 rpm to remove large
particles and the supernatant was stored at -20.degree. C. For the
FISH analysis and lactic acid measurements, the samples were thawed
in ice water, diluted 10.times. (w/v) in phosphate buffered saline,
pH 7.4 (PBS) and homogenized for 10 minutes using a stomacher. The
homogenised faeces were stored at -20.degree. C.
Fluorescence In Situ Hybridisation
[0129] FISH analysis was performed as described (Langendijk et al,
1995, Appl. Environ. Microbiol. 61:3069-3075.) with some slight
modifications. Paraformaldehyde fixed samples were applied to
gelatin coated glass slides (PTFE coated 8-wells [1 cm2/well]
object slides, CBN labsuppliers, Drachten, The Netherlands) and
air-dried. The dried samples were dehydrated in 96% ethanol for 10
minutes. Hybridisation buffer (20 mm Tris-HCl, 0.9 M NaCl, 0.1% SDS
[pH 7.1[) with 10 ng/l Cy.sub.3 Labeled Bifidobacterium specific
probe Bif164mod (5'-CAT CCG GYA TTA CCA CCC), was preheated and
added to the dried samples. Bif 164 mod is modified version of
probe S-G-Bif-a-0164-a-A-18 ((Langendijk et al, 1995, Appl.
Environ. Microbiol. 61:3069-3075.). The slides were incubated
overnight in a dark moist chamber at 50.degree. C. After
hybridisation the slides were washed for 30 minutes in 50 ml
preheated washing buffer (20 mM Tris-HCl, 0.9 M NaCl [pH 7.2]) and
briefly rinsed in MilliQ. For staining all bacteria, the samples
were incubated with 0.25 ng/l 4',6-diamidino-2-phenylindole (DAPI)
in PBS for 5 minutes at room temperature. After DAPI staining the
slides were briefly rinsed in MilliQ, dried, mounted with
Vectashield (Vector Laboratories, Burlingame, Calif., U.S.A.) and
covered with a coverslip. The slides were automatically analysed
using an Olympus AX70 epifluorescence microscope with automated
image analysis software (Analysis 3.2, Soft Imaging Systems GmbH,
Munster, Germany). The percentage of bifidobacteria per sample was
determined by analysing 25 randomly chosen microscopic positions.
At each position the percentage of bifidobacteria was determined by
counting all cells with a DAPI filter set (SP 100, Chroma
Technology Corp., Brattleboro, U.S.A.) and counting all
bifidobacteria using a Cy3 filter set (41007, Chroma Technology,
Brattleboro, U.S.A.).
Short Chain Fatty Acids Analysis
[0130] The short chain fatty acids (SCFA) acetic, propionic,
n-butyric, iso-butyric and n-valeric acids were quantitatively
determined by a Varian 3800 gas chromatograph (GC) (Varian Inc.,
Walnut Creek, U.S.A.) equipped with a flame ionisation detector.
0.5 .mu.l of the sample was injected at 80.degree. C. in the column
(Stabilwax, 15.times.0.53 mm, film thickness 1.00 .mu.m, Restek
Co., U.S.A.) using helium as a carrier gas (3.0 psi). After
injection of the sample, the oven was heated to 160.degree. C. at a
speed of 16.degree. C./min, followed by heating to 220.degree. C.
at a speed of 20.degree. C./min and finally maintained at a
temperature of 220.degree. C. for 1.5 minutes. The temperature of
the injector and detector was 200.degree. C. 2-ethylbytyric acid
was used as an internal standard.
Lactate Analysis
[0131] Homogenised faeces was thawed on ice and centrifuged for 5
minutes ant 14,000 rpm 100 .mu.l supernatant was heated for 10
minutes at 100.degree. C. to inactivate all enzymes. Lactate was
determined enzymatically, using a L-lactate acid detection kit with
D- and L-lactate-dehydrogenase (Boehringer Mannheim, Mannheim,
Germany). Lactate was only determined in those faecal samples which
were large enough.
pH Analysis
[0132] After storage at -20.degree. C., faecal samples were thawed
and the pH was directly measured in the faeces at room temperature
using a Handylab pH meter (Scott Glas, Mainz, Germany) equipped
with an Inlab 423 pH electrode (Mettler-Toledo, Columbo,
U.S.A.)
Data Analysis
[0133] Prior to the study, power calculations showed that to detect
a difference in percentage of bifidobacteria between the
intervention formula group and the standard formula group of 30%
with a SD of 25%, 13 infants per group should be included. Because
of an expected drop out of 30% in the formula groups, more infants
than calculated were included in the study. Statistical package
SPSS (version 11/0) was used for statistical analysis of the
results. All values were checked for normality by visual inspection
of the normal probability plots. Differences in percentage
bifidobacteria, pH, relative amounts of SCFA and lactate between
the groups were tested for significance using analysis of variance.
In case of a significant difference (p<0.05), groups were
compared by using the Bonferroni post hoc test. Because it is not
possible to double blind assign breast and bottle-feeding and to
ensure adequate randomisation, no statistical analysis were
performed to compare the breast-feeding with any of the formula
feeding groups. Data from the breast-fed group are only given when
the infant was only fed breast milk at that time point.
2.2 Results
[0134] In total, 120 (-pro group infants were included. 57 infants
started on formula feeding directly after birth and were equally
divided among the formula groups. Of the 63 infants that were fed
breast milk directly after birth, 24 switched to formula feeding
before the age of 16 weeks and 5 infants dropped out. The
characteristics of the study subjects are shown in Table 4. In the
formula groups, 9 infants dropped out of the study within the first
16 weeks after birth (4 in the SF, 5 in the OSF group. Reasons for
drop out included: colics, suspicion of cow's milk allergy,
constipation and practical problems.
TABLE-US-00004 TABLE 4 Characteristics of study objects Standard
Prebiotic Formula, SF formula, OSF Breast milk, N = 19 N = 19 BF N
= 63 Sex Male 5 12 33 Female 14 7 30 Place of birth At home 7 8 40
Hospital 12 11 23 Mode of Vaginal 14 16 59 delivery Caesarean 5 3 4
Birth weight 3600 .+-. 501 3318 .+-. 602 3651 .+-. 601
Faecal Bifidobacteria
[0135] The percentages of bifidobacteria in faeces at the age of 5
days, 10 days, 4, 8, 12, and 16 weeks of the feeding groups are
shown in Table 5 and the amounts in Table 5. The OSF group tends to
have a higher bifidobacteria % than the SF group from total
bacterial count at all ages, but the differences were not
statistically different. Unexpectedly, the percentage of
Bifidobacteria in breast fed babies was also relatively low and
were in line with the formula fed groups.
[0136] Preliminary data also show an increase in Lactobacilli in
the BF and OSF group, but the amounts of Lactobacilli in the faecal
flora are at least one order of magnitude lower than the
Bifidobacteria, the overall pattern is changes very little.
pH Results
[0137] The pH values measured in the faeces of the formula-fed
infants are shown in table 6. Lowest pH was found in infants fed on
breast milk. Faecal pH of faeces of infants fed the OSF formula
were lower than the SF group (p<0.045 at all ages except day
5).
SCFA Results
[0138] The total amount of SCFA in the faeces is shown in Table 5
below.
[0139] The percentage of the different SCFA from total SCFA are
shown in Table 6. There are no statistically significant
differences found in total SCFA concentration between the formula
groups. Also the amount of SCFA is comparable to those of the other
feeding groups. However, already after 10 days, differences in the
SCFA profiles can be seen between infants fed on OSF or breast milk
compared to infants fed on standard formula. Infants fed the
formula containing GOS and polyfructose and fed breast milk, have
higher percentages of acetate and lower percentages of propionate,
butyrate, iC4-6 SCFA when compared to infants fed the standard
formula.
Lactate Results
[0140] The concentrations lactate (mmol/kg faeces) of all groups
are shown in Table 5. Already from 5 days of age, the OS formula
(not sign.) and the groups of breast milk have higher amounts of
lactate compared to the standard formula group. The relative amount
of lactate (as a percentage of the sum of SCFA and lactate) is
highest in breast fed babies and lowest in standard formula fed
babies. Babies fed a formula containing TOS/inulin have an
intermediate relative amount of acetate. The percentage of lactate
in OSF fed babies at 16 weeks (relative to total acids)
significantly differs from that of SF babies.
TABLE-US-00005 TABLE 5 Concentration of lactate and total SCFA
(mmol/kg faeces) and Bifidobacteria (*1.10.sup.13/kg faeces) pH
between birth and 16 weeks of age. Lactate SCFA pH Bifidobacteria 5
days SF 13.5 .+-. 7.7 54.7 .+-. 12.6 5.93 .+-. 0.15 0.58 .+-. 0.49
OSF 10.7 .+-. 4.3 56.5 .+-. 7.7 5.49 .+-. 0.15 1.20 .+-. 2.24 BF
13.3 .+-. 2.8 48.7 .+-. 4.4 5.27 .+-. 0.07 0.47 .+-. 0.39 10 days
SF 4.6 .+-. 3.0 62.0 .+-. 7.9 6.88 .+-. 0.15 0.96 .+-. 0.83 OSF 9.7
.+-. 3.6 62.3 + 7.2 5.95 .+-. 0.20 1.10 .+-. 0.99 BF 15.1 .+-. 3.2
54.7 .+-. 4.9 5.35 .+-. 0.07 0.48 .+-. 0.61 4 SF 2.6 .+-. 1.4 68.3
.+-. 10.3 6.77 .+-. 0.21 0.97 .+-. 0.96 weeks OSF 9.9 .+-. 3.4 83.1
.+-. 8.8 5.88 + 0.18* 1.20 .+-. 0.55 BF 22.8 .+-. 4.4 59.8 .+-. 4.8
5.45 .+-. 0.12 0.56 .+-. 0.64 8 SF 7.6 .+-. 6.8 76.5 .+-. 13.2 6.80
+ 0.20 0.89 .+-. 0.56 weeks OSF 24.4 .+-. 5.3 76.0 + 8.4 5.68 .+-.
0.18* 1.00 .+-. 0.52 BF 30.9 .+-. 5.3 62.8 .+-. 5.4 5.27 .+-. 0.15
0.58 .+-. 0.59 12 SF 14.1 .+-. 9.4 73.9 .+-. 11.9 6.88 .+-. 0.20
0.91 .+-. 0.80 weeks OSF 18.4 .+-. 7.0 76.1 .+-. 12.1 5.60 .+-.
0.18* 1.30 .+-. 0.99 BF 42.1 .+-. 7.1 60.4 .+-. 4.9 5.29 .+-. 0.17
1.40 + 1.38 16 SF 1.7 .+-. 1.2 68.6 .+-. 14.0 7.09 .+-. 0.15 1.00
.+-. 0.80 weeks OSF 18.5 .+-. 5.7 67.7 .+-. 11.7 5.60 .+-. 0.20*
1.30 .+-. 0.76 BF 45.1 .+-. 9.0 59.2 .+-. 6.9 5.68 .+-. 0.24 0.89
.+-. 0.78 Mean .+-. SEM. Except for pH, no statistically
differences were found.
TABLE-US-00006 TABLE 6 relative amounts of SCFA (% of total SCFA),
lactate (% of total acids), % Bifidobacteria between birth and 16
weeks of age. Day/ Sum week Acetate Propionate Butyrate iC4-C5 %
Bifidobacteria lactate 5 d SF 84.3 .+-. 3.4 12.9 .+-. 3.2 1.7 .+-.
0.5 1.1 .+-. 0.4 45 .+-. 3.6 13.8 .+-. 18.5 OSF 85.8 + 5.1 12.0
.+-. 4.7 0.5 .+-. 0.5 1.7 .+-. 0.7 50 .+-. 8.6 8.7 .+-. 13.3 BF
89.5 .+-. 1.8 7.0 .+-. 1.5 1.6 .+-. 0.4 2.0 .+-. 0.4 54 .+-. 4.1
12.5 .+-. 14.0 10 d SF 70.9 .+-. 2.0 21.3 .+-. 2.6 4.6 .+-. 1.1 3.2
.+-. 0.5 65 .+-. 6.0 4.3 .+-. 10.8 OSF 84 .+-. 2.4* 13.5 .+-. 2.3
1.4 .+-. 0.4* 1.1 .+-. 0.4 61 .+-. 6.3 8.2 .+-. 9.8 BF 89.3 .+-.
1.9 5.8 .+-. 1.3 2.3 .+-. 0.3 2.6 .+-. 0.4 42 .+-. 4.1 13.4 .+-.
13.8 4 w SF 71.8 .+-. 2.8 17.8 .+-. 3.3 5.0 .+-. 1.1 5.5 .+-. 2.6
52 .+-. 5.4 6.9 .+-. 16.4 OSF 77.7 .+-. 2.2 15.4 .+-. 2.0 5.8 .+-.
2.2 1.1 .+-. 0.3 71 .+-. 4.5 8.4 .+-. 10.7 BF 91.0 .+-. 1.8 4.3
.+-. 1.2 2.6 .+-. 0.6 2.1 .+-. 0.4 47 .+-. 5.4 19.6 .+-. 17.0 8 w
SF 74.6 .+-. 2.9 16.4 .+-. 2.0 6.1 .+-. 1.2 2.9 .+-. 0.7 50 .+-.
6.3 3.5 .+-. 9.9 OSF 83.5 .+-. 2.7 11.4 .+-. 2.1 3.7 .+-. 1.2 1.4
.+-. 0.4 64 .+-. 4.1 17.7 .+-. 15.2 BF 91.2 .+-. 1.6 5.4 .+-. 1.4
1.9 .+-. 0.5 1.6 .+-. 0.3 41 .+-. 4.5 21.7 .+-. 13.4 12 w SF 73.9
.+-. 2.9 17.8 .+-. 3.3 5.0 .+-. 1.1 3.2 .+-. 0.5 56 .+-. 5.4 5.4
.+-. 11.9 OSF 86.5 .+-. 2.1 11.2 .+-. 1.8 1.2 .+-. 0.3* 1.0 .+-.
0.4 60 .+-. 5.0 13.6 .+-. 13.2 BF 86.1 .+-. 3.3 7.5 .+-. 2.2 3.0
.+-. 0.7 3.5 .+-. 0.8 59 .+-. 4.5 30.2 .+-. 16.3 16 w SF 69.9 .+-.
3.9 19.6 .+-. 2.7 5.6 .+-. 0.9 4.9 .+-. 0.8 52 .+-. 6.3 0.6 .+-.
0.9 OSF 82.2 .+-. 5.3 14.3 .+-. 4.9 2.1 .+-. 0.5* 1.5 .+-. 0.4* 69
.+-. 7.7 16.4 .+-. 12.0* BF 89.7 .+-. 2.7 6.4 .+-. 2.1 1.6 .+-. 0.4
2.2 .+-. 0.5 47 .+-. 6.3 35.6 .+-. 20.4 P < 0.05 compared to SF;
SF = standard formula fed, OSF = SF supplemented with GOS + inulin;
BF = breast fed.
[0141] The above demonstrates that in faeces of infants fed with
this combination of GOS and polyfructose the pH is lowered, that
more lactate is formed, and that an acid (acetate and lactate)
pattern is formed which is more similar to that of breast fed
babies and that this effect cannot be attributed to the
quantitative increase of Bifidobacteria.
Example 3
Beneficial Effects of Lactate and SCFA Mixture on Muc-2, PGE1 and
PGE2 expression
3.1 Material and Methods
[0142] The effect of lactate and a SCFA mixture was analysed as
described in Willemsen, L E M, Koetsier M A, van Deventer S J H,
van Tol E A F (2003), Gut 52:1442-1447, with the following
modifications: Lactate and a mixture of acetate/propionate/butyrate
90/5/5 was tested. For the mucus production experiments a
co-culture of CCD 18 and T84 cells was used, whereas for the PGE1
and PGE2 production experiments a monoculture of CCD18 cells was
used.
3.2 Results
[0143] SCFA, in a mixture of 90/5/5 (acetate/propionate/butyrate),
and L-lactate dose dependently induces MUC-2 expression in a
co-culture of CCD18 and T84 cells as can be seen in FIG. 4A. Also
the concentration of PGE1 and PGE2 increases in CCD18 cells as can
be seen in FIG. 4B. At higher concentrations of the added organic
acids the increases reaches statistically significance.
Example 4
Effect of Lactate on Colon Contraction
4.1 Material and Methods
[0144] Male Wistar rats (CKP/Harlan, Wageningen/Horst, Netherlands)
were housed under conditions of controlled temperature and light
cycle and were provided free access to food pellets and water.
Animals were anaesthetised by a mixture of N.sub.2O, O.sub.2 and
isofluran, the abdomen was opened and the colon was removed
immediately. The tissue was placed in Krebs-Henseleit buffer pH 7.4
(composition in mM: 118.0 NaCl, 4.75 KCl, 1.18 MgSO.sub.4, 2.5
CaCl.sub.2, 10 glucose, 1.17 KH.sub.2PO.sub.4 and 24.9
NaHCO.sub.3).
[0145] The colon was cut into a distal and a proximal portion and
was rinsed with Krebs-Henseleit buffer while gently squeezing out
faecal content. To approximate in vivo conditions as closely as
possible, 1 cm fully intact segments were attached longitudinally
to an isometric force transducer (F30 type 372, HSE, Germany) in 20
ml water-jacketed (37.degree. C.) organ baths (Schuler, HSE,
Germany) containing Krebs-Henseleit buffer gassed continuously with
95% O.sub.2-5% CO.sub.2. The segments were gradually stretched to a
resting tension of 1 g and allowed to equilibrate for 45 minutes
with intermittent washings. The tensions of the segments in rest
and in response to different stimuli were amplified by a transducer
amplifier module (HSE, Germany) and recorded on a multi-pen
recorder (Rikadenki, HSE, Germany).
[0146] The segments were incubated with 40 mM KCl for 5 minutes and
the contractile responses were measured. KCl was washed out by
three consecutive washes at 5 minutes intervals. The segments were
then incubated with increasing concentrations up to 100 mM of
acetate or sodium-L-lactate. The acid solutions were prepared
freshly in distilled water. NaOH was added to acetate to obtain a
neutral pH. At the end of the incubation with a fatty acid, 40 mM
KCl was added to determine whether the contractile response to KCl
was influenced by the fatty acid. Before a new incubation the
segments were allowed to equilibrate for 45 minutes in fresh
Krebs-Henseleit buffer with intermittent washings.
[0147] The experimental protocol consisted of two proximal and two
distal sections of the colon. For data analysis (n=3), the
contraction level induced by the stimuli was defined as the tension
in g after 5 minutes incubation. Data obtained from identical
segments (proximal or distal) were used to calculate a mean value
and each segment served as its own control sample.
[0148] 4.2 Results
[0149] As can be seen from FIG. 5, sodium acetate and especially
sodium-L-lactate decrease the tension of tonic contractions. The
relaxation effects are higher in the distal part of the colon than
in the proximal part of the colon.
[0150] Also the number of spontaneous contractions, the phasic
contractions, decrease in the proximal part of the colon upon
addition of the sodium acetate and sodium-L-lactate, whereas no
effects are observed in the distal part of the colon.
[0151] In the proximal part of the colon the tonic contractions as
a response to KCl, however, are comparable in the presence or
absence of 25 mM sodium actetate or sodium L-lactate. At higher
concentrations a significant relaxation is observed even after
addition of KCl.
* * * * *