NZ619099B2 - Probiotic compositions and methods - Google Patents
Probiotic compositions and methods Download PDFInfo
- Publication number
- NZ619099B2 NZ619099B2 NZ619099A NZ61909912A NZ619099B2 NZ 619099 B2 NZ619099 B2 NZ 619099B2 NZ 619099 A NZ619099 A NZ 619099A NZ 61909912 A NZ61909912 A NZ 61909912A NZ 619099 B2 NZ619099 B2 NZ 619099B2
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- NZ
- New Zealand
- Prior art keywords
- composition
- food product
- cfu
- paracasei cba
- fermented
- Prior art date
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
- 235000020985 whole grains Nutrition 0.000 description 1
- 235000008939 whole milk Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
- A23C9/1275—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss using only lactobacteriaceae for fermentation in combination with enzyme treatment of the milk product; using enzyme treated milk products for fermentation with lactobacteriaceae
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A23Y2220/63—
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C12R1/225—
Abstract
Disclosed is a probiotic bacterium, Lactobacillus paracasei CBA L74, International Depository Accession Number LMG P-24778; and methods of using it in a food product and a medicament for treating gastrointestinal disorders.
Description
/042959
Probiotic Compositions and s
Field of the Invention
The present ion relates to probiotic sms, food products
prepared with probiotic organisms and pharmaceutical compositions comprising
probiotic sms. These compositions are useful in stimulating the mucosal immune
system and for treatment of disorders associated with immaturity of the mucosal
immune system.
Background of the Invention
The intestinal epithelium is constantly exposed to foreign materials that
can be either harmful or beneficial to the host. As a result, the intestinal immune
system must strike a delicate balance between: 1) protective immune responses that
are induced by inal ens or toxins and 2) avoidance of immune responses
against both food antigens and the 1014 commensal beneficial microorganisms that
ly reside in the gut. Disruption of either the protective responses or the tolerance
responses can result in a wide array of disorders including, for example, infections,
inflammation, food allergies, food hypersensitivity, inflammatory bowel disease, Crohn’s
disease, celiac disease, periodontal disease, rheumatoid arthritis, atherosclerosis and
colon cancer.
The immunoregulatory network sing the intestinal immune system
changes with age. The network is poorly developed in human newborns and is
established gradually over the first few years of life. The immaturity of the immune
system plays a role in the prevalence of infections and food-related disorders in infants
and young children. Conversely, the ability of the intestinal immune system to respond
to new challenges declines in the elderly.
Gastrointestinal disorders, for example, infections, inflammatory disorders
and food-related disorders such as food allergies, food rance or food
hypersensitivity have a significant impact on the health and quality of life in both children
and adults. Infectious enteritis is the most common pediatric gastrointestinal
disorder. About 1 billion episodes occur worldwide each year, most commonly in
ping countries among children under 5 years of age. Worldwide death rates for
infectious gastroenteritis average from 3 to 6 million children per year. In the United
States, 25 to 35 million new cases occur annually, resulting in 300 to 400 deaths. in
addition, infectious gastroenteritis in the US results in an estimated 0
hospitalizations and 1.5 million outpatient visits at a cost in excess of 1 n dollars.
Food-related disorders such as allergies also have a substantial effect on health or both
children and adults. Symptoms of food allergies can vary depending upon the ty
of the allergy and can range from a mild tingling sensation around the mouth and lips to
life-threatening anaphylaxis. It is estimated that food ies affect between 140% of
the population in the U.S. The Center for Disease Control found that in 2007,
approximately 3 million children under age 18 years (3.9%) were reported to have a
food or digestive allergy in the previous 12 months. For some children, food allergies
become less severe with age, for , they remain a lifelong concern. Infants who
suffer from allergy early in life may develop “allergic ” For example, many
individuals who have severe allergic ons to cow’s milk in infancy at risk for the
development of asthma later in ood. There are indications that the prevalence of
food allergies is increasing ide.
Regardless of the etiology, gastrointestinal disorders not only adversely
affect a child’s health, but can have a serious impact on family economics, social
interactions and school and parental work attendance. There is a continuing need for
therapeutic strategies that promote gastrointestinal health, particularly in individuals who
are risk for or who suffer from gastrointestinal disorders.
Summagy of the Invention
W0 2012/1 77556 PCT/U52012/042959
The present invention provides compositions comprising a ted food
product, wherein the food product has been ted by the probiotic bacterium,
Lactobacillus paracasei CBA L74, Internationai Depository Accession Number LMG P-
24778. The food product can be a dairy product or a cereal t. Also ed are
compositions comprising the probiotic bacterium, Lactobacillus paracasei CBA L74,
International Depository Accession Number LMG P-24778 and a physiologically
acceptable carrier. The physiologically acceptable carrier can be a food product or a
pharmaceutical carrier. Also provided are methods of making a nutritional ition,
the method comprising: providing a food product; combining the food product with an
effective amount of the probiotic bacterium, Lactobacillus paracasei CBA L74,
International Depository ion Number LMG P-24778 and, optionally, a co-
inoculum, to form a mixture; and incubating the mixture at a temperature and for a time
sufficient for fermentation to occur. The nutritional composition may be dried. The
nutritional composition may be combined with one or more additional food ts.
For any of the compositions and methods described herein, the Lactobacillus paracasei
CBA L74 cells can be subjected to treatments that render them non-replicating. The
concentration of Lactobacillus paracasei CBA L74 in the compositions can vary
depending upon the intended use, e.g., as a nutritional ition or a pharmaceutical
composition. Useful ranges include the equivalent of about 1 x 102 colony-forming units
per gram (“cfu/g”) to about 1 x 1012 colony-forming units per gram (“cfu/g") dry weight.
Also provided are methods of treating a subject at risk for a developing a
gastrointestinal disorder, the method comprising: identifying a t at risk for a
gastrointestinal disorder; stering an ive amount of a composition comprising
a food product wherein the food product has been fermented by the probiotic bacterium,
Lactobacillus paracasei CBA L74, International tory Accession Number LMG P-
24778. The gastrointestinal disorder can be a mucosal immune system deficit, for
examine, an re immune system, a food allergy, a disorder ated with
diarrhea, a bacterial or viral infection, irritable bowel syndrome, inflammatory bowel
disease, Crohn’s disease, necrotizing enterocolitis or aging, particularly aging of the
gastrointestinal system.
W0 2012/1 77556
Also ed are s of treating a subject having a gastrointestinal
disorder. The methods include: identifying a subject having a gastrointestinal er;
administering an effective amount of a composition comprising a food product wherein
the food product has been fermented by the probiotic bacterium, Lactobacillus
paracasei CBA L74, International Depository Accession Number LMG P-24778. In
some embodiments, the methods include : identifying a subject having a gastrointestinal
disorder; administering an effective amount of a pharmaceutical composition comprising
the probiotic bacterium, Lactobacillus paracasei CBA L74, International Depository
Accession Number LMG P-24778. The intestinal disorder can be a mucosal
immune system deficit, for examine, an immature immune system, a food allergy, a
disorder associated with diarrhea, a ial or viral ion, irritable bowel syndrome,
inflammatory bowel disease, Crohn’s disease or necrotizing enterocolitis.
Also ed are and methods of modulating the immune system in a
subject. The methods include: identifying a subject in need of immune system
modulation and administering an effective amount of a composition comprising a food
product wherein the food product has been fermented by the probiotic ium,
acillus paracasei CBA L74, ational Depository Accession Number LMG P-
24778.
Articles of manufacture are also provided. These can include kits
comprising a measured amount of a ional composition comprising a fermented
food product, wherein the food product has been fermented by the probiotic bacterium,
Lactobacillus paracasei CBA L74, International Depository Accession Number LMG P-
24778 and one or more items selected from the group consisting of packaging material,
a package insert comprising instructions for use, a sterile fluid, and a sterile container.
In some embodiments, the kit can e a measured amount of a pharmaceutical
composition comprising Lactobacillus paracasei CBA L74, ational Depository
Accession Number LMG P-24778 and one or more items selected from the group
consisting of packaging material, a package insert comprising instructions for use, a
sterile fluid, and a sterile container.
W0 2012/177556 PCT/U82012/042959
The s of one or more embodiments of the invention are set forth in
the accompanying drawings and the description below. Other features, objects and
advantages of the invention will be apparent from the description and drawings and
from the .
Brief Description of the Drawings
These and other features and advantages of the present invention will be
more fully sed in, or rendered obvious by, the following detailed description of the
preferred embodiment of the invention, which is to be considered together with the
accompanying drawings wherein like numbers refer to like parts and further wherein:
is a table showing an analysis of the effect of L. paracasei CBA L74
on DC cell ype.
is a graph depicting iL-‘IO production in DOS co-cultured with
CacoZ exposed to L. paracasei CBA L74. is a graph depicting IL-12 tion
in DOS co-cultured with Caco2 exposed to L. paracasei CBA L74.
is a graph depicting proliferation of T cells exposed to DCs ured
with CaCoZ cells.
is a graph depicting IL—1 [5 production in intestinal mucosa of mice
supplemented with L. paracasei CBA L74.
is a graph depicting IL—4 production in intestinal mucosa of mice
mented with L. paracasei CBA L74.
is a graph depicting IgA production in intestinal mucosa of mice
supplemented with L. paracasei CBA L74.
is depicts an analysis of levels of TLR2, TLR4 and TLR9 in
intestinal mucosa of mice supplemented with L. paracasei CBA L74.
PCT/U52012/042959
is depicts an analysis of levels of PPARy in intestinal mucosa of
mice supplemented with L. paracasei CBA L74.
is a graph depicting serum lL-1B levels in mice supplemented with
L. paracasei CBA L74. is a graph depicting serum lL-4 levels in mice
supplemented with L. paracasei CBA L74.
is a table showing an analysis of the effect of L. sei CBA
L74 on DC phenotype in mice supplemented with L. paracasei CBA L74.
a is a graph depicting intestinal CD4+ lymphocyte phenotypes in
mice supplemented with L. paracasei CBA L74. FlG. 11b is a graph depicting intestinal
CD8+ lymphocyte phenotypes in mice supplemented with L. paracasei CBA L74
is a graph depicting lL-10 production in intestinal mucosa of mice
supplemented with milk ted by L. paracasei CBA L74
is a graph depicting lL-1B production in intestinal mucosa of mice
supplemented with milk fermented by L. sei CBA L74
is a graph depicting lgA production in intestinal mucosa of mice
mented with milk fermented by L. paracasei CBA L74
depicts an analysis of leveis of TLR2, TLR4, TLR9 and PPARy in
intestinal mucosa of mice mented with milk fermented by L. paracasei CBA L74.
depicts an analysis of levels of pNF-kB and lKBor in intestinal
mucosa of mice supplemented with milk fermented by L. paracasei CBA
is depicts an analysis of the effect of L. paracasei CBA L74 on DC
cell phenotype in mice supplemented with milk fermented by L. paracasei CBA.
F lG. 18 is an is of the effect of L. sei CBA L74 on DC cell
phenotype after exposure to LPS or CpG in mice supplemented with milk fermented by
L. paracasei CBA.
W0 2012/177556 PCT/U52012/042959
is a graph depicting intestinal CD4+ lymphocyte phenotypes in
mice supplemented with milk fermented by L. paracasei CBA L74.
is a graph depicting intestinal CD8+ lymphocyte phenotypes in
mice supplemented with milk fermented by L. paracasei CBA L74
shows histological evaluation of ileal mucosa in mice
supplemented with milk fermented by L. paracasei CBA L74
is a graph depicting lL-1B and IL~4 production in intestinal mucosa
of mice mented with rice fermented by L. sei CBA L74
is a graph depicting IL—10 production in intestinal mucosa of mice
supplemented with rice fermented by L. paracasei CBA L74
is an analysis of levels of TLR2 and TLR4 in intestinal mucosa of
mice supplemented with rice fermented by L. paracasei CBA L74.
a is an analysis of levels of PPARy in intestinal mucosa of mice
supplemented with rice fermented by L. sei CBA L74. b is an analysis of
levels of pNF-kB and IKBa in intestinal mucosa of mice mented with rice
fermented by L. paracasei CBA L74
is a table showing an analysis of the effect of L. paracasei CBA
L74 on DC phenotype in mice supplemented with rice fermented by L. paracasei CBA
L74.
is a table showing an is of the effect of L. paracasei CBA
L74 on DC phenotype after exposure to LPS or CpG in mice supplemented with rice
fermented by L. paracasei CBA L74.
is a graph ing intestinal CD4+ lymphocyte phenotypes in
mice supplemented with rice fermented by L. paracasei CBA L74
PCT/U52012/042959
is a graph depicting intestinal CD8+ lymphocyte phenotypes in
mice supplemented with rice fermented by L. sei CBA L74
FIG 30 is a graph depicting an analysis of the effect of L. paracasei CBA
L74 cells and cell supernatant on lL-10 production in human MoDCs presence of
Salmonella typhimurium.
FIG 31 is a graph depicting an is of the effect of L. paracasei CBA
L74 cells and cell supernatant on IL-12p70 production in human MoDCs in the presence
of Salmonella typhimurium.
FIG 32 is a graph depicting an analysis of the effect of L. paracasei CBA
L74 fermented milk on IL—10 production in human MoDCs presence of Salmonella
typhimurium and the effect of inactivation of L. paracasei CBA L74 on IL-10 production
in human MoDCs in the presence of ella typhimurium.
FIG 33 is a graph depicting an analysis of the effect of L. paracasei CBA
L74 fermented milk on IL-12p70 production in human MoDCs presence of Salmonella
typhimurium and the effect of vation of L. paracasei CBA L74 on IL-12p70
production in human MoDCs in the presence of ella typhimun'um.
FIG 34 is a graph depicting an analysis of the effect of L. paracasei CBA
L74 fermented rice on IL-1B, TNF-d and IL—10 in a gut tissue explant model in the
presence of Salmonella typhimurium.
Detailed Description
The present ion is based, in part, on the inventors’ discovery that
foods fermented by the tic organism Lactobaci/lus paracasei, strain CBA L74, can
have immunomodulatory properties. This strain was isolated by the inventors and
deposited under the Budapest Treaty on the ational Recognition of the Deposit of
Micro-organisms for the Purposes of Patent Procedure on September 9, 2008 at the
PCT/U52012/042959
Belgian Coordinated Collections of Micro—organisms (BCCM) Laboratorium voor
Microbiologie (LMG), Ghent, Belgium. The Accession Number given by the
International Depositary Authority is LMG P-24778. For ease of reading, we will not
repeat the phrase “Accession Number LMG P~24778” on every occasion. it is to be
understood that where we refer to L. paracasei, strain CBA L74, we refer to the
deposited strain having the Accession Number LMG P-24778.
The compositions of the invention e the probiotic organism, L.
paracasei CBA L74. The World Health Organization has defined probiotics as: "Live
microorganisms which when administered in adequate amounts confer a health benefit
on the host.” In some embodiments, the L. paracasei CBA L74 can be subjected to
treatments that render them non—replicating, for example, exposure to heat, dessication,
y—irradiation, or adiation. A non-replicating L. paracasei CBA L74 can be a dead
cell or a living cell that has been rendered incapable of cell division. A non—replicating L.
paracasei CBA L74 can be an intact cell or a cell that has undergone partial or complete
lysis. In some embodiments, the non—replicating cells can include a mixture of intact
and lysed cells.
While we e we tand certain events that occur upon
administration of itions comprising or made by fermentation with L. paracasei
CBA L74, the compositions of the present invention are not limited to those that work by
affecting any particular cellular mechanism. Our working hypothesis is that tic
organisms or compositions fermented with probiotic organisms may provide an
increased barrier to translocation of bacteria and ial products across mucosa,
itively e potential pathogens, modify of host response to microbial
products, and enhance enteral ion in ways that inhibits the growth of pathogens.
The beneficial effects of compositions comprising non-replicating probiotic organisms
may derive for example, from metabolites produced during fermentation, for example,
organic acids such as lactic acid, butyric acid or acetic acid. Alternatively or in addition,
microbial DNA, 9.9, unmethylated CpG dinucleotides, bacterial cell wall fragments and
other sub-cellular bacterial components, such as proteins, carbohydrates and lipids,
may exert immunomodulatory effects on the l immune system.
W0 2012/177556 PCT/U$2012/042959
The inventors have found that isolated L. paracasei CBA L74 modulated
the levels of both pro- and anti—inflammatory markers when assayed in vitro and in vivo.
Moreover, immunomodulatory effects were also ed ing administration of
foods that had been fermented by L. paracasei CBA L74. Such immunomodulatory
effects were noted even when the fermented foods had been treated, e.g., by heat, to
render the L. paracasei CBA L74 non-replicating. Accordingly, the invention features
compositions and methods that can be used to stimulate the intestinal immune system.
The compositions can include food products that have been fermented by L. paracasei
CBA L74. The food products can be any of a wide range of foods that are amenable to
fermentation by L. paracasei CBA L74. In some embodiments, the compositions can
include isolated L. paracasei CBA L74 and a physiological carrier. The carrier may be a
food product, but the invention is not so limiting and in some embodiments the carrier
may be a pharmacological carrier. Also ed are s of making and using the
compositions. The methods of the invention include methods of producing
itions comprising L. paracasei CBA L74, methods of fermenting food products
with L. paracasei CBA L74 and methods of stering the compositions to generate
an immunomodulatory response in a subject. The compositions may be administered to
a subject having an immature immune , a subject at risk for a gastrointestinal
disorder or who has a gastrointestinal disorder. The methods can be used on human
subjects, for e, infants and children, or in veterinary medicine. Regardless of
the t (whether human or non-human), any of the present methods can include a
step of identifying the subject. For example, the methods can include a step of
determining whether the subject is in need of treatment.
itions
Fermented foods
The compositions of the invention include nutritional compositions, i.e.,
food products fermented by the probiotic organism, L. paracasei CBA L74. Any food
t le to fermentation by L. paracasei CBA L74 may be used. The food
product can be a dairy product, for example, milk or a milk-based product. Exemplary
W0 2012/177556 2012/042959
milk sources include, without limitation, , sheep, goats, yaks, water buffalo, horses,
donkeys, er and camels. Regardless of the source, the milk or milk products can
be in any form suitable for fermentation by L. paracasei CBA L74. For example, the
milk can be whole milk or milk that has been sed to remove some or all of the
butterfat, e.g., 2% milk, 1% milk or no-fat milk. Alternatively or in addition, the milk can
be previously pasteurized and or homogenized, dried and reconstituted, condensed or
evaporated. Fractions of milk products including casein, whey protein or lactose may
also be used. In some embodiments, the milk product can be from about 1% to about
% reconstituted skim milk powder, for example about 2%, about 5%, about 7%,
about 9%, about 10%, about 12%, about 15%, about 20%, about 25%, about 30%
reconstituted skim milk powder. Prior to fermentation the milk product can be combined
with one or more of the following: a) a carbohydrate (e.g., a disaccharide such as
dextrose or a starch; b) a lipid; c) a n and d) a l. For e, skim milk
powder may be combined with dextrose to about 2%, e.g., about 0.25%, about 0.50%,
about 0.75%, about 1.0%, about 1.5% or about 2.0%.
The food product can be a cereal product, for example, rice, wheat, oats,
barley, corn, rye, m, millet, or ale. The cereal product can be a whole grain
or be milled into a flour. The food product can be a single kind of cereal or a mixture of
two or more kinds of cereals, e.g., oat flour plus malted barley flour. The cereal
products can be of a grade and type suitable for human consumption or can be
products suitable for consumption by domestic animals. Generally, the cereal product is
hydrated prior to fermentation. The concentration of cereal can vary, but useful ranges
include from about 5% to about 50% weight/volume, for example, about 8%
weight/volume, about 10% weight/volume, about 12% weight/volume, about 15%
weight/volume, about 18% weight/volume, about 20% weight/volume, about 22%
/volume, about 25% weight/volume, about 30% weight/volume, about 35%
weight/volume, about 40% weight/volume, about 45% weight/volume or about 50%
weight/volume. Exemplary concentrations include 15% weight/volume of rice or a
mixture of 18.5% weight/volume oat flour plus 5% weight/volume of malted barley flour.
The pH of the hydrated s may be adjusted using any acid suitable for
consumption. The acid can be, for example, an organic acid. Useful organic acids
W0 2012/177556
include acetic acid, citric acid, lactic acid, adipic acid, malic acid and tartaric acid. Any
combination of two or more acids can be used. In some embodiments, the pH may be
ed to about 4.0 using citric acid.
The food product can also be a vegetable or a fruit product, for example, a
juice, a puree, a concentrate, a paste, a sauce, a pickle or a ketchup. Exemplary
vegetables and fruits include, without limitation, squashes, e.g., ni, yellow squash,
winter squash, pumpkin; potatoes, asparagus, broccoli, Brussels sprouts, beans, e.g.,
green beans, wax beans, lima beans, fava beans, soy beans, e, carrots,
cauliflower, cucumbers, kohlrabi, leeks, scallions, onions, sugar peas, English peas,
peppers, turnips, rutabagas, tomatoes, apples, pears, peaches, plums, strawberries,
raspberries, blackberries, blueberries, lingonberries, boysenberries, gooseberrles,
grapes, currants, oranges, lemons, grapefruit, bananas, mangos, kiwifruit, and
carambola.
The food t can also be a ”milk“ made from grains (barley, oat or
spelt “milk”) tree nuts (almond, cashew, t, hazelnut or walnut “milk”), legumes
(soy, peanut, pea or lupin “milk”) or seeds (quinoa, sesame seed or sunflower seed
Also contemplated are food products sing animal proteins, for
example, meat, for example, sausages, dried meats, fish and dried fish products.
Regardless of the type of food product that is used, the t is
ed with L. paracasei CBA L74 and incubated at a temperature and for a time
sufficient for fermentation to occur. Any standard fermentation method known in the art
may be used. Specific fermentation ions will vary according to many factors
including, for example, the type of food product, the concentration of the food product,
the instrumentation that is used, the sample volume, the initial concentration of the L.
paracasei CBA L74 inoculum, the presence, if any, of a co-inoculum, the organoleptic
properties of the fermented food, and the intended use of the fermented food.
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Both the instrumentation and the substrate (i.e., the food t to be
fermented) are sterilized prior to inoculation with L. paracasei CBA L74 in order to
decrease the level of, or eliminate, viable bacteria and/or fungi and/or infectious viruses.
The instrumentation can be sterilized using standard methods or according to the
manufacturer’s instructions. Choice of a particular method for ization of the
substrate will depend, in part, on the stability of the substrate to the ization method.
For example, the substrate can be sterilized by steam and pressure, 9.9. by
autoclaving, repeated cycles of heating and cooling (e.g., tyndalization) exposure to
high pressures (e.g., pascalization), ultrafiltration, or radiation (e.g., re to
gamma-, x~, e—beam, and/or ultra-violet ength of 10 nm to 320 nm, e.g., 50 nm to
320 nm, 100 nm to 320 nm, 150 nm to 320 nm, 180 nm to 320 nm, or 200 nm to 300
nm). Aliquots of the ate can be d following treatment and plated on
suitable media to confirm the absence of bacterial and/or fungal contaminants. If the
ate has been sterilized by exposure to high temperatures, it should be cooled to
at least 37°C prior to inoculation with L. paracasei CBA L74.
The substrate can be inoculated with L. paracasei CBA L74 according to
standard methods, for example, from fresh liquid culture or a freeze-dried culture that
has been resuspended in aqueous medium for a short time prior to inoculation. in
general, L. sei CBA L74 are added at concentrations of about 0.5 x 1 06 to about
1 x 1 Oscfu/ml of substrate, 9.9., about 1 x 106cfu/ml, about 2 x /ml, about 5 x
106cfu/ml, 7 x loscfu/ml 8x 106cfu/ml. The culture should be agitated sufficiently to
produce a relatively uniform distribution of ia and substrate, but not excessively
since L. paracasei CBA L74 is an anaerobic bacterium. For example, a five liter culture
may be agitated at about 150 rpm. Fermentation temperature is generally at 37°C.
Various parameters, for example, the pH, the partial pressure of 02, stirrer speed,
temperature, gas , foam level and substrate concentration can be monitored
during during fermentation and adjusted accordingly. Growth of the L. paracasei CBA
L74 can be monitored using standard microbiological methods. Fermentation is carried
out until the concentration of L. paracasei CBA L74 is about between about 108/ml and
about tog/ml. Depending upon the substrate and other conditions, this concentration
PCT/U82012/042959
may be reached in about 10 to about 30 hours after inoculation, e.g., about 12 hours,
about 15 hours, about 18 hours, about 24 hours, about 30 hours.
Samples of the substrate can be assayed before, during and after
fermentation for quality assurance using rd iological methods. Exemplary
s include, but are not limited to, growth on Rogosa agar for L. paracasei CBA
L74, growth on plate count agar (FDA) for total aerobes, growth on McConkay agar for
coliforms, growth on reinforced clostridial agar (RCM) for 'dia. In addition to
colony counts, colony morphologies can be observed and compared to reference
samples.
In some ments, a co-inoculum can be added along with the L.
paracasei CBA L74 in order to help initiate fermentation. Useful co—inocula for
fermentation of milk products include, for example, without limitation, Streptococcus
thermophilus, Lactobacillus paracasei, Lactobaciilus salivarious, Lactobacillus
rhamnosus, Lactobacillus casei, Lactobacillus lactis, Lactobacillus delbrueckii, subsp.
Bulgaricus, Lactobacillus acidophi/us, Lactobaciifus brevis, or ostoc
mesenteroides. In general, the concentration of the oo-inoculum will be lower than that
of L. paracasei CBA L74, for example, about 1 x 104/ml x105/ml. The final concentration
of S. thermophi/us can range from about 0.5 x 108Iml to about 2.5 x 108/ml.
Food Products
Once suitable concentrations of L. paracasei CBA L74 have been
reached, the fermented food can be further processed for use. In some ments,
the pH of the fermented food can be ed, for example from about 3.0 to nearer to
neutrality, e.g., 6.5, with the addition of NaOH or KOH. In some embodiments the
fermented food can be dried. The ted food product can be dried by any method
known in the art that will result in the retention of immunomodulatory properties of the
fermented food. Exemplary drying methods include spray drying, freeze-drying e.g.,
lyophilization, or drum-drying. The final water content of the ted food product
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may vary but can be between about 1% and about 10% or more. In some
embodiments, the drying process can render the L. paracasei CBA L74 non-replicating.
The dried fermented foods can be ed before use. Depending on the
amount of liquid used in the hydration, the fermented food products may contain the
equivalent of about 102 and 1012 cfu/ml of L. paracasei CBA L74. The dried L.
paracasei CBA L74 do not form colonies, so it is understood that this amount is
calculated based on the number of live bacteria that were present in the fermented
foods prior to the drying step. In some embodiments, the fermented food products may
include the equivalent of about 107 to about 1012 cfu/g, e.g., about 5 x107 cfu/g, about 1
x108 cfu/g, about 5 x108 cfu/g, about 1 x109 cfu/g, about 5 x109 cfu/g, about 1 x1010
cfu/g, about 5 x1010 cfu/g, about 1 x1011 cfu/g, about 5 x1011 cfu/g of dry weight.
Two or more fermented food products prepared by the methods of the
invention may be combined prior to administration. For example, fermented milk
products may be combined with fermented cereal products. Alternatively, the fermented
food product can be ed with other food ts, for example, rmented
food products or food ts ted using other bacterial strains. Any
ation can be used provided that the immunomodulatory properties of the
fermented food are retained. Exemplary food products include, without limitation, dairy
products, e.g., milk, yoghurt, curd, cheese and cheese-based products, fermented
milks, milk-based fermented products, milk-based powders, infant formulae, milk-based
strained infant foods, ice cream, gelato, puddings, soups, sauces, purees, or dressings,
ional formulas for the elderly; cereal products e.g., pablum, cereal—based strained
infant foods, oatmeal, farina, semolina, polenta, pasta, biscuits, crackers, energy bars;
vegetable products, e.g., purees, vegetable-based strained infant foods, pickled
vegetables ing cucumbers, cabbage, carrots, beans, peppers, or relishes; fruit
products, e.g., fruit—based strained infant foods, tomato products, purees, sauces,
pastes, ketchups, fruit purees; or a protein—based products, e.g., legumes, sausages,
lunch meats, hot dogs, or pureed meats. ln some embodiments the fermented food
may be combined with pet foods or animal feeds.
PCT/U82012/042959
In some embodiments, the compositions can include L. paracasei CBA
L74 fermentates, from which all or substantially all, of the L. paracasei CBA L74 cells
have been d. Methods for separating cells from growth media are well known in
the art and can rely upon physical methods, for example, centrifugation to produce a
cell pellet and a culture supernatant, tion, ultrafiltration, tangential flow—filtration,
normal flow filtration or reverse osmosis. Alternatively or in addition, the separation
method can be ligand-based and include, for example, an antibody that ically
binds to L. paracasei CBA L74. The dy can be coupled to a solid support such as
a magnetic bead.
Isolated L. paracasei CBA L74
In some embodiments, the compositions of the invention e L.
paracasei CBA L74 that are partially or substantially isolated from the media in which
they were grown. The L. paracasei CBA L74 can be live or non-replicating, e.g.,
inactivated, for example, by heat-treatment. The cells can be lyophilized or freeze-dried
under conditions that preserve cell ity. Methods of lyophilization are well known in
the art.
Physiological carriers
In some embodiments, the compositions of the invention may include
isolated L. paracasei CBA L74 in combination with a logically acceptable carrier.
The L. paracasei CBA L74 can be live or non—replicating, e.g., inactivated, for example,
by heat—treatment. The dosage may vary, but can range from the equivalent of about
102 to about 1012 cfulg, e.g., 1 x1 02 cfu/g, 5 x102 cfu/g, 1 x103 cfu/g, 5 x103 cfu/g, 1
x104 cfu/g, 5 x104 cfulg, 1 x105 cfulg, 5 x105 cfulg, 1 x106 cfu/g, 5 x106 cfu/g, 1 x107
cfu/g, 5 x107 cfu/g, 1 x1 08 cfu/g, 5 x108 cfu/g, 1 x109 cfu/g, 5 x1 09 cfu/g, 1 x1010
cfu/g, 5 x1010 cfu/g, 1 x1011cfu/g, 5 x1011cfu/g, 1 x1012 cfu/g.ofdry .
The physiologically acceptable r can be a food or food product.
Isolated L. paracasei CBA L74 can be added to a food or food product prior to
packaging or processing. Alternatively or in addition, isolated L. paracasei CBA L74
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can be added to a food or food product prior to consumption. For example, isolated L.
sei CBA L74 can be ed with any of the foods or food products described
above. The food product can be a fermented food product or an unfermented food
product. For example, isolated L. paracasei CBA L74 can be added to an unfermented
dairy or cereal product. In some embodiments, the L. paracasei CBA L74 can be added
to a food or food product to include the equivalent of about 107 to about 1012 cfu/g, e.g.,
about 5 x107 cfu/g, about 1 x108 cfu/g, about 5 x108 cfu/g, about 1 x109 cfu/g, about 5
x109 cfu/g, about 1 x’lO10 cfu/g, about 5 x1010 cfu/g, about 1 x1011 cfu/g, about 5 x1011
cfu/g of dry weight.
Pharmaceutical carriers
The itions also include a pharmaceutically acceptable carrier. We
use the terms “pharmaceutically acceptable” (or “pharmacologically acceptable”) to refer
to molecular entities and compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal or a human, as appropriate. The
term “pharmaceutically acceptable carrier,” as used herein, includes any and all
solvents, dispersion media, coatings, antibacterial, ic and absorption delaying
agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may
be used as media for a pharmaceutically acceptable substance.
This invention also includes pharmaceutical compositions which contain,
as the active ingredient, the L. paracasei CBA L74 described herein, in combination with
one or more pharmaceutically able carriers. In some embodiments, the L.
paracasei CBA L74 can be sterilized using conventional ization ques before
or after it is ed with the pharmaceutically acceptable carrier. In making the
compositions of the invention, the L. paracasei CBA L74 is typically mixed with an
ent, diluted by an excipient or enclosed within such a carrier in the form of, for
example, a capsule, tablet, sachet, paper, or other ner. When the excipient serves
as a diluent, it can be a solid, lid, or liquid material (9.9., normal saline), which
acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs,
W0 77556 PCT/U52012/042959
suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium),
ointments, soft and hard gelatin capsules, itories, sterile able solutions, and
sterile packaged powders. As is known in the art, the type of diluent can vary
depending upon the intended route of administration. The resulting compositions can
include additional agents, such as preservatives. The excipient or carrier is selected on
the basis of the mode and route of administration. Suitable pharmaceutical carriers, as
well as pharmaceutical ities for use in ceutical formulations, are
described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known
reference text in this field, and in the USP/NF (United States Pharmacopeia and the
National Formulary). Some examples of suitable excipients include lactose, dextrose,
sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,
tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,
ose, water, syrup, and methyl cellulose. The formulations can additionally include:
lubricating agents such as talc, magnesium stearate, and mineral oil; g agents;
emulsifying and suspending agents; ving agents such as methyl- and
propylhydroxy-benzoates; sweetening agents; and flavoring agents. The pharmaceutical
compositions can be formulated so as to provide quick, sustained or delayed release of
the active ingredient after stration to the patient by employing procedures known
in the art.
Pharmaceutically acceptable compositions for use in the present methods,
including those in which L. paracasei CBA L74 is entrapped in a colloid for oral delivery,
can be prepared according to rd techniques. The L. paracasei CBA L74 can be
dried and compacted by grinding or pulverizing and inserted into a capsule for oral
administration. in some embodiments, the L. paracasei CBA L74 can be combined one
or more excipients, for example, a disintegrant, a filler, a glidant, or a vative.
Suitable capsules e both hard shell es or soft—shelled capsules. Any lipid-
based or polymer-based colloid may be used to form the capusule. Exemplary
polymers useful for colloid preparations include gelatin, plant polysaccharides or their
derivatives such as carrageenans and ed forms of starch and cellulose, e.g.,
hypromellose. Optionally, other ingredients may be added to the gelling agent solution,
for example plasticizers such as glycerin and/or sorbitol to se the capsule's
W0 2012/177556 PCT/U82012/042959
ss, ng agents, preservatives, disintegrants, lubricants and surface
treatment. In some embodiments, the capsule does not include gelatin. In other
embodiments, the capsule does not include plant polysaccharides or their derivatives.
Regardless of their original source or the manner in which they are
obtained, the L. paracasei CBA L74 of the invention can be formulated in accordance
with their use. These compositions can be prepared in a manner well known in the
pharmaceutical art, and can be administered by a variety of routes, depending upon
whether local or systemic treatment is desired and upon the area to be treated.
Administration may be oral or topical (including ophthalmic and to mucous membranes
including asal, vaginal and rectal delivery). In some embodiments, administration
can be pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by
nebulizer; lntratracheal, intranasal, epidermal and transdermal) or ocular. Methods for
ocular delivery can include topical administration (eye drops), subconjunctival,
periocular or intravitreal ion or introduction by balloon er or ophthalmic
inserts ally placed in the conjunctival sac. Parenteral administration es
intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or
infusion; or intracranial, e.g., intrathecal or entricular administration. Parenteral
administration can be in the form of a single bolus dose, or may be, for example, by a
continuous perfusion pump. Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions, creams, gels,
drops, suppositories, sprays, s, powders, and the like. Conventional
pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable.
The compositions can be ated in a unit dosage form, each dosage
containing, for example, from about 0.005 mg to about 2000 mg of L. paracasei CBA
L74 per daily dose. The term "unit dosage forms" refers to physically discrete units
le as unitary dosages for human subjects and other mammals, each unit
containing a predetermined quantity of active material calculated to produce the desired
therapeutic effect, in association with a suitable pharmaceutical ent. For preparing
solid compositions such as tablets, the pal active ingredient is mixed with a
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pharmaceutical excipient to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present ion. When referring to these
preformulation compositions as homogeneous, the active ingredient is typically
dispersed evenly throughout the composition so that the composition can be y
ided into equally effective unit dosage forms such as tablets, pills and capsules.
This solid preformulation is then subdivided into unit dosage forms of the type described
above containing from, for example, 0.005 mg to about 1000 mg of the L. paracasei
CBA L74 of the present ion.
The compositions can be formulated in a unit dosage form, each dosage
containing, for example, from about 0.1 mg to about 50 mg, from about 0.1 mg to about
40 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from
about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to
about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg,
from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg,, from about 0.5
mg. to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5
mg; from about 1 mg from to about 50 mg, from about 1 mg to about 30 mg,, from about
1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg;
from about 5 mg to about 50 mg, from about 5 mg to about 20 mg, from about 5 mg to
about 10 mg; from about 10 mg to about 100 mg, from about 20 mg to about 200 mg,
from about 30 mg to about 150 mg, from about 40 mg to about 100 mg, from about 50
mg to about 100 mg of the active ingredient.
In some embodiments, tablets or pills of the present invention can be
coated or otherwise compounded to provide a dosage form affording the advantage of
prolonged action. For example, the tablet or pill can comprise an inner dosage and an
outer dosage component, the latter being in the form of an envelope over the former.
The two components can be separated by an enteric layer which serves to resist
egration in the stomach and permit the inner component to pass intact into the
duodenum or to be delayed in release. A variety of als can be used for such
c layers or coatings, such materials including a number of polymeric acids and
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mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose
The liquid forms in which the compositions of the present invention can be
incorporated for administration orally or by ion include aqueous solutions, suitably
flavored , aqueous or oil suspensions, and flavored emulsions with edible oils
such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and
similar pharmaceutical vehicles.
The proportion or concentration of the compositions of the invention in a
pharmaceutical composition can vary depending upon a number of factors including
dosage, chemical characteristics (e.g., hydrophobicity), and the route of stration.
For example, the L. paracasei CBA L74 of the invention can be provided in a capsule
containing from about 0.005 mg gram to about 1000 mg for oral stration.
Alternatively or in addition, the dosage can be expressed as cfu/g of dry weight. The
dosage may vary, but can range from the equivalent of about 102 to about 1012 cfu/g,
e.g., 1 x102 cfu/g, 5 x102 cfu/g, 1 x103 cfu/g, 5 x103 cfu/g, 1 x104 cfu/g, 5 x104 cfu/g, 1
x105 cfu/g, 5 x105 cfu/g, 1 x106 cfu/g, 5 x106 cfu/g, 1 x107 cfu/g, 5 x107 cfu/g, 1 x108
cfu/g, 5 x108 cfu/g, 1 x1 09 cfu/g, 5 x1 09 cfu/g, 1 x1010 cfu/g, 5 x1010 cfu/g, 1 x1011
cfu/g, 5 x1011 cfu/g, 1 x1012 cfu/g.of dry weight.
Methods of use
The compositions disclosed herein are generally and variously useful for
ation of an immunomodulatory se in the mucosal immune system.
Subjects for whom such stimulation is beneficial include those have a mucosal immune
system deficit, for example, those having an immature immune system, such as infants
or small children, those having a depressed immune system, such as the elderly,
patients taking immunosuppressive drugs, radiation or chemotherapy, those having a
hyperactivated immune system due to allergies or autoimmune disorders and those
suffering from gastrointestinal disorders. Gastrointestinal disorders can include
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infections due to viruses, e.g., rotaviruses; pathogenic bacteria, e.g., ella,
Yersinia, Shigella, Listeria, Closfridium, E. coil, E. sakazaki, H,pylori; or pathogenic
oa, e.g., Entamoeba histolytica, Cn/ptosporidium spp, Campy/obacter spp.
Gastrointestinal disorders can also include, for example, food allergies, food
hypersensitivity, irritable bowel me, inflammatory bowel disease, pouchitis,
Crohn’s disease, ulcerative colitis, celiac disease, necrotizing enterocolitis, and aging,
particularly aging of the intestinal system,
A subject is effectively treated whenever a clinically beneficial result
ensues. This may mean, for example, a complete resolution of the symptoms
associated with a mucosal immune system deficit, a se in the ty of the
symptoms associated with a mucosal immune system deficit, or a slowing of the
progression of ms associated with a mucosal immune system deficit. These
methods can further include the steps of a) identifying a subject (9.9., a patient and,
more specifically, a human patient) who has a mucosal immune system deficit; and b)
providing to the subject a composition sing L. paracasei CBA L74 described
herein, such as any fermented food product or composition sing L. sei
CBA L74 in a physiologically acceptable carrier. An amount of such a composition
provided to the t that results in a te resolution of the symptoms associated
with a mucosal immune system deficit, a decrease in the severity of the symptoms
associated with a mucosal immune system deficit, or a slowing of the progression of
symptoms associated with a mucosal immune system deficit is considered a
therapeutically effective amount. The present methods may also include a monitoring
step to help optimize dosing and scheduling as well as predict outcome.
The methods disclosed herein can be applied to a wide range of species,
e.g., humans, non-human primates (e.g., monkeys), horses, pigs, cows or other
livestock, dogs, cats or other mammals kept as pets, rats, mice, or other laboratory
animals. The compositions described herein are useful in therapeutic compositions and
ns or for the manufacture of a medicament for use in treatment of conditions as
described herein (9.9., a mucosal immune system t due to immaturity, aging,
infection, food allergies, an inflammatory or autoimmune disorder.)
PCT/U52012/042959
The nutritional itions described herein can be administered orally
as part of the ry daily diet of a subject. The food itions may be
administered as nutritional support to both en and adults. When ated as
pharmaceuticals, the compositions can be administered to any part of the host’s body
for subsequent delivery to a target cell. A composition can be delivered to, without
limitation, the brain, the ospinal fluid, joints, nasal mucosa, blood, lungs,
intestines, muscle tissues, skin, or the peritoneal cavity of a mammal. In terms of routes
of delivery, a composition can be administered by intravenous, intracranial,
intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal,
intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal
administration, or by gradual perfusion over time. In a further example, an aerosol
ation of a composition can be given to a host by inhalation.
Regardless of whether the compositions are formulated as food products
or as pharmaceuticals, the dosage required will depend on the route of administration,
the nature of the formulation, the nature of the subject’s condition, 9.9., immaturity of the
immune system or a gastrointestinal disorder, the subject’s size, , surface area,
age, and sex, other drugs being administered, and thejudgment of the attending
clinicians. Suitable dosages are in the range of 0.01-1,000 mg/kg. Some typical dose
ranges are from about 1 ug/kg to about 1 g/kg of body weight per day. In some
embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body
weight per day. In some embodiments, the dose can be, for example, 1 mg/kg, 2 mg/kg,
mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg or 100 mg/kg. The dosage is likely to depend
on such variables as the type and extent of progression of the disease or disorder, the
overall health status of the particular patient, the relative biological efficacy of the
compound selected, formulation of the excipient, and its route of administration.
Effective doses can be extrapolated from dose-response curves derived
from in vitro or animal model test systems. For example, in vitro analysis of cytokine
production by peripheral blood mononuclear cells (PBMCs) can be a useful for assaying
pro- and anti-inflammatory responses, e.g., secretion of lL-1B, lL-12, IL-4, TNF-d, or IL-
respectively. Compositions can also be ed for s in animal models, for
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example, lgA production, cytokine production by explants of Peyer’s patches, and
dendritic cell and T-cell responses.
Wide variations in the needed dosage are to be expected in view of the
variety of cellular targets and the ing efficiencies of various routes of
administration. Variations in these dosage levels can be ed using standard
empirical routines for optimization, as is well understood in the art. Administrations can
be single or multiple (9.9., 2— or 3—, 4—, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).
Encapsulation of the nds in a le delivery vehicle (e.g., polymeric
microparticles or implantable devices) may se the ency of delivery.
The duration of ent with any composition provided herein can be
any length of time from as short as one day to as long as the life span of the host (e.g.,
many years). For example, a composition can be administered once a week (for, for
example, 4 weeks to many months or years); once a month (for example, three to
twelve months or for many ; or once a year for a period of 5 years, ten years, or
longer. It is also noted that the frequency of treatment can be variable. For example,
the t compositions can be administered once (or twice, three times, etc.) daily,
weekly, monthly, or yearly. When the compositions are formulated as food product, for
example, the compositions can be administered daily at every meal.
Any method known to those in the art can be used to determine if a
particular response is induced. Clinical methods that can assess the degree of a
particular e state can be used to determine if a response is induced. For
example, a subject can be monitored for symptomatic relief, e.g., relief from colic,
diarrhea, constipation, nausea, ng, abdominal pain, cramping, heartburn,
nal distention, flatulence, or incontinence. Alternatively or in addition, serum
markers, imaging techniques, e.g., ultrasound, x-rays, and endoscopic methods can be
used.
The compositions may also be administered in conjunction with other
therapeutic modalities. Other therapeutic modalities will vary according to the particular
disorder, but can include, for example, anti-diarrhea medications, anti-emetics, anti—
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cholinergic agents, anti-inflammatory agents, antibiotics anti—histamines and other
dietary treatments, for example, hypoallergenic infant formulas. Concurrent
administration of two or more therapeutic agents does not require that the agents be
stered at the same time or by the same route, as long as there is an overlap in
the time period during which the agents are exerting their therapeutic .
Simultaneous or sequential administration is contemplated, as is administration on
different days or weeks.
es of manufacture
The compositions described herein can also be assembled in kits,
together with instructions for use. ingly, packaged products (e.g., containers
containing one or more of the L. paracasei CBA L74 compositions described herein and
packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations)
and kits, including at least one compound of the invention and instructions for use, are
also within the scope of the invention. In any of the packaged products or kits, the L.
paracasei CBA L74 compositions can include L. paracasei CBA L74 that have been
rendered non—replicating. For example, the kits can include measured amounts of a
nutritional composition including one or more food products ted with L. paracasei
CBA L74. The instructions for use can be conveyed by any suitable media. For
e, they can be printed on a paper insert in one or more ges or supplied
y or visually (e.g., on a compact disc). The packaging materials can include
packaging materials, for example, vials, s, containers. In some embodiments,
the kits can include measured amounts of a composition comprising L. paracasei CBA
L74 in a logically acceptable carrier along with packaging materials and
instructions for use in any of the formats described above. in some embodiments, the
kits can include containers containing one or more L. sei CBA L74 compositions,
e.g., L. paracasei CBA L74 and a pharmaceutical carrier, and one or more of a suitable
stabilizer, carrier molecule, flavoring, and/or the like, as appropriate for the intended
use. A product can include a container (e.g., a vial, jar, bottle, bag, or the like)
containing one or more L. paracasei CBA L74 compositions. In addition, an article of
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manufacture further may include, for example, packaging materials, instructions for use,
syringes, buffers or other control reagents for treating or monitoring the condition for
which prophylaxis or treatment is ed. The product may also include a legend
(e.g., a printed label or insert or other medium describing the product's use (e.g., an
audio- or videotape». The legend can be associated with the container (e.g., affixed to
the container) and can describe the manner in which the compound therein should be
administered (e.g., the ncy and route of administration), indications therefor, and
other uses. The ents of the kit may be suitable for immediate use. The
compounds can be ready for administration (e.g., present in ppropriate units),
and may include a pharmaceutically acceptable adjuvant, carrier or other diluent and/or
an additional therapeutic agent. The invention encompasses kits, however, that include
concentrated ations and/or als that may require dilution prior to use.
atively, the compounds can be provided in a concentrated form with a diluent and
instructions for dilution. The components of the kit may be le for immediate use.
The invention encompasses kits, however, that include concentrated ations
and/or materials that may require dilution prior to use.
Examples
Example 1: Isolation and characterization of L.paracasei CBA-174
We analyzed different strains of Lactobacilli for their ability to ferment
aqueous sions containing different concentrations of rice flour or wheat flour.
L.paracasei CBA L74 was selected for further analysis based on the low pH values and
high CFU counts. This strain was deposited under the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the Purposes of Patent
Procedure on September 9, 2008 at the Belgian Coordinated Collections of Micro-
organisms (BCCM) Laboratorium voor Microbiologie (LMG), Ghent, Belgium. The
Accession Number given by the International Depositary Authority is LMG P-24778.
e 2: Preparation of L.paracasei CBA L74 fermented milk
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Conditions:
. ate: 9% reconstituted skim milk powder, dextrose added at 0.25%
. Substrate heat treatment: UHT — 135°C for 33 or equivalent F0
. Co-lnoculum: 5 x 106 for Lactobacilius paracesei CBA-L74
x 104 for Streptococcus thermophi/us (as starter of the
fermentation)
o Fermentation Temperature: 37°C
0 Fermentation time: 15 h hours
0 pH during fermentation: no ment
- At the end of the fermentation pH adjustment to 6.5 with NaOH solution
o Spray drying with inlet ature 190°C and outlet temperature 90°C.
. Analysis: Cells count on the fermentate to ine Streptococcus thermophilus
and Lactobacillus paracesei CBA-L74
Eating; Lactobacilli selective agar (LBS) was used for detection of
Lactobacillus paracesei CBA-L74. L-M17 agar was used for Streptococcus
thermophi/us counts. Both were incubated at 37°C anaerobically. Plate count agar
(PCA) was used for detection of contaminants and incubated at 30°C aerobically.
Fermentation: L. paracesei CBA L74 and S. philus 1773 co-
inoculum were added as fresh cultures. Fermentation was carried out for 15 hours, to a
concentration of 108 cfu/mL of L. sei CBA-L74. The initial pH was 6.6. At the
end of the fermentation the pH was 5.1. The pH was adjusted to 6.5 by adding 2.5N
NaOH. The initial concentration of L. paracesei 08-74 was 5 x 106 CFU/ml; the final
concentration was more than 108 . The initial concentration of Streptococcus
thermophilus was 5 x 104 CFU/ml; the final concentration was 1 x 108 CFU/ml. The
initial total bacterial count on PCA was 0 in the milk prior to inoculation and at T0 and
too few es to count (TFTC) after the 15 hour fermentation period. was 5 x 104
CFU/ml; the final concentration was 1 x 108 CFU/ml.
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Dying: The fermentate was dried at an inlet temperature of 190°C and an
outlet temperature of 90°C. The re content of the powder after spray drying was
4.87 %.
Example 3: Preparation of L.paracasei CBA L74 fermented oat and barley flour
We prepared a one liter solution of 18.5 %(w/vol) oat flour + 5 %(w/w)
malted barley flour, using 185 g oat flour and 9.25 g malted barley flour. The mixture of
flour and water was adjusted to pH 4,00 with 0.5 M citric acid. The fermenter was
sterilized by autoclaving., Then the mixture of flour + water + citric acid was added to
the fermenter.
The mixture was heat-treated at 80°C for 30 minutes then cooled down to
37°C. Three different sets of fermentations conditions were tested. All tations
were terminated after 16 hours, a time that coincided with the end of log phase growth,
TRlAL #1: L. paracasei CBA L74 was added to the heat-treated cereal
solution to a final concentration of 2.3 x 106 CFU/ml and incubated with agitation at
37°C. After 16 hours count plate in MRS was 7.6 x 108 UFC/ml (lactic acid bacteria);
contaminants measured in PCA, MC and SB were below 1000 UFC/ml. The final pH
was 3.8. After 20 hours count plate in MRS was 1.2 x 108 UFC/ml (lactic acid bacteria);
contaminants were absent. After 24 hours count plate in MRS was 5 x 108 UFC/ml
(lactic acid bacteria); inants were absent. Because log phase stop 6
hours, it was preferable to stop fermentations at 16 hours.
TRIAL #2 (pH ized) This fermentation was carried out by keeping
the pH at 4 using 2N NaOH L.paracasei CBA L74 was added to the heat-treated
cereal solution to a final concentration of 2.1 x 106 CFU/ml and incubated with agitation
at 37°C. After 16 hours count plate in MRS was 7,5 x 108 UFC/ml (lactic acid bacteria);
contaminants measured in PCA, MC and SB were below 1000 UFC/ml.
TRIAL # 3 (pH stabilized) This fermentation was d out by keeping
the pH at 4 using 2N NaOH L.paracasei CBA L74 was added to the heat-treated
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cereal on to a final tration of 5.1 x 106 CFU/ml and incubated with agitation
at 37°C. After 16 hours count plate in MRS was 2.1 x 109 UFC/ml (lactic acid bacteria);
inants measured in PCA, MC and SB were below 1000 UFC/ml
Example 4: Preparation of L.paracasei CBA L74 fermented rice and wheat flour
We prepared a one liter solution of 15% weight/volume of rice by
combining 150 g of rice and 900 ml of water. The mixture was prepared at room
temperature and mixed by shaking for several minutes at 1000- 1300 rpm. The rice
mixture was treated by tyndalization by heating of the mixture inside the instrument at
70°C, starvation at 70°C per 20-30 minutes, cooling at C, starvation at 30-37°C
per 20-30 minutes, heating at 70°C, starvation at 70°C per 20-30 minutes, cooling at the
fermentation temperature (37°C) while shaking at 150-600 rpm.
casei CBA L74 was added from a freeze-dried sample to a final
tration of 1x106 CFU/ml. The freezevdried sample was resuspended in water
and incubated briefly at 37°C to activate the bacteria. After the inoculation, the mixture
was homogenized by shaking briefly at 300-600 rpm; during fermentation the solution
was shaken at 150 rpm. Fermentation was d out at 37°C for 24 hours at a p02 of
< 15%. Aliquots were collected at the time of inoculation (T0), at 16 hours (T16), 18
hours (T18), 21 hours (T21) and at 24 hours (T24). After fermentation, the cereal was
heated to 50°C with continuous mixing. The heated cereal was then spray dried at T
airin 80°C, T airout 210°C. The final moisture content was 6%.
Samples were analyzed on Rogosa agar (+ vancomycin 12 microgrjml)
(48 h at 37°C), for quantification of the L.paracasei CBA—174), on PCA for total aerobes
(24 h at 37°C), on McConkay agar for coliformes and RCM agar for clostridia.
The results of this fermentation were as s:
inoculum (L.paracasei CBA—174): 1x106 (+1— ‘/2 log) CFU/ml (on the instrument)
L.paracasei CBA L74 concentration after 24 hours of fermentation : 1 x 108 (+/- 1/2 log)
CFU/ml
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Contaminants on PCA before inoculum:< 104 CFU/ml
Contaminants on McConkay before um: < 104 CFU/ml
Contaminants on RCM before inoculum: <10 CFU/ml
Contaminants on PCA after um: < 104 CFU/mi
Contaminants on PCA after 24 hours of fermentation: < 104 CFU/ml
pH before the addition of inoculum: 6 (+/- 0.20)
pH at 16—18 hours: 3.70 (+/—0.20)
pH at 24 hours: 3.60 (+/-0.20).
Example 5: Effects of L.paracasei CBA L74 on dendritic cells in a Cac02 cell coculture
system
We analyzed the effect of live and non-replicating L.paracasei CBA L74 in
a co-culture of intestinal epithelial cells (Cac02 cells) and human dendritic cells (DCs).
Cac02 cells were seeded on a transwell membrane and after about 3 weeks, when the
trans- epithelial ance was adequate, were supplemented with L. paracasei CBA
L74 for 96 hours. Human DCs were differentiated from peripheral blood monocytes and
seeded in the basal compartment of the co-culture chamber. The trans-epithelial
resistance remained constant during the experiment.
To characterize DCs phenotype, we red DC cell surface expression
of co-stimulatory molecules (CD80 and CD86), MHC-II and adhesion molecules (CD40)
by cytofluorimetric analysis. To determine the cytokine e produced by DCs co-
cultured with intestinal epithelial cells exposed to L. paracasei CBA L74, we ted
the culture medium and quantified lL—10 and 70 by ELISA. To verify the ability of
CacoZ cells exposed to L. paracasei CBA L74 to condition DCs to promote the
proliferation of T cells we performed FACS analysis and mixed lymphocyte cultures.
As shown in the Table in Figure 1 incubation of Cac02 cells for 24 hrs with
L. paracasei CBA L74 alive or inactivated (thermal inactivation) d the phenotype
of co-cultured dendritic cells. Furthermore the ce of L. paracasei CBA L74
modulated LPS-mediated changes in DC phenotype.
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We then quantified the cytokines released from DC co—cultured with
Cac02 cells conditioned with L. paracasei (ative or inactivated). As shown in Figure 2a,
DCs co-cultured with Cac02 exposed to L. paracasei CBA L74 alive or inactivated
showed statistically significant increase in lL—10 production. We did not observe a
significant se in lL-12 production in the absence of LPS (Figure 2b). However, the
DCs retained the y to respond to LPS challenge by enhanced lL-12 production,
suggesting that exposure to L. paracasei CBA L74 did not affect the overall ability of
DCs to respond to pathogens.
We then conducted a functional assay to verify the y of DC exposed
to CaC02 cells ed in presence of medium or medium supplemented with L.
paracasei to modulate the ability of T cells to erate following a mixed lymphocyte
reaction. As shown in Figure 3, we did not observe significant differences in the ability of
DC oo-cultured with CaC02 cells to modify s proliferation.
Taken together, the in vitro data indicate that L. paracasei CBA L74, alive
or heat inactivated, can influence the environment generated by intestinal epithelial cells
that in turn modulates the activity of other immune cells such as DC. The l e
indicates that DCs exposed to CacoZ-conditioned medium reduces the expression of
activation markers, produce anti—inflammatory cytokines as IL-10 while retaining the
ability to respond to LPS by enhancing lL-12 tion.
Example 6: Effects of L.paracasei CBA L74 on morphology, cytokine expression
and innate immunity in whole intestinal mucosa
We examined the in vivo effects by administering L.paracasei CBA L74
(live and heat—inactivated) as a dietary supplement in mice. After two weeks of
supplementation, the animals were sacrificed and the whole intestinal mucosa was
analyzed.
Mucosal morphology: We performed a haematoxylin and eosin staining of
in embedded ileal ns. None of the supplements had icant effects on
intestinal architecture or caused an infiltrate of inflammatory cells.
PCT/U82012/042959
Cytokine and lgA expression: We then determined whether administration
of L. paracasei CBA L74 (alive or inactivated) affected the level of anti- and pro-
inflammatory cytokines in the intestinal mucosa. As shown in Figure 4, the
administration of L. paracasei CBA L74 alive or inactivated significantly decreased the
level of iL-1B, a potent pro-inflammatory mediator, in the intestinal mucosa of mice. As
shown in Figure 5, we also observed a reduction in basal mucosal iL-4 level following
administration of L. paracasei CBA L74 alive or inactivated. Finally, we ined the
effect of different regimen supplementation on mucosal lgA level. ing two weeks
of diet supplementation with L. paracasei CBA L74, animals were sacrificed and the
intestinal mucosa collected and homogenized. Total lgA was then measured by ELISA
and values normalized to total mucosal proteins. As shown in Figure 6, heat killed L.
paracasei CBA L74 significantly increased mucosal lgA.
innate immunity; We analyzed the effect of dietary supplementation on levels of Toll-
Like or 2, 4 and 9. These receptors are involved in the recognition of conserved
bacterial structures and thus play a key role in modulating the vity of immune and
non-immune cells toward microbial conserved structures. As shown in Figure 7, diet
supplementation with L. sei CBA L74, live or heat inactivated, increased levels of
protein expression of TLR2 and TLR4. We also assayed the effect of dietary
supplementation on levels of PPARy in the . As shown in Figure 8, Live L.
paracasei CBA L74 significantly sed the level of PPARy.
e 7: Effects of L.paracasei CBA L74 on levels of circulating cytokines
To assess whether the diet supplements were able to modify the level of
circulating cytokines, we measured the effects of the dietary administration of the strain
(alive or inactivated) on anti- and pro-inflammatory cytokines level in the serum. As
shown in Figures 9a and 9b, Live L.paracasei CBA L74 d a statistically significant
decrease in lL-1B and a modest increase in circulating IL—4, respectively.
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Example 8: Effects of L.paracasei CBA L74 on dendritic cell and T-lymphocyte
activity
] We next focused on the impact of dietary supplementation with L.
sei CBA L74 on immune cells relevant to the activity of mucosaI-associated
immune system, namely dendritic cells and lymphocytes. We evaluated the phenotype
of DCs within the Peyers Patches (PP), since these cells are instrumental in
establishing the fate of an antigen and contribute to the environment that will ine
the nature of the adaptive immune response.
As shown in the Table in Figure 10, L. paracasei CBA L74
supplementation (live or inactivated) decreased the expression of the mulatory
molecule CD80 and of the adhesion molecule CD40 whereas MHCII expression was
up—regulated. These data suggested that the DCs from L. paracasei CBA L74
supplemented animals seemed less ready to interact with s and to mount an
immune response but their ability to process and present antigens was preserved.
We then determined the ability of diet mentation with L. paracasei
CBA L74 to modify the reactivity of DCs to pro-inflammatory stimuli (such as ial
LPS and CpG). Exposure of DCs from control mice to LPS or CpG induced a strong
ulation of CD80 in control DCs. Supplementation with heat inactivated L.
paracasei CBA L74 DC did not modify the reactivity to inflammatory stimuli.
Supplementation with live bacteria significantly reduced LPS— and CpG-induced CD80
up-regulation.
Finally, we investigated whether dietary supplementation with L. paracasei
CBA L74 (live or inactivated) affected intestinal T—Iymphocytes (either CD4+ and CD8+)
polarization toward a Th1 or Th2 phenotype. For these studies, Peyer’s Patches were
exposed to PHA, a strong, non-specific stimulus and then lymphocyte zation was
evaluated by intrakine ng for IL-4 and IFN—y. As shown in Figure 11a, in the
absence of PHA (the “basal ion”) in CD4+ lymphocytes, lL-4 and IFN were almost
in equilibrium. About 10-12% of the cells were positive for these cytokines. As shown in
Figure 11b, for CD8+ lymphocytes in the basal condition, there was a slight
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predominance of lL-4 over IFN expressing cells. Exposure of either CD4+ or CD8+
cytes to PHA caused a strong increase in intracellular staining for lL-4 and IFN-y
with bias toward IFN-y tion.
] In CD4+ cells, dietary supplementation with live L. paracasei CBA L74
increased IFN—y levels in the basal condition. ing PHA exposure there were no
significant differences in the response among the supplemented groups (Figure 11a).
In CD8+ lymphocytes, oral supplementation with inactivated L. sei CBA L74
favored a Th2 profile with a stimulation of lL-4 production over lFN-v production (Figure
11b) The anti-inflammatory profile is further supported by the blunted response to PHA.
A similar trend was evident also in CD8+ lymphocytes isolated from mice supplemented
with live L. paracasei CBA—L74, although less pronounced.
Example 9: Effect of milk fermented by L. paracasei CBA L74 on immune system
markers in the intestinal mucosa
Mice were mented twice a day for two weeks with either: 1) Control
(PBS); 2) Skim milk (not fermented); 3) Skim milk fermented with L. sei CBA L74
(1x108 cfu/ml); 4) Skim milk fermented with s. philus (6.7x106 cfu/ml); 5) Skim
milk fermented with L. paracasei CBA L74 (1x108 ) and S. philus (1 .18x107
cfu/ml); 6) Skim milk fermented with L. sei CBA L74 (1 .9x109 cfu/die) and S.
thermophilus (2.2x108 cfu/ml). At the end of the treatment animals were sacrificed and
intestinal mucosa and Peyer's Patches were collected and processed for analysis as
described below.
Levels of cytokines in the intestinal mucosa. As shown in Figure 12,
supplementation with skim milk fermented with L. paracasei CBA L74 caused a
significant increase lL—10 levels in the intestinal mucosa. ,Administration of skim milk
fermented with S. thermophilus induced a slight reduction as compared to controls.
Administration of milk fermented with both strains at higher dosage induced a 2.1-fold
increase in mucosal lL—10. None of the treatments had any significant effect on
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mucosal lL-1B gh supplementation with skim milk fermented with L. paracasei
CBA L74 caused a slight increase in mucosal levels of this cytokine.
eroxidase activity in the intestinal mucosa. eroxidase (MPO)
activity was d in intestinal mucosa nates. Administration of non
fermented or fermented milk with different bacteria did not induce statistically significant
differences in the myeloperoxidase activity, although there was a slight increase in MPO
activity observed in the mucosa of animals receiving non fermented milk.
Levels of lgA in the intestinal mucosa. As shown in Figure 14,
supplementation with non-fermented skim milk caused a icant se in the
intestinal mucosa lgA. This increase was not observed in animals mented with
skim milk fermented with either L. paracasei CBA L74 or S. thermophilus alone.
Administration of milk ted with both strains at higher dosage induced an increase
in mucosal lgA.
Levels of TLR2. TLR4I TLR9 and PPARy in the intestinal mucosa. We
next determined by WB the mucosal levels of key receptors of innate immune system.
As shown in Figure 15, none of the treatments had any significant effect on TLR4
receptor expression. Non-femtented milk caused a modest increase in TLR2.
Administration of skim milk fermented with either L. paracasei CBA L74 or S.
thermophilus alone or in combination at the lower dose prevented this effect. A modest
increase, comparable to milk alone, in TLR2 was also evident in mice receiving milk
fermented with both strains at higher dosage. Administration of skim milk alone had
profound effects on TLR9 levels, whereas administration of fermented milk with both
strains re—established mucosal levels comparable to controls. As shown in Figure 15,
administration of non-fermented milk per se caused a strong increase in mucosal
PPARy. A similar effect was t in mice supplemented with skim milk fermented
with L. sei CBA L74 and the lower dose of milk fermented with both strains.
Levels of pNF-kB and IKB in the intestinal mucosa. We determined the
activation status of NF—kB, a key transcription factor involved in both intestinal
inflammation and epithelial cell survival. As shown in Figure 16, activated NF-kB (pNF-
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KB) was detectable in the control, normal mucosa. Following the different ents,
we observed only a slight increase in pNF-kB levels in the mucosa of mice treated with
skim milk fermented with either L. paracasei CBA L74 or S. thermophilus alone,
whereas in mice ing skim milk fermented with both s, pNF-KB was
comparable to controls. The increase in pNF—KB eled the disappearance of the
inhibitory subunit of NF-KB, IKB, that was not detectable in the mucosa of mice
supplemented with skim milk ted with either L. paracasei CBA L74 or S.
thermophilus alone. Mice receiving skim milk fermented with both s showed lkB
levels comparable to controls.
Example 10: Effect of milk fermented by L. paracasei CBA L74 on dendritic cell
ype in the intestinal mucosa
Phenotype of dendritic cells extracted from Peyer’s Patches. We
examined the effect of diet supplementation on the phenotype of intestinal DCs. The
s of this analysis are shown in Figure 17. Administration of milk fermented with L.
paracasei alone as well as with L. paracasei and S. philus at lower and higher
doses produced a statistically significant decrease in CD80 and CD86 sion as
compared to control mice (* means p<0.05 vs control), whereas HLAll and CD40 levels
overall remained stable. Data in Figure 17 are expressed as meaniSE. of three
separate experiments.
Responses of dendritic cells exposed to LPS/CpG. We determined the
ability of diet supplementation to modify the reactivity of DCs to pro-inflammatory stimuli
(such as bacterial LPS and CpG). As shown in Figure 18, exposure of isolated DCs
isolated from control animals to LPS and CpG caused a significant increase in the
expression of CD80 (* means p<0.05 vs un—stimulated 003 from control mice). A similar
effect was observed in DOS purified from mice receiving rmented milk (° means
p<0.05 vs un-stimulated DCs from mice treated with non—fermented milk).
Administration of skim milk fermented with 1.. paracasei CBA L74 alone completely
prevented LPS-mediated effects in mice treated with non-fermented milk, although it
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was less effective on CpG-induced CD80 upregulation. Skim milk fermented with S.
thermophi/us alone prevented LPS and CpG effects and reduced CD80 expression.
Finally, administration of skim milk fermented with both strains prevented LPS- and
CpG—induced CD80 up-regulation and maintained CD80 expression at basal levels.
Data in Figure 18 are expressed as meaniSE. of three separate experiments.
Example 11: Effect of milk fermented by L. sei CBA L74 on T-lymphocyte
phenotype in the intestinal mucosa
Responses of intestinal lymphocytes CD4+ e CD8+ exposed to PHA. We
asked whether dietary supplementation with milk (either fermented or unfermented)
would affect intestinal T-lymphocytes (CD4+ and CD8+) polarization toward a Th1 or
Th2 phenotype. Peyer’s Patches derived cytes were exposed to PHA, and then
cyte zation was evaluated by staining for intracellular lL—4 and lFN-y.
As shown in Figure 19, exposure to PHA significantly increased lL4 and
lFNy tion in CD4+ cytes from control mice (* means p<0.05 vs un-
stimulated cytes from control mice). This ion was not observed in mice
treated with either non-fermented or fermented milk. Following supplementation with
non—fermented milk, CD4+ lymphocytes showed increased basal levels of lL4 and lFN-y
as compared to control animals (° means p<0.05 vs un- ated lymphocytes from
control mice). These data suggested that supplementation with fermented milk
prevented non-specific activation and maintained lL4 and lFN-v production at basal
levels.
As shown in Figure 20, exposure to PHA in CD8+ intestinal lymphocytes
from l mice increased production of lL4 and lFNv (* means p<0.05 vs non—
stimulated lymphocytes from control mice). This induction was not observed in mice
treated with either non-fermented or fermented milk. Administration of milk fermented
with L. paracasei and S. thermophilus at higher dose decreased the basal production of
lL4 s following stimulation with PHA, the levels of both lL4 and lFNy were
reduced as compared to stimulated lymphocytes from control mice (§ means p<0.05).
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Example 12: Effect of milk fermented by L. paracasei CBA L74 on inal
mucosa histology
To determine the distribution of proliferating cells in the intestinal mucosa,
we performed an immunohistochemcal analysis of expression of Ki67, an antigen
expressed by dividing cells. In control mice there was a clear immunoreactivity in the
inal crypts, whereas there was no staining on the villi. in the tissue from mice
ing non-fermented milk there was a stronger expression in the crypts, although
there was no ectopic expression of the antigen. In mice receiving the different kind of
fermented milk we observed comparable staining patterns. We have performed a
histological evaluation of ileal mucosa of the different experimental groups. Results of
this study are shown in Figure 21. Administration of non-fermented milk d the
number and length of intestinal villi, whereas the intestinal morphology was preserved in
the animals receiving the various forms of ted milk.
Example 13: Effect of rice fermented by L. paracasei CBA L74 on immune system
markers in the intestinal mucosa
These experiments were designed to evaluate the immuno-modulatory
ties of L. paracasei CBA—L74-fermented cereals. Studies included daily
astric administration for two weeks of: 1) Non fermented rice; 2) Fermented rice
(with L. paracasei CBA L74) 100 mg day (corresponding to 2 x108 cfu/L of L.paracasei
CBA—L74) and 3) Fermented rice (with L. paracasei CBA L74) 500 mg day
(corresponding to 109 cfu/L of L.paracasei CBA-L74) Mice during the treatment period
did not show any sign of illnesses or diarrhoea. At the end of the treatment mice were
killed and the Peyer‘s patches (PP) were collected aseptically. Epithelial cells were
removed from PP by incubation in HBSS with no Ca2+ plus EDTA and then s
were subjected to enzymatic digestion. The single cell sion was washed,
resuspended in cold RMPI and used for the following tests.
2012/042959
We initially examined the effect of different diet supplements on the whole
intestinal mucosa. First, we performed haematoxylin and eosin staining of paraffin
embedded ileal sections. None of the diet supplements used had significant effects on
intestinal architecture or caused an infiltrate of inflammatory cells.
We then determined whether administration of L. paracasei CBA L74
fermented rice affected the level of anti- and pro-inflammatory nes in the intestinal
mucosa. As shown in Figure 22, administration of rice or fermented rice increased both
mucosal lL-1B. and lL-4. Supplementation with fermented rice at the highest dose
caused a reduction in IL-10 as shown in Figure 23.
We then determined the effect of ent regimen supplementation on
l lgA level. Following two weeks of diet supplementation, animals were
sacrificed and the intestinal mucosa collected and homogenized. Total lgA was
measured by ELISA and values normalized to total l ns. None of the
supplements had significant effects on mucosal lgA.
We ed the effects of rice or L. paracasei CBA L74-fermented rice on
intestinal mucosa innate immunity. As shown in Figure 24, diet mentation with
rice and fermented rice (low dose) had only moderate effects on mucosal TLRs. As
shown in Figure 25a, diet supplementation with non-fermented rice drastically increased
mucosal PPARy levels. High doses of fermented rice also increased mucosal PPARy
levels. As shown in Figure 25b, two weeks of diet supplementation with rmented
rice reduced NF-KB activation and increased IKB in the mucosa. Supplementation with
fermented rice at low doses had no effect compared to control, whereas the higher dose
had effects able to those observed with non—fermented rice.
We also measured the effects of the dietary administration on anti- and
pro-inflammatory cytokines level in the serum. Dietary supplementation with either rice
orfermented rice (low dose) had no influence on serum lL-1B and lL-4 levels.
W0 2012/177556
Example 14: Effect of rice fermented by L. paracasei CBA L74 on tic cell
phenotype in the intestinal mucosa
We next focused on the impact of dietary supplementation with nonferrnented
rice or rice fermented with L. paracasei CBA L74 on immune cells nt to
the activity of mucosal— associated immune system, namely dendritic cells and
cytes. First we labeled the single cells sion with anti—CD1 (to identify DC)
and with either anti CD-80 and CD-86, MHC-ll and 00-40 to determine the level of
ty and maturity of intestinal DC in these lymphoid organs. As shown in the table in
Figure 26, we observed a significant effect of fermented rice at high dose on DCs
phenotype. Non-fermented rice supplementation (in these experiments we used two
different doses) had no effect, whereas the effects of fermented rice on CD80, CD40
and MHC-ll level were evident at 100 mg/day and more pronounced at 500 mg/day.
] We next determined the y of diet supplementation with rice or L.
sei CBA L74 fermented rice to modify the reactivity of DCs to pro-inflammatory
stimuli (such as bacterial LPS and CpG). As shown in the table in Figure 27, exposure
of DCs from control mice to LPS or CpG induced an up— regulation of CD80 in control
DCs. Supplementation with non-fermented rice did not modify the reactivity to
inflammatory stimuli. Supplementation with the tested dose of fermented rice reduced
LPS— and CpG-induced CD80 up-regulation.
Example 15: Effect of rice fermented by L. paracasei CBA L74 on T-lymphocyte
phenotype in the intestinal mucosa
We investigated whether dietary supplementation with rice or L. paracasei
CBA L74 fermented rice was able to influence inal T-lymphocyte (either CD4+ and
CD8+) polarization toward a Th1 or Th2 phenotype. Peyer’s Patches derived
lymphocytes were exposed to PHA and then lymphocyte polarization was evaluated by
intrakine staining for lL—4 and IFN-v.
The s of this experiment for CD4+ and CD8+ lymphocytes are shown
in Figures 28 and 29, respectively. As shown in Figure 28, in the basal condition, in
W0 2012/177556 PCT/U$2012/042959
CD4+ lymphocytes IL—4 and lFNv were almost in equilibrium since about 10-12% of the
cells are positive for these cytokines. As shown in Figure 29, in CD8+, in the basal
ion there was a slight predominance of lL-4 over IFNv expressing cells. Exposure
of either CD4+ or CD8+ lymphocytes to PHA caused a strong increase in ellular
staining for lL-4 and IFNy, with a predominance of lFNy production.
In mice receiving non-fermented rice we observed a preponderance of IL-
4 production (Th2 phenotype) either in basal condition and following PHA stimulation for
both CD4+ and CD8+ lymphocytes. However, lL-4 production was associated to a
persistence of lFNy production in basal condition and, following PHA stimulation we
observed an increased expression of this cytokine although the response was blunted
as compared to the response in control cells. In mice receiving the fermented rice in
basal condition we observed a preponderance of lFNy positive cells over lL—4, although
in basal condition the percentage was comparable (for CD4+) or slightly lower (for
CD8+) as compared to controls. Following PHA stimulation, in both cellular populations
the cytokine response was d as ed to control mice, although directed
toward a Th1 phenotype (preponderance of lFNy positive cells).
e 16: Effect of L. paracasei CBA L74 on lL-10 production in monocyte-
derived dendritic cells ) in the presence of ella urium
We tested the ability of both 1.. paracasei CBA L74 cells and L. paracasei
CBA L74 cell supernatants to induce production of the anti—inflammatory cytokine, lL-10,
in human MoDCs in the presence of the bacterial pathogen, Salmonella urium.
Human MoDCs were obtained from at least four, and in some cases, seven . As
shown in Figure 30, Salmonella typhimurium (“F862”), the control acillus strain,
L.paracasei 21060 (“821060”) and L. paracasei CBA L74 (“CBAL74”) cells all induced
lL-10 production. ln contrast, although supernatant from Salmonella typhimurium (“sn
fb62”) induced lL—10 production, supernatants from both Lactobacilli, Lactobacillus
strain, L.paracasei 21060 (“sn b21060”) and L. paracasei CBA L74 (“sn cbal74”) did not.
Co-incubation of L. paracasei CBA L74 supernatant with Salmonella typhimurium (“sn
cbal74 + F862”) induced lL-1O to levels over and above that seen with ella
typhimurium alone (“sn fb62 + FB62”). Interestingly, a similar effect was observed even
W0 2012/177556 PCT/U82012/042959
if the MoDCs were preconditioned with L. paracasei CBA L74 supernatant for only one
hour and then washed to remove the supernatant (“1 h an cbal74 + F862").
Example 17: Effect of L. paracasei CBA L74 on lL-12p70 production in monocyte-
derived dendritic cells (MoDCs) in the presence of Salmonella typhimurium
We tested the ability of both L. paracasei CBA L74 cells and L. paracasei
CBA L74 cell supernatants to induce production of the pro-inflammatory cytokine, IL-
12p70, in human MoDCs in the presence of the bacterial pathogen, ella
typhimurium. Human MoDCs were obtained from at least four, and in some cases,
seven donors. The results of this experiment are shown in Figure 31. In contrast to the
results ed in Example 16 for the anti—inflammatory cytokine, we found that
Salmonella typhimurium (“F862") induced high levels of lL-12p70 while the control
Lactobacillus strain, L.paracasei 21060 (“821060”) and L. paracasei CBA L74
(“CBAL74”) cells induced only low levels of lL-12p70. Here again, supernatant from
Salmonella typhimurium (“sn fb62”) induced lL—12p70 production, but supernatants from
both Lactobacillus strains, L.paracasei 21060 (“sn b21060”) and L. paracasei CBA L74
(“sn cbal74”) did not. Co-incubation of L. paracasei CBA L74 supernatant with
Salmonella typhimurium (“sn cbal74 + F362”) caused a striking reduction in lL-12p70
production. The effect was observed even if the M0003 were preconditioned with L.
paracasei CBA L74 supernatant for only one hour and then washed to remove the
atant (“1h sn cbal74 + FB62"). This reduction exceeded that observed for the
control acillus, L.paracasei 21060 (“1h sn b21060 + . These data
suggest that both L. paracasei CBA L74 and culture supernatant from L. paracasei CBA
L74 have anti-inflammatory ties that can mitigate the inflammation induced by the
bacterial pathogen, Salmonella typhimurium.
Example 18: Effect of L. paracasei CBA L74 fermented milk on lL-10 production
in te-derived dendritic cells (MoDCs) in the ce of Salmonella
urium
We tested the ability of L. sei CBA rmented milk to induce
production of the anti-inflammatory ne, lL-10, in human MoDCs in the presence of
the bacterial pathogen, Salmonella typhimurium. The results of this experiment are
W0 2012/177556 2012/042959
shown in Figure 32. Although unactivated MoDCs do not usually produce lL-10, we
found that tion of MoDCs with L. paracasei CBA L74-fermented milk induced
dose-dependent lL-10 production (see “matrice CBAL74 1.4tgr/100mi” and ce
CBAL74 0.14gr/100ml”). The capacity was retained even in the presence of Salmonella
typhimurium (see “matrice CBAL74 1.41gr/100ml + F862” and “matrice CBAL74
0.14gr/100ml + F862”).
Figure 32 also shows that L. paracasei CBA L74 that had been activated
by heat (“6.3*106 CFU CBA74 boiled”), paraformaldehyde treatment (“6.3*106 CFU
CBA74 PFA”) or freeze-thawing (“6.3*106 CFU CBA74 F&T”) ed the ability to
induce lL—10.
As we observed for L. paracasei CBA L74 culture media, L. paracasei
CBA L74-fennented milk caused a significant, dose-dependent reduction in production
of lL—12p70 induced by Salmonella typhimurium. (Figure 33). S. thermophi/us-
fermented milk caused an increase in lL-12p70 tion in the presence of
Salmonella urium. However, milk fermented by L. paracasei CBA L74 did not
stimulate 70 production, consistent with the anti-inflammatory properties of L.
paracasei CBA L74. These data suggest that L. paracasei CBA L74 ed its anti—
inflammatory properties even in the presence of more pro-inflammatory species.
Example 19: Effect of L. paracasei CBA L74 cell supernatants on lL-10 and
lL12p70 production in monocyte-derived dendritic cells (MoDCs) in the presence
of Enterobacter sakazaki
We tested the ability of L. paracasei CBA L74 cell supernatants to induce
production of the anti—inflammatory ne, lL—10 and the pro-inflammatory cytokine,
70, in human MoDCs in the presence of the bacterial pathogen, Enterobacter
ki. We tested two strains of E. sakazaki, N9 and N13. E. sakazaki induced the
production of both IL-10 and lL-12p70 in human MoDCs. The on of L. paracasei
CBA L74 cell supernatants resulted in an increase in lL—1O and a significant decrease in
lL-12p70.
W0 2012/1 77556 PCT/U82012/042959
Example 20: Effect of L. paracasei CBA L74 fermented rice on cytokine
production in a tissue explant model in the presence of Salmonella typhimurium
Mouse gut epithelium as used to set up three-dimensional co-culture
system as described in Abud (Exp. Cell Res. 303: 252-262 ). Tissue explants
were ed for 24 hour in the presence of 100% 02 with a pressure of one
atmosphere. The lower chamber contained 1 mi of hEC DMEM plus ITS-X and EGF.
The upper chamber contained 200 ml of medium plus either S.typhimurium, L.
sei CBA L74 fermented rice, or a combination of S.typhimurium and L. paracasei
CBA L74 fermented rice. atants were harvested and levels of lL—1B, TNF-d, and
|L-10 were assayed by ELlSA. As shown in Figure 33, the addition of fermented rice
significantly reduced production of the pro-inflammatory cytokines, |L-1[3 and TNF-(x in
the presence of S.typhimurium, with only a moderate effect on the levels of the anti—
infiammatory ne, lL-10.
It is to be understood that the present invention is by no means limited
only to the particular constructions herein disclosed and shown in the drawings, but also
comprises any cations or equivalents within the scope of the claims.
Claims (59)
1. A composition comprising a fermented food product, wherein the food product has been fermented by the tic bacterium, Lactobaci/lus paracasei CBA L74, international Depository Accession Number LMG P- 24778.
2. The composition of claim 1, wherein the food product is a dairy t or a cereal product.
3. The composition of claim 2, wherein the dairy product is milk, yoghurt, curd, cheese or an infant a.
4. The composition of claim 2, wherein the cereal product is rice, wheat, oats, barley, corn, rye, sorghum, millet, or triticale.
5. The composition of any one of claims 1-4, wherein the food product is dried.
6. The composition of any one of claims 1—5, wherein the dried food product is rehydrated.
7. The composition of claim 6, wherein the dried, rehydrated food product is modulatory.
8. The composition of any one of claims 1-7, wherein the probiotic bacterium is non-replicating.
9. The composition of any one of claims 1-8, wherein the food product comprises from about 1 x102 cfu/g to about 1012 cfu/g of tic bacteria per gram of dry weight. 2260695V1
10. A composition comprising the probiotic bacterium, Lactobaci/Ius paracasei CBA L74, international Depository Accession Number LMG P-24778 and a physiologically acceptable carrier.
11. The composition of claim 10, wherein the physiologically acceptable carrier is a food product.
12. The composition of claim 11, wherein the food product is a dairy product or a cereal t.
13. The composition of claim 12, wherein the dairy product is milk, yoghurt, curd, cheese or an infant formula.
14. The composition of claim 12, wherein the cereal t is rice, wheat, oats, barley, corn, rye, sorghum, millet, ortriticale.
15. The composition of any one of claims 10-14, wherein the food product is dried.
16. The composition of any one of claims 10—15, n the dried food product is rehydrated.
17. The composition of claim 16, wherein the dried, rehydrated food product is modulatory.
18. The composition of any one of claims 10—17, wherein the probiotic bacterium is non-replicating.
19. The composition of any one of claims 10-18, n the food product comprises from about 1 x102 cfu/g to about 1012 cfu/g of probiotic bacteria per gram of dry weight.
20. The ition of claim 10, wherein the physiologically acceptable carrier is a pharmaceutically acceptable carrier. 2260695v1
21. The composition of claim 20, wherein the pharmaceutically acceptable carrier carrier ses a lipid—based or polymer-based colloid.
22. The composition of claim 21, wherein the colloid is a liposome, a hydrogel, a microparticle, a nanoparticle or a block copolymer micelle.
23. The composition of claim 21, wherein the polymer-based colloid is a
24. The composition of any one of claims 20-23, wherein the probiotic ium is non-replicating.
25. The composition of any one of claims 20-24, n the tic bacterium is in a unit dosage form of about 1 x 102 cfu/g to about 1 x 1012 cfu/g dry weight.
26. The composition of any one of claims 20-25, wherein the composition is formulated for oral stration.
27. A method of making a nutritional composition, the method comprising: a) providing a food product; b) combining the food product with an effective amount of the probiotic bacterium, Lactobacillus paracasei CBA L74, International Depository Accession Number LMG P-24778 and, optionally, a co-inoculum, to form a mixture; c) incubating the mixture at a temperature and for a time ient for fermentation to occur.
28. The method of claim 27, wherein the food product is a dairy product or a cereal product.
29. The method of claim 27 or 28, wherein the amount of the probiotic bacterium is from about 1 x 106 cfu/ml to about 5 x 106 cfu/ml. 2260695v1
30. The method of any one of claims 27—29, n the culum is Streptococcus thermophilus.
31. The method of claim 30 wherein the amount of the co-inoculum is from about 1 x 104 cfu/ml to about 1 x 105 .
32. The method of any one of claims 27-31, wherein the incubating step continues until the level of probiotic bacteria in the mixture reaches about 1 x 108 cfu/ml to about 1 x 1010 cfu/ml.
33. The method of any one of claims 27-32, further comprising drying the nutritional ition.
34. The method of claim 33, wherein the drying step is spray drying.
35. The method of any one of claims 27-34, wherein the nutritional composition is further combined with one or more additional food products.
36. The method of claim 35, wherein the additional food product comprises a dairy product, a cereal product, a vegetable-based product, a fruit-based product or a protein-based product.
37. The method of claim 36, wherein the fruit-based product comprises a tomato product.
38. Use of a food product that has been fermented by the probiotic bacterium Lactobacillus paracasei CBA L74, International Depository Accession Number LMG P—24778, in the preparation of a composition for modulating the immune system of a subject, wherein said composition contains an effective amount of said food product to modulate the immune system of the t. 2260695vl
39. The use of claim 38, wherein the food product comprises the food product of any one of claims 1-19.
40. The use of claim 38 or claim 39, wherein the subject is a human infant.
41. The use of any one of claims 38-40, n the composition is for treatment of a subject at risk of developing a gastrointestinal disorder.
42. The use of claim 41, wherein the gastrointestinal disorder is a mucosal immune system deficit, a food allergy, a disorder associated with diarrhea, a bacterial or viral infection, irritable bowel syndrome, inflammatory bowel disease, Crohn’s disease, celiac disease or necrotizing enterocolitis.
43. The use of claim 42, wherein the mucosal immune system deficit is an immature immune system.
44. The use of any one of claims 38-40, wherein the composition is for treatment of a subject having a gastrointestinal disorder.
45. The method of claim 44, wherein the gastrointestinal disorder is a mucosal immune system t, a food allergy, a er associated with diarrhea, a bacterial or viral infection, irritable bowel syndrome, inflammatory bowel disease, Crohn’s disease, celiac disease or necrotizing colitis.
46. The method of claim 45, wherein the mucosal immune system deficit is an immature immune system.
47. Use of probiotic bacterium, Lactobacillus sei CBA L74, international Depository ion Number LMG 8 for the preparation of a food product for treatment of a gastrointestinal disorder.
48. The use of claim 47, wherein the food product comprises the food product of any one of claims 1-19. 2260695v1
49. The use of claims 47 or 48, wherein the gastrointestinal disorder is a l immune system deficit, a food allergy, a disorder associated with diarrhea, a ial or viral infection, irritable bowel syndrome, inflammatory bowel disease, Crohn’s disease, celiac disease or necrotizing enterocolitis.
50. The use of claim 49, wherein the mucosal immune system deficit is an immature immune system.
51. The use of any one of claims 47-50, wherein the subject is a human infant.
52. Use of probiotic bacterium, LactobaciI/us sei CBA L74, International Depository Accession Number LMG P~24778 for the preparation of a medicament for treatment of a gastrointestinal disorder.
53. The use of claim 52, wherein the pharmaceutical composition comprises the composition of any one of claims 20-26.
54. The use of claim 52 or claim 53, wherein the gastrointestinal disorder is a mucosal immune system t, a food allergy, a disorder associated with diarrhea, a bacterial or viral infection, irritable bowel syndrome, inflammatory bowel disease, Crohn’s disease, celiac disease or necrotizing enterocolitis.
55. The use of claim 54, wherein the mucosal immune system deficit is an re immune .
56. The use of any one of claims 52-55, wherein the subject is a human infant.
57. The nutritional composition made by the method of any one of claims 27-
58. A kit comprising a ed amount of a nutritional ition comprising a fermented food product, wherein the food product has been 2260695vl fermented by the probiotic bacterium, acillus paracasei CBA L74, International Depository Accession Number LMG P-24778 and one or more items selected from the group ting of packaging material, a package insert comprising instructions for use, a sterile fluid, a syringe and a sterile container.
59. A kit comprising a measured amount of a pharmaceutical composition comprising Lactobacillus paracasei CBA L74, International Depository Accession Number LMG P-24778 and one or more items ed from the group consisting of packaging material, a package insert comprising ctions for use, a sterile fluid, and a sterile container. 2260695v1
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US201161498910P | 2011-06-20 | 2011-06-20 | |
US61/498,910 | 2011-06-20 | ||
PCT/US2012/042959 WO2012177556A2 (en) | 2011-06-20 | 2012-06-18 | Probiotic compositions and methods |
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NZ619099A NZ619099A (en) | 2015-08-28 |
NZ619099B2 true NZ619099B2 (en) | 2015-12-01 |
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