U.S. patent application number 13/319477 was filed with the patent office on 2012-10-25 for growing up milks containing probiotic micro-organisms.
This patent application is currently assigned to NESTEC S.A.. Invention is credited to Annick Mercenier, Sophie Nutten, Guenolee Prioult.
Application Number | 20120269789 13/319477 |
Document ID | / |
Family ID | 42646295 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120269789 |
Kind Code |
A1 |
Mercenier; Annick ; et
al. |
October 25, 2012 |
GROWING UP MILKS CONTAINING PROBIOTIC MICRO-ORGANISMS
Abstract
The present invention relates to the field of nutrition for
infants and young children. In particular, the present invention
relates to growing-up milks comprising probiotic micro-organisms to
be administered to infants and young children older than 10 months.
These probiotic micro-organisms may be non-replicating probiotic
micro-organisms such as bioactive heat treated probiotic
micro-organisms, for example.
Inventors: |
Mercenier; Annick;
(Bussigny, CH) ; Nutten; Sophie; (Lausanne,
CH) ; Prioult; Guenolee; (Lausanne, CH) |
Assignee: |
NESTEC S.A.
Vevey
CH
|
Family ID: |
42646295 |
Appl. No.: |
13/319477 |
Filed: |
May 11, 2010 |
PCT Filed: |
May 11, 2010 |
PCT NO: |
PCT/EP10/56399 |
371 Date: |
January 12, 2012 |
Current U.S.
Class: |
424/93.45 ;
424/93.4; 424/93.48 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
9/04 20180101; A61P 31/10 20180101; A61P 37/00 20180101; A23V
2002/00 20130101; A61P 11/06 20180101; A61P 7/00 20180101; A61P
35/00 20180101; A23Y 2300/29 20130101; A23Y 2300/49 20130101; A61P
3/00 20180101; A61P 15/00 20180101; A61P 31/22 20180101; Y02A
50/473 20180101; A61P 31/06 20180101; C12N 1/005 20130101; A23Y
2220/15 20130101; A23Y 2220/17 20130101; A61P 11/04 20180101; A23L
33/40 20160801; A23Y 2220/71 20130101; A61K 35/74 20130101; A61P
11/02 20180101; A61P 17/02 20180101; A61P 17/00 20180101; C12N 1/20
20130101; A23Y 2240/75 20130101; A23Y 2300/55 20130101; A61P 31/18
20180101; A61P 37/06 20180101; A23K 10/18 20160501; A61P 1/12
20180101; A61P 3/02 20180101; A61P 31/12 20180101; A61P 33/00
20180101; A61P 29/00 20180101; A61P 37/08 20180101; A23Y 2220/43
20130101; A61P 31/00 20180101; A61P 31/04 20180101; A23Y 2220/03
20130101; A61P 13/02 20180101; A61P 27/16 20180101; A61K 35/744
20130101; A61K 35/745 20130101; A61P 1/14 20180101; A23Y 2220/63
20130101; A61P 11/00 20180101; A61P 27/02 20180101; A61P 37/02
20180101; A61P 37/04 20180101; C12N 1/36 20130101; A61K 35/747
20130101; A61P 1/00 20180101; Y02A 50/30 20180101; A61P 1/02
20180101; A61P 21/02 20180101; A23Y 2240/41 20130101; A61P 15/02
20180101; A23L 33/135 20160801; A23Y 2220/73 20130101; A61P 43/00
20180101 |
Class at
Publication: |
424/93.45 ;
424/93.4; 424/93.48 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61P 29/00 20060101 A61P029/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2009 |
EP |
09159925.8 |
May 11, 2009 |
EP |
09159929.0 |
Claims
1. Growing-up-milk composition comprising probiotic micro-organisms
to be administered to infants or young children starting at the age
of 10 months.
2. Composition in accordance with claim 1 having a caloric density
of from 70-80 kcal/100 ml, and comprising a protein source of 2-3
g/100 kcal, a carbohydrate source of 12-15 g/100 kcal, and a lipid
source of 3-4.6 g/100 kcal.
3. Composition in accordance with claim 1, comprising 0.15-0.25 g
LC-PUFA/100 g fatty acids, wherein the LC-PUFA are selected from
the group consisting of ARA, DHA and combinations thereof.
4. Composition in accordance with claim 1, comprising 1.5-2.5 mg
nucleotides per 100 mL formula.
5. Composition in accordance with claim 1, wherein the probiotic
micro-organisms comprise non-replicating probiotic
micro-organisms.
6. Composition in accordance with claim 1, comprising probiotic
micro-organisms in an amount corresponding to about 10.sup.6 to
10.sup.12 cfu.
7. Composition in accordance with claim 5, wherein the
non-replicating probiotic micro-organisms were rendered
non-replicating by a heat-treatment.
8. Composition in accordance with claim 7, wherein the heat
treatment is a high temperature treatment at about 71.5-150.degree.
C. for about 1-120 seconds.
9. (canceled)
10. Composition in accordance with claim 7, wherein the heat
treatment is performed in a temperature range of about
70-150.degree. C. for about 3 minutes-2 hours.
11. (canceled)
12. Composition in accordance with claim 1, wherein at least 90% of
all of the probiotics are non-replicating.
13. Composition in accordance with claim 1, wherein the probiotic
micro-organisms are selected from the group consisting of
bifidobacteria, lactobacilli, propionibacteria, or combinations
and/or mixtures thereof.
14. Composition in accordance with claim 1, wherein the probiotic
micro-organisms are selected from the group consisting of
Bifidobacterium longum NCC 3001, Bifidobacterium longum NCC 2705,
Bifidobacterium breve NCC 2950, Bifidobacterium lactis NCC 2818,
Lactobacillus johnsonii La1, Lactobacillus paracasei NCC 2461,
Lactobacillus rhamnosus NCC 4007, Lactobacillus reuteri DSM17983,
Lactobacillus reuteri ATCC55730, Streptococcus thermophilus NCC
2019, Streptococcus thermophilus NCC 2059, Lactobacillus casei NCC
4006, Lactobacillus acidophilus NCC 3009, Lactobacillus casei
ACA-DC 6002 (NCC 1825), Escherichia coli Nissle, Lactobacillus
bulgaricus NCC 15, Lactococcus lactis NCC 2287, and combinations
thereof.
15. Composition in accordance with claim 1 containing about 0.005
mg-1000 mg non-replicating micro-organisms per daily dose.
16. A method of providing nutrition to infants or young children
starting from the age of 10 months comprising administering a
composition comprising probiotic micro-organisms to the
individual.
17. Method in accordance with claim 16 having a caloric density of
from 70-80 kcal/100 ml, and comprising a protein source of 2-3
g/100 kcal, a carbohydrate source of 12-15 g/100 kcal, and a lipid
source of 3-4.6 g/100 kcal.
18. Method in accordance with claim 16, comprising 0.15-0.25 g
LC-PUFA/100 g fatty acids, wherein the LC-PUFA are selected from
the group consisting of ARA, DHA and combinations thereof.
19. Method in accordance with claim 16, comprising 1.5-2.5 mg
nucleotides per 100 mL formula.
20. Method in accordance with claim 16, wherein the probiotic
micro-organisms comprise non-replicating probiotic
micro-organisms.
21. Method in accordance with claim 16, comprising probiotic
micro-organisms in an amount corresponding to about 10.sup.6 to
10.sup.12 cfu.
22. Method in accordance with claim 20, wherein the non-replicating
probiotic micro-organisms were rendered non-replicating by a
heat-treatment.
23. Method for the prevention or treatment of inflammatory
disorders in an individual 10 months or older comprising
administering a composition comprising probiotic
micro-organisms.
24. Method for the prevention or treatment disorders related to a
compromised immune defense in an individual 10 months or older
comprising administering a composition comprising probiotic
micro-organisms.
25. Method in accordance with claim 16 containing about 0.005
mg-1000 mg non-replicating micro-organisms per daily dose.
Description
[0001] The present invention relates to the field of nutrition for
infants and young children. In particular, the present invention
relates to growing-up milks comprising probiotic micro-organisms to
be administered to infants and young children older than 10 months.
These probiotic micro-organisms may be non-replicating probiotic
micro-organisms such as bioactive heat treated probiotic
micro-organisms, for example.
[0002] Breast milk is the ideal food for healthy growth and
development of babies. In 2001 the World Health Organization (WHO)
changed its recommended duration of exclusive breastfeeding from 4
to 6 months to 6 months, therefore breastfeeding should be
encouraged and promoted accordingly.
[0003] Beginning from the 6th month onwards the behaviour of
infants changes. They sit up for the first time or grab a toy with
only one hand. An infant's nutritional requirements change as the
infant develops. Nutrients like iron and calcium become even more
important.
[0004] From the age of one onwards, drinks based on cow's milk
become an important item in the baby's diet. But cow's milk itself
is not adapted to the nutritional requirements of young children.
In particular, cow's milk contains too much protein, too many
minerals, too little iron, too much saturated fatty acids and not
enough unsaturated fats.
[0005] Growing up milks (GUM) were developed to assist in bridging
this step. GUM ensures a steady transition from the lower protein
level of breast milk to the high protein level present in cow's
milk and normal dairy products used by adults.
[0006] Growing Up Milk (GUM) aims at helping Junior Stage toddlers
to maximize their potential in terms of physical and mental
development. GUM fulfils the toddler's protein requirements without
overloading the still immature metabolic system.
[0007] One important function of early nutrition of infants and
young children is to generate a healthy gut flora and to develop a
strong immune system.
[0008] A healthy gut flora will contribute to a functional GI
tract, which in turn will help to properly digest ingested food and
will reduce stomach ache in infants and young children.
[0009] Infants and young children typically have around 10 colds
per year. Such colds are uncomfortable and may even have more
serious consequences.
[0010] It would hence be desirable to further improve the immune
boosting effect of growing up milks.
[0011] It would also be desirable to further improve the
anti-inflammatory effect of growing up milks.
[0012] Hence, there is a need in the art for a growing up milk that
ensures a steady transition from the lower protein level of breast
milk to the high protein level present in cow's milk. Such a
growing up milk should have an improved immune boosting effect, an
anti-inflammatory effect and/or should facilitate digestion. It
would be preferred if this was achieved by using natural
ingredients that are safe to administer without side effects and
that are easy to incorporate into growing up milk compositions
using state of the art industrial techniques.
[0013] The present inventors have addressed this need. It was hence
the objective of the present invention to improve the state of the
art and to provide a GUM composition that satisfies the needs
expressed above.
[0014] The present inventors were surprised to see that they could
achieve this object by the subject matter of the independent claim.
The dependant claims further develop the idea of the present
invention.
[0015] Accordingly, the present inventors propose to provide a
growing-up-milk composition comprising probiotics.
[0016] Growing-up-milk compositions are nutritional compositions.
Growing-up-milks are in particular specific nutritional
compositions having a composition that allows a steady transition
from the composition of breast milk to the composition of cow milk.
This adjustment may be made stepwise. Several different
growing-up-milks may be foreseen to accompany this transition. For
example, one growing-up-milk composition may be to be administered
beginning at the age of 10 months, a second one growing-up-milk
composition may be to be administered beginning at the age of 12
months and a third growing-up-milk composition may be to be
administered beginning at the age of 24 months.
[0017] Probiotics were found to be able to provide their health
benefits in the framework of growing-up-milks. Additionally, e.g.
bifidobacteria, are present in breast milk and are part of what
gives breast milk its naturally protective properties.
[0018] Hence, adding probiotic micro-organisms to the nutritional
formulas of infants and young children would allow them to more
closely resemble breast milk.
[0019] However, as in particular powdered formulas to be
reconstituted with water usually have a shelf life that exceeds the
shelf life of, e.g., yoghurt drinks comprising probiotics,
probiotics are usually not added to such formulas, because of
uncertainties that the viability of the probiotics can be ensured
during an extended shelf life, for example.
[0020] The present inventors were now able to show that even
non-replicating probiotics can provide the health benefits of
probiotics and may even have improved benefits.
[0021] Consequently, the present invention relates to a
growing-up-milk composition comprising probiotic micro-organisms to
be administered to infants or young children starting from the age
of 10 months.
[0022] Growing up Milk provides the right amount of lipids with a
balanced fatty acid composition. Lipids are the best energy
provider (9 kcal/g), needed for the increasing activity of the
toddler. But fatty acids are also required to build cell membranes,
and are important precursors for some physiologically active
factors (e.g. hormones, coagulation factors etc.).
[0023] FDA regulations define infants as persons not more than 12
months old (Title 21, Code of Federal Regulations 21 CFR
105.3(e)).
[0024] The growing up milk composition of the present invention
comprises a balanced protein content.
[0025] On the one hand, protein is needed, especially during growth
periods, e.g. to build body tissues such as muscles. On the other
hand, high amounts of protein burden the toddler's still immature
kidneys' function. If kidney function has not matured, a high
protein intake can compromise renal function. Research suggests
that children over one year of age tend to have a protein intake
which is higher than paediatric recommendations. A high protein
intake has also been implicated in obesity in later life.
[0026] Cow's milk has a Sub-optimal fatty acid composition for the
nutrition of infants and young children. High in saturated fatty
acids and low in unsaturated fatty acids, cow's milk does not
provide the optimal and most healthy fatty acid composition.
Unsaturated fatty acids are needed for growth, e.g. for building
cells in the nervous system, and studies show that many toddlers do
not receive the recommended levels. Brain, for instance, is
extremely rich in DHA (n-3 unsaturated fatty acid). Therefore, the
growing up milk composition of the present invention may have a
good balance between the two families of essential fatty acids,
with an approximate 4-10 ratio between n-6 fatty acids like
linolenic acid and n-3 fatty acids like alpha-linolenic acid.
[0027] If provided as a dried composition it is preferred that the
GUM composition has a water activity of below 0.2, preferably below
0.15 to further increase shelf stability. Most bacteria, for
example, do not grow at water activities below 0.91, and most molds
cease to grow at water activities below 0.80.
[0028] Water activitiy (a.sub.w) is a measurement of the energy
status of the water in a system. It is defined as the vapour
pressure of the water divided by that of pure water. Consequently,
distilled water has a water pressure of 1.
[0029] The growing up milk composition of the present invention may
have a caloric density in the range of 70-80 kcal/100 ml, and
comprises a protein source in an amount of 2-3 g/100 kcal, a
carbohydrate source in an amount of 12-15 g/100 kcal, and a lipid
source in an amount of 3-4.6 g/100 kcal.
[0030] A growing up milk composition of the present invention to be
administered to infants at the age of 10-12 months may have a
caloric density in the range of about 70 kcal/100 ml, and comprises
a protein source in an amount of 2.2-2.3 g/100 kcal, a carbohydrate
source in an amount of about 13 g/100 kcal, and a lipid source in
an amount of 4.3 to 4.4 g/100 kcal.
[0031] A growing up milk composition of the present invention to be
administered to children at the age of 12-24 months may have a
caloric density in the range of about 70-80 kcal/100 ml, and
comprises a protein source in an amount of 2.2-2.9 g/100 kcal, a
carbohydrate source in an amount of about 11.9-13.9 g/100 kcal, and
a lipid source in an amount of 3.9 to 4.6 g/100 kcal.
[0032] A growing up milk composition of the present invention to be
administered to children at the age of above 24 months may have a
caloric density of about 70 kcal/100 ml, and comprises a protein
source in an amount of about 2.5 g/100 kcal, a carbohydrate source
in an amount of about 14.6 g/100 kcal, and a lipid source in an
amount of 3.9 to 4.6 g/100 kcal.
[0033] The protein source may consist of whey proteins and casein.
For example a ratio of whey to casein in the range of about 20:80
to 80:20, e.g., 40:60 to 60:40 may be used.
[0034] The carbohydrate source may consist essentially of Lactose.
However, also ratios of Lactose and Maltodextrin in the range of
3:1 to 1:1 may be used, for example.
[0035] The feeding formula of the present invention may comprise
0.2-0.3 g LC-PUFA/100 g fatty acids. The LC-PUFA may be selected
from ARA, DHA or combinations thereof. For example, the LC-PUFA may
comprise a combination of ARA and DHA. Formulas containing DHA and
ARA have been shown to provide visual and mental development
similar to that of the breastfed infant.
[0036] The feeding formula of the present invention may also
contain 1.5-2.5 mg nucleotides per 100 mL formula. Nucleotides and
their bases are not considered `essential` because they can be
synthesised by the infant body from simpler compounds. At certain
times, however, the processes of synthesis may not be able to meet
demand, for example, during periods of rapid cell turnover as in
normal growth or in gut disease. At these times, the body relies
more heavily on dietary sources of nucleotides.
[0037] The composition may comprise in part or only non-replicating
probiotic micro-organisms.
[0038] The inventors were surprised to see that, e.g., in terms of
an immune boosting effect and/or in terms of an anti-inflammatory
effect non-replicating probiotic microorganisms may even be more
effective than replicating probiotic microorganisms.
[0039] This is surprising since probiotics are often defined as
"live micro-organisms that when administered in adequate amounts
confer health benefits to the host" (FAO/WHO Guidelines). The vast
majority of published literature deals with live probiotics. In
addition, several studies investigated the health benefits
delivered by non-replicating bacteria and most of them indicated
that inactivation of probiotics, e.g. by heat treatment, leads to a
loss of their purported health benefit (Rachmilewitz, D., et al.,
2004, Gastroenterology 126:520-528; Castagliuolo, et al., 2005,
FEMS Immunol. Med. Microbiol. 43:197-204; Gill, H. S, and K. J.
Rutherfurd, 2001, Br. J. Nutr. 86:285-289; Kaila, M., et al., 1995,
Arch. Dis. Child 72:51-53.) Some studies showed that killed
probiotics may retain some health effects (Rachmilewitz, D., et
al., 2004, Gastroenterology 126:520-528; Gill, H. S, and K. J.
Rutherfurd, 2001, Br. J. Nutr. 86:285-289), but clearly, living
probiotics were regarded in the art so far as more performing.
[0040] The composition according to the present invention may
comprise probiotic micro-organisms in any effective amount, for
example in an amount corresponding to about 10.sup.6 to 10.sup.12
cfu/g dry weight.
[0041] The probiotic micro-organisms may be non-replicating
probiotic micro-organisms.
[0042] "Non-replicating" probiotic micro-organisms include
probiotic bacteria which have been heat treated. This includes
micro-organisms that are inactivated, dead, non-viable and/or
present as fragments such as DNA, metabolites, cytoplasmic
compounds, and/or cell wall materials.
[0043] "Non-replicating" means that no viable cells and/or colony
forming units can be detected by classical plating methods. Such
classical plating methods are summarized in the microbiology book:
James Monroe Jay, Martin J. Loessner, David A. Golden. 2005. Modern
food microbiology. 7th edition, Springer Science, New York, N.Y.
790 p. Typically, the absence of viable cells can be shown as
follows: no visible colony on agar plates or no increasing
turbidity in liquid growth medium after inoculation with different
concentrations of bacterial preparations (`non replicating`
samples) and incubation under appropriate conditions (aerobic
and/or anaerobic atmosphere for at least 24 h).
[0044] Probiotics are defined for the purpose of the present
invention as "Microbial cell preparations or components of
microbial cells with a beneficial effect on the health or
well-being of the host." (Salminen S, Ouwehand A. Benno Y. et al
"Probiotics: how should they be defined" Trends Food Sci. Technol.
1999:10 107-10).
[0045] The possibility to use non-replicating probiotic
micro-organisms offers several advantages. In severely
immuno-compromised children, the use of live probiotics may be
limited in exceptional cases due to a potential risk to develop
bacteremia. Non-replicating probiotics may be used without any
problem.
[0046] Additionally, the provision of non-replicating probiotic
micro-organisms allows the hot reconstitution while retaining
health benefit.
[0047] The compositions of the present invention comprise probiotic
micro-organisms and/or non-replicating probiotic micro-organisms in
an amount sufficient to at least partially produce a health
benefit. An amount adequate to accomplish this is defined as "a
therapeutically effective dose". Amounts effective for this purpose
will depend on a number of factors known to those of skill in the
art such as the weight and general health state of the child, and
on the effect of the food matrix.
[0048] In prophylactic applications, compositions according to the
invention are administered to a consumer susceptible to or
otherwise at risk of a disorder in an amount that is sufficient to
at least partially reduce the risk of developing that disorder.
Such an amount is defined to be "a prophylactic effective dose".
Again, the precise amounts depend on a number of factors such as
the childs state of health and weight, and on the effect of the
food matrix.
[0049] Those skilled in the art will be able to adjust the
therapeutically effective dose and/or the prophylactic effective
dose appropriately.
[0050] In general the composition of the present invention contains
probiotic micro-organisms and/or non-replicating probiotic
micro-organisms in a therapeutically effective dose and/or in a
prophylactic effective dose.
[0051] Typically, the therapeutically effective dose and/or the
prophylactic effective dose is in the range of about 0.005 mg-1000
mg probiotic micro-organisms and/or non-replicating, probiotic
micro-organisms per daily dose.
[0052] In terms of numerical amounts, the "short-time high
temperature" treated non-replicating micro-organisms may be present
in the composition in an amount corresponding to between 10.sup.4
and 10.sup.12 equivalent cfu/g of the dry composition. Obviously,
non-replicating micro-organisms do not form colonies, consequently,
this term is to be understood as the amount of non replicating
micro-organisms that is obtained from 10.sup.4 and 10.sup.12 cfu/g
replicating bacteria. This includes micro-organisms that are
inactivated, non-viable or dead or present as fragments such as DNA
or cell wall or cytoplasmic compounds. In other words, the quantity
of micro-organisms which the composition contains is expressed in
terms of the colony forming ability (cfu) of that quantity of
micro-organisms as if all the micro-organisms were alive
irrespective of whether they are, in fact, non replicating, such as
inactivated or dead, fragmented or a mixture of any or all of these
states.
[0053] Preferably the non-replicating micro-organisms are present
in an amount equivalent to between 10.sup.4 to 10.sup.9 cfu/g of
dry composition, even more preferably in an amount equivalent to
between 10.sup.5 and 10.sup.9 cfu/g of dry composition.
[0054] The probiotics may be rendered non-replicating by any method
that is known in the art.
[0055] The technologies available today to render probiotic strains
non-replicating are usually heat-treatment, .gamma.-irradiation, UV
light or the use of chemical agents (formalin,
paraformaldehyde).
[0056] It would be preferred to use a technique to render
probiotics non-replicating that is relatively easy to apply under
industrial circumstances in the food industry.
[0057] Most products on the market today that contain probiotics
are heat treated during their production. It would hence be
convenient, to be able to heat treat probiotics either together
with the produced product or at least in a similar way, while the
probiotics retain or improve their beneficial properties or even
gain a new beneficial property for the consumer.
[0058] However, inactivation of probiotic micro-organisms by heat
treatments is associated in the literature generally with an at
least partial loss of probiotic activity.
[0059] The present inventors have now surprisingly found, that
rendering probiotic micro-organisms non-replicating, e.g., by heat
treatment, does not result in the loss of probiotic health
benefits, but--to the contrary--may enhance existing health
benefits and even generate new health benefits.
[0060] Hence, one embodiment of the present invention is a
composition wherein the non-replicating probiotic micro-organisms
were rendered non-replicating by a heat-treatment.
[0061] Such a heat treatment may be carried out at at least
71.5.degree. C. for at least 1 second.
[0062] Long-term heat treatments or short-term heat treatments may
be used.
[0063] In industrial scales today usually short term heat
treatments, such as UHT-like heat treatments are preferred. This
kind of heat treatment reduces bacterial loads, and reduces the
processing time, thereby reducing the spoiling of nutrients.
[0064] The inventors demonstrate for the first time that probiotics
micro-organisms, heat treated at high temperatures for short times
exhibit anti-inflammatory immune profiles regardless of their
initial properties. In particular either a new anti-inflammatory
profile is developed or an existing anti-inflammatory profile is
enhanced by this heat treatment.
[0065] It is therefore now possible to generate non replicating
probiotic micro-organisms with anti-inflammatory immune profiles by
using specific heat treatment parameters that correspond to typical
industrially applicable heat treatments, even if live counterparts
are not anti-inflammatory strains.
[0066] Hence, for example, the heat treatment may be a high
temperature treatment at about 71.5-150.degree. C. for about 1-120
seconds. The high temperature treatment may be a high
temperature/short time (HTST) treatment or a ultra-high temperature
(UHT) treatment.
[0067] The probiotic micro-organisms may be subjected to a high
temperature treatment at about 71.5-150.degree. C. for a short term
of about 1-120 seconds.
[0068] More preferred the micro-organisms may be subjected to a
high temperature treatment at about 90-140.degree. C., for example
90.degree.-120.degree. C., for a short term of about 1-30
seconds.
[0069] This high temperature treatment renders the micro-organisms
at least in part non-replicating.
[0070] The high temperature treatment may be carried out at normal
atmospheric pressure but may be also carried out under high
pressure. Typical pressure ranges are form 1 to 50 bar, preferably
from 1-10 bar, even more preferred from 2 to 5 bar. Obviously, it
is preferred if the probiotics are heat treated in a medium that is
either liquid or solid, when the heat is applied. An ideal pressure
to be applied will therefore depend on the nature of the
composition which the micro-organisms are provided in and on the
temperature used.
[0071] The high temperature treatment may be carried out in the
temperature range of about 71.5-150.degree. C., preferably of about
90-120.degree. C., even more preferred of about 120-140.degree.
C.
[0072] The high temperature treatment may be carried out for a
short term of about 1-120 seconds, preferably, of about 1-30
seconds, even more preferred for about 5-15 seconds.
[0073] This given time frame refers to the time the probiotic
micro-organisms are subjected to the given temperature. Note, that
depending on the nature and amount of the composition the
micro-organisms are provided in and depending on the architecture
of the heating apparatus used, the time of heat application may
differ.
[0074] Typically, however, the composition of the present invention
and/or the micro-organisms are treated by a high temperature short
time (HTST) treatment, flash pasteurization or a ultra high
temperature (UHT) treatment.
[0075] A UHT treatment is Ultra-high temperature processing or a
ultra-heat treatment (both abbreviated UHT) involving the at least
partial sterilization of a composition by heating it for a short
time, around 1-10 seconds, at a temperature exceeding 135.degree.
C. (275.degree. F.), which is the temperature required to kill
bacterial spores in milk. For example, processing milk in this way
using temperatures exceeding 135.degree. C. permits a decrease of
bacterial load in the necessary holding time (to 2-5 s) enabling a
continuous flow operation.
[0076] There are two main types of UHT systems: the direct and
indirect systems. In the direct system, products are treated by
steam injection or steam infusion, whereas in the indirect system,
products are heat treated using plate heat exchanger, tubular heat
exchanger or scraped surface heat exchanger. Combinations of UHT
systems may be applied at any step or at multiple steps in the
process of product preparation.
[0077] A HTST treatment is defined as follows (High
Temperature/Short Time): Pasteurization method designed to achieve
a 5-log reduction, killing 99.9999% of the number of viable
micro-organisms in milk. This is considered adequate for destroying
almost all yeasts, molds and common spoilage bacteria and also
ensure adequate destruction of common pathogenic heat resistant
organisms. In the HTST process milk is heated to 71.7.degree. C.
(161.degree. F.) for 15-20 seconds.
[0078] Flash pasteurization is a method of heat pasteurization of
perishable beverages like fruit and vegetable juices, beer and
dairy products. It is done prior to filling into containers in
order to kill spoilage micro-organisms, to make the products safer
and extend their shelf life. The liquid moves in controlled
continuous flow while subjected to temperatures of 71.5.degree. C.
(160.degree. F.) to 74.degree. C. (165.degree. F.) for about 15 to
30 seconds.
[0079] For the purpose of the present invention the term "short
time high temperature treatment" shall include high-temperature
short time (HTST) treatments, UHT treatments, and flash
pasteurization, for example.
[0080] Since such a heat treatment provides non-replicating
probiotics with an improved anti-inflammatory profile, the
composition of the present invention may be for use in the
prevention or treatment of inflammatory disorders.
[0081] The inflammatory disorders that can be treated or prevented
by the composition of the present invention are not particularly
limited. For example, they may be selected from the group
consisting of acute inflammations such as sepsis; burns; and
chronic inflammation, such as inflammatory bowel disease, e.g.,
Crohn's disease, ulcerative colitis, pouchitis; necrotizing
enterocolitis; skin inflammation, such as UV or chemical-induced
skin inflammation, eczema, reactive skin; irritable bowel syndrome;
eye inflammation; allergy, asthma; and combinations thereof.
[0082] If long term heat treatments are used to render the
probiotic micro-organisms non-replicating, such a heat treatment
may be carried out in the temperature range of about 70-150.degree.
C. for about 3 minutes-2 hours, preferably in the range of
80-140.degree. C. from 5 minutes-40 minutes.
[0083] While the prior art generally teaches that bacteria rendered
non-replicating by long-term heat-treatments are usually less
efficient than live cells in terms of exerting their probiotic
properties, the present inventors were able to demonstrate that
heat-treated probiotics are superior in stimulating the immune
system compared to their live counterparts.
[0084] The present invention relates also to an composition
comprising probiotic micro-organisms that were rendered
non-replicating by a heat treatment at at least about 70.degree. C.
for at least about 3 minutes.
[0085] The immune boosting effects of non-replicating probiotics
were confirmed by in vitro immunoprofiling. The in vitro model used
uses cytokine profiling from human Peripheral Blood Mononuclear
Cells (PBMCs) and is well accepted in the art as standard model for
tests of immunomodulating compounds (Schultz et al., 2003, Journal
of Dairy Research 70, 165-173; Taylor et al., 2006, Clinical and
Experimental Allergy, 36, 1227-1235; Kekkonen et al., 2008, World
Journal of Gastroenterology, 14, 1192-1203)
[0086] The in vitro PBMC assay has been used by several
authors/research teams for example to classify probiotics according
to their immune profile, i.e. their anti- or pro-inflammatory
characteristics (Kekkonen et al., 2008, World Journal of
Gastroenterology, 14, 1192-1203). For example, this assay has been
shown to allow prediction of an anti-inflammatory effect of
probiotic candidates in mouse models of intestinal colitis
(Foligne, B., et al., 2007, World J. Gastroenterol. 13:236-243).
Moreover, this assay is regularly used as read-out in clinical
trials and was shown to lead to results coherent with the clinical
outcomes (Schultz et al., 2003, Journal of Dairy Research 70,
165-173; Taylor et al., 2006, Clinical and Experimental Allergy,
36, 1227-1235).
[0087] Allergic diseases have steadily increased over the past
decades and they are currently considered as epidemics by WHO. In a
general way, allergy is considered to result from an imbalance
between the Th1 and Th2 responses of the immune system leading to a
strong bias towards the production of Th2 mediators. Therefore,
allergy can be mitigated, down-regulated or prevented by restoring
an appropriate balance between the Th1 and Th2 arms of the immune
system. This implies the necessity to reduce the Th2 responses or
to enhance, at least transiently, the Th1 responses. The latter
would be characteristic of an immune boost response, often
accompanied by for example higher levels of IFN.gamma., TNF-.alpha.
and IL-12. (Kekkonen et al., 2008, World Journal of
Gastroenterology, 14, 1192-1203; Viljanen M. et al., 2005, Allergy,
60, 494-500)
[0088] The composition of the present invention allows it hence to
treat or prevent disorders that are related to a compromised immune
defence.
[0089] Consequently, the disorders linked to a compromised immune
defence that can be treated or prevented by the composition of the
present invention are not particularly limited.
[0090] For example, they may be selected from the group consisting
of infections, in particular bacterial, viral, fungal and/or
parasite infections; phagocyte deficiencies; low to severe
immunodepression levels such as those induced by stress or
immunodepressive drugs, chemotherapy or radiotherapy; natural
states of less immunocompetent immune systems such as those of the
neonates; allergies; and combinations thereof.
[0091] The composition described in the present invention allows it
also to enhance a childs response to vaccines, in particular to
oral vaccines.
[0092] Any amount of non-replicating micro-organisms will be
effective. However, it is generally preferred, if at least 90%,
preferably, at least 95%, more preferably at least 98%, most
preferably at least 99%, ideally at least 99.9%, most ideally all
of the probiotics are non-replicating.
[0093] In one embodiment of the present invention all
micro-organisms are non-replicating.
[0094] Consequently, in the composition of the present invention at
least 90%, preferably, at least 95%, more preferably at least 98%,
most preferably at least 99%, ideally at least 99.9%, most ideally
all of the probiotics may be non-replicating.
[0095] All probiotic micro-organisms may be used for the purpose of
the present invention.
[0096] For example, the probiotic micro-organisms may be selected
from the group consisting of bifidobacteria, lactobacilli,
propionibacteria, or combinations thereof, for example
Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium
animalis, Bifidobacterium breve, Bifidobacterium infantis,
Bifidobacterium adolescentis, Lactobacillus acidophilus,
Lactobacillus casei, Lactobacillus paracasei, Lactobacillus
salivarius, Lactobacillus reuteri, Lactobacillus rhamnosus,
Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus
fermentum, Lactococcus lactis, Streptococcus thermophilus,
Lactococcus lactis, Lactococcus diacetylactis, Lactococcus
cremoris, Lactobacillus bulgaricus, Lactobacillus helveticus,
Lactobacillus delbrueckii, Escherichia coli and/or mixtures
thereof.
[0097] The composition in accordance with the present invention
may, for example comprise probiotic micro-organisms selected from
the group consisting of Bifidobacterium longum NCC 3001,
Bifidobacterium longum NCC 2705, Bifidobacterium breve NCC 2950,
Bifidobacterium lactis NCC 2818, Lactobacillus johnsonii La1,
Lactobacillus paracasei NCC 2461, Lactobacillus rhamnosus NCC 4007,
Lactobacillus reuteri DSM17983, Lactobacillus reuteri ATCC55730,
Streptococcus thermophilus NCC 2019, Streptococcus thermophilus NCC
2059, Lactobacillus casei NCC 4006, Lactobacillus acidophilus NCC
3009, Lactobacillus casei ACA-DC 6002 (NCC 1825), Escherichia coli
Nissle, Lactobacillus bulgaricus NCC 15, Lactococcus lactis NCC
2287, or combinations thereof.
[0098] All these strains were either deposited under the Budapest
treaty and/or are commercially available.
[0099] The strains have been deposited under the Budapest treaty as
follows:
Bifidobacterium longum NCC 3001: ATCC BAA-999 Bifidobacterium
longum NCC 2705: CNCM I-2618 Bifidobacterium breve NCC 2950 CNCM
I-3865 Bifidobacterium lactis NCC 2818: CNCM I-3446 Lactobacillus
paracasei NCC 2461: CNCM I-2116 Lactobacillus rhamnosus NCC 4007:
CGMCC 1.3724 Streptococcus themophilus NCC 2019: CNCM I-1422
Streptococcus themophilus NCC 2059: CNCM I-4153 Lactococcus lactis
NCC 2287: CNCM I-4154 Lactobacillus casei NCC 4006: CNCM I-1518
Lactobacillus casei NCC 1825: ACA-DC 6002 Lactobacillus acidophilus
NCC 3009: ATCC 700396 Lactobacillus bulgaricus NCC 15: CNCM I-1198
Lactobacillus johnsonii La1 CNCM I-1225 Lactobacillus reuteri
DSM17983 DSM17983 Lactobacillus reuteri ATCC55730 ATCC55730
Escherichia coli Nissle 1917: DSM 6601
[0100] Those skilled in the art will understand that they can
freely combine all features of the present invention described
herein, without departing from the scope of the invention as
disclosed.
[0101] Further advantages and features of the present invention are
apparent from the following Examples and Figures.
[0102] FIGS. 1A and B show the enhancement of the anti-inflammatory
immune profiles of probiotics treated with "short-time high
temperatures".
[0103] FIG. 2 shows non anti-inflammatory probiotic strains that
become anti-inflammatory, i.e. that exhibit pronounced
anti-inflammatory immune profiles in vitro after being treated with
"short-time high temperatures".
[0104] FIGS. 3 A and B show probiotic strains in use in
commercially available products that exhibit enhanced or new
anti-inflammatory immune profiles in vitro after being treated with
"short-time high temperatures".
[0105] FIGS. 4 A and B show dairy starter strains (i.e. Lc1 starter
strains) that exhibits enhanced or new anti-inflammatory immune
profiles in vitro upon heat treatment at high temperatures.
[0106] FIG. 5 shows a non anti-inflammatory probiotic strain that
exhibits anti-inflammatory immune profiles in vitro after being
treated with HTST treatments.
[0107] FIG. 6: Principal Component Analysis on PBMC data (IL-12p40,
IFN-.gamma., TNF-.alpha., IL-10) generated with probiotic and dairy
starter strains in their live and heat treated (140.degree. C. for
15 second) forms. Each dot represents one strain either live or
heat treated identified by its NCC number or name.
[0108] FIG. 7 shows IL-12p40/IL-10 ratios of live and heat treated
(85.degree. C., 20 min) strains. Overall, heat treatment at
85.degree. C. for 20 min leads to an increase of IL-12p40/IL-10
ratios as opposed to "short-time high temperature" treatments of
the present invention (FIGS. 1, 2, 3, 4 and 5).
[0109] FIG. 8 shows the enhancement of in vitro cytokine secretion
from human PBMCs stimulated with heat treated bacteria.
[0110] FIG. 9 shows the percentage of diarrhea intensity observed
in OVA-sensitized mice challenged with saline (negative control),
OVA-sensitized mice challenged with OVA (positive control) and
OVA-sensitized mice challenged with OVA and treated with
heat-treated or live Bifidobacterium breve NCC2950. Results are
displayed as the percentage of diarrhea intensity (Mean.+-.SEM
calculated from 4 independent experiments) with 100% of diarrhea
intensity corresponding to the symptoms developed in the positive
control (sensitized and challenged by the allergen) group.
EXAMPLE 1
Methodology
Bacterial Preparations
[0111] The health benefits delivered by live probiotics on the host
immune system are generally considered to be strain specific.
Probiotics inducing high levels of IL-10 and/or inducing low levels
of pro-inflammatory cytokines in vitro (PBMC assay) have been shown
to be potent anti-inflammatory strains in vivo (Foligne, B., et
al., 2007, World J. Gastroenterol. 13:236-243).
[0112] Several probiotic strains were used to investigate the
anti-inflammatory properties of heat treated probiotics. These were
Bifidobacterium longum NCC 3001, Bifidobacterium longum NCC 2705,
Bifidobacterium breve NCC 2950, Bifidobacterium lactis NCC 2818,
Lactobacillus paracasei NCC 2461, Lactobacillus rhamnosus NCC 4007,
Lactobacillus casei NCC 4006, Lactobacillus acidophilus NCC 3009,
Lactobacillus casei ACA-DC 6002 (NCC 1825), and Escherichia coli
Nissle. Several starter culture strains including some strains
commercially used to produce Nestle Lc1 fermented products were
also tested: Streptococcus thermophilus NCC 2019, Streptococcus
thermophilus NCC 2059, Lactobacillus bulgaricus NCC 15 and
Lactococcus lactis NCC 2287.
[0113] Bacterial cells were cultivated in conditions optimized for
each strain in 5-15 L bioreactors. All typical bacterial growth
media are usable. Such media are known to those skilled in the art.
When pH was adjusted to 5.5, 30% base solution (either NaOH or
Ca(OH).sub.2) was added continuously. When adequate, anaerobic
conditions were maintained by gassing headspace with CO.sub.2. E.
coli was cultivated under standard aerobic conditions.
[0114] Bacterial cells were collected by centrifugation
(5,000.times.g, 4.degree. C.) and re-suspended in phosphate buffer
saline (PBS) in adequate volumes in order to reach a final
concentration of around 10.sup.9-10.sup.10 cfu/ml. Part of the
preparation was frozen at -80.degree. C. with 15% glycerol. Another
part of the cells was heat treated by: [0115] Ultra High
Temperature: 140.degree. C. for 15 sec; by indirect steam
injection. [0116] High Temperature Short Time (HTST): 74.degree.
C., 90.degree. C. and 120.degree. C. for 15 sec by indirect steam
injection [0117] Long Time Low Temperature (85.degree. C., 20 min)
in water bath
[0118] Upon heat treatment, samples were kept frozen at -80.degree.
C. until use.
In Vitro Immunoprofiling of Bacterial Preparations:
[0119] The immune profiles of live and heat treated bacterial
preparations (i.e. the capacity to induce secretion of specific
cytokines from human blood cells in vitro) were assessed. Human
peripheral blood mononuclear cells (PBMCs) were isolated from blood
filters. After separation by cell density gradient, mononuclear
cells were collected and washed twice with Hank's balanced salt
solution. Cells were then resuspended in Iscove's Modified
Dulbecco's Medium (IMDM, Sigma) supplemented with 10% foetal calf
serum (Bioconcept, Paris, France), 1% L-glutamine (Sigma), 1%
penicillin/streptomycin (Sigma) and 0.1% gentamycin (Sigma).
[0120] PBMCs (7.times.10.sup.5 cells/well) were then incubated with
live and heat treated bacteria (equivalent 7.times.10.sup.6
cfu/well) in 48 well plates for 36 h. The effects of live and heat
treated bacteria were tested on PBMCs from 8 individual donors
splitted into two separated experiments. After 36 h incubation,
culture plates were frozen and kept at -20.degree. C. until
cytokine measurement. Cytokine profiling was performed in parallel
(i.e. in the same experiment on the same batch of PBMCs) for live
bacteria and their heat-treated counterparts.
[0121] Levels of cytokines (IFN-.gamma., IL-12p40, TNF-.alpha. and
IL-10) in cell culture supernatants after 36 h incubation were
determined by ELISA (R&D DuoSet Human IL-10, BD OptEIA Human
IL12p40, BD OptEIA Human TNF.alpha., BD OptEIA Human IFN-.gamma.)
following manufacturer's instructions. IFN-.gamma., IL-12p40 and
TNF-.alpha. are pro-inflammatory cytokines, whereas IL-10 is a
potent anti-inflammatory mediator. Results are expressed as means
(pg/ml) +/-SEM of 4 individual donors and are representative of two
individual experiments performed with 4 donors each. The ratio
IL-12p40/IL-10 is calculated for each strain as a predictive value
of in vivo anti-inflammatory effect (Foligne, B., et al., 2007,
World J. Gastroenterol. 13:236-243).
[0122] Numerical cytokine values (pg/ml) determined by ELISA (see
above) for each strain were transferred into BioNumerics v5.10
software (Applied Maths, Sint-Martens-Latem, Belgium). A Principal
Component Analysis (PCA, dimensioning technique) was performed on
this set of data. Subtraction of the averages over the characters
and division by the variances over the characters were included in
this analysis.
Results
Anti-Inflammatory Profiles Generated by Ultra High Temperature
(UHT)/High Temperature Short Time (HTST)-Like Treatments
[0123] The probiotic strains under investigation were submitted to
a series of heat treatments (Ultra High Temperature (UHT), High
Temperature Short Time (HTST) and 85.degree. C. for 20 min) and
their immune profiles were compared to those of live cells in
vitro. Live micro-organisms (probiotics and/or dairy starter
cultures) induced different levels of cytokine production when
incubated with human PBMC (FIGS. 1, 2, 3, 4 and 5). Heat treatment
of these micro-organisms modified the levels of cytokines produced
by PBMC in a temperature dependent manner. "Short-time high
temperature" treatments (120.degree. C. or 140.degree. C. for 15'')
generated non replicating bacteria with anti-inflammatory immune
profiles (FIGS. 1, 2, 3 and 4). Indeed, UHT-like treated strains
(140.degree. C., 15 sec) induced less pro-inflammatory cytokines
(TNF-.alpha., IFN-.gamma., IL-12p40) while maintaining or inducing
additional IL-10 production (compared to live counterparts). The
resulting IL-12p40/IL-10 ratios were lower for any UHT-like treated
strains compared to live cells (FIGS. 1, 2, 3 and 4). This
observation was also valid for bacteria treated by HTST-like
treatments, i.e. submitted to 120.degree. C. for 15 sec (FIGS. 1,
2, 3 and 4), or 74.degree. C. and 90.degree. C. for 15 sec (FIG.
5). Heat treatments (UHT-like or HTST-like treatments) had a
similar effect on in vitro immune profiles of probiotic strains
(FIGS. 1, 2, 3 and 5) and dairy starter cultures (FIG. 4).
Principal Component Analysis on PBMC data generated with live and
heat treated (140.degree. C., 15'') probiotic and dairy starter
strains revealed that live strains are spread all along the x axis,
illustrating that strains exhibit very different immune profiles in
vitro, from low (left side) to high (right side) inducers of
pro-inflammatory cytokines. Heat treated strains cluster on the
left side of the graph, showing that pro-inflammatory cytokines are
much less induced by heat treated strains (FIG. 6). By contrast,
bacteria heat treated at 85.degree. C. for 20 min induced more
pro-inflammatory cytokines and less IL-10 than live cells resulting
in higher IL-12p40/IL-10 ratios (FIG. 7).
Anti-Inflammatory Profiles are Enhanced or Generated by UHT-Like
and HTST-Like Treatments.
[0124] UHT and HTST treated strains exhibit anti-inflammatory
profiles regardless of their respective initial immune profiles
(live cells). Probiotic strains known to be anti-inflammatory in
vivo and exhibiting anti-inflammatory profiles in vitro (B. longum
NCC 3001, B. longum NCC 2705, B. breve NCC 2950, B. lactis NCC
2818) were shown to exhibit enhanced anti-inflammatory profiles in
vitro after "short-time high temperature" treatments. As shown in
FIG. 1, the IL-12p40/IL-10 ratios of UHT-like treated
Bifidobacterium strains were lower than those from the live
counterparts, thus showing improved anti-inflammatory profiles of
UHT-like treated samples. More strikingly, the generation of
anti-inflammatory profiles by UHT-like and HTST-like treatments was
also confirmed for non anti-inflammatory live strains. Both live L.
rhamnosus NCC 4007 and L. paracasei NCC 2461 exhibit high
IL-12p40/IL-10 ratios in vitro (FIGS. 2 and 5). The two live
strains were shown to be not protective against TNBS-induced
colitis in mice. The IL-12p40/IL-10 ratios induced by L. rhamnosus
NCC 4007 and L. paracasei NCC 2461 were dramatically reduced after
"short-time high temperature" treatments (UHT or HTST) reaching
levels as low as those obtained with Bifidobacterium strains. These
low IL-12p40/IL-10 ratios are due to low levels of IL-12p40
production combined with no change (L. rhamnosus NCC 4007) or a
dramatic induction of IL-10 secretion (L. paracasei NCC 2461) (FIG.
2).
[0125] As a consequence: [0126] Anti-inflammatory profiles of live
micro-organisms can be enhanced by UHT-like and HTST-like heat
treatments (for instance B. longum NCC 2705, B. longum NCC 3001, B.
breve NCC 2950, B. lactis NCC 2818) [0127] Anti-inflammatory
profiles can be generated from non anti-inflammatory live
micro-organisms (for example L. rhamnosus NCC 4007, L. paracasei
NCC 2461, dairy starters S. thermophilus NCC 2019) by UHT-like and
HTST-like heat treatments. [0128] Anti-inflammatory profiles were
also demonstrated for strains isolated from commercially available
products (FIGS. 3 A & B) including a probiotic E. coli
strain.
[0129] The impact of UHT/HTST-like treatments was similar for all
tested probiotics and dairy starters, for example lactobacilli,
bifidobacteria and streptococci.
[0130] UHT/HTST-like treatments were applied to several
lactobacilli, bifidobacteria and streptococci exhibiting different
in vitro immune profiles. All the strains induced less
pro-inflammatory cytokines after UHT/HTST-like treatments than
their live counterparts (FIGS. 1, 2, 3, 4, 5 and 6) demonstrating
that the effect of UHT/HTST-like treatments on the immune
properties of the resulting non replicating bacteria can be
generalized to all probiotics, in particular to lactobacilli and
bifidobacteria and specific E. coli strains and to all dairy
starter cultures in particular to streptococci, lactococci and
lactobacilli.
EXAMPLE 2
Methodology
Bacterial Preparations
[0131] Five probiotic strains were used to investigate the immune
boosting properties of non-replicating probiotics: 3 bifidobacteria
(B. longum NCC3001, B. lactis NCC2818, B. breve NCC2950) and 2
lactobacilli (L. paracasei NCC2461, L. rhamnosus NCC4007).
[0132] Bacterial cells were grown on MRS in batch fermentation at
37.degree. C. for 16-18 h without pH control. Bacterial cells were
spun down (5,000.times.g, 4.degree. C.) and resuspended in
phosphate buffer saline prior to be diluted in saline water in
order to reach a final concentration of around 10E10 cfu/ml. B.
longum NCC3001, B. lactis NCC2818, L. paracasei NCC2461, L.
rhamnosus NCC4007 were heat treated at 85.degree. C. for 20 min in
a water bath. B. breve NCC2950 was heat treated at 90.degree. C.
for 30 minutes in a water bath. Heat treated bacterial suspensions
were aliquoted and kept frozen at -80.degree. C. until use. Live
bacteria were stored at -80.degree. C. in PBS-glycerol 15% until
use.
In Vitro Immunoprofiling of Bacterial Preparations
[0133] The immune profiles of live and heat treated bacterial
preparations (i.e. the capacity to induce secretion of specific
cytokines from human blood cells in vitro) were assessed. Human
peripheral blood mononuclear cells (PBMCs) were isolated from blood
filters. After separation by cell density gradient, mononuclear
cells were collected and washed twice with Hank's balanced salt
solution. Cells were then resuspended in Iscove's Modified
Dulbecco's Medium (IMDM, Sigma) supplemented with 10% foetal calf
serum (Bioconcept, Paris, France), 1% L-glutamine (Sigma), 1%
penicillin/streptomycin (Sigma) and 0.1% gentamycin (Sigma). PBMCs
(7.times.10.sup.5 cells/well) were then incubated with live and
heat treated bacteria (equivalent 7.times.10.sup.6 cfu/well) in 48
well plates for 36 h. The effects of live and heat treated bacteria
were tested on PBMCs from 8 individual donors splitted into two
separate experiments. After 36 h incubation, culture plates were
frozen and kept at -20.degree. C. until cytokine measurement.
Cytokine profiling was performed in parallel (i.e. in the same
experiment on the same batch of PBMCs) for live bacteria and their
heat-treated counterparts.
[0134] Levels of cytokines (IFN-.gamma., IL-12p40, TNF-.alpha. and
IL-10) in cell culture supernatants after 36 h incubation were
determined by ELISA (R&D DuoSet Human IL-10, BD OptEIA Human
IL12p40, BD OptEIA Human TNF, BD OptEIA Human IFN-.gamma.)
following manufacturer's instructions. IFN-.gamma., IL-12p40 and
TNF-.alpha. are pro-inflammatory cytokines, whereas IL-10 is a
potent anti-inflammatory mediator. Results are expressed as means
(pg/ml) +/-SEM of 4 individual donors and are representative of two
individual experiments performed with 4 donors each.
In Vivo Effect of Live and Heat Treated Bifidobacterium Breve
NCC2950 in Prevention of Allergic Diarrhea
[0135] A mouse model of allergic diarrhea was used to test the Th1
promoting effect of B. breve NCC2950 (Brandt E. B et al. JCI 2003;
112(11): 1666-1667). Following sensitization (2 intraperitoneal
injections of Ovalbumin (OVA) and aluminium potassium sulphate at
an interval of 14 days; days 0 and 14) male Balb/c mice were orally
challenged with OVA for 6 times (days 27, 29, 32, 34, 36, 39)
resulting in transient clinical symptoms (diarrhea) and changes of
immune parameters (plasma concentration of total IgE, OVA specific
IgE, mouse mast cell protease 1, i.e MMCP-1). Bifidobacterium breve
NCC2950 live or heat treated at 90.degree. C. for 30 min, was
administered by gavage 4 days prior to OVA sensitization (days -3,
-2, -1, 0 and days 11, 12, 13 and 14) and during the challenge
period (days 23 to 39). A daily bacterial dose of around 10.sup.9
colony forming units (cfu) or equivalent cfu/mouse was used.
Results
[0136] Induction of Secretion of `Pro-Inflammatory` Cytokines after
Heat Treatment
[0137] The ability of heat treated bacterial strains to stimulate
cytokine secretion by human peripheral blood mononuclear cells
(PBMCs) was assessed in vitro. The immune profiles based on four
cytokines upon stimulation of PBMCs by heat treated bacteria were
compared to that induced by live bacterial cells in the same in
vitro assay.
[0138] The heat treated preparations were plated and assessed for
the absence of any viable counts. Heat treated bacterial
preparations did not produce colonies after plating.
[0139] Live probiotics induced different and strain dependent
levels of cytokine production when incubated with human PBMCs (FIG.
8). Heat treatment of probiotics modified the levels of cytokines
produced by PBMCs as compared to their live counterparts. Heat
treated bacteria induced more pro-inflammatory cytokines
(TNF-.alpha., IFN-.gamma., IL-12p40) than their live counterparts
do. By contrast heat treated bacteria induced similar or lower
amounts of IL-10 compared to live cells (FIG. 8). These data show
that heat treated bacteria are more able to stimulate the immune
system than their live counterparts and therefore are more able to
boost weakened immune defences. In other words the in vitro data
illustrate an enhanced immune boost effect of bacterial strains
after heat treatment.
[0140] In order to illustrate the enhanced effect of heat-treated
B. breve NCC2950 (compared to live cells) on the immune system,
both live and heat treated B. breve NCC2950 (strain A) were tested
in an animal model of allergic diarrhea.
[0141] As compared to the positive control group, the intensity of
diarrhea was significantly and consistently decreased after
treatment with heat treated B. breve NCC2950 (41.1%.+-.4.8) whereas
the intensity of diarrhea was lowered by only 20.+-.28.3% after
treatment with live B. breve NCC2950. These results demonstrate
that heat-treated B. breve NCC2950 exhibits an enhanced protective
effect against allergic diarrhea than its live counterpart (FIG.
9).
[0142] As a consequence, the ability of probiotics to enhance the
immune defences was shown to be improved after heat treatment.
FURTHER EXAMPLES
[0143] The following growing up milk compositions may be
prepared:
TABLE-US-00001 >10 months Protein (g/100 kcal) 2.23 Whey/Casein
50/50 CHO (g/100 kcal) 13.0 Lactose (g/100 kcal) 7.7 Maltodextrine
(g/100 kcal) 4.0 Starch (g/100 kcal) 1.0 Sucrose (g/100 kcal) Fat
(g/100 kcal) 4.31 Prebiotics (g/100 kcal) -- Energy Kcal/100 ml 70
The growing up milk composition contains 10.sup.9 cfu Lactobacillus
johnsonii La1/g dry weight
TABLE-US-00002 >1 year Protein (g/100 kcal) 2.7 2.22 2.23 2.3
2.9 2.26 Whey/Casein 23/77 40/60 40/60 40/60 77/23 60/40 CHO (g/100
kcal) 12.2 13.5 13.1 13.0 11.9 13.9 Lactose (g/100 kcal) 5.05 6.7
6.1 4.9 4.42 5.33 Maltodextrine (g/100 kcal) 4.99 5.8 5.5 4.9 2.31
2.35 Starch (g/100 kcal) -- 1.0 1.0 2.9 2.29 3.17 Sucrose (g/100
kcal) 1.93 2.66 2.41 Fat (g/100 kcal) 4.5 4.14 4.31 4.3 4.53 3.93
Prebiotics (g/100 kcal) -- 0.58 0.58 0.52 -- 0.49 Energy Kcal/100
ml 70 70 70 79 80 The growing up milk compositions contain 10.sup.9
cfu heat treated (75.degree. C., 20 min) Bifidobacterium longum NCC
3001/g dry weight
TABLE-US-00003 >2 years Protein (g/100 kcal) 2.5 2.5 Whey/Casein
50/50 40/60 CHO (g/100 kcal) 14.6 14.6 Lactose (g/100 kcal) 8.7 6.9
Maltodextrine (g/100 kcal) 4.6 6.6 Starch (g/100 kcal) 1.0 1.0
Sucrose (g/100 kcal) Fat (g/100 kcal) 3.5 3.5 Prebiotics (g/100
kcal) -- 0.58 Energy Kcal/100 ml 70 70 The growing up milk
compositions contain 10.sup.9 cfu heat treated UHT treated
Lactobacillus johnsonii La1/g dry weight
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