U.S. patent application number 12/997527 was filed with the patent office on 2011-07-28 for immune system stimulating nutrition.
This patent application is currently assigned to N.V. Nutricia. Invention is credited to Johan Garssen, Leon Matthieu Johannes Knippels, Alma Jildou Nauta, Belinda Potappel-van 'T Land.
Application Number | 20110182934 12/997527 |
Document ID | / |
Family ID | 40935729 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110182934 |
Kind Code |
A1 |
Potappel-van 'T Land; Belinda ;
et al. |
July 28, 2011 |
IMMUNE SYSTEM STIMULATING NUTRITION
Abstract
Disclosed is a nutritional composition comprising a combination
of non-digestible oligosaccharides and a product obtained by
incubating an aqueous substrate by bifidobacteria and optionally a
product obtained by incubating an aqueous substrate by S.
thermophilus. Said combination synergistically improves the immune
system by stimulating the Th1 response, and/or decreasing the Th2
response, improving the vaccination response, improving the
resistance against infections and/or decreasing allergy.
Inventors: |
Potappel-van 'T Land; Belinda;
(Kootwijk, NL) ; Knippels; Leon Matthieu Johannes;
(Utrecht, NL) ; Nauta; Alma Jildou; (Driebergen,
NL) ; Garssen; Johan; (Nieuwegein, NL) |
Assignee: |
N.V. Nutricia
|
Family ID: |
40935729 |
Appl. No.: |
12/997527 |
Filed: |
June 12, 2009 |
PCT Filed: |
June 12, 2009 |
PCT NO: |
PCT/NL2009/050333 |
371 Date: |
April 8, 2011 |
Current U.S.
Class: |
424/203.1 ;
424/234.1 |
Current CPC
Class: |
A61K 38/018 20130101;
A23L 33/40 20160801; A23V 2002/00 20130101; A61K 31/702 20130101;
A61K 38/017 20130101; A61P 37/00 20180101; A61P 37/08 20180101;
A61P 29/00 20180101; A61K 31/733 20130101; A61P 11/06 20180101;
A61K 35/744 20130101; A61P 37/04 20180101; A61P 31/00 20180101;
A61K 35/745 20130101; A61P 17/00 20180101; A23L 33/135 20160801;
A61P 1/12 20180101; A61P 1/14 20180101; A61K 35/744 20130101; A61K
2300/00 20130101; A61K 35/745 20130101; A61K 2300/00 20130101; A61K
38/017 20130101; A61K 2300/00 20130101; A23V 2002/00 20130101; A23V
2200/324 20130101; A23V 2200/3202 20130101; A23V 2200/3204
20130101; A23V 2250/28 20130101 |
Class at
Publication: |
424/203.1 ;
424/234.1 |
International
Class: |
A61K 39/116 20060101
A61K039/116; A61K 39/02 20060101 A61K039/02; A61P 37/04 20060101
A61P037/04; A61P 11/06 20060101 A61P011/06; A61P 37/08 20060101
A61P037/08; A61P 29/00 20060101 A61P029/00; A61P 31/00 20060101
A61P031/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2008 |
NL |
PCT/NL2008/050375 |
Oct 31, 2008 |
EP |
08168054.8 |
Claims
1-21. (canceled)
22. A process for the manufacture of a preparation, comprising: (a)
incubating bifidobacteria with at least one aqueous substrate
selected from the group consisting of milk, milk protein, whey,
whey protein, whey protein hydrolysate, casein hydrolysate, and
lactose to obtain a first incubated mixture; (b) combining the
incubated mixture with at least two different non-digestible
carbohydrates, at least one of which is selected from the group
consisting of fructo-oligosaccharides, galacto-oligosaccharides,
gluco-oligosaccharides, arabino-oligosaccharides,
mannan-oligosaccharides, xylo-oligosaccharides,
fuco-oligosaccharides, arabinogalacto-oligosaccharides,
glucomanno-oligosaccharides, galactomanno-oligosaccharides,
raffinose, lactosucrose, sialic acid comprising oligosaccharides
and uronic acid oligosaccharides.
23. The process according to claim 22, further comprising
inactivating and/or removing the bifidobacteria from the first
incubated mixture.
24. The process according to claim 23, wherein the bifidobacteria
are inactivated by heating the first incubated mixture.
25. The process according to claim 23, wherein the bifidobacteria
are removed from the first incubated mixture by centrifugation,
filtration, or both.
26. The process according to claim 22, wherein two of the at least
two different non-digestible carbohydrates, are selected from the
group consisting of fructo-oligosaccharides,
galacto-oligosaccharides, gluco-oligosaccharides,
arabino-oligosaccharides, mannan-oligosaccharides,
xylo-oligosaccharides, fuco-oligosaccharides,
arabinogalacto-ologosaccharides, glucomanno-oligosaccharides,
galactomanno-oligosaccharides, raffinose, lactosucrose, sialic acid
comprising oligosaccharides and uronic acid oligosaccharides.
27. The process according to claim 22, wherein the bifidobacteria
belong to species B. breve.
28. The process according to claim 27, wherein the species B. breve
comprises strain B. breve CNCM I-2219.
29. The process according claim 22, further comprising: (i)
incubating Streptococcus thermophilus, with a substrate selected
from the group consisting of milk, milk protein, whey, whey
protein, whey protein hydrolysate, casein, casein hydrolysate, and
lactose, to obtain a second incubated mixture; (ii) optionally
inactivating the S. thermophilus by heating the second incubated
mixture of (i) and/or removing S. thermophilus by centrifugation,
filtration or both; and (iii) combining the first and second
incubated mixtures.
30. The process according to claim 29, wherein the Streptococcus
thermophilus comprises strain S. thermophilus CNCM I-1620.
31. The process according to claim 22, wherein one or both of the
at least two different non-digestible carbohydrates are selected
from the group consisting of galacto-oligosaccharides and
fructo-oligosaccharides.
32. The process according to claim 22, further comprising
inoculating the substrate with bifidobacteria in amount of between
1.times.10.sup.2 to 1.times.10.sup.11 cfu per ml substrate at a
temperature of between 20.degree. C. to 50.degree. C., for at least
2 h and at a pH of between 4 and 8.
33. The process according to claim 29, further comprising
inoculating the substrate of (i) with Streptococci in amount of
between 1.times.10.sup.2 to 1.times.10.sup.11 cfu per ml substrate
at a temperature of between 20.degree. C. to 50.degree. C., for at
least 2 h and at a pH of between 4 and 8.
34. The process according to claim 29, wherein the first or the
second incubated mixtures are inactivated by heating for at least 5
minutes at a temperature above 50.degree. C.
35. The process according to claim 22, further comprising a drying
step.
36. The process according to claim 22, wherein only one
non-digestible carbohydrate is added, said non digestible
carbohydrate being fructo-oligosaccharide.
37. A nutritional composition obtained by the process of claim
22.
38. The nutritional composition according to claim 37, having a
viscosity of 1 to 60 mPas at a shear rate of 100 s.sup.-1 at
20.degree. C.
39. A nutritional composition comprising, based on total dry weight
of the composition: (a) 0.5 to 10 wt. % of the sum of
galacto-oligosaccharides and fructo-oligosaccharides, and (b) 5 to
99.5 wt. % of the preparation obtained by the process of claim 22,
wherein the bifidobacteria in step a belong to the species B.
breve.
40. The nutritional composition of claim 39, further comprising (c)
2 to 94.5 wt. % the preparation obtained by: (i) incubating
Streptococcus thermophilus, with a substrate selected from the
group consisting of milk, milk protein, whey, whey protein, whey
protein hydrolysate, casein, casein hydrolysate, and lactose, to
obtain a second incubated mixture; (ii) optionally inactivating the
S. thermophilus by heating the second incubated mixture of (i)
and/or removing S. thermophilus by centrifugation, filtration or
both; and (iii) combining the first and second incubated
mixtures.
41. The nutritional composition according to claim 39, comprising
less than 10.sup.3 du living bifidobacteria per g dry weight of the
preparation.
42. A method of treating a disease and/or infection and/or
enhancing response to vaccination, comprising administering to a
subject in need thereof a composition according to claim 39.
43. The method according to claim 38, wherein the disease is an
immune disorder selected from the group consisting of asthma,
allergy, atopic dermatitis, allergic conjunctivitis, dust mite
allergy, urticaria and allergic rhinitis.
44. The method according to claim 42, wherein the subject suffers
from at least one of the following: (a) immunosenescence, (b) HIV
and/or AIDS, (c) cancer, (d) chronic obstructive pulmonary disease
and (e) diabetes.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a nutrition for improving
the immune system response and a process for its preparation.
BACKGROUND OF THE INVENTION
[0002] The immune system has different possible ways of reacting. A
decisive step for the type of immune response is the stimulation of
different T-cell subpopulations. So called Th1 cells predominantly
produce cytokines that stimulate a cellular immune response (IFN-y,
IL-12, IL-2), for instance to pathogens and vaccines. In contrast,
Th2 cells predominantly produce IL-4, IL-5 and IL-10. These
cytokines boost an IgE-mediated allergic reaction and inflammation.
Th1- and Th2-related cytokines act antagonistically and the Th1 and
Th2 responses are under normal physiological circumstances in a
well-controlled balance. Neither the Th 1 nor the Th2 response
prevails. If the responses are in imbalance, the dominance of
either Th1 or Th2 immune responses play a role in several
pathological conditions.
[0003] WO 2005/039319 and WO 2006/091103 relate to a preparation
comprising Bifidobacterium breve and a mixture of non-digestible
carbohydrates for non- or partially breast-fed infants as well as
the use thereof for the treatment or prevention of immune disorders
in non- or partially breast-fed infants. WO 2005/039597 relates to
the use of acid oligosaccharide and neutral oligosaccharide for
enhancing the immune system and the treatment and/or prevention of
immune system related disorders. WO 01/642255 relates to a
nutritional composition comprising a prebiotic for enhancement of
an immune response. WO 2004/0938998 and WO 2004/0938999 relate to
an immunomodulatory products obtained from a Bifidobacterium
culture.
[0004] Much effort is dedicated to find further solutions for
balancing and stimulating the immune system.
SUMMARY OF THE INVENTION
[0005] The present inventors have surprisingly found that a
combination of i) a product obtained by incubating an aqueous
substrate with bifidobacteria, wherein the substrate is at least
one selected from the group consisting of milk, milk protein, whey,
whey protein, whey protein hydrolysate, casein hydrolysate, and
lactose and subsequently inactivating the bifidobacteria by heating
the incubated mixture and/or removing the bifidobacteria cells from
the incubated mixture by centrifugation and/or filtration and ii)
at least two different non-digestible carbohydrates, wherein at
least one, preferably two, is selected from the group consisting of
fructo-oligosaccharides, galacto-oligosaccharides,
gluco-oligosaccharides, arabino-oligosaccharides,
mannan-oligosaccharides, xylo-oligosaccharides,
fuco-oligosaccharides, arabinogalacto-oligosaccharides,
glucomanno-oligosaccharides, galactomanno-oligosaccharides,
raffinose, lactosucrose, sialic acid comprising oligosaccharides
and uronic acid oligosaccharides synergistically stimulates the
immune-system. It was found that the present preparation, i.e. the
preparation comprising the combination as described above, lowered
the Th2 response and/or increased the Th1 response. The effect
observed was unexpectedly higher than the sum of the effects of the
single components.
[0006] In two different experimental animal models, the
unexpectedly higher effect was found. An increased response in
delayed type hypersensitivity reaction, indicative for an increased
Th1 response, was observed after vaccination with an influenza
vaccine in animals having consumed the present preparation,
compared to animals having consumed the single components. A
combination of a product obtained by incubating a substrate with
bifidobacteria and a product obtained by incubating with
streptococci plus two non-digestible carbohydrates resulted in an
even higher response in delayed type hypersensitivity reaction.
[0007] A decreased response in immediate type hypersensitivity
reaction, indicative for a decreased Th2 response, was observed
after exposure to an allergen via the lungs, in animals having
consumed the present preparation, compared to animals having
consumed the single components.
[0008] The synergistic effect between the combination of the
ingredients i) and ii) as defined above is surprising. It cannot be
explained by a symbiotic effect, wherein the non-digestible
carbohydrates (ii)) are specifically stimulating the growth of the
beneficial micro-organisms present in the same preparation, since
no living cells of bifidobacteria are present in the present
preparation. Removal and/or inactivation of living bifidobacteria
cells has the advantage that the preparation can be pasteurised
and/or sterilised, consequently reducing the chance of
contamination with harmful micro-organisms. This is especially
advantageous in infants, since infants have an increased intestinal
permeability. Additionally, since the bifidobacteria are removed or
inactivated they cannot cause infections themselves.
[0009] A further advantage is that the dose of bioactive components
received by each human subject can be better controlled. Also
advantageously storage of the product is more easily and with
reduced costs. Furthermore, advantageously no post-acidification
occurs in stored products, thereby avoiding adverse effects
relating to coagulation of proteins and adverse taste. Still a
further advantage is that inactivated and/or removed bifidobacteria
no longer able to breakdown and consume the non-digestible
carbohydrates.
[0010] The present preparation is especially advantageous for human
subjects shaving a reduced Th1 response in comparison with healthy
adults, in particular newborn infants, elderly humans suffering
from immunosenescence, humans suffering from AIDS or being infected
with the Human Immunodeficiency Virus, and cancer patients that are
or have been subjected to chemotherapy, radiation and cancer
patients that are cachectic.
[0011] The present preparation is especially suitable for the
treatment and/or prevention of diseases which can be prevented
and/or treated by a decrease in Th2 response, in particular
allergy, atopic dermatitis, asthma, food allergy, allergic rhinitis
(e. g. pollen allergy), dust mite allergy and other forms of
hypersensitivity like systemic anaphylaxis and acute urticaria.
[0012] The present preparation is suitable for treatment and/or
prevention of infections, and/or for supporting vaccination
response before, during and/or after vaccination.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The present invention concerns a process for the manufacture
of a preparation comprising the steps of:
[0014] a: incubating an aqueous substrate with bifidobacteria,
wherein the substrate comprises at least one selected from the
group consisting of milk, milk protein, whey, whey protein, whey
protein hydrolysate, casein hydrolysate, and lactose to obtain an
incubated mixture;
[0015] b: inactivating the bifidobacteria by heating the incubated
mixture and/or removing bifidobacteria cells from the incubated
mixture by centrifugation and/or filtration; and
[0016] c: combining a composition comprising the mixture obtained
in step a or obtained in step b, preferably obtained in step b with
at least two different non-digestible carbohydrates, wherein at
least one, preferably two, is selected from the group consisting of
fructo-oligosaccharides, galacto-oligosaccharides,
gluco-oligosaccharides, arabino-oligosaccharides,
mannan-oligosaccharides, xylo-oligosaccharides,
fuco-oligosaccharides, arabinogalacto-oligosaccharides,
glucomanno-oligosaccharides, galactomanno-oligosaccharides,
raffinose, lactosucrose, sialic acid comprising oligosaccharides
and uronic acid oligosaccharides.
[0017] In one aspect, the present invention concerns a preparation
obtainable by the process according to the present invention. In
one embodiment the invention concerns a nutritional composition
comprising or consisting of the preparation obtainable by the
process according to the present invention.
[0018] Also the invention concerns a method for the treatment
and/or prevention of a disease in a mammal, said method comprising
administering the present preparation to the mammal.
[0019] Also the invention concerns a method for providing nutrition
to an infant, said method comprising administering the present
preparation or nutritional composition comprising the present
preparation to the infant.
Process Comprising Incubating a Cubstrate with Bifobacteria
[0020] The present invention concerns a preparation obtainable or
obtained by incubating an aqueous substrate with bifidobacteria,
wherein the aqueous substrate comprises at least one selected from
the group consisting of milk, milk protein, whey, whey protein,
whey protein hydrolysate, casein hydrolysate, and lactose
(hereinafter referred to as step (a)). The incubated mixture
obtained or obtainable in step (a) is subjected to a step (b)
comprising an inactivation step by heat treatment and/or removal
step of the bifidobacteria cells by centrifugation and/or
filtration. Step (b) is performed in order to reduce the amount of
living bifidobacteria in the preparation, preferably by at least
90%, more preferably by at least 99%.
[0021] In one embodiment the incubation step comprises a
fermentation step and/or bioconversion step. During fermentation
the aqueous substrate is fermented by the bifidobacteria. During
bioconversion the aqueous substrate is bioconversed by the
bifidobacteria.
[0022] The preparation obtained or obtainable by (hereafter
wherever only `obtained` is mentioned, also `obtainable by` is
meant) the present process preferably comprises bacterial cell
fragments like glycoproteins, glycolipids, peptidoglycan,
lipoteichoic acid (LTA), lipoproteins, DNA, and/or capsular
polysaccharides. These fragments evoke an immunological response.
It is of advantage to use the product obtained by incubating an
aqueous substrate comprising at least one selected from the group
consisting of milk, milk protein, whey, whey protein, whey protein
hydrolysate, casein hydrolysate and lactose with bifidobacteria and
subsequently inactivating and/or removing the bifidobacteria, since
this will result in a higher concentration of bacterial cell
fragments. Upon incubation of the aqueous substrate with the
bifidobacteria, additional bio-active compounds may be formed, such
as organic acids, bioactive peptides and/or oligosaccharides, which
stimulate the immune system. When commercial preparations of
probiotics are used, the probiotic bacterial cells are usually
washed and separated from the aqueous growth medium that comprised
the bacterial cell fragments, thereby strongly reducing or even
eliminating the supernatant of the incubates substrate comprising
the bacterial cell fragments. In the present invention this is not
the case. The presence of intact cells (living or dead) is not
necessary for the immune response; the aqueous substrate itself,
after the present incubation step with bifidobacteria, has already
beneficial effects on the immune system.
Bifidobacteria
[0023] Bifidobacteria used for the present process are preferably
provided as a mono- or mixed culture. Bifidobacteria are
Gram-positive, anaerobic, rod-shaped lactic acid producing
bacteria. The present Bifidobacterium species preferably have at
least 95% identity of the 16 S rRNA sequence when compared to the
type strain of the respective Bifidobacterium species, more
preferably at least 97% identity as defined in handbooks on this
subject for instance Sambrook, J., Fritsch, E. F., and Maniatis, T.
(1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold
Spring Harbor (N.Y.) Laboratory Press. The Bifodobacteria
preferably used are also described by Scardovi, V. Genus
Bifidobacterium. p. 1418-p. 1434. In: Bergey's manual of systematic
Bacteriology. Vol. 2. Sneath, P. H. A., N. S. Mair, M. E. Sharpe
and J. G. Holt (ed.). Baltimore: Williams & Wilkins. 1986. 635
p. Preferably the bifidobacteria used in producing the present
preparation is at least one Bifidobacterium selected from the group
consisting of B. breve, B. infantis, B. bifidum, B. catenulatum, B.
adolescentis, B. thermophilum, B. gallicum, B. animalis or lactis,
B. angulatum, B. pseudocatenulatum, B. thermacidophilum and B.
longum more preferably B. breve, B. infantis, B. bifidum, B.
catenulatum, B. longum, more preferably B. longum and B. breve,
even more preferably B. breve, most preferably B. breve I-2219
deposited at the CNCM in Paris, France. B. breve CNCM I-2219 was
deposited under the Budapest Treaty at the Collection Nationale de
Cultures de Microorganisms van Institute Pasteur, Paris, France on
31 May 1999 by Compagnie Gervais Danone. This strain was published
in WO 2004/093899.
[0024] Preferably the composition also comprises a product obtained
by incubating an aqueous substrate comprising at least one selected
from the group consisting of milk, milk protein, whey, whey
protein, whey protein hydrolysate, casein, casein hydrolysate and
lactose, preferably the group of whey and lactose with streptococci
and preferably subsequently inactivating and/or removing the
streptococci. Streptococci are Gram-positive, anaerobic,
coccoid-shaped lactic acid producing bacteria. The Streptococcus
species preferably have at least 95% identity of the 16 S rRNA
sequence when compared to the type strain of the respective
Streptococcus species, more preferably at least 97% identity as
defined in handbooks on this subject for instance Sambrook, J.,
Fritsch, E. F., and Maniatis, T. (1989), Molecular Cloning, A
Laboratory Manual, 2nd ed., Cold Spring Harbor (N.Y.) Laboratory
Press. Preferably production of a product obtained by incubating an
aqueous substrate with streptococci and subsequently inactivating
the streptococci is performed with Streptococcus species selected
from the group consisting of S. salivarius and S. thermophilus,
more preferably S. thermophilus, even more preferably strain S.
thermophilus CNCM I-1620 or strain CNCM I-1470, most preferably
strain CNCN I-1620. S. thermophilus CNCM I-1620 and I-1470
advantageously produces high amounts of beta-galactosidase. S.
thermophilus CNCM I-1620 was deposited under the Budapest Treaty on
23 Aug. 1995 at Collection Nationale de Cultures de Microorganisms
van Institute Pasteur, Paris, France by Compagnie Gervais Danone.
S. thermophilus CNCM I-1470 was deposited under the Budapest Treaty
on 25 Aug. 1994 at Collection Nationale de Cultures de
Microorganisms van Institute Pasteur, Paris, France by Compagnie
Gervais Danone. These strains were published in EP 778885.
Process Step a) Incubation the Aqueous Substrate
[0025] Step (a) is preferably performed by:
[0026] a1 inoculating bifidobacteria in the aqueous substrate in
amount of between 1.times.10.sup.2 to 1.times.10.sup.11 cfu
bifidobacteria/ml, said aqueous substrate having a pH of between 4
and 8, and comprising at least one selected from the group
consisting of milk, whey, whey protein, whey protein hydrolysate,
casein hydrolysate, and lactose,
[0027] a2 incubating said bifidobacteria in said aqueous medium,
under aerobic or anaerobic conditions and at a temperature of
20.degree. C. to 50.degree. C., for at least 2 h.
[0028] The aqueous substrate to be incubated with bifidobacteria
comprises at least one, more preferably at least two, selected from
the group consisting of milk, whey, whey protein, whey protein
hydrolysate, casein hydrolysate, and lactose. Preferably the
substrate does not comprise intact casein. It was found that less
immunostimulatory substances were formed when the aqueous substrate
comprised high amounts of intact casein. Therefore the aqueous
substrate comprises preferably less than 25 g/l casein, more
preferably less than 15 g/l, even more preferably less than 5 g/l,
most preferably less than 1 g/l intact casein. The aqueous
substrate therefore even more preferably comprises whey and/or whey
protein and/or whey protein hydrolysate.
[0029] Milk can be whole milk, semi-skimmed milk and/or skimmed
milk. Preferably skimmed milk is used. Whey can be sweet whey, acid
whey or whey from which the casein has been removed for example by
filtration or whey permeate. Preferably the whey is present in a
concentration of 3 to 80 g dry weight per liter (l) aqueous
substrate, more preferably 40 to 60 g per l. Preferably whey
protein concentrate is used. Preferably whey protein hydrolysate is
used and is present in an amount of 2 to 80 g dry weight per l
aqueous substrate, more preferably 5 to 15 g/l. Preferably lactose
is present in an amount of 5 to 50 g dry weight per l aqueous
substrate, more preferably 1 to 30 g/l. Preferably the aqueous
substrate comprises buffer salts in order to keep the pH within a
desired range. Preferably sodium or potassium dihydrogen phosphate
is used as buffer salt, preferably in an amount of 0.5 to 5 g/l,
more preferably 1.5 to 3 g per l. Preferably the aqueous substrate
comprises cysteine in amount of 0.1 to 0.5 g per l aqueous
substrate, more preferably 0.2 to 0.4 g/l. The presence of cysteine
results in low redox potential of the substrate which is
advantageous for activity of lactic acid producing bacteria,
particularly bifidobacteria. Preferably the aqueous substrate
comprises yeast extract in an amount of 0.5 to 5 g/l aqueous
substrate, more preferably 1.5 to 3 g/l. Yeast extract is a rich
source of enzyme co-factors and growth factors for lactic acid
producing bacteria. The presence of yeast extract will enhance the
bioconversion and/or fermentation by bifidobacteria.
[0030] Preferably the aqueous substrate to be incubated comprises a
high concentration of solids, preferably more than 20 wt. % solids
based on volume, more preferably more than 40 wt. % solids. A high
concentration is advantageous when performing the further
processing steps, such as for example spray drying, centrifugation
or filtration.
[0031] Suitably the aqueous substrate is pasteurised before the
incubation step, in order to eliminate the presence of unwanted
living bacteria. Suitably the product is pasteurised after
incubation, in order to inactivate enzymes. Suitably the enzyme
inactivation takes place at 75.degree. C. for 1 min. Suitably the
enzyme inactivation takes place at 75.degree. C. for 3 min Suitably
the aqueous substrate is homogenised before and/or after the
incubation step. Homogenisation results in a more stable product,
especially in the presence of fat (lipids).
[0032] The inoculation density is preferably between
1.times.10.sup.2 to 1.times.10.sup.11, preferably between
1.times.10.sup.4 to 1.times.10.sup.10 cfu bifidobacteria per ml
aqueous substrate, more preferably between 1.times.10.sup.7 to
1.times.10.sup.9 cfu bifidobacteria/ml aqueous substrate. Methods
for obtaining a concentrated starter culture of bifidobacteria to
be inoculated in the aqueous substrate are known in the art. The
final bacteria density of bifidobacteria after incubation is
preferably between 1.times.10.sup.3 to 1.times.10.sup.11, more
preferably between 1.times.10.sup.4 to 1.times.10.sup.9 cfu/ml
aqueous substrate.
[0033] The incubation with bifidobacteria is preferably performed
at a temperature of approximately 20.degree. C. to 50.degree. C.,
more preferably 30.degree. C. to 45.degree. C., even more
preferably approximately 37.degree. C. to 42.degree. C. The optimum
temperature for growth and/or activity for bifidobacteria is
between 37.degree. C. and 42.degree. C.
[0034] The incubation with bifidobacteria is preferably under
anaerobic conditions, since the growth of bifidobacteria and the
enzymatic activity of many enzymes of bifidobacteria are impaired
under aerobic conditions. However acidification is not always
desired. Thus, in one embodiment, the incubation step suitably
takes place under aerobic conditions.
[0035] The incubation with bifidobacteria is preferably performed
at a pH of 4 to 8, more preferably 5.6 to 7.5, even more preferably
6 to 7.5. This pH does not induce protein precipitation and/or an
adverse taste, while at the same time bifidobacteria are able to
interact with the aqueous substrate.
[0036] The incubation time is preferably at least 2 h, preferably
between 4 and 48 h, more preferably between 6 and 24 h, even more
preferably between 6 and 15 h. A sufficient long time enables the
interaction between the bifidobacteria and the aqueous substrate
and/or the production of cell fragments such as glycoproteins,
glycolipids, peptidoglycan, lipoteichoic acid (LTA), lipoproteins,
DNA and/or capsular polysaccharides to take place to a large
extent, whereas the incubation time need not be unnecessarily long
for economical reasons.
Methods of Inactivation and/or Physically Removal of Living Cells
of Bifidobacteria
[0037] In step (b) of the present process living cells
bifidobacteria are after incubation in step a) preferably
essentially all eliminated, for example by inactivation by heat
treatment and/or physical removal. The cells are preferably
inactivated by heat treatment. Preferably the bifidobacteria are
heat killed after incubation step a). Preferable ways of heat
killing are pasteurization, sterilization, ultra high temperature
treatment, spray cooking and/or spray drying at temperatures
bifidobacteria do not survive. The heat treatment is preferably
performed at at least 50.degree. C., more preferably at at least
65.degree. C. The heat treatment is preferably performed for at
least 5 minutes, more preferably for at least 10 minutes. The heat
treatment is preferably performed for at least 5 minutes at at
least 50.degree. C., more preferably for at least 10 minutes at at
least 65.degree. C. The heat treatment is preferably performed for
at least 1 minutes at at least 75.degree. C., more preferably for
at least 3 minutes at at least 75.degree. C.
[0038] Preferably intact cells of bifidobacteria are removed from
the incubated product by physical elimination such as filtration
and/or centrifugation, for example centrifugation for 1 h at 3000
g, with the intact cells remaining in the pellet or retentate and
the product obtained by incubating a milk and/or milk-derived
substrate with bifidobacteria and subsequently inactivating the
bifidobacteria cell fragments remaining in the supernatant and/or
filtrate, respectively.
[0039] The heat inactivation and/or physical removal of living
cells is such that the amount of living bifidobacteria after
treatment is below the detection limit as used by conventional
plating techniques known in the art. This detection limit is less
than 10.sup.4 cfu living cells of bifidobacteria based on g dry
weight composition, more preferably less than 10.sup.3 cfu/g.
Preferably the heat inactivation and/or removal step is such that
at least 90, more preferably at least 99% of the cells present in
the incubated mixture after step a) is eliminated.
[0040] The requirement that living cells are inactivated has the
advantage that, after production, the final nutritional composition
can be pasteurised and/or sterilised, consequently reducing the
chance of contamination with harmful micro-organisms. So the
present invention enables liquid, ready-to-use formula to be
prepared and stored at room temperature. Furthermore, the dose of
bioactive components received by each human subject can be more
easily controlled, since no further growth in a liquid product
occurs, nor growth in the intestinal tract of the human subject.
The latter is a variable factor depending on the individual's
intestinal environment, and thereby leads to variations in the
extent of beneficial effects in individual subjects. Still a
further advantage is that inactivated and/or removed bifidobacteria
and streptococci no longer are able to breakdown and consume the
non-digestible carbohydrates.
[0041] Additional advantages are that the nutritional composition
can be stored more easily and with reduced costs, since no special
precautions have to be taken to maintain the viability of
bifidobacteria at an acceptable level. This is especially the case
in products with a water activity above 0.3. Also no
post-acidification occurs in stored products with a high water
activity and/or in powdered nutritional compositions in the period
after reconstitution with water and before consumption. Adverse
effects relating to coagulation of proteins and adverse taste are
avoided in this way.
Addition of Additional Components and Other Optional Process
Steps
[0042] Optionally one or more of the following steps may follow the
above process step b): [0043] i) Ultrafiltrating the product after
incubation through filtration membranes having a cut-off threshold
between 100 and 300 kDa, so as to obtain a concentrated retentate.
The membranes are preferably polyethersulfone membranes and
filtration is preferably performed at a temperature below
60.degree. C. [0044] ii) Washing the concentrated retentate with
water. [0045] iii) Dehydrating the concentrated retentate,
preferably by lyophilisation. [0046] iv) Dissolving the dehydrated
retentate in a buffer, preferably a Tris buffer with pH 6-8. [0047]
v) Performing gel exclusion chromatography of the retentate
solution, on a column having an exclusion threshold of 600 kDa,
preferably a Dextran or agarose column such as Superdex.RTM. 200.
[0048] vi) Recovery of the filtered or excluded fraction at the end
of the chromatography [0049] vii) Desalting the product with a
membrane with a cut-off of 10 kDa. Recovering the excluded fraction
at the end of the chromatography.
[0050] These steps are preferably performed under sterile
conditions. Additional ingredients that may be beneficial for
obtaining the desired final nutritional composition may be added
after process step a), preferably immediately prior to step b) or
after process step b). Preferably these are added after step b).
For an infant milk formula, ingredients such as skimmed milk, whey,
lactose, vegetable fat, minerals, vitamins, as known in the art may
be added.
[0051] Preferably, an aqueous substrate comprising whey, whey
protein and/or whey protein hydrolysate, is pasteurized, cooled and
incubated with one or more Bifidobacterium strains, preferably B.
breve strain CNCM I-2219, upon which the incubated product is heat
treated and stored. Optionally the incubated product is mixed with
other components making up the nutritional composition. A fat
component may or may not be included, but preferably a fat
component is not yet included at this stage. Preferably, the
mixture is preheated, and subsequently fat (also the term `lipids`
is used herein) is added in-line, homogenized, heat-treated and
dried.
[0052] Another preferred method for preparing the incubated product
of the present invention is disclosed in WO 01/01785, more
particular in example 1 and 2. Another preferred method for
preparing the incubated product of the present invention is
described in WO 2004/093899, more particularly in example 1.
[0053] Preferably the final nutritional composition comprises from
5 to 100 wt. % based on dry weight of the preparation obtained by
step b, more preferably from 5 to 99.5 wt. %, more preferably from
5 to 95 wt. %, even more preferably from 5 to 80 wt. %, even more
preferably from 5 to 40 wt. %, most preferably from 10 to 40 wt. %.
Preferably, the final nutritional composition comprises from 0.5 to
20 wt. % of a product obtained by step b per 100 ml, more
preferably 0.5 to 14 wt. %, more preferably 1 to 10 wt. %, even
more preferably 1 to 5 wt. % per 100 ml.
[0054] Preferably the present final nutritional composition
comprises inactivated bifidobacteria and/or bacterial fragments
derived from bifidobacteria obtained from more than
1.times.10.sup.3 cfu bifidobacteria per g, based on dry weight of
the final composition, more preferably more than
1.times.10.sup.4cfu, even more preferably more than
1.times.10.sup.5 cfu. Preferably the inactivated bifidobacteria
and/or bacterial fragments derived from bifidobacteria are obtained
from less than 1.times.10.sup.11 cfu bifidobacteria per g, based on
dry weight of the final composition, more preferably less than
1.times.10.sup.10 cfu, even more preferably less than
1.times.10.sup.9 cfu. These numbers can be calculated by
determining the amount of bifidobacteria in the mixture after
incubation as in step a) and before step b), and subsequently
taking into account how many gram of the present preparation is
present in the final composition based on dry weight.
[0055] Additional ingredients that may be beneficial for obtaining
the desired final nutritional composition may be added after
process step a) or b). Preferably these are added after step a).
For an infant milk formula, ingredients such as skimmed milk, whey,
lactose, vegetable fat, minerals, vitamins, as known in the art may
be added.
[0056] Preferably the process comprises the additional steps of
[0057] d: incubating a substrate with Streptococcus thermophilus,
preferably strain S. thermophilus CNCM I-1620 or strain CNCM
I-1470, wherein the substrate is selected from the group consisting
of milk, milk protein, whey, whey protein, whey protein
hydrolysate, casein, casein hydrolysate, and lactose, to obtain an
incubated mixture and
[0058] e: inactivating the S. thermophilus by heating the incubated
mixture of step e and/or removing S. thermophilus cells from the
incubated mixture of step e by centrifugation and/or filtration.
Step e and b are preferably performed simultaneously. Preferably
intact cells of streptococci are removed from the incubated product
by physical elimination such as filtration and/or centrifugation,
for example centrifugation for 1 h at 3000 g, with the intact cells
remaining in the pellet or retentate and the product obtained by
incubating a milk and/or milk-derived substrate with streptococci
and subsequently inactivating the streptococcal cell fragments
remaining in the supernatant and/or filtrate, respectively.
[0059] The heat inactivation and/or physical removal of living
cells is such that the amount of living streptococci after
treatment is below the detection limit as used by conventional
plating techniques known in the art. This detection limit is less
than 10.sup.4 cfu living cells of streptococci based on g dry
weight composition, more preferably less than 10.sup.3 cfu/g.
Hence, preferably in one embodiment according to the invention the
preparation after step e comprises less than 10.sup.3 cfu living
streptococci per g dry weight of the preparation. Preferably the
heat inactivation and/or removal step is such that at least 90,
more preferably at least 99% of the cells present in the incubated
mixture after step d) is eliminated.
[0060] Another preferred method for preparing the incubated product
with S. thermophilus strains of the present invention is disclosed
in EP 0778885, more particular in example 5 and 6. Another
preferred method for preparing the incubated product with S.
thermophilus strains of the present invention is disclosed in
FR2723960 examples 2 to 6.
[0061] Preferably, an aqueous substrate comprising whey, whey
protein and/or whey protein hydrolysate, is pasteurized, cooled and
incubated with one or more Bifidobacterium strains, preferably B.
breve strain CNCM I-2219, upon which the incubated product is heat
treated and stored. Preferably a second aqueous substrate
comprising whey and/or lactose is incubated with S. thermophilus,
preferably strain CNCM I-1620 or strain CNCM I-1470. Subsequently,
the two incubated products are preferably mixed together and mixed
with other components making up the nutritional composition. A fat
component may or may not be included, but preferably a fat
component is not yet included at this stage. Preferably, the
mixture is preheated, and subsequently fat (also the term `lipids`
is used herein) is added in-line, homogenized, heat-treated and
dried.
[0062] The incubation step d may be performed simultaneously with
the incubation step with bifidobacteria in step a. Preferably the
incubation with S. thermophilus is performed in a separate process
step from the incubation with bifidobacteria. Separate incubation
allows optimum conditions for each of the different bacteria and/or
prevents unwanted interference of the different bacteria with the
release of immunostimulatory components. Preferably the incubated
mixture obtained after incubation of the substrate with
streptococci is added to the mixture obtained in step a, step b, or
step c, more preferably after step a). An improved effect on
delayed hyper hypersensitivity response is observed when the
present composition also comprised a mixture obtained after
incubation with S. thermophilus. Thus in one embodiment the process
according to the invention comprises the further step of:
[0063] f: combining the incubated mixture obtained in step d or e,
preferably step d, with the incubated mixture obtained in step a,
b, or c, preferably in step a. In one embodiment the incubated
mixture obtained in step d is combined with the incubated mixture
obtained in step a and steps b and e are performed
simultaneously.
[0064] Preferably the final nutritional composition comprises from
2 to 94.5% based on dry weight of the preparation obtained by step
d, more preferably from 5 to 80 wt. %, even more preferably from 5
to 40 wt. %. Preferably, the final nutritional composition
comprises from 0.2 to 20 wt. % of a product obtained by step d per
100 ml, more preferably 0.5 to 14 wt. %, more preferably 1 to 10
wt. %, even more preferably 1 to 5 wt. % per 100 ml.
Non-Digestible Carbohydrates
[0065] The preparation obtained by the present process comprises at
least two different non-digestible carbohydrates. These
non-digestible carbohydrates are added in process step c). The
non-digestible carbohydrates advantageously stimulate the immune
system. This stimulation may occur via an improvement of the
intestinal microbiota and/or via a direct effect on the immune
system. The presence of two different non-digestible carbohydrates
synergistically improves the intestinal flora and/or
synergistically stimulates the immune system.
[0066] The presence of both two non-digestible carbohydrates and a
product obtained by incubating an aqueous substrate with
bifidobacteria and subsequently inactivating and/or removing the
bifidobacteria acts synergistically on the immune response. The
combination unexpectedly synergistically results in a higher Th1
response, a lower Th2 response and/or an increased Th1/Th2 response
ratio. The combination synergistically increases vaccination
response, synergistically improves allergic reaction and/or
increases resistance against infections. The synergistic effect
between these two compounds was unexpected and cannot be explained
by a symbiotic effect, wherein the non-digestible carbohydrates are
specifically stimulating the growth of the beneficial
micro-organisms present in the same preparation, since no living
bifidobacteria are present in the incubated milk and/or
milk-derived product.
[0067] The term "oligosaccharide" as used in the present invention
refers to carbohydrates with a degree of polymerization (DP) of 2
to 250, preferably a DP 2 to 100, more preferably 2 to 60, even
more preferably 2 to 10. If the oligosaccharide with a DP of 2 to
100 is included in the present preparation, this includes
compositions which contain oligosaccharides with a DP between 2 and
5, a DP between 50 and 70 and a DP of 7 to 60. The term
"non-digestible carbohydrate" as used in the present invention
refers to carbohydrates which are not digested in the intestine by
the action of acids or digestive enzymes present in the human upper
digestive tract (small intestine and stomach) but which are
preferably fermented by the human intestinal microbiota. For
example, sucrose, lactose, maltose and maltodextrins are considered
digestible.
[0068] Preferably the present non-digestible carbohydrate is
soluble. The term "soluble" as used herein, when having reference
to a carbohydrate, means that the carbohydrate is soluble according
to the method described by L. Prosky et al., J. Assoc. Off. Anal.
Chem. 71, 1017-1023 (1988).
[0069] Different non-digestible carbohydrates in the present
invention relates to non-digestible carbohydrates differing in
monosaccharide unit composition, or differing in degree of
polymerization (DP) or both. Two non-digestible carbohydrates
differ in monosaccharide composition when there is at least 30 mol
% difference, more preferably at least 50 mol % difference in
monosaccharide composition based on total mol monosaccharide units.
For instance galacto-oligosaccharides with an average composition
of Glu-Gal.sub.3 and fructo-oligosaccharides with an average
composition of Glu-Fru.sub.3 differ for 75 mol %. Two
non-digestible carbohydrates differ in DP if the average DP of the
two carbohydrates differs more than 5 monosaccharide units,
preferably more than 10 units, even more preferably more than 15
units. For example hydrolysed inulin with an average DP of 4 and
long chain inulin with an average DP of 25 have a difference in DP
of 21 units.
[0070] The non-digestible carbohydrates are at least one, more
preferably at least two, selected from the group consisting of
fructo-oligosaccharides, galacto-oligosaccharides,
gluco-oligosaccharides, arabino-oligosaccharides,
mannan-oligosaccharides, xylo-oligosaccharides,
fuco-oligosaccharides, arabinogalacto-oligosaccharides,
glucomanno-oligosaccharides, galactomanno-oligosaccharides, sialic
acid comprising oligosaccharides and uronic acid oligosaccharides.
Preferably the present preparation comprises
fructo-oligosaccharides, galacto-oligosaccharides and/or
galacturonic acid oligosaccharides, more preferably
galacto-oligosaccharides, most preferably
beta-galacto-oligosaccharides. The group of fructo-oligosaccharides
includes inulin, the group of galacto-oligosaccharides includes
transgalacto-oligosaccharides or beta-galacto-oligosaccharides, the
group of gluco-oligosaccharides includes gentio-, nigero- and
cyclodextrin-oligosaccharides and polydextrose, the group of
arabinogalacto-oligosaccharides includes gum acacia, and the group
of galactomanno-oligosaccharides includes partially hydrolysed guar
gum.
[0071] For further improvement, the present non-digestible
carbohydrates preferably have a relatively high content of short
chain oligosaccharides, as these strongly stimulate the growth of
bifidobacteria. Hence, preferably at least 10 wt. % of the
non-digestible carbohydrates in the present preparation has a DP of
2 to 5 (i.e. 2, 3, 4, and/or 5) and at least 5 wt. % has a DP of 10
to 60. Preferably at least 50 wt. %, more preferably at least 75
wt. % of the non-digestible carbohydrates has a DP of 2 to 9 (i.e.
2, 3, 4, 5, 6, 7, 8, and/or 9).
[0072] More preferably the preparation obtained by the present
process comprises galacto-oligosaccharides. The
galacto-oligosaccharides are preferably selected from the group
consisting of beta-galacto-oligosaccharides, lacto-N-tetraose
(LNT), lacto-N-neotetraose (neo-LNT), fucosyl-lactose, fucosylated
LNT and fucosylated neo-LNT. In a particularly preferred embodiment
the present preparation comprises beta-galacto-oligosaccharides.
Beta-galacto-oligosaccharides as used in the present invention
refers to oligosaccharides composed of over 50%, preferably over
65% galactose units based on monomeric subunits, with a degree of
polymerization (DP) of 2 to 20, in which at least 50%, more
preferably at least 75%, even more preferably at least 90%, of the
galactose units are linked together via a beta-glycosidic linkage,
preferably a beta-1,4 glycosidic linkage. Beta-linkages are also
predominant in human milk oligosaccharides. The average DP is
preferably in the range of 3 to 6. A glucose unit may be present at
the reducing end of the chain of galactose units.
Beta-galacto-oligosaccharides are sometimes also referred to as
transgalacto-oligosaccharides (TOS). A suitable source of
beta-galacto-oligosaccharides is Vivinal.RTM.GOS (commercially
available from Borculo Domo Ingredients, Zwolle, Netherlands).
Other suitable sources are Oligomate (Yakult), Cupoligo, (Nissin)
and Bi2muno (Classado). Beta-galacto-oligosaccharides were found to
stimulate the growth of lactic acid producing bacteria, especially
bifidobacteria.
[0073] Preferably the preparation obtained by the present process
comprises fructo-oligosaccharides. Fructo-oligosaccharides as used
in the present invention refers to carbohydrates composed of over
50%, preferably over 65% fructose units based on monomeric
subunits, in which at least 50%, more preferably at least 75%, even
more preferably at least 90%, of the fructose units are linked
together via a beta-glycosidic linkage, preferably a beta-2,1
glycosidic linkage. A glucose unit may be present at the reducing
end of the chain of fructose units. Preferably the
fructo-oligosaccharide has a DP or average DP in the range of 2 to
250, more preferably 2 to 100, even more preferably 10 to 60.
Fructo-oligosaccaride comprises levan, hydrolysed levan, inulin,
hydrolysed inulin, and synthesised fructo-oligosaccharides.
Preferably the preparation comprises short chain
fructo-oligosaccharides with an average degree of polymerization
(DP) of 3 to 6, more preferably hydrolysed inulin or synthetic
fructo-oligosaccharide. Preferably the preparation comprises long
chain fructo-oligosaccharides with an average DP above 20.
Preferably the preparation comprises both short chain and long
chain fructo-oligosaccharides. Fructo-oligosaccharide suitable for
use in the process of the invention is also readily commercially
available, e.g. RaftilineHP (Orafti).
[0074] More preferably the preparation obtained by the process
according to the invention comprises a combination of
galacto-oligosaccharides and fructo-oligosaccharides, more
preferably long chain fructo-oligosaccharides. Such a mixture
synergistically stimulates the growth of a healthy intestinal
microbiota, particularly bifidobacteria.
[0075] The preparation obtained by the process according to the
invention preferably comprises uronic acid oligosaccharides, more
preferably mannonuric acid and/or galacturonic acid
oligosaccharides, even more preferably galacturonic acid. The term
uronic acid oligosaccharide as used in the present invention refers
to an oligosaccharide wherein at least 50% of the monosaccharide
units present in the oligosaccharide is uronic acid. The term
galacturonic acid oligosaccharide as used in the present invention
refers to an oligosaccharide wherein at least 50% of the
monosaccharide units present in the oligosaccharide is galacturonic
acid. The galacturonic acid oligosaccharides used in the invention
are preferably prepared from degradation of pectin, pectate, and/or
polygalacturonic acid Preferably the degraded pectin is prepared by
hydrolysis and/or beta-elimination of fruit and/or vegetable
pectins, more preferably apple, citrus and/or sugar beet pectin,
even more preferably apple, citrus and/or sugar beet pectin
degraded by at least one lyase. In a preferred embodiment, at least
one of the terminal galacturonic acid units of the galacturonic
acid oligosaccharide has a double bond. The double bond effectively
protects against attachment of pathogenic bacteria to intestinal
epithelial cells. Preferably one of the terminal galacturonic acid
units comprises a C.sub.4-C.sub.5 double bond. The galacturonic
acid oligosaccharide can be derivatised. The galacturonic acid
oligosaccharide may be methoxylated and/or amidated. Preferably the
galacturonic acid oligosaccharides are characterised by a degree of
methoxylation above 20%, preferably above 50% even more preferably
above 70%. Uronic acid oligosaccharides advantageously reduce the
adhesion of pathogenic micro-organisms to the intestinal epithelial
cells. Furthermore, uronic acid oligosaccharides stimulate the
immune system by increasing the Th1 response.
[0076] Thus, in one embodiment the preparation obtained by the
process according to the invention and for use according to the
present invention preferably comprises at least
beta-galacto-oligosaccharides. In one embodiment the preparation
obtained by the process according to the invention and for use
according to the present invention preferably comprises at least
short chain fructo-oligosaccharides and/or long chain
fructo-oligosaccharides, preferably long chain
fructo-oligosaccharides. In one embodiment the preparation obtained
by the process according to the invention and for use according to
the present invention preferably comprises at least uronic acid
oligosaccharides. In one embodiment the preparation for use
according to the present invention preferably comprises at least
beta-galacto-oligosaccharides and at least short chain
fructo-oligosaccharides or long chain fructo-oligosaccharides or
both. In one embodiment the preparation for use according to the
present invention preferably comprises at least
beta-galacto-oligosaccharides and at least uronic acid
oligosaccharides. In one embodiment the preparation for use
according to the present invention preferably comprises at least
short chain fructo-oligosaccharides and uronic acid
oligosaccharides or long chain fructo-oligosaccharides and uronic
acid oligosaccharides. In one embodiment the preparation for use
according to the present invention preferably comprises at least
beta-galacto-oligosaccharides and short chain
fructo-oligosaccharides and uronic acid oligosaccharides or at
least beta-galacto-oligosaccharides and long chain
fructo-oligosaccharides and uronic acid oligosaccharides. A
combination of uronic acid oligosaccharides together with a a
combination of galacto-oligosaccharides and/or
fructo-oligosaccharides results in a higher Th1 response than the
uronic acid oligosaccharides or galactose and/or
fructo-oligosaccharides alone.
[0077] Preferably the weight ratio between the mixture of two
different non-digestible carbohydrates, preferably
beta-galacto-oligosaccharides and fructo-oligosaccharide, is
between 20 and 0.05, more preferably between 20 and 1.
Beta-galacto-oligosaccharides are more reminiscent to the human
milk oligosaccharides. Preferably the present preparation comprises
beta-galacto-oligosaccharides with a DP of 2-10 and/or
fructo-oligosaccharides with a DP of 2-60. This combination was
found to synergistically increase bifidobacteria and lactobacilli.
The presence of these three non-digestible oligosaccharides even
further stimulates the bifidobacteria. The weight ratio
transgalacto-oligosaccharide:fructo-oligosaccharide:uronic acid
oligosaccharide is preferably (20 to 2) : 1 : (1 to 20), more
preferably (12 to 7) : 1 : (1 to 3).
[0078] Preferably, the final nutritional composition consisting of
or comprising the preparation obtained by the process according to
the invention comprises 80 mg to 2 g non-digestible carbohydrates
per 100 ml, more preferably 150 mg to 1.50 g, even more preferably
300 mg to 1 g. Based on dry weight, the nutritional composition
preferably comprises 0.25 wt. % to 20 wt. %, more preferably 0.5
wt. % to 10 wt. %, even more preferably 1.5 wt. % to 7.5 wt. %
non-digestible carbohydrates. A lower amount of non-digestible
carbohydrate will be less effective in stimulating the immune
system and/or beneficial bacteria in the microbiota, whereas a too
high amount will result in side-effects of bloating and abdominal
discomfort.
[0079] The two different non-digestible carbohydrates are added
(i.e. step c) after step a) preferably immediately prior to step b)
or after step b), preferably step c) is conducted after step b),
preferably step c) is conducted after step e), i.e. after
inactivation by heat treatment and/or removal of the bifidobacteria
and optionally S. thermophilus.
[0080] Preferably the preparation obtained by the process according
to the invention comprises an aqueous substrate comprising at least
one of the group selected from milk, milk protein, whey, whey
protein, whey protein hydrolysate and lactose incubated with B.
breve, more preferably strain CNCM I-2219, and at least one,
preferably two non-digestible carbohydrates from the group
consisting of galacto-oligosaccharides and
fructo-oligosaccharides.
[0081] In one aspect the present invention concerns the present
process, wherein in step c only one non-digestible carbohydrate is
added. It is particularly advantageous that the only one non
digestible carbohydrate is fructo-oligosaccharide.
[0082] In one aspect, the invention concerns a preparation
obtainable by the process according to the present invention as
described above. In one embodiment the final nutritional
composition consisting of or comprising the preparation obtained by
the present process comprises 0.5 to 10 g non-digestible
carbohydrate as defined above per 100 g dry weight of the
composition. In one embodiment the final nutritional composition
consisting of or comprising the preparation obtained by the present
process has a viscosity of 1 to 60 mPas at a shear rate of 100
s.sup.-1 at 20 .degree. C.
[0083] Preferably the above process comprises a drying step. Drying
steps are known in the art. A suitable drying step is spray drying.
Preferably the drying step is performed in such a way that the
dried product is a powder comprising less than 10 wt. % water, more
preferably less than 5 wt. %. Preferably the drying step is
performed after step c. Alternatively, the drying step may be
performed after step b and/or after step e, after which the
non-digestible oligosaccharides are dry blended in the product.
Nutrition
[0084] It was found that the present preparation can be
advantageously applied in food, such as baby food and clinical
food. The present preparation or composition comprising the present
preparation is preferably enterally administered, more preferably
orally. Preferably the composition is a complete nutrition.
[0085] Preferably the nutrition is suitable for administration to
infants. More preferably the present nutritional composition is an
infant or follow on formula. The present composition can be
advantageously applied as a complete nutrition for infants.
[0086] Preferably the present composition is an infant nutrition
comprising based on dry weight of the infant nutrition
[0087] i) from 0.5 to 10 wt. % of the sum of
galacto-oligosaccharides and fructo-oligosaccharides, and
[0088] ii) from 5 to 99.5 wt. % of the preparation obtained after
step b according to the present process, wherein the bifidobacteria
in step a belong to the species B. breve, preferably strain B.
breve CNCM I-2219
[0089] iii) and optionally 2 to 94.5 wt. % of the preparation
obtained after step e.
[0090] Such nutrition preferably comprises lipid, protein and
carbohydrate and is preferably administered in liquid form. The
term "liquid food" as used in the present invention includes dry
food (e. g. powders) which are accompanied with instructions as to
admix said dry food mixture with a suitable liquid (e. g.
water).
[0091] Hence, the nutritional composition of the present invention
preferably comprises between 5 and 60% lipids based on total of
calories, between 5 and 60% protein based on total calories,
between 15 and 90% digestible carbohydrate based on total calories.
Preferably the present nutritional composition comprises between 5
and 30% lipid based on total calories, between 15 and 40% protein
based on total calories and between 25 and 75% digestible
carbohydrate based on total calories when intended for adult human
subjects. Preferably the present nutritional composition comprises
between 30 and 60% lipid based on total calories, between 5 and 15%
protein based on total calories and between 25 and 75% digestible
carbohydrate based on total calories, more preferably 35 to 50%
lipids based on total calories, 7.5 to 12.5% proteins based on
total calories, and 40 to 55% digestible carbohydrate based on
total calories when intended for infants. For calculation of the %
protein based on total calories, the total of calories provided by
proteins, peptides and amino acids needs to be taken into
account.
[0092] Preferably the lipids comprise vegetable oils. The vegetable
lipid is preferably at least one selected from the group consisting
of soy oil, palm oil, coconut oil, safflower oil, sunflower oil,
corn oil, canola oil and lecithins. Preferably a combination of
vegetable lipids and at least one oil selected from the group
consisting of fish oil and omega-3 containing vegetable, algae or
bacterial oil is used. In a preferred embodiment, the present
method further comprises the administration of long-chain
polyunsaturated acid (LC-PUFA). As it is believed that these act on
the immune system via a mechanism different from the non-digestible
carbohydrates and the product obtained by incubating a milk and/or
milk-derived substrate with bifidobacteria and subsequently
inactivating the bifidobacteria, the combination of the present
invention with the LC-PUFA is deemed to act synergistically.
[0093] The nutritional composition of the present invention
preferably comprises between 5 and 60% lipids based on total of
calories, preferably between 5 and 30% lipid based on total
calories when intended for adults, preferably between 30 and 60%
lipid based on total calories, more preferably 35 to 50% lipids
based on total calories, when intended for infants.
[0094] The proteins used in the nutritional preparation are
preferably selected from the group of non-human animal proteins
(such as milk proteins, meat proteins and egg proteins), vegetable
proteins (such as soy protein, wheat protein, rice protein, and pea
protein), hydrolysates thereof, free amino acids and mixtures of
proteins, hydrolysates and free amino acids. Cow milk proteins such
as casein and whey proteins are particularly preferred. As the
present composition is suitably used to reduce the allergic
reactions, especially in infants, the protein of is preferably
selected from the group consisting of hydrolyzed milk protein.
Preferably the present composition comprises hydrolyzed casein
and/or hydrolyzed whey protein, hydrolyzed vegetable protein and/or
free amino acids, most preferably hydrolyzed whey protein. The use
of these proteins further reduced the allergic reactions. The use
of these hydrolysed proteins advantageously improves the absorption
of the dietary protein component. This is especially advantageous
for infants and for subjects suffering from a disorder.
[0095] The nutritional composition of the present invention
preferably comprises between 5 and 60% protein based on total
calories, preferably between 15 and 40% protein based on total
calories when intended for adult human subjects, and preferably
between 5 and 15% protein based on total calories and more
preferably 7.5 to 12.5% proteins based on total calories, calories
when intended for infants. For calculation of the % protein based
on total calories, the total of calories provided by proteins,
peptides and amino acids needs to be taken into account.
[0096] A source of digestible carbohydrate may be added to the
nutritional formula. Any suitable (source of) digestible
carbohydrate may be used, for example sucrose, lactose, glucose,
fructose, corn syrup solids, and maltodextrins, and mixtures
thereof. Hence, the nutritional composition of the present
invention preferably comprises between 15 and 90% carbohydrate
based on total calories, more preferable between 25 and 75%
carbohydrate based on total calories, more between 40 to 55%
carbohydrate based on total calories.
[0097] The nutritional composition of the present invention is
preferably is in liquid form. It preferably has a limited
viscosity. It was found that the present process provides a liquid
nutrition with sufficiently low viscosity so it can be applied as
e. g. liquid baby foods and liquid clinical food which can be fed
through a teat, a tube or a straw, while retaining the low
viscosity. In a preferred embodiment, the present composition has a
viscosity below 600 mPas, preferably below 250 mPas, more
preferably below 60 mPas, even more preferably below 35 mPas, most
preferably below 6 mPas, at a shear rate of 100 s.sup.-1 at
20.degree. C. Whenever the term viscosity used in the present
document, this refers to the physical parameter which is determined
according to the following method: The viscosity may be determined
using a Carri-Med CSL rheometer. The used geometry is of conical
shape (6 cm 2 deg acrylic cone) and the gap between plate and
geometry is set on 55 um. A linear continuous ramp shear rate is
used from 0 to 150 s.sup.-1 in 20 seconds. It is noted that a
composition in powder form with the instruction to prepare an
aqueous solution, e.g. by adding water in a certain ratio and which
then results in a viscosity as specified is also encompassed by the
invention.
[0098] Stool irregularities (e. g. hard stools, insufficient stool
volume, diarrhoea) is a major problem in many babies and ill
subjects that receive liquid foods. It was found that stool
problems may be reduced by administering the present preparation in
liquid food which has an osmolality between 50 and 500 mOsm/kg,
more preferably between 100 and 400 mOsm/kg.
[0099] In view of the above, it is also important that the liquid
food does not have an excessive caloric density, however still
provides sufficient calories to feed the subject. Hence, the liquid
food preferably has a caloric density between 0.1 and 2.5 kcal/ml,
even more preferably a caloric density of between 0.5 and 1.5
kcal/ml. When used as an infant formula the caloric density is most
preferably between 0.6 and 0.8 kcal/ml.
Application
[0100] The present preparation obtained by the present process was
found to synergistically stimulate the immune-system. In
particularly the Th2 response was lowered, and/or the Th1 response
was increased. The effect of the combination of these two
components is higher than the sum of the effects of the single
components.
[0101] The present preparation can advantageously be used in the
treatment and/or prevention of a disease, and thus the invention
concerns a method for the treatment and/or prevention of a disease
in a mammal, said method comprising administering the present
preparation to the mammal. In other words, the invention also
concerns the use of a preparation according to the present
invention for the manufacture of a composition, preferably a
nutritional composition, for the treatment and/or prevention of a
disease. In other words the invention concerns a preparation or
nutritional composition comprising a prepration according to the
present invention for use in the treatment and/or prevention of a
disease. Preferably the mammal is a human, even more preferably a
human infant. Thus the invention also concerns the use of a
preparation according to the present invention for the manufacture
of a composition, preferably a nutritional preparation, for the
treatment and/or prevention of a disease in an infant. Or in other
words the invention concerns a preparation or nutritional
composition comprising a prepration according to the present
invention for use in the treatment and/or prevention of a disease
in an infant. In the context of this invention, an infant is in the
age of 0 to 6 years, preferably in the age of 0 to 4 year Th1/s,
preferably in the age of 0 to 2 years, preferably in the age of 0
to 1 year.
[0102] Also the invention concerns a method for providing nutrition
to an infant, said method comprising administering the present
preparation or nutritional composition to the infant. In other
words, the invention also concerns the use of a preparation
according to the present invention for the manufacture of a
nutritional composition for providing nutrition to an infant. In
other words the invention concerns a preparation or nutritional
composition comprising a prepration according to the present
invention for use in providing nutrition to an infant.
[0103] The present preparation can advantageously be used to
increase the Th1 response, decrease the Th2 response, restore
imbalance in the Th1/Th2 responses, maintain a favorable Th1/Th2
balance and/or for the treatment and prevention of disorders which
are associated with an Th1/Th2 imbalance. Hence, compositions which
are advertised to e. g. simulate maturation of the immune system,
enhance the resistance to pathogens by enhancing the immune system
and/or support the immune system are part of the present invention.
In a further aspect, the present invention provides a method for
the treatment and/or prevention of an immune system related
disorder, said method comprising administering to said mammal a
composition comprising a therapeutically effective amount of the
present preparation. In a further aspect, the present invention
provides a method of enhancing the immune response in a mammal said
method comprising administering to the mammal the present
preparation.
[0104] The immune system of newborn human infants is characterized
by an excess of Th2 response. During maturation of the immune
system, the Th1 response increases and the Th1/Th2 balance shifts
to values observed for healthy adults. Hence, the present
preparation is especially advantageous for human infants. The
present invention supports the maturation of the immune system in
infants. In a further embodiment, the method of the invention
relates to the administration of the present preparation to humans
in the age of 0 to 6 years, preferably in the age of 0 to 4 years,
preferably in the age of 0 to 2 years, more preferably in the age
of 0 to 1 year.
[0105] In a preferred embodiment the present method relates to the
stimulation of the maturation of the immune system in human
subjects in the age of 0-6 years, preferably in the age of 0 to 4
years, preferably in the age of 0 to 2 years, more preferably in
the age of 0 to 1 year.
[0106] An excessive Th2 response leads to extreme sensitivity
towards foreign components which should not lead to any
immunological reaction, e. g. allergies and related diseases such
as atopic dermatitis, asthma, food allergy, allergic rhinitis (e.
g. pollen allergy), dust mite allergy and other forms of
hypersensitivity like systemic anaphylaxis and acute urticaria.
Hence, the present preparation is especially advantageous for
treatment and/or prevention of a disorder selected from the group
consisting of allergy, food allergy, atopic dermatitis, asthma,
allergic rhinitis, dust mite allergy and urticaria. The present
invention decreases the Th2 response.
[0107] An increase in Th1 response leads to an increase in the
response against pathogenic bacteria and/or viruses. Hence, the
present preparation is suitable for the treatment and/or prevention
of infections. The present preparation can be advantageously used
for the treatment and/or prevention of intestinal infections,
systemic infections and/or respiratory tract infections.
[0108] It was also found that the present preparation can suitably
be used to support vaccination processes, e. g. enhance the effects
of a vaccination process. The present preparation is suitable for
supporting vaccination response before, during and/or after
vaccination. Particularly the effects of vaccinations for
diptheria-tetanus, pertussis, polio vaccine, measles/mumps/rubella,
pneumococcal conjugate, haemophilus B conjugate, hepatitis B,
hepatitis A, varicella, influenza can suitably be enhanced. Hence,
the present preparations are advantageously used in the treatment
and/or prevention of infections, and/or for use in enhancement of
vaccination response.
[0109] In the context of the present invention, `prevention` of a
disease or cetain disorder also means `treatment of a person at
risk` of a disease or certain disorder.
[0110] Hence, the present preparation is advantageous for humans
subjects suffering from immune deficiencies, in particular elderly
humans suffering from immunosenescence, humans suffering from AIDS
or being infected with the Human Immunodeficiency Virus, and cancer
patients, more particular cancer patients that are or have been
subjected to chemotherapy, radiation and cancer patients that are
cachectic, patients suffering from chronic obstructive pulmonary
disease and/or patients suffering from diabetes.
EXAMPLE 1
Preparation of an Incubated Mixture Using B. breve
[0111] Pasteurized skimmed cow's milk was concentrated by
evaporation to 43 wt. % dry matter based on weight of the skimmed
cow's milk. The concentrate was cooled to 37.degree. C., was then
inoculated with 10% (v/w) B. breve CNCM I-2219 culture comprising
3.times.10.sup.9 cfu per ml. This inoculum was prepared as
described below. The initial pH was between 6-6.1. After incubating
for 8 h at 37.degree. C., in a tank with periodic stirring for 10
minutes every 2 hours, the pH stayed between 6-6.1 and the B. breve
population was 10.sup.6 bacteria/ml (step (a)). The concentrate was
pasteurized (step b) and subsequently spray-dried and called
BbC50cf.
[0112] The inoculum was prepared as follows: A starter culture in
the form of concentrated pellets was added to pasteurized skimmed
milk and incubated for 8 h at 37.degree. C. under anaerobic
conditions. During the process the number of B. breve cells
increased to about 3'10.sup.9 cfu per ml and an acidification from
about pH 6.7 to a pH of between 4.5 and 5.0 occurred. Suitably
cysteine in amount of 0.1 to 0.5 g per 1 and/or yeast extract in an
amount of 0.5 to 5 g/l aqueous substrate, was present.
EXAMPLE 2
Preparation of an Incubated Mixture Using S. thermophilus
[0113] A pre-warmed inoculum was prepared from S. thermophilus CNCM
I-1620 culture by maintaining a frozen inoculum for about 7 h at
about 40.degree. C. Pasteurized lactose solution (between 350 and
450 g/l) was cooled to about 45 and 55.degree. C., and then
inoculated with about 10% (v/w) S. thermophilus CNCM I-1620
pre-warmed inoculum comprising about 3.times.10.sup.9 cfu per ml.
The initial pH was about pH 6. After incubating for about 7 h at
about 50.degree. C., in a tank with periodic stirring for 10
minutes every 2 hours, the pH was kept constant between 6-8 and the
S. thermophilus population was about 10.sup.6 bacteria/ml (step
(d)). The concentrate was pasteurized (step e) and subsequently
spray-dried and called St065cf.
Example 3
Synergistic Effect of the Incubated Mixture and Non-Digestible
Carbohydrates on Th1 Response Increase
Methods:
[0114] The effect of diets comprising (a) BbC50cf prepared
according to example 1; (b) a combination of
galacto-oligosaccharides (Elixor) and fructo-oligosaccharides
(Raftilin HP); and (c) a combination of (a) and (b), i.e.
comprising step c) of the present process, were tested in a mouse
model wherein a response to an antigen is measured by a
delayed-type hypersensitivity (DTH) response. This DTH response in
the ears after local challenge with an antigen present in a vaccine
is a measure of Th1 cell proliferation. During response to
infection and/or vaccination Th1 cells proliferate in response to
the challenge with the antigen. These Th1 cells infiltrate the ear
when the ear is subsequently challenged with the antigen and cause
swelling. Infiltration with the Th1 cells in the ear takes about 24
h and the swelling is therefore delayed. The more Th1 cells
proliferated during initial vaccination and/or infection, the more
DTH upon challenging with the antigen is observed.
[0115] The BbC50cf was lyophilized and used in the mouse diet in a
final concentration of 3 wt. %. Non-digestible oligosaccharide
mixture (GF) containing transgalacto-oligosaccharides (GOS) (source
Vivinal-GOS (Borculo Domo Ingredients, Netherlands)) and
fructo-oligosaccharide (FPS) (source RaftilineHP, Orafti, Tiense,
Belgium) were used in a weight ratio GOS:FPS of 9:1. Diets
containing 1 wt. % GF based on total weight of the mouse diet were
tested. The effects of a combination of non-digestible
oligosaccharides and B. breve incubated mixture (GF plus BbC50cf)
was tested in a diet containing 1 wt. % GF and 3 wt. % BbC50cf
based on total weight of the diet.
[0116] Female, 6 weeks old C57B1/6 mice (Harlan Nederland BV,
Horst, the Netherlands) were group-housed under a regular 12 hours
light/dark regime. Group size was 10 animals per group and 3
animals in the negative control groups. The animals were given
semi-synthetic diets (Research Diet Services, Wijk bij Duurstede,
the Netherlands). Control diets were made to the AIN93G
specifications (Reeves et al (1993) J Nutrition 123 (11): 1923-31),
oligosaccharide supplemented diets were based on these
specifications.
[0117] Vaccinations were started after a period of 20 days of
adaptation to the new housing and diets. At day 0, a blood sample
was collected prior to vaccination. At day 1, the first vaccination
was administered subcutaneously. After three weeks, a booster
vaccination was given (day 22). Nine days after booster injection
(day 31), basal ear thickness was measured with a Digimatic outside
micrometer (Mitutoyo, Veenendaal, the Netherlands) and a
delayed-type hypersensitivity (DTH) response was induced by
injecting antigen solution i. c. (intracutaneous) in the mouse ear
pinnae. 24 h thereafter (day 32), the DTH response was measured, a
blood sample was taken and the mice were sacrificed. This is the
ear thickness after 24 h subtracted with the ear thickness at
t=0.
[0118] The vaccinations consisted of a 100 .mu.l i. c.
(intracutaneous) injection of a 1:1 mix of antigen solution and
Stimune adjuvant (Specol, Cedi-diagnostics BV, Lelystad, the
Netherlands). The antigen solution was a 1:100 dilution of Influvac
2002/2003 (Solvay Pharmaceuticals, Weesp, the Netherlands) in PBS.
Influvac is a trivalent protein vaccine, containing 3.times.3011
g/ml haemagglutinin of three different influenza strains. For the
DTH responses, mice were i. c. injected with 25 .mu.l dialysed
Influvac in both ears as a DTH challenge.
Results:
[0119] The diets containing dosages of 1 wt. % GF or 3 wt. %
BbC50cf both induced a small non-statistically significant increase
in the DTH response. The combination of 1 wt. % GF and 3 wt. %
BbC50cf induced a statistically significant increase of 89% in the
DTH response (see Table 1). As the effect is significantly higher
than the DTH responses from diets containing the oligosaccharides
alone, and also much higher than based on the additive effect of GF
and BbC50cf, which can be calculated to be 49% increase of DTH,
these results are indicative for the synergistic effect provided by
the administration of non-digestible oligosaccharides and BbC50cf
on Th1 response increase. The observed effect is indicative for the
advantageous use of a combination of at least two different
non-digestible carbohydrates and a product obtained by incubating
an aqueous substrate with bifidobacteria, subsequently heating the
incubated mixture and/or removing the bifidobacteria cells by
centrifugation and/or centrifugation in the present uses.
TABLE-US-00001 TABLE 1 DTH response Mean DTH .DELTA. DTH Relative
Group: .mu.m (S.E.) .mu.m DTH Sham 1.7 (4.6) 0 0 Placebo 67.6
(14.9) 69.3 1.00 GF 73.55 (8.6) 75.25 1.09 BbC50cf 95.55 (5.5)
97.25 1.40 GF + BbC50cf 129.4* (17.1) 131.1 1.89 GF + BbC50cf
theoretically 101.50 103.2 1.49 *significantly different from
control (P < 0.01).
[0120] The results of this experiment are an indication that the
present invention can advantageously be used for support in
vaccination response. The results of this experiment are also an
indication that it can advantageously be used in subjects with a
low Th1 response, in particular infants. The results of this
experiment are also an indication that it can advantageously be
used in subjects with a low Th1 response, in particular elderly
suffering or at risk for suffering from immunosenescence, HIV
patients, AIDS patients and/or cancer patients that are or have
been subjected to chemotherapy and/or radiation or that are
cachectic, patients suffering from COPD and/or patients suffering
from diabetes. This model is indicative for basic immunological
changes, which can be beneficial in all disorders with
malfunctioning immune system. It is known that infants, elderly,
HIV infected, cancer patients, COPD patients and/or diabetes
patients have an immune system that does not function at full
capacity. For all the above is an additional help possibly
beneficial.
EXAMPLE 4
Improved Effect of the Incubated Mixtures Obtained with B. breve
and S. thermophilus and Non-Digestible Carbohydrates on Th1
Response Increase
Methods:
[0121] The effect of diets comprising (a) BbC50cf (3 wt. %)
prepared according to example 1 and a combination of
galacto-oligosaccharides (Elixor) and fructo-oligosaccharides
(Raftilin HP); (b) BbC50cf (3 wt. %) prepared according to example
1 and St065cf (3 wt. %) prepared according to example 2 and (c) a
combination of BbC50 (3 wt. %), St065f (3 wt. %) and
galacto-oligosaccharides and fructo-oligosaccharides were tested in
a separate experiment, using the same mouse model as in example
3.
[0122] Galacto-oligosaccharides (Elixor) and
fructo-oligosaccharides (Raftilin HP) (GF) were present in a 9:1
weight ratio and in 1 wt. % based on total diet.
Results:
[0123] The diets containing a combination of 1 wt. % GF and 3 wt. %
BbC50cf induced a statistically significant increase of 100% in the
DTH response (see Table 2).
[0124] The combination of BbC50cf and St065cf also induced a
statistically significant increase of 112 in the DTH response.
[0125] Surprisingly a combination of all three ingredients showed
the highest response increase of 192%.
[0126] These results are indicative for a further improved effect
provided by the administration of non-digestible oligosaccharides
plus BbC50cf and St065cf on Th1 response increase. The observed
effect is indicative for the advantageous use of a combination of
i) at least two different non-digestible carbohydrates, ii) a
product obtained by incubating an aqueous substrate with
bifidobacteria, subsequently inactivating by heating the incubated
mixture and/or removing the bifidobacteria cells by centrifugation
and/or centrifugation and iii) a product obtained by incubating an
aqueous substrate with streptococci, subsequently inactivating by
heating the incubated mixture and/or removing the streptococci
cells by centrifugation and/or centrifugation, in the present
uses.
TABLE-US-00002 TABLE 2 DTH response Mean DTH .DELTA. DTH Relative
Group: .mu.m (S.E.) .mu.m DTH Sham 24.3 (0.6) 0 0 Placebo 66.5
(0.3) 42.2 1.00 BbC50cf + GF 108.9 (0.3).sup.* 84.6 2.00 BbC50cf +
St065cf 113.6 (0.3).sup.* 89.3 2.12 BbC50cf + GF + St065cf 147.6
(0.2).sup.* a 123.2 2.92 *p < 0.05 compared with placebo .sup.ap
< 0.05 compared with Bbc50f + GF and with Bbc50f + St065cf
EXAMPLE 5
Synergistic Effect of the Incubated Product and Non-Digestible
Carbohydrates on Decrease of Th2 Response
Methods
[0127] The effect of diets comprising (a) BbC50cf prepared
according to example 1; (b) a combination of non-digestible
carbohydrates (GF) containing galacto-oligosaccharides (Elixor) and
fructo-oligosaccharides (Raftilin HP); and (c) a combination of (a)
and (b), i.e. comprising step c) of the present process, were
tested in a mouse model wherein a response to an allergen is
measured by an immediate type hypersensitivity (ITH) response. This
ITH response in the ears after challenge with an allergen in the
lungs is a measure of an increased Th2 response. During response to
the allergen in the lungs, masts cell almost immediately
degranulate systemically, including in the ears. These reactions
all involve IgE, which in its turn requires a Th2 response during
helper T cell development. Therefore an enhanced ITH is indicative
of increased IgE and hence increased Th2 response.
[0128] Specific pathogen free male BALB/c mice were obtained from
Charles River (Maastricht, the Netherlands). Food and water was
provided ad libitum and the mice were used when 6-9 weeks of age.
Ovalbumin (grade V) and acetyl-P-methylcholine chloride
(methacholine) were purchased from Sigma Chemical Co. (St. Louis,
Mo., USA). Aluminium hydroxide (AlumImject) was purchased from
Pierce (Rockford, Ill., USA).
[0129] The same diets as in example 2 were tested for 14 days prior
to OVA sensitisation until the end of the experiment. Mice were
sensitised by two i.p. injections with 10 .mu.g ovalbumin adsorbed
onto 2.25 mg aluminium hydroxide in 100 .mu.l saline or saline
alone on days 0 and 7. Mice were challenged on days 35, 38, and 41
by inhalation of ovalbumin aerosols in a plexiglass exposure
chamber for 20 minutes. The aerosols were generated by nebulising
an ovalbumin solution (10 mg/ml) in saline using a Pari LC Star
nebulizer (Pari respiratory Equipment, Richmond, VA, USA). ITH was
measured after 1 h after challenge with the allergen ovalbumin
Results
[0130] The results on ITH are shown in table 3:
TABLE-US-00003 TABLE 3 ITH response Mean ITH .DELTA. ITH Relative
Group: .mu.m (S.E.) .mu.m ITH control 104.3 (3.8) 0 0 Placebo 184.7
(8.7) 80.4 1 GF 159.8 (17.9)* 55.5 0.69 BbC50cf 173.1 (20.3) 68.8
0.86 GF + BbC50cf 136.6 (14.4)** 32.3 0.40 GF + BbC50cf
theoretically 151.7 47.4 0.59 *significantly different from control
(*P < 0.05 and **P < 0.01).
[0131] The diets containing dosages of 1 wt. % GF or 3 wt. %
BbC50cf both induced a small decrease in the ITH response. The
combination of 1 wt. % GF plus 3 wt. % BbC50cf induced a
statistically significant decrease of about 60% in the ITH response
(see Table 3). As the effect is significantly higher than the ITH
responses from diets containing the oligosaccharides alone, and
also much higher than based on the additive effect of GF and
BbC50cf, which can be calculated to be about 40% decrease of ITH,
these results are indicative for the synergistic effect provided by
the administration of non-digestible oligosaccharides and incubated
product on lowering of the Th2 response.
[0132] The observed effect is indicative for the advantageous use
of a combination of non-digestible carbohydrates and a product
obtained by incubating a substrate with bifidobacteria and
subsequently inactivating and/or removing the bifidobacteria in the
present uses. The results of this experiment are an indication that
the present invention can advantageously be used for support in
prevention and/or treatment of in particular asthma, allergy,
atopic dermatitis, allergic conjunctivitis, dust mite allergy,
urticaria and allergic rhinitis.
EXAMPLE 6
Infant Milk Formula
[0133] An infant milk formula was prepared by mixing the following
ingredients: demineralised whey, vegetable fat, lactose, skim milk,
non-digestible carbohydrates, whey protein concentrate, fish oil,
minerals, vitamins.
[0134] The skim milk was pre-incubated in the manner described in
example 1.
[0135] The concentration of the preparation obtained by incubation
with Bifidobacterium breve in step a) and b) was 15.6 wt. % based
on dry weight of the infant formula.
[0136] No living bifidobacteria were detected in the final
product.
[0137] The final composition of the infant formula comprised per
100 ml: [0138] 66 kcal [0139] 1.3 g milk protein (casein and whey
protein). [0140] 7.3 g digestible carbohydrates (mainly lactose)
[0141] 3.5 g fats (mainly vegetable fats) [0142] 0.8 g
trans-galactoligosaccharides (source VivinalGOS) and polyfructose
(source raftilinHP) [0143] Trace elements, minerals, vitamins and
other micronutrients as known in the art.
[0144] The infant milk formula claims to strengthen the immune
system and/or to lower the incidence of atopic eczema and/or to
reduce the incidence of infections and/or reduce the incidence on
allergy.
EXAMPLE 7
Infant Milk Formula
[0145] Pasteurized skimmed cow's milk was concentrated by
evaporation to about 43 wt. % dry matter based on weight of the
skimmed cow's milk. The concentrate was cooled to about 37 .degree.
C., was then inoculated with about 10% (v/w) B. breve CNCM I-2219
culture comprising 3.times.10.sup.9 cfu per ml. This inoculum was
prepared as known in the art. The initial pH was between 6-7.1.
After incubating for 8 h at 37.degree. C., in a tank with periodic
stirring for 10 minutes every 2 hours, the pH stayed between 6-7.1
and the B. breve population was about 10.sup.6 bacteria/ml (step
(a)).
[0146] A pre-warmed inoculum was prepared from S. thermophilus CNCM
I-1620 culture by maintaining a frozen inoculum for about 7 h at
about 40 .degree. C. Pasteurized lactose solution (between 350 and
450 g/l) was cooled to about 45 and 55.degree. C., and then
inoculated with about 10% (v/w) S. thermophilus CNCM I-1620
pre-warmed inoculum comprising about 3.times.10.sup.9 cfu per ml.
The initial pH was about pH 6. After incubating for about 7 h at
about 50.degree. C., in a tank with periodic stirring for 10
minutes every 2 hours, the pH was kept constant between 6-8 and the
S. thermophilus population was about 10.sup.6 bacteria/ml (step
(d)).
[0147] Both incubated preparations, skim milk, vegetable fat,
malto-dextrin, trans-galacto-oligosaccharides and
fructo-oligosaccharides oligosaccharides, and other ingredients
well known for infant milk formula (such as vitamins, minerals,
trace elements) were mixed (step c, f).
[0148] The mixture was pasteurized (step b, e) and subsequently
spray-dried.
[0149] Final composition of the infant formula comprising per 100
ml: [0150] 68 kcal [0151] 1.45 g protein (casein and whey protein
from milk; partially hydrolysed) [0152] 8.6 g digestible
carbohydrates (mainly lactose and maltodextrin) [0153] 3.1 g fats
(mainly vegetable fats) [0154] 0.8 g trans-galactoligosaccharides
(source VivinalGOS) and polyfructose (source raftilinHP) [0155]
Trace elements, minerals, vitamins and other micronutrients
(taurine, choline, inositol, nucleotides, carnitine) as known in
the art.
* * * * *