U.S. patent application number 15/458677 was filed with the patent office on 2017-07-06 for bacteroides cect 7771 and the use thereof in the prevention and treatment of excess weight, obesity and metabolic and immunological alterations.
The applicant listed for this patent is Consejo Superior de Investigaciones Cientificas. Invention is credited to Paola Gauffin Cano, Yolanda Sanz Herranz, Moises Laparra Llopis, Angela Moya Perez, Yolanda Arlette Santacruz.
Application Number | 20170189456 15/458677 |
Document ID | / |
Family ID | 49623202 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170189456 |
Kind Code |
A1 |
Herranz; Yolanda Sanz ; et
al. |
July 6, 2017 |
Bacteroides CECT 7771 and the Use Thereof in the Prevention and
Treatment of Excess Weight, Obesity and Metabolic and Immunological
Alterations
Abstract
The invention relates to a strain of Bacteroides uniformes with
registration number CECT 7771, and to the cellular constituents,
metabolites and/or secreted molecules thereof. The invention also
relates to a composition (nutritional or pharmaceutical) comprising
at least one of the previous products. The invention further
relates to the use of a strain of Bacteroides uniforms, preferably
CECT 7771, or of the cellular constituents, metabolites and/or
secreted molecules of said strain, or of a composition comprising
same, for the prevention and/or treatment of alterations such as
excess weight, obesity, adipocyte hypertrophy, hepatic steatosis or
fatty liver, dyslipidemia, hyperglycemia, insulin resistance and
diabetes, metabolic syndrome, hypertension, cardiovascular
diseases, dysfunction of the immune system, reduced defences
against infections, and imbalance in the composition of the
intestinal microbiota.
Inventors: |
Herranz; Yolanda Sanz;
(Paterna, ES) ; Cano; Paola Gauffin; (Paterna,
ES) ; Santacruz; Yolanda Arlette; (Paterna, ES)
; Perez; Angela Moya; (Paterna, ES) ; Llopis;
Moises Laparra; (Paterna, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Consejo Superior de Investigaciones Cientificas |
Madrid |
|
ES |
|
|
Family ID: |
49623202 |
Appl. No.: |
15/458677 |
Filed: |
March 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14403930 |
Apr 10, 2015 |
9636366 |
|
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PCT/ES2013/070309 |
May 16, 2013 |
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15458677 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 9/12 20180101; C12N 1/20 20130101; A61K 35/74 20130101; A23L
2/52 20130101; A61K 35/741 20130101; A61P 9/00 20180101; A61K 45/06
20130101; A23C 9/152 20130101; A23L 33/135 20160801; A61P 3/00
20180101; A23V 2002/00 20130101; A61P 37/00 20180101; A61P 1/16
20180101; A61P 3/04 20180101; A61P 3/06 20180101; A61P 31/00
20180101; C12R 1/01 20130101; A61P 3/10 20180101 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A23L 2/52 20060101 A23L002/52; A23L 33/135 20060101
A23L033/135; A61K 45/06 20060101 A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2012 |
ES |
201230796 |
Claims
1-49. (canceled)
50. A method for the treatment and/or prevention of excess weight
or obesity in a subject comprising administering to said subject a
composition comprising a Bacteroides uniformis strain with deposit
number CECT 7771 and a carrier and/or excipient.
51. The method according to claim 50, wherein the composition
further comprises at least an additional microorganism.
52. The method according to claim 51, wherein the additional
microorganism is an intestinal bacterium or lactic acid
bacterium.
53. The method according to claim 50, wherein the composition
further comprises at least a bioactive component.
54. The method according to claim 50, wherein the composition is a
pharmaceutical or a nutritional composition.
55. The method according to claim 54, wherein said nutritional
composition is a food, a supplement, a nutraceutical, a probiotic,
or a symbiotic.
56. The method according to claim 55, wherein said food is selected
from dairy products, vegetable products, meat products, snacks,
chocolate, baby food and drink.
57. The method according to claim 50, wherein the composition has a
strain concentration between 10.sup.3 and 10.sup.14 colony-forming
units (cfu) per gramme or millilitre of final composition.
58. The method according to claim 50, wherein the cells obtained
from the strain are in viable or non-viable form.
59. The method according to claim 50, wherein the composition is in
a form adapted for oral, sublingual, nasal, intrathecal, bronchial,
lymphatic, rectal, transdermal, inhaled or parenteral
administration.
Description
FIELD OF THE ART
[0001] The present invention falls within the field of
pharmaceuticals and food. Specifically, the present invention
relates to the Bacteroides uniformis CECT 7771 strain, to its
cellular components, metabolites and secreted molecules, and to
compositions which, comprising at least one of the foregoing
products, can also comprise other microorganisms or other compounds
with biological activity. Likewise, the present invention also
relates to the use of a B. uniformis strain or to the use of the
CECT 7771 strain in the prevention and/or treatment of alterations
such as excess weight, obesity, hepatic steatosis or fatty liver,
dyslipidemia and, in particular, hypercholesterolemia and/or
hypertriglyceridemia; hyperglycemia, insulin resistance and
diabetes, preferably
[0002] Type 2 diabetes mellitus and gestational diabetes; metabolic
syndrome, hypertension, cardiovascular diseases, immune system
dysfunction associated or not associated with these pathologies,
preferably inflammation in peripheral tissues (adipose and
pancreas) and reduced defence against infections, and imbalance in
the composition of the intestinal microbiota.
STATE OF THE PRIOR ART
[0003] Excess weight and obesity currently constitute one of the
major public health concerns due to their increasing prevalence and
comorbidities. These include, for example, dyslipidemia, diabetes,
cardiovascular diseases, arteriosclerosis, hepatic steatosis or
fatty liver, metabolic syndrome, hypertension and some types of
cancer.
[0004] Obesity is produced as a consequence of a positive and
prolonged imbalance between intake and energy expenditure, which
entails excessive gain in weight and body fat. Control of energy
balance involves peptides and hormones synthesized by the
neuroendocrine system that allow communication between various
peripheral tissues and organs and the central nervous system which,
globally, contribute to body weight regulation. The signals
emanating from adipose tissue (leptin) and the pancreas (insulin)
are essential in long-term control of food intake (Konturek et al.,
2004. J Physiol Parmacol., 55: 137-154). Insulin is the most
important hormone in the regulation of the proper functioning of
adipose tissue and the accumulation of triglycerides therein, and
in glucose uptake. In normal, insulin-sensitive adipose tissue, fat
storage takes place here in response to insulin and other hormones
(leptin) by stimulating lipoprotein lipase activity and inhibiting
lipolysis. However, the excessive accumulation of fatty acids in
the adipose tissue associated with obesity reduces insulin
sensitivity, which promotes the accumulation of free fatty acids in
the form of triglycerides in other organs and tissues (liver,
muscle, heart, etc.), and causes alterations in leptin production
or sensitivity, and an increase in the synthesis of proinflammatory
cytokines, which in turn entails a greater risk of developing
associated diseases (metabolic syndrome, hypertension, diabetes,
cardiovascular diseases, etc.). In the central nervous system,
insulin signalling is also essential for controlling the energy
balance and glucose homeostasis, and is dependent upon its
interaction with other regulating factors, such as leptin, which
act jointly as anorexigenic factors, reducing intake (Gerozissis
K., 2004. Eur J Pharmacol., 490(1-3): 59-70). Leptin is a
hormone/adipokine synthesized mainly by adipose tissue and, to a
lesser extent, by other tissues such as the stomach, and its
secretion is stimulated by insulin. At central nervous system
level, leptin suppresses appetite, increases energy expenditure and
intervenes in vital processes such as pancreatic .beta.-cell
function, favouring the secretion of insulin (La Cava A, Matarese
G. The weight of leptin in immunity. Nat Rev Immunol. 2004
May;4(5):371 -9). At peripheral level, leptin acts reducing the
synthesis of fatty acids and triglycerides, and increasing lipid
oxidation. However, in obese subjects, the peripheral
concentrations of this adipokine are abnormally high and produce
resistance thereto. The high leptin concentrations in obese
subjects, in addition to being a marker of metabolic disturbances,
can alter the immune response and contribute to the inflammatory
state associated with obesity.
[0005] Hepatic steatosis or non-alcoholic fatty liver is an
alteration with a high degree of association with obesity and
appears in up to 50% of obese individuals, both children and
adults, constituting the main current liver disease. Likewise,
dyslipidemias (hypertriglyceridemia and hypercholesterolemia) are
associated with obesity, Type 2 diabetes mellitus and hypertension,
and constitute the main risk factor for cardiovascular pathologies.
Lowering triglyceride and cholesterol levels in the serum of
subjects with abnormally high levels of these biochemical
parameters is beneficial and, particularly, lowering LDL
cholesterol, as it is considered a clear risk factor for
cardiovascular pathologies and a decrease therein is related to a
reduction in morbidity and total mortality as a result of long-term
cardiovascular pathologies. In this context, the liver plays an
important role because it is the main organ principally responsible
for maintaining cholesterol homeostasis (maintenance of
physiological concentrations). The liver synthesizes 15% of novo
cholesterol and this process is, in turn, regulated by dietary
cholesterol. Cholesterol levels are maintained at a constant level
by means of various mechanisms, including (i) regulation of the
activity and concentration of the 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase enzyme, (ii) regulation of the
acyl-CoA:cholesterol acyltransferase (ACAT) enzyme, which controls
excess intracellular free cholesterol and its transformation into
cholesterol esters, which is the form in which they are
transported, and (iii) regulation of the expression of the hepatic
LDL receptors which allow absorption of plasma cholesterol and
reverse transport thereof by HDL. The cholesterol just created in
the liver is initially released into the bloodstream in the form of
very low-density lipoproteins (VLDL) and can contribute to the
increase thereof. However, the liver also contributes to the
elimination of blood cholesterol through several mechanisms: (i)
conversion into bile acids, (ii) transport of excess cholesterol to
the intestine for faecal excretion and (iii) conversion of VLDL to
LDLc and TG, which shall be used as sources of energy for
extra-hepatic tissues. Alterations in lipid metabolism that affect
the blood lipid profile and their accumulation in peripheral
tissues can also occur in non-obese subjects, preceding obesity, or
occur for causes other than obesity, including those of genetic
(e.g: congenital diseases), infectious (for example, viral
hepatitis), self-immune or nutritional origin (for example,
malnutrition) or those arising from other clinical situations or
pharmacological treatments (for example, use of drugs).
[0006] Obesity is also considered a state of mild chronic
inflammation, characterised in that there is a high production of
cytokines, adipokines and other pro-inflammatory proteins in the
adipose tissue and in other peripheral tissues and at systemic
level, that contribute to the metabolic alterations which can be
permanently suffered by these individuals, such as Type 2 diabetes
mellitus and cardiovascular pathologies (Tilg y Moschen, 2006. Nat
Rev Immunol., 6: 772-783). The inflammatory factors related to
obesity and metabolic alterations include, most notably,
pro-inflammatory cytokine TNF-.alpha.. In particular, TNF-.alpha.
reduces the expression of the genes involved in the action of
insulin (for example, that of the insulin receptor gene),
attenuates insulin signalling and inhibits lipoprotein lipase
activity stimulated by the insulin. This favours the development of
insulin resistance and hepatic steatosis. The function of the
pro-inflammatory cytokines in this process is also evident in the
use of drugs based on anti-TNF-.alpha. to improve pathologies such
as hepatic steatosis and Type 2 diabetes mellitus (Tilg y Moschen,
2006. Nat Rev Immunol., 6: 772-783).
[0007] Obesity is also characterised by alterations in the
functions of various immune system cells, such as macrophages,
dendritic cells and T cells, associated with reduced defences
against pathogens and other antigens, and with a higher risk of
infections and post-operative complications. Adipose tissue
macrophages have less phagocytic capability and reduced respiratory
burst, which are processes involved in the innate immune system's
response to infectious agents (Zhou et al., 2009. Proc Nati Acad
Sci USA, 106(26): 10740-5.). Additionally, dendritic cells have
reduced capability to stimulate T cells, which are involved in the
adaptive immune response responsible, for example, for antibody
production in vaccination and for memory T cell response to
infection (Karlsson et al., 2010. J Immunol., 184:3127-33).
[0008] Social changes associated with the steady increase in intake
of high-energy-dense food and a low level of physical activity are
considered to be the main causes of the increase in global obesity
rates. However, traditional treatments based on hypocaloric diets
and increased physical activity are less effective at controlling
obesity and, in general, lead to limited and temporary weight loss.
Neither has the use of pharmacological strategies been
satisfactory, as they entail side effects. Consequently, the search
for new intervention strategies aimed at improving the treatment
and enabling the prevention of these pathologies continues.
[0009] The microbiota that colonise the human intestine are
considered a new factor involved in obesity and associated diseases
through their capability to regulate the individual's metabolic and
immunological functions (Sanz et al., 2010. Proc Nutr Soc, 14:
1-8.). In recent years, various studies have established an
association between an increase in the proportion of members of the
phylum Bacteroidetes and a thin phenotype or weight loss and, on
the contrary, a decrease therein has been associated with an obese
phenotype (Ley et al., 2006. Nature, 444: 1022-1023; Nadal et al.,
2008. Int J Obes., 33(7): 758-67); however, direct evidence of the
possible effect of strains of the genus Bacteroides or of strains
of the species Bacteroides uniformis administered orally in obesity
has not been provided. Patent WO/2008/076696 proposes the use of
changes in the intestinal microbiota to diagnose obesity and
modification thereof as a way of treating obesity by increasing the
proportion of the phylum Bacteroidetes and reducing that of the
phylum Firmicutes. However, these phylogenetic groups integrate
more than 90 and 200 different species and subspecies,
respectively, whose individual effects could be very different and
contradictory. In fact, WO/2008/076696 does not prove that no
specific species or strain of the phylum Bacteroides has a
beneficial effect in this context and, on the contrary, the only
species evaluated in animal models, Bacteroides thetaiotaomicron,
causes increase in body weight and adipose tissue and insulin
resistance (Samuel y Gordon. Proc Nati Acad Sci USA. 2006; 27;
103(26): 10011-6). Patent US 2009/01 10664A1 proposes the use of
the genus Bacteroides in body weight loss, but administering the
bacterium after cleansing or removing the components themselves
from the intestinal tract, as opposed to the present invention.
Additionally, this patent does not disclose the results of the
effects of any species or strain of this genus on body weight.
[0010] Other strategies based on the use of certain food
ingredients or supplements only partially address the problem of
obesity or of the pathologies arising from alterations in lipid and
glucose metabolism, as in the case of stanols and phytosterols,
which only act by reducing absorption of dietary cholesterol, which
is not the only cause of elevation in plasma cholesterol. Likewise,
lipid-lowering drugs such as statins that inhibit endogenous
cholesterol synthesis do not achieve the required effectiveness due
to being monotherapies focused on a single mechanism of action.
[0011] Therefore, the problem of finding specific components of
commensal intestinal microbiota which can be used to prevent and/or
treat diseases such as excess weight, obesity and metabolic
pathologies associated or not associated to obesity and related to
alterations in lipid and glucose metabolism, such as for example
dyslipidemia, hepatic steatosis, metabolic syndrome, insulin
resistance, Type 2 diabetes mellitus, gestational diabetes,
hypertension and cardiovascular pathologies, in a more suitable
manner by acting jointly on the immune system and metabolism
alterations, responsible for chronic pathologies, remains
unsolved.
Explantion of the Invention
[0012] The present invention relates to the strain Bacteroides
uniformis CECT 7771, to the cellular components, metabolites,
molecules secreted by said strain and combinations thereof; and to
the compositions comprising at least one of the aforementioned
products and which can comprise other microorganisms and/or other
bioactive components, as well as to their use in the prevention
and/or treatment of excess weight and/or obesity, and of the
associated metabolic alterations, such as dyslipidemia, hepatic
steatosis, insulin resistance and diabetes, metabolic syndrome,
hypertension, cardiovascular diseases or immune system dysfunction
with consequences on these or other pathologies such as infections.
The present invention also relates to the use of said strain to
prevent and/or treat these alterations when not associated to a
problem of excess weight and/or obesity.
[0013] The CECT 7771 strain belonging to the species B. uniformis,
has comparatively more favourable immunological properties than
other strains of the same species and other species of the genus
Bacteroides. The CECT 7771 strain induces significantly less
production of pro-inflammatory cytokine TNF-.alpha. in macrophages
than other strains of the same genus that form part of human
intestinal microbiota, except B. dorei SS1 and B. thetaiotaomicrom
SAC4, where the differences do not become significatives (Example
2, Table 1). The strain CECT 7771 also induces greater synthesis of
anti-inflammatory cytokine
[0014] IL-10 than the other evaluated strains (Example 2, Table 1).
Other evaluated strains of the same species (.beta.. uniformis)
induced a significantly higher proportion of the TNF-.alpha./IL-10
ratio than the strain object of the patent (CECT 7771), indicating
that the balance of pro- and anti-inflammatory cytokines induced by
the latter is more favourable than that induced by the other
strains (Example 2, Table 1). As argued in the section on the state
of the art, the synthesis of TNF-.alpha. by macrophages has been
directly linked to obesity, dyslipidemia, hepatic steatosis,
diabetes, hypertension and the risk of cardiovascular pathologies.
The capability of the strain of the invention to increase synthesis
by means of anti-inflammatory cytokine IL-10 is also a relevant
property because it can contribute to reduce the chronic
inflammation associated with obesity and metabolic alterations.
Studies conducted on hepatocytes also indicate that the CECT 7771
strain reduces the accumulation of triglycerides and cholesterol
and improves sensitivity to insulin and to the use of glucose in
comparison with other species of the genus Bacteroides and with
other strains of the species B. uniformis (Example 2, FIG. 1). All
of these results show the greater suitability of the strain object
of the patent to control inflammation and lipid and glucose
metabolism than other species and strains of the genus Bacteroides.
Studies conducted by our group also reveal that breastfeeding
promotes an increase in the prevalence of the species object of the
patent (.beta.. uniformis) in the microbiota of children in the
early stages of life but not in those subjected to artificial
feeding (Sanchez et al. 2011. Appl Environ Microbiol. 201 1;
77(15):5316-23) and, in turn, breastfeeding protects them against
the development obesity and metabolic alterations.
[0015] Globally, the results obtained with macrophage and
hepatocyte cultures indicate that the species B. uniformis is
particularly suited for use in these pathologies, in comparison to
the rest of the species found in humans that do not have these
properties (for example, but not limited to, B. thetaiotaomicron)
and, in particular, the strain B. uniformis CECT 7771.
[0016] In addition to the specific selection of the strain object
of the invention and as opposed to the state of the art, the
present invention addresses the treatment of obesity from a
multifactorial perspective and acts on new key targets for
preventing and/or treating this pathology and other metabolic
alterations associated or not associated to obesity, not described
for any known strain of the species Bacteroides uniformis. The most
interesting fact is that none of the known strains of this species
has proven to be useful in the simultaneous and effective treatment
of all the pathologies indicated throughout the present
invention.
[0017] Therefore, the present invention contributes a highly
valuable strain of the species B. uniformis to the state of the art
for treating excess weight and/or obesity, in addition to certain
pathologies such as, for example, but not limited to, hepatic
steatosis, dyslipidemia, insulin resistance, diabetes, metabolic
syndrome, hypertension or cardiovascular diseases associated or not
associated with obesity. Likewise, the present invention
contributes a strain of the species B. uniformis to the state of
the art that improves immune system alterations and, in particular,
the inflammation of the peripheral tissues associated with the
aforementioned chronic pathologies and reduced defences against
infections, in addition to restoring the composition of the
intestinal microbiota, which also contributes to the aforementioned
pathologies.
[0018] Essentially, the advantages of using the strain B. uniformis
CECT 7771 of the present invention are the following:
[0019] Administration of the strain object of the invention
produces a reduction in body weight in obese subjects (Example 3,
Table 2).
[0020] Administration of the strain object of the invention gives
rise to a reduction in fat accumulated in the liver in obese and
non-obese subjects (Example 3, FIG. 2). Specifically, in
normal-weight subjects the strain B. uniformis CECT 7771 produces
an increase in the number of hepatocytes without steatosis (grade
0) and a decrease in the number of hepatocytes with steatosis grade
1 and 2. In obese subjects the strain produces an increase in the
number of hepatocytes with a lower grade of steatosis (grade 0 and
1) and a decrease in the number of hepatocytes with a higher grade
of steatosis (grade 2 and 3); however, in obese subjects to which
the strain is not administered, the proportion of the type of
hepatocytes is reversed, with a predominance of those with maximum
fat content. This demonstrates that the administration of the
strain reduces the total accumulation of fat in the liver, induced
or not induced dietetically. Histology sections of hepatic tissue
also demonstrated these effects (Example 3, FIG. 2). The strain B.
uniformis CECT 7771 also reduces triglyceride and cholesterol
levels in the liver in obese subjects (Example 3, Table 2).
[0021] Administration of the strain B. uniformis CECT 7771 produces
a reduction in adipocyte size in obese subjects (Example 3, FIG.
3). In particular, administration of the strain CECT 7771 to
animals gives rise to an increase in small-sized adipocytes
(<2000 .mu.m.sup.2) at the expense of a decrease in larger-sized
adipocytes in epidydimal tissue, while all large-sized adipocytes
(>2000-7000 .mu.m.sup.2) in obese animals to which the strain
was not administered increased (Example 3, FIG. 3). Histology
sections of the adipose tissue also demonstrated these effects
(Example 3, FIG. 3).
[0022] The fact that the CECT 7771 strain reduces the size of the
adipocytes demonstrates that it is useful for treating adipocyte
hypertrophy, which is maintained over time and occurs in a large
number of adipocytes, can cause excess weight and obesity and
insulin resistance. This is because larger-sized adipocytes secrete
a higher concentration of growth factors that trigger adipogenesis
through the differentiation of preadipocytes, generating a feedback
process. Additionally, hypertrophic adipocytes produce an
abnormally high concentration of inflammatory cytokines and
chimiokines (TNF-.alpha., MCP-1, resistin, etc.) that inhibit
insulin signalling in the hepatocytes and give rise to insulin
resistance and alter the corporal distribution of lipids. For
example, an increase in adipocyte size is also related to hepatic
fatty acid intake, which gives rise to hepatic steatosis and its
complications. Therefore, the strain can also contribute to
preventing or improving these associated pathologies.
[0023] The B. uniformis CECT 7771 strain reduces the number of fat
globules in the enterocytes, i.e. it reduces the amount of dietary
fat that can be absorbed and passed to the lymphatic system and
bloodstream in the form of chylomicrons and, thus, to peripheral
tissues (Example 3, FIG. 4).
[0024] The increased absorption of dietary fat, in addition to
giving rise to excess weight and/or obesity on causing an increase
in the accumulation thereof in adipose tissues, can be associated
with other pathologies without causing excess weight or obesity,
such as for example, and without limiting the scope of the
invention, dyslipidemia, metabolic syndrome, arterial hypertension,
cardiovascular pathologies and other alterations arising from the
relationship between lipid and glucose metabolism. Therefore, the
CECT 7771 strain can be effective in the prevention and/or
treatment of diseases related to the excessive absorption of
dietary fat.
[0025] The B. uniformis CECT 7771 strain reduces dyslipidemia and,
in particular, peripheral blood triglyceride and cholesterol levels
in obese subjects (Example 3, Table 2), thereby reducing the risk
of developing cardiovascular diseases. This effect could be
partially due to the strain's capability to inhibit the amount of
dietary fat absorbed.
[0026] Dyslipidemia can also be a consequence not only of absorbed
dietary fat but also of other metabolic alterations such as
adipocyte insulin resistance which, without being necessarily
associated with obesity, causes the adipocytes to release fatty
acids that will be used in the liver for triglyceride and
cholesterol synthesis, and can also be secreted and their
concentration in peripheral blood increased. Dyslipidemia can also
appear in subjects with a genetic predisposition to develop this
metabolic alteration, without necessarily being associated with
obesity, with insulin resistance or an increase in the absorption
of dietary fat. Therefore, the CECT 7771 strain can be effective in
the prevention and/or treatment of dyslipidemia (for example,
hypertriglyceridemia and hypercholesterolemia) and related
pathologies, such as hypertension and cardiovascular
pathologies.
[0027] The B. uniformis CECT 7771 strain reduces serum glucose in
parallel to fasting insulin and resistance to insulin index HOMA
(Homeostasis Model Assessment), which makes it possible to estimate
insulin resistance (a high index indicates low insulin sensitivity)
and pancreatic beta-cell function. Additionally, the strain object
of the invention reduces postprandial glycemic response after
ingestion of oral glucose, which also indicates an improvement in
glucose metabolism and insulin sensitivity (Example 3, Table 2).
The strain object of the invention also reduces adipokine leptin
concentrations in obese subjects, indicating an improvement in the
metabolic function of said adipokine, which in turn can contribute
to improving glucose metabolism and insulin production or
sensitivity (Example 4, Table 3).
[0028] An increase in serum concentrations of glucose, due to the
development of insulin resistance, is frequently associated with
excess weight and obesity, although it can also occur in the
absence of obesity, and can lead to the development of Type 2
diabetes mellitus and gestational diabetes. Therefore, the CECT
7771 strain can be effective in the prevention and/or treatment of
related glucose metabolism alterations that can lead to the
development of insulin resistance and, finally, diabetes.
[0029] The CECT 7771 strain is capable of reducing the synthesis of
pro-inflammatory proteins in peripheral tissue in normal-weight
subjects treated with said strain with respect to those not treated
with said strain. Therefore, the strain object of the invention
reduces the synthesis of the inflammatory cytokine TNF-.alpha. and
increases the synthesis of the inflammatory cytokine IL-10 in
adipose tissue, while the levels of this cytokine decreases in
obese subjects not treated with the strain. TNF-.alpha. synthesis
increases with obesity and other pathologies and contributes to the
development of insulin and leptin resistance, inhibiting its
anorexigenic effects (reduction in the sense of hunger) and its
function in the regulation of body weight and lipid and glucose
metabolism (Example 4, Table 3). Additionally, the strain object of
the invention reduces the concentration of the inflammatory
cytokine TNF-.alpha. in the pancreas, improving the function of
this organ in the regulation of glucose metabolism (Example 4,
Table 3). The strain object of the invention also reduces the
concentration of adipokine leptin, which can also favour
inflammation in the context of excess weight and obesity (Example
4, Table 3).
[0030] Therefore, the CECT 7771 strain regulates the production of
cytokines and adipokines, whose synthesis is altered in the case of
obesity and in certain diseases associated therewith, such as for
example, but not limited to, dyslipidemia, metabolic syndrome,
insulin resistance, hypertension, cardiovascular diseases and
steatosis, both in peripheral blood and in tissues, and in other
diseases not necessarily associated with excess weight and/or
obesity and, therefore, can be used in the treatment and prevention
of these pathologies.
[0031] The CECT 7771 strain improves innate and adaptive immune
system cell function, increasing their capability to respond to
infectious agents, antigens or allergens in obese and non-obese
subjects. In particular, the administration of the strain to animal
models of obesity induced by a fat-rich diet improves, inter alia,
macrophage function in phagocytosis and in cytokine synthesis in
response to pathogen stimuli (Example 4, FIG. 5). The strain object
of the invention also improves adaptive immune system dendritic
cells and Ts cell function (Example 4, FIG. 6).
[0032] Therefore, the CECT 7771 strain has an additional positive
effect because it can be useful in the prevention and treatment of
infections and improvement in protective responses, for example in
vaccination and immunisation processes, due to the fact that these
immune system functions are altered in subjects with excess weight
and obesity. Additionally, the strain of the invention can be
useful in the treatment or prevention of other diseases accompanied
by immunosuppression (essentially of macrophages, dendritic cells
and T cells), associated or not associated with obesity and excess
weight, as these effects are also demonstrated in non-obese
subjects.
[0033] The CECT 7771 strain also restores the composition of the
intestinal microbiota, normalising the alterations associated with
excess weight and obesity (reduced abundance of the group C.
coccoides of the genus Bifidobacterium and increased abundance of
the Enterobacteriaceae family) and attenuating the inflammatory
effect caused by said alterations, and which has been related to
weight gain, insulin resistance, metabolic endotoxemia, hepatic
steatosis and alterations of the intestinal barrier (Example 4,
Table 4 and Example 3, FIG. 7). The strain of the invention also
increases the number of Bacteroides spp. and Bifidobacterium spp.
in normal-weight subjects and can be used to restore these
microbial populations in the intestine, which may be altered due to
conditions other than obesity and excess weight. Therefore, the
CECT 7771 strain is also applicable in the prevention and treatment
of diseases associated with alterations of the intestinal
microbiota and Enterobacter infections.
[0034] One aspect of the present invention relates to a B.
uniformis strain with deposit number CECT 7771. Said strain was
deposited with the Spanish Type Culture Collection (CECT) on 21
Jul. 2010 and assigned deposit number CECT 7771. The address of
said international deposit Authority is: Universidad de
Valencia/Edificio de investigacion/Campus de Burjassot/46100
Burjassot (Valencia, Spain).
[0035] The scientific classification of the CECT 7771 strain of the
present invention is: Kingdom: Bacteria/Phylum:
Bacteroidetes/Order: Bacteroidales/Family: Bacteroidaceae/Genus:
Bacteroides/Species: uniformis.
[0036] The characteristics of said strain are the following:
[0037] The substrates oxidised or fermented by the B. Uniformis
CECT 7771 strain are: lactose, sucrose, maltose, salicin, xylose,
arabinose, esculin, cellobiose, mannose and raffinose.
[0038] The B. uniformis CECT 7771 strain grows in a temperature
range between 31 and 42.degree. C., with an optimum at 37.degree.
C.
[0039] Additionally, the B. uniformis CECT 7771 strain is stable
under conditions of gastrointestinal stress (acid pH and high
concentration of bile). Its viability after incubation under
gastric conditions (3g/l pepsin at pH 3 and 2.5) during average
gastric emptying time (2 h) is 50-70% and, after incubation in the
presence of bile salts (0.5 and 1%), remains above 90%. It is also
resistant to the conditions of technological preservation processes
(freezing, freeze drying, etc.) and food processing conditions
(cooling, freeze drying, fermentation, etc.). All of these
properties guarantee its viability and persistence and
effectiveness in the intestine.
[0040] Another aspect of the present invention relates to a strain
derived from the B. uniformis CECT 7771 strain, wherein said strain
maintains or improves the capabilities described throughout the
present invention. The microorganism derived can be naturally
occurring or produced intentionally by mutagenesis methods known in
the prior art such as, but not limited to, growth of the original
microorganism in the presence of mutagenic agents or stressors, or
directed genetic engineering of specific genes. According to a
preferred embodiment, the strain derived from the B. uniformis CECT
7771 strain is a genetically modified mutant. The terms "mutant
strain" or "derived strain" can be used interchangeably.
[0041] The B. uniformis CECT 7771 strain or any mutant or
derivative thereof may be used in any way to exert the effects
described, for example, according to a preferred embodiment of the
present invention, the B. uniformis CECT 7771 strain is formed of
viable cells (cultivable or uncultivable) or, according to another
preferred embodiment of the invention, the strain is in the form of
non-viable cells ("dead" cells inactivated by any technique known
in the art, such as for example, but not limited to, heat, freezing
or ultraviolet radiation).
[0042] Hereinafter, any of the bacterial strains of the previously
described species B. uniformis (B. uniformis CECT 7771 strain or
any mutant or derivative thereof) may be referred to as the "strain
of the present invention" or the "strain of the invention."
[0043] Another aspect of the present invention relates to cellular
components, metabolites, secreted molecules or any combination
thereof, obtained from the strain of the invention or from a
mixture of microorganisms comprising at least one strain of the
invention.
[0044] The cellular components of the bacterium may include the
components of the cell wall (such as, but not limited to,
peptidoglican), nucleic acids, membrane components, or others such
as proteins, lipids and carbohydrates and combinations thereof,
such as lipoproteins, glycolipids or glicoproteins. The metabolites
include any molecule produced or modified by the bacterium as a
consequence of their metabolic activity during growth, their use in
technological processes (for example, but not limited to, food or
drug elaboration processes) during product storage or during
gastrointestinal transit. Examples of these metabolites are, but
not limited to, organic and inorganic acids, proteins, peptides,
amino acids, enzymes, lipids, carbohydrates, lipoproteins,
glycolipids, glycoproteins, vitamins, salts, metals or nucleic
acids. The secreted molecules include any molecule exported or
released by the bacterium during growth thereof, its use in
technological processes (for example, preparation of food or
drugs), product storage or gastrointestinal transit. Examples of
these molecules include, but not limited to, organic and inorganic
acids, proteins, peptides, amino acids, enzymes, lipids,
carbohydrates, lipoproteins, glycolipids, glycoproteins, vitamins,
salts, metals or nucleic acids.
[0045] Another aspect of the present invention relates to a
composition comprising the strain of the invention, and/or cellular
constituents, metabolites, molecules secreted by the strain of the
invention or any previously defined combination thereof.
[0046] The composition, generally defined, is a set of components
which is formed at least by the strain of the invention at any
concentration, or at least by the cellular components, metabolites,
molecules secreted by the strain of the invention or any of its
combinations, or a combination thereof.
[0047] According to the invention, the previous composition may
further comprise at least one additional microorganism other than
the strain of the invention and/or its cellular components,
metabolites or secreted molecules, or any combination thereof. For
example, but not limited to, the additional microorganism that may
form part of said composition is selected from among at least one
of the following groups:
[0048] at least one strain of another species of the genus
Bacteroides or of the species B. uniformis;
[0049] at least one lactic acid bacterium or intestinal
bifidobacterium, of alimentary or environmental origin. The lactic
bacterium is selected from the list comprising, but not limited to,
bacteria of the genus Bifidobacterium, Lactobacillus, Lactococcus,
Enterococcus, Propionibacterium, Leuconostoc, Weissella,
Pediococcus, or Streptococcus;
[0050] at least one strain of other phylogenetic groups, genera or
species of intestinal prokaryotes of intestinal, alimentary or
environmental origin, such as, but not limited to, Archaea,
Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria,
Verrucomicrobia, Fusobacteria, Metanobacteria, Spirochaetes,
Fibrobacteres, Deferribacteres, Deinococcus, Thermus,
Cyanobacteria, Methanobrevibacterium, Peptostreptococcus,
Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia,
Anaerofustis, Gemella, Roseburia, Catenibacterium, Dialister,
Anaerotruncus, Staphylococcus, Micrococcus, Propionibacterium,
Enterobacteriaceae, Faecalibacterium, Bacteroides, Parabacteroides,
Prevotella, Eubacterium, Akkermansia, Bacillus, Butyrivibrio, or
Clostridium;
[0051] at least one strain of fungus or yeast such as, but not
limited to, belonging to the genus Saccharomyces, Candida, Pichia,
Debaryomyces, Torulopsis, Aspergillus, Rhizopus, Mucor or
Penicillium.
[0052] Said additional microorganism may be a strain of the same
species or different species or taxonomic group of microorganisms
corresponding to the strain of the invention. The cells comprising
the composition may be viable or non-viable, and be in any stage of
development or growth (latent, exponential, stationary, etc.),
regardless of their morphology. Preferably, said additional
microorganism further comprises at least one intestinal bacterium
or lactic bacterium.
[0053] Optionally, the composition according to any of those
defined above may further comprise at least one bioactive component
(active substance, active ingredient or therapeutic agent) such as,
for example, other food, plant and/or pharmaceutical
components.
[0054] The term "bioactive component" relates to a compound having
biological activity in the scope of applicability of the patent
that can enhance or supplement the activity of a strain of the
species B. uniformis and preferably the CECT 7771 strain object of
the invention, including food ingredients or components (such as
but not limited to: poly-unsaturated fatty acids, conjugated
linoleic acid, prebiotics, fibre, guar gum, glucomannan, chitosan
picolinate, copper, calcium, etc.), plants, plant extracts or
components (for example, but not limited to, polyphenols, ephedrine
or Ephedra spp., green tea [Camellia sinensis], bitter orange
[Citrus aurantium]), and drugs (for example, but not limited to,
statins, orlistat, sibutramine, liraglutido etc.).
[0055] In a preferred embodiment, the composition as defined above
is a pharmaceutical composition. The pharmaceutical composition is
a set of components which is formed at least by the strain of the
invention at any concentration, or at least by the cellular
components, metabolites, molecules secreted by the strain of the
invention or any combination thereof, having at least one
application in improving the physical or physiological or
psychological well-being of a subject, which implies an improvement
in the general state of health or reduced risk of disease. Said
pharmaceutical composition can be a drug.
[0056] The meaning of the term "drug" is more limited than the
meaning of "pharmaceutical composition", as defined herein, as a
drug necessarily implies a preventive or therapeutic effect. The
drug to which the present invention relates may be for human or
veterinary use. The "drug for human use" is any substance or
combination of substances presented as having properties for
treating or preventing diseases in human beings or that can be used
in humans or administered to human beings either with a view to
restoring, correcting or modifying physiological functions by
exerting a pharmacological, immunological or metabolic action, or
to making a medical diagnosis. The "drug for veterinary use" is any
substance or combination of substances presented as having
properties for curing or preventing animal diseases or which may be
administered to animals with a view to restoring, correcting or
modifying their physiological functions by exerting a
pharmacological, immunological or metabolic action, or to make a
veterinary diagnosis. "Veterinary drugs" shall also be considered a
"premix for medicated feed" prepared for incorporation into
feed.
[0057] In addition to the requirement of therapeutic effectiveness
where said pharmaceutical composition may require the use of other
therapeutic agents, there may be additional basic reasons that
oblige or recommend using a combination of a compound of the
invention and a bioactive component to a large extent, wherein said
bioactive component is attributed an appropriate activity for
constituting a drug. Said compound of the invention obviously
relates to any of Bacteroides uniformis strains of the invention or
to the cell components, metabolites, secreted molecules or any
combination thereof, derived from one of the strains of the
invention.
[0058] In a preferred embodiment, the pharmaceutical composition
further comprises, at least, one vehicle and/or a pharmaceutically
acceptable excipient. The term "excipient" relates to a substance
that aids the absorption of any of the components of the
composition of the present invention, stabilises said components or
aids the preparation of the pharmaceutical composition in the sense
of giving consistency or contributing flavours that make it more
enjoyable. Thus, excipients may have the function of holding the
components together, such as starches, sugars or celluloses, a
sweetening function, colouring function, drug protection function
such as to isolate from the air and/or humidity, the function of
filling a tablet, capsule or other form of presentation such as,
for example, dibasic calcium phosphate, a disintegrating function
to facilitate the dissolution of the components and their
absorption in the intestine, without excluding any other type of
excipients not mentioned in this paragraph. Therefore, the term
"excipient" is defined as the material included in the galenic
forms, is added to the active ingredients or their associations to
enable their preparation and stability, modify their organoleptic
properties or determine the physico-chemical properties of the
pharmaceutical composition and its bioavailability. A
"pharmaceutically acceptable" excipient must allow the activity of
the compounds of the pharmaceutical composition, i.e. to be
compatible with said components.
[0059] The "galenic form or pharmaceutical form" is the provision
to which the active ingredients and excipients are adapted to
constitute a drug. It is defined by the combination of the form in
which the pharmaceutical composition is presented by the
manufacturer and the form in which it is administered.
[0060] The "vehicle" or carrier is preferably an inert substance.
The function of the vehicle is to facilitate the incorporation of
other compounds, allow better dosage and administration or give
consistency and shape to the pharmaceutical composition. Therefore,
the vehicle is a substance used in the drug to dilute the any of
the components of the pharmaceutical composition of the present
invention to a given volume or weight; or while not diluting said
components, is capable of allowing better dosage and administration
or giving the drug consistency and shape. When the form of
presentation is liquid, the pharmaceutically acceptable vehicle is
the diluent.
[0061] Additionally, the excipient and the vehicle must be
pharmaceutically acceptable, i.e. the excipient and the vehicle is
allowed and evaluated so as not to cause damage to the bodies to
which it is administered.
[0062] In each case the galenic form of the pharmaceutical
composition and, therefore, the drug, will be adapted to the dosage
form used. Therefore, the composition of the present invention can
be provided in the form of solutions or any other clinically
permitted dosage form and in a therapeutically effective quantity.
The pharmaceutical composition of the invention may be formulated
in solid, semisolid, liquid or gaseous forms, such as tablet,
capsule, powder, granule, ointment, solution, suppository,
injection, inhalant, gel, microsphere or aerosol. According to an
even more preferred embodiment of the present invention, the
pharmaceutical composition is in a form adapted for oral
administration.
[0063] The form adapted for oral administration relates to a
physical state which would permit its oral administration. Said
form adapted for oral administration is selected from the list
comprising, but not limited to, drops, syrup, herbal tea, elixir,
suspension, extemporaneous suspension, drinkable phial, tablet,
capsule, granule, wafer, pill, tablet, lozenge, troche or
lyophilised.
[0064] Alternatively, the pharmaceutical composition may also be
presented in a form adapted for sublingual, nasal, intrathecal,
bronchial, lymphatic, rectal, transdermal, inhaled or parenteral
administration. The strain of the invention; the cellular
components, metabolites, secreted molecules or any combination
thereof, obtained from the strain of the invention, or the
composition of the invention may, for example, be associated with,
but not limited to, liposomes or micelles.
[0065] In the sense used in this description, the expression
"therapeutically effective amount" relates to a certain amount of
the component of the pharmaceutical composition which, when
administered to a mammal, preferably a human, is sufficient to
result in prevention and/or treatment, as defined later in the
text, of a disease or pathological condition of interest in the
mammal, preferably a human. Said component of the pharmaceutical
composition relates to the strain of the invention; or to the
cellular components, metabolites, secreted molecules; or a
combination thereof, that may optionally be comprised in said
composition in combination with an additional bioactive component,
and contributing to the therapeutic effect of the pharmaceutical
composition. The therapeutically effective amount will vary, for
example, according to the activity of the strain of the invention;
the additional microorganism or additional microorganisms
comprising the composition of the invention, cellular components,
metabolites, secreted molecules or any combination thereof, in any
dosage form; the therapeutically effective amount will also vary
according to the metabolic stability and duration of action of that
compound; the patient's age, body weight, general state of health,
sex and diet, the route and time of administration , the rate of
excretion, drug combination; the severity of the particular
alteration or pathological condition; and the subject undergoing
therapy, but may be determined by a person skilled in the art based
on their own knowledge and that description.
[0066] In another preferred embodiment, the composition defined
according to the invention is a nutritional composition.
[0067] In a more preferred embodiment, the nutritional composition
is selected from a food (which may be a food for specific
nutritional purposes or a medicinal food), a supplement, a
nutraceutical, a probiotic or a symbiotic.
[0068] The term "nutritional composition" of the present invention
relates to a food that, regardless of providing nutrients to the
subject eating it, has a beneficial effect on one or more bodily
functions, so as to provide a better state of health and
well-being. Accordingly, such nutritional composition may be
destined for the prevention and/or treatment of a disease or for
the reduction of disease risk factors.
[0069] The term "supplement", which is synonymous with any of the
terms "dietary supplement", "nutritional supplement" or "food
supplement" is a component or components destined for supplementing
the diet and may be a food. Examples of dietary supplements are,
but not limited to, vitamins, minerals, botanical products, amino
acids and food components such as enzymes and glandular extracts.
They are not presented as a substitute for a conventional food or
as the sole component of a meal or diet, but rather as a dietary
supplement.
[0070] The term "nutraceutical" as used herein relates to the
isolated substances of a food used in dosage form and having a
beneficial effect on human health. Said nutraceutical can be a
supplement.
[0071] The term "probiotic" as used herein relates to
microorganisms which, when administered in adequate amounts, have
beneficial effects on the health of the host organism.
[0072] The term "symbiotic" as used herein relates to those foods
which contain a mixture of prebiotics and probiotics. As a general
rule, they contain a prebiotic component to enhance the growth
and/or metabolic activity and, ultimately, the effect of the
probiotic with which it is combined, such as for example, but not
limited to, the association of fructooligosaccharides and
galactooligosaccharides with an intestinal bacterium such as a
strain of the species B. uniformis.
[0073] According to a more preferred embodiment of the foregoing,
the food is selected from the list comprising: dairy product,
vegetable product, meat product, snack, chocolate, baby food or
drink. The dairy product is selected from the list comprising, but
not limited to, fermented milk by-products (for example, but not
limited to, yoghurt or cheese) or non-fermented milk by-products
(for example, but not limited to, ice cream, butter, margarine,
whey). The vegetable product is, for example, but not limited to, a
grain in any form of presentation, fermented or non-fermented. The
drink may be, but not limited to, any fruit juice or non-fermented
milk.
[0074] Another more preferred embodiment of the present invention
relates to any of the compositions described in the invention,
wherein said composition has a concentration of the strain of
between 10.sup.3 and 10.sup.14 colony-forming units (cfu) per
gramme or millilitre of final composition. The concentration of the
strain is the therapeutically effective or nutritionally effective
concentration, as appropriate. The nutritional composition and the
pharmaceutical composition may be formulated, but not limited to,
in solid, semi-solid, liquid or gaseous forms, such as tablet,
capsule, microcapsule, powder, granule, ointment, solution, paste,
suppository, injection, inhalant, gel, microsphere or aerosol.
[0075] Hereinafter, reference may be made to any of the
compositions, general composition, pharmaceutical composition or
nutritional composition, defined in the preceding paragraphs using
the term "composition of the present invention" or "composition of
the invention."
[0076] Another aspect of the invention relates to the use of the
strain of the invention; or the cellular component, metabolite,
secreted molecule or any combination thereof, of the invention in
the manufacture of a pharmaceutical composition, a drug or a
nutritious composition.
[0077] Another aspect of the invention relates to a strain of the
species Bacteroides uniformis for uses other than the treatment
and/or prevention of excess weight or obesity. The present
invention demonstrates how a strain of the species Bacteroides
uniformis (such as the B. uniformis CECT 7771 strain) may be used
to treat and/or prevent other lipid and glucose metabolism
alterations, not necessarily associated with excess weight or
obesity, such adipocyte hypertrophy; hepatic steatosis or fatty
liver, dyslipidemia (for example, hypertriglyceridemia and/or
hypercholesterolemia), hypertension, cardiovascular diseases,
hyperglycaemia, insulin resistance and/or diabetes (for example,
gestational diabetes or Type 2 diabetes mellitus); or metabolic
syndrome. Likewise, it was observed that said strain of Bacteroides
uniformis can be used to improve the function of the immune system,
for example, reducing inflammation in peripheral tissues (adipose
and pancreas) caused by the previously described chronic metabolic
alterations, and to increase defences against infection and the
response to vaccination. In addition, the Bacteroides uniformis
strain can be used to restore the composition of the intestinal
microbiota and prevent pathologies related to the alteration
thereof, for example, reducing the concentration of enterobacteria
in intestinal contents.
[0078] In a preferred embodiment of use in medicine, a strain of
Bacteroides uniformis; or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used to reduce
adipocyte size in a subject and, therefore, is also used in the
treatment and/or prevention of adipocyte hypertrophy.
[0079] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used to reduce the
accumulation of fat in hepatocytes and, therefore, is also used in
the treatment and/or prevention of hepatic steatosis or fatty
liver.
[0080] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used to reduce blood
triglyceride and cholesterol levels and, therefore, is also used in
the treatment and/or prevention of dyslipidemia, more preferably of
a dyslipidemia selected from among hypertriglyceridemia and/or
hypercholesterolemia.
[0081] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used in the treatment
and/or prevention of a cardiovascular disease.
[0082] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used to reduce the
blood glucose levels and, therefore, can also be used in the
treatment and/or prevention of hyperglycemia and/or a pathology
associated with higher levels of blood glucose amounts.
[0083] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used in the treatment
and/or prevention of insulin resistance and/or diabetes, and, more
preferably, the diabetes is selected from among gestational
diabetes or Type 2 diabetes mellitus.
[0084] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used in the treatment
and/or prevention of metabolic syndrome.
[0085] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used in the treatment
and/or prevention of hypertension.
[0086] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used to improve the
function of the immune system of a subject with respect to an
untreated control.
[0087] Among the improvements of immune function, a strain of
Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, can reduce inflammation in
peripheral tissues that causes the chronic metabolic alterations
object of the patent including, inter alia, dyslipidemia, hepatic
steatosis, adipocyte hypertrophy, insulin resistance and diabetes,
hypertension, metabolic syndrome and cardiovascular diseases. Also,
another enhancement of the immune system provided by a strain of
Bacteroides uniformis is its ability to stimulate responses of
immunocompetent cells (macrophages, dendritic cells and lymphocyte
T-cells) and, thus, defences against pathogens and antigens.
[0088] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used to reduce
inflammation in peripheral tissues, preferably adipose and/or
pancreatic tissue.
[0089] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used in the treatment
and/or prevention of infection and/or to improve the response to
vaccination. The examples show how a strain of B. uniformis
improves the function of the innate and adaptive immune system
against pathogens and antigens, therefore improving the response to
infection of an individual to whom it is administered.
[0090] In another preferred embodiment of use in medicine, a strain
of Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, is used to restore the
composition of the intestinal microbiota and, preferably, to reduce
the concentration of potential pathogens, such as enterobacteria in
the intestinal contents of a subject with respect to an untreated
control.
[0091] According to the present description, a strain of
Bacteroides uniformis, or one of its cellular components,
metabolites, secreted molecules or a combination thereof; or a
composition comprising any of the above, for use in the treatment
and/or prevention of different diseases or metabolic alterations,
to improve the function of the immune system or to reduce the
concentration of enterobacteria, can obviously be understood as a
method for treating and/or preventing such diseases or alterations,
or a method for improving the function of the immune system, or a
method for reducing the concentration of enterobacteria, which
comprises administering a therapeutically effective amount of said
strain to a subject. Likewise, the present invention also protects
the use of said strain; or its cellular components, metabolites,
secreted molecules, or a combination thereof, for the manufacture
of a nutritional composition, a pharmaceutical composition or a
drug for the treatment and/or prevention of such diseases or
metabolic alterations, for improving the function of the immune
system or for reducing the concentration of enterobacteria. Another
aspect of the invention relates to the strain of the invention, or
the cellular component, metabolite, secreted molecule or any
combination thereof of the invention; or the composition of the
invention for use in medicine.
[0092] The term "treatment", as understood herein, relates to fight
the effects of a disease or pathological condition of interest in a
subject (preferably a mammal and, more preferably, a human) that
includes:
[0093] (i) inhibiting the disease or pathological condition, i.e.
arresting its development;
[0094] (ii) relieving the disease or the pathological condition,
i.e. causing regression of the disease or the pathological
condition or its symptoms;
[0095] (iii) stabilizing the disease or pathological condition.
[0096] The term "prevention" as understood in the present invention
consists of preventing the onset of the disease, that is,
preventing the disease or pathological condition from occurring in
a subject (preferably a mammal and, more preferably, a human),
particularly when said subject is predisposed to develop the
pathological condition.
[0097] The term "excess weight" relates to a disease characterised
in that the subject has a body mass index (BMI) equal to or greater
than 25. BMI is a measure of association between the weight and
height of an individual. BMI is calculated using the following
formula: Mass (kg)/height.sup.2 (m). Excess weight is characterised
by a BMI of .gtoreq.25 to <30.
[0098] The term "obesity" relates to a disease characterised in
that the subject has a BMI equal or greater than 30. Obesity is
classified into different levels, considering that subjects having
a BMI>40 have morbid obesity. Other parameters used to determine
if an individual has central obesity are the absolute waist
circumference (subject is obese when>102 cm in men [central
obesity] and >88 cm in women) or the waist-hip ratio (the
subject is obese when >0.9 for men and >0.85 for women). An
alternative way to determine obesity is to measure the percentage
of body fat (the subject is obese when approximately >25% of
body fat in men and approximately >30% of body fat in
women).
[0099] In an example of use in medicine, a strain of the invention;
or the cellular components, metabolites, secreted molecules, or any
combination thereof of the invention; or a composition of the
invention, is used in the treatment and/or the prevention of excess
weight or obesity and, preferably, when caused by diet. As
demonstrated in the present invention, administration of the B.
uniformis CECT 7771 strain in animals with diet-induced obesity
causes a reduction in weight gain (Example 3, Table 2). Overall,
this means that the strain can be used for treating or preventing
excess weight or obesity.
[0100] Given that the administration of the B. uniformis CECT 7771
strain produces a reduction in weight gain (Example 3, Table 2),
said strain can also be used in cosmetic applications to reduce
weight gain. Therefore, it is understood that a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention for reducing weight gain is also
equivalent to a method for reducing weight gain (both for
therapeutic and cosmetic purposes) comprising the administration of
said strain, said components, metabolites or secreted molecules, or
said composition to a subject.
[0101] Other alterations of lipid and glucose metabolism, wherein
the immune system may also be affected, such as, but not limited
to, diabetes mellitus type 2 and gestational diabetes, dyslipidemia
(preferably hyperlipidemia and hypercholesterolemia),
cardiovascular pathologies, hypertension, fatty liver (preferably
non-alcoholic fatty liver or hepatic steatosis, nonalcoholic
steatohepatitis, cirrhosis or hepatitis), metabolic syndrome,
cancer, infections, etc. can occur in both normal-weight subjects
and subjects with excess weight or obesity.
[0102] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used to decrease the growth and
differentiation of adipose tissue in subjects with obesity or
excess weight and at an earlier stage of excess weight or obesity,
and, therefore relates to the use in the prevention and/or
treatment of adipocyte hypertrophy. As demonstrated in example 3
and in FIG. 3, the strain object of the invention reduces the size
of adipocytes, the increase (hypertrophy) of which at certain
stages of life (particularly in childhood and adolescence)
especially favors the development of excess weight and obesity in
adulthood and other associated complications such as insulin
resistance. In particular, administration of the CECT 7771 strain
to obese animals gives rise to an increase in the number of small
adipocytes (Example 3, FIG. 3).
[0103] In another example of use in medicine, a strain of the
invention, or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used in the treatment and/or
prevention of hepatic steatosis or fatty liver. As demonstrated
herein, administration of the B. uniformis CECT 7771 strain to both
animal obesity models and control animals (non-obese) produces a
reduction in the number of hepatocytes with high accumulation of
fat (Example 3, FIG. 2). Overall, this means that the strain of the
invention reduces fat accumulation in the liver.
[0104] The present invention also relates to the prevention and/or
treatment of pathologies related to the aggravation of hepatic
steatosis, such as, but not limited to, non-alcoholic hepatitis,
steatohepatitis, fibrosis, cirrhosis, end stage liver disease or
hepatocellular carcinoma. A strain of the species B. uniformis or
the strain of the invention can be used for these or other
pathologies accompanied by lipid accumulation in the liver and
inflammation, which may be associated with obesity or excess weight
or be a consequence of other alterations. These include, for
example but not limited to, nutritional alterations (for example,
but not limited to, malabsorption, protein-calorie malnutrition or
parenteral nutrition); inherited or non-inherited metabolic
alterations (for example, but not limited to, diabetes mellitus
type 2, abetalipoproteinemia, or systemic carnitine deficiency);
diseases caused by drug (for example, but not limited to,
corticosteroids or ibuprofen) or toxic (for example, but not
limited to, alcohol) exposure, chronic or acute hepatitis due to
infection, cirrhosis, fibrosis, end stage liver disease; hepatic
carcinoma or alterations of the pituitary gland. In particular,
steatosis affects approximately 50% of patients with type 2
diabetes mellitus.
[0105] In another example of use in medicine, a strain of the
invention, the cellular components, metabolites, secreted molecules
or any combination thereof, of the invention; a composition of the
invention, is used in the prevention and/or treatment of a disease
caused by changes in blood lipid levels (for example, dyslipidemia)
and preferably in blood triglyceride and/or cholesterol levels, due
to which it is used to normalise said levels. Preferably the drug
or nutritional composition is used to treat dyslipemia (synonymous
with dyslipidemia). Preferably dyslipiedemia is
hypertriglyceridemia and/or hypercholesterolemia. Dyslipidemia is a
pathological condition whose only common element is an altered
metabolism of lipids, with the consequent alteration of blood lipid
and lipoprotein levels. Dyslipidemia may or may not be associated
with obesity and the intake of high-fat diets and increased fat
absorption. In turn, these changes are associated with an increased
risk of cardiovascular disease, hypertension and diabetes, among
other pathologies. The strain of the invention reduces the
absorption of lipids and blood triglyceride and cholesterol levels,
proving effective in the applications described, as demonstrated in
Example 3, Table 2.
[0106] In another example of use in medicine, the strain of the
invention; or the cellular components, metabolites, secreted
molecules, or any combination thereof; or the composition of the
invention, is used to reduce the amount of absorved lipids from the
diet with respect to an untreated control. As shown in Example 3,
the strain of the invention reduces the number of fat micelles that
form chylomicra in intestinal enterocytes, i.e. it reduces the
amount of dietary fat that is absorbed by more than 80% (Example 3,
FIG. 4). Chylomicra are the way whereby dietary lipids are packaged
and transported from the intestine to the lymphatic system and
bloodstream to be used by the peripheral tissues, and the mechanism
wherethrough the strain administered would limit its absorption and
accumulation in the body. The absorption of dietary fat, in
addition to leading to excess weight and/or obesity by causing an
increase in accumulation in adipose tissue, may be the cause of
other pathologies without causing obesity, such as, without
limiting the scope of the invention, atherosclerosis, which is
characterised by a thickening of the tunica intima of an artery
with plaques where the fat is embedded, and dyslipidemia,
characterised by alterations in the plasma concentrations of lipids
(triglycerides and/or cholesterol and associated lipoproteins);
pathologies associated with increased cardiovascular risk; or other
alterations derived from the lipid-glucose metabolism ratio (for
example, but not limited to, insulin resistance or diabetes).
[0107] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules, or any combination thereof, of the invention; or a
composition of the invention, is used in the prevention and/or
treatment of a cardiovascular disease. Cardiovascular diseases are
those that affect the heart and blood vessels, including
atherosclerosis, aneurysm, angina, stroke, cerebrovascular disease,
congestive heart failure, coronary artery disease, acute myocardial
infarction and peripheral vascular disease. Chronic inflammation
and altered lipid metabolism (dyslipidemia) and glucose against
which a Bacteroides uniformis strain, and preferably the strain of
the invention, B. uniformis CECT 7771, is effective, dyslipidemia
(hypercholesterolemia and hypertriglyceridemia), insulin resistance
and diabetes, increased body fat or adipocyte hypertrophy, are risk
factors for cardiovascular diseases and, therefore, their treatment
and prevention may avoid the development of this other group of
pathologies.
[0108] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules, or any combination thereof, of the invention; or a
composition of the invention, is used in the prevention and/or
treatment a disease caused by increased blood glucose levels, with
respect to a control, and therefore is used to decrease the
concentration of blood glucose levels (hyperglycemia) with respect
to an untreated subject. This reduction in the concentration of
glucose is produced in parallel with the reduction in insulin
concentration and the reduction in the insulin resistance index,
demonstrating that it improves insulin sensitivity and can
therefore be used to treat or prevent metabolism alterations caused
by insulin resistance and reduced insulin production.
[0109] The increase in blood glucose levels (hyperglycemia) can be
diet-induced or arise from the development of insulin resistance
(subjects who produce sufficient insulin but whose body fails to
respond normally) or from the lack of insulin synthesis, with or
without obesity, due to other metabolic alterations or drug
interactions. Example 3 and Table 2 of the present invention
provide experimental support to this preferred embodiment. The term
"disease caused by increased blood glucose levels" relates to a
health alteration caused by higher blood glucose levels than would
be expected in a healthy individual with normal glucose levels,
i.e. approximately 72-110 mg/dl or 4-7 mmol/l (fasting), or
approximately <180 mg/dl (or 10 mmol/l) when measured 1.5 hours
after meals. Said values are approximate average values, as the
variation experienced and individual condition of each subject must
be taken into account. The disease caused or associated with higher
blood glucose levels is selected from the list comprising, but not
limited to, neuropathy (nerve damage in limbs and/or organs);
retinopathy (retinal damage in eyes), nephropathy (kidney damage
that may cause renal failure), cardiovascular diseases (myocardial
infarction); cerebrovascular disease (for example, cerebral
thrombosis).
[0110] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used in the prevention and/or
treatment of insulin resistance and/or diabetes (preferably
gestational diabetes or type 2 diabetes mellitus). An example of
more preferred use relates to the prevention and/or treatment of
gestational diabetes or type 2 diabetes mellitus, a pathology
associated, but not necessarily, with excess weight and/or
obesity.
[0111] Type 2 diabetes mellitus is characterised by a relative
deficit in insulin production and sensitivity in tissues and, thus,
poor use of peripheral glucose. Type 2 diabetes mellitus accounts
for 80%-90% of all diabetic patients. It often develops in adult
life stages, and is very frequently associated with obesity.
Several drugs and other causes can, however, cause this type of
diabetes. For example, diabetes associated with prolonged use of
steroids is very frequent, often associated with untreated
hemochromatosis, and gestational diabetes not always associated
with obesity.
[0112] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used in the prevention and/or
treatment of metabolic syndrome. The term "metabolic syndrome"
refers to the set of metabolic alterations that jointly increase
the risk of diabetes and cardiovascular disease, including the
combination of obesity, dyslipidemia (for example, triglycerides
and hipecolesterolemia) and hyperglycaemia. As demonstrated in
previous examples (Example 3, Table 2), the strain of the invention
is useful in the simultaneous prevention and treatment of these
alterations and, therefore, of metabolic syndrome.
[0113] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used in the prevention and/or
treatment of hypertension. Hypertension is caused by changes in
blood flow due to dysfunction of the inner layer of blood vessels.
The factors that contribute to arterial hypertension include
obesity, insulin resistance (insulin does not exert its vasodilator
effect correctly), and dyslipidemia and chronic inflammation, which
favour the deposition of lipids in the arteries and infiltration of
inflammatory cells that cause vasoconstriction and, ultimately,
atherosclerotic plaques. This invention demonstrates that a
Bacteroides uniformis strain and preferably the strain of the
invention, B. uniformis CECT7771, improves all these alterations
and, therefore, may help prevent and treat the causes of
hypertension.
[0114] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof of the invention; or a
composition of the invention, is used for improving the function of
the immune system and, in particular, in the prevention and/or
treatment of an inflammation-associated disease caused by an
increased production of pro-inflammatory molecules and a reduction
in anti- inflammatory molecules with respect to a control. In this
regard, experimental data are shown in Example 4 and Table 3.
Examples of pro-inflammatory proteins are, but not limited to,
cytokines and chemokines and adipokines. Preferably, the
pro-inflammatory proteins are selected from the list comprising,
IL-1, IL-6, IL-8, IL-12, IL-16, C-reactive protein, TNF-.alpha. or
MCP1 and leptin, or any combination thereof. More preferably, the
pro-inflammatory proteins are selected from the list comprising
TNF-.alpha. and leptin or any combination thereof. Examples of
anti-inflammatory proteins that can reduce pro-inflammatory
proteins include, but not limited to, IL-10 cytokine.
[0115] The term "disease associated with an increased production of
pro-inflammatory proteins" relates to diseases caused by at least
the production of a protein involved in inflammation
(pro-inflammatory) of various types of tissues. Some of the
diseases associated with increased production of pro-inflammatory
proteins are also associated with excess weight and/or obesity,
such as for example, but not limited to, type-2 diabetes,
gestational diabetes, metabolic syndrome, fatty liver,
non-alcoholic hepatitis, hypertension, dyslipemia, cardiovascular
diseases, steatohepatitis or cancer. Other diseases associated with
the increased production of pro-inflammatory proteins are not
associated with excess weight and/or obesity, or may occur in the
absence of obesity, such as for example, but not limited to, the
aforementioned diseases (for example, diabetes) and others such as
allergic inflammation.
[0116] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used to reduce inflammation of
peripheral tissues, preferably of adipose and/or pancreatic
tissue.
[0117] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used in the prevention and/or
treatment of a disease associated with decreased innate and
adaptive immune response, with respect to that of the control
subjects. The term "associated with decreased innate and adaptive
immune response" relates to diseases or physiological situations
characterised by immunosuppression of the function of the innate
and adaptive immune system, which may lead to a higher
susceptibility to develop certain pathologies such as infections.
This disease associated with a decreased innate and adaptive immune
response is preferably disease excess weight, obesity and
associated disorders that involve an alteration of these immune
functions. In this regard, experimental data are shown in Example 4
(FIGS. 5 and 6).
[0118] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used in the prevention and/or
treatment of an infection or to enhance vaccination response and,
therefore, the degree of protection of the subject against this
antigen.
[0119] In another example of use in medicine, a strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or a
composition of the invention, is used to restore the composition of
the intestinal microbiota and, preferably, to reduce the
concentration of potential pathogens such as enterobacteria in the
intestinal contents of a subject, with respect to an untreated
control. In an even more preferred example, the restoration of the
composition of the intestinal microbiota, or a reduction in the
concentration of enterobacteria in intestinal contents is carried
out in a subject with excess weight, obesity or any disease
associated therewith.
[0120] The restoration of the intestinal microbiota can be based,
for example but not limited to, on the increase in the abundance of
the genus Bifidobacterium and on the decrease in the abundance of
bacteria of the Enterobaceriaceae family, whose concentrations are
altered in obesity. This fact also implies a reduction in the risk
of enterobacteria infections and a reduction in the
pro-inflammatory signals that may be transmitted from the intestine
to peripheral tissues (for example, liver) that may be affected in
obese or non-obese subjects by various pathologies. As demonstrated
in Example 4 and in FIG. 7, the administration of the strain object
of the invention reduces the capacity of the microbiota (faeces) of
obese animals to stimulate the synthesis of the inflammatory
cytokine TNF-.alpha. in macrophages and dendritic cells. The
inflammatory effect that causes alterations of the intestinal
microbiota in obese subjects has been associated with insulin
resistance, metabolic endotoxemia, hepatic steatosis, and
alteration of the intestinal barrier function, which could be
attenuated by the strain of the invention. Moreover, the strain of
the invention also increases the number of Bacteroides spp. and
Bifidobacterium spp in normal-weight subjects and can be used to
increase or restore these microbial populations in the intestine,
which may be altered due to conditions other than obesity and
excess weight. Therefore, the CECT 7771 strain is additionally
applicable to the prevention and treatment of diseases associated
with alterations in the composition of the intestinal
microbiota.
[0121] According to the present description, the strain of the
invention; or the cellular components, metabolites, secreted
molecules or any combination thereof, of the invention; or the
composition of the invention, for use in the treatment and/or
prevention of various diseases or metabolic alterations, for
improving the function of the immune system or restoring the
composition of the intestinal microbiota or reducing the
concentration of enterobacteria, can obviously be understood as a
method of treatment and/or prevention of such diseases or
alterations, or a method for improving the immune function, or a
method for restoring the composition of the intestinal microbiota,
or a method for reducing the concentration of enterobacteria, which
comprises administering a therapeutically effective amount of such
a strain; or of said cell components, metabolites, secreted
molecules, or any combination thereof, or of said composition, to a
subject. Also, the present invention also covers the use of said
strain, or of said cell components, metabolites, secreted
molecules, or any combination thereof, for the manufacture of a
nutritional composition, a pharmaceutical composition or a drug (as
previously described), for the treatment and/or prevention of such
diseases or metabolic alterations, for improving the function of
the immune system or for restoring the composition of the
intestinal microbiota or reducing the concentration of
enterobacteria.
[0122] Another aspect of the present invention relates to a method
of improving the bodily appearance of a subject, which comprises
administering to said subject a strain of the invention; or the
cellular component, metabolite, secreted molecule or any
combination thereof of the invention; or a nutritional composition
of the invention, for reducing body weight gain or contribute to
weight loss, for cosmetic purposes. Within the scope of the present
invention, it is understood that the subject to whom the strain of
the invention or the cell component, metabolite, secreted molecule
or any combination thereof of the invention; or the nutritional
composition of the invention, is administered for cosmetic purposes
is a healthy subject.
[0123] Throughout the description and claims the word "comprises"
and its variants are not intended to exclude other technical
features, additives, components or steps. For persons skilled in
the art, other objects, advantages and features of the invention
will become apparent partly from the description and partly from
the practice of the invention. The following figures and examples
are provided by way of illustration, and are not intended to be
limiting of the present invention.
DESCRIPTION OF THE DRAWINGS
[0124] FIG. 1 shows the differential effect of strains of different
species of the genus Bacteroides and of the species B. Uniformis on
the accumulation of triglycerides (A) and cholesterol (B) in the
hepatocytes and in the use of glucose (C). Results are expressed as
means and their standard deviation. *Statistically significant
differences with respect to the B. uniformis CECT 7771 strain
(equivalent to CAY1) applying ANOVA and Tukey's Test
(p<0.05).
[0125] FIG. 2 shows the effect of administering the B. uniformis
CECT 7771 strain (5.times.10.sup.8 cfu/day) to obese C57BL/6 mice
(n=6/group) for 7 weeks on the development of steatosis
(accumulation of lipids in the liver). Panel A: SD, control animals
with a standard diet; SD+B, control animals with a standard diet+B.
uniformis CECT 7771; HFD, high-fat diet, HFD+B, with a high-fat
diet+B. uniformis CECT 7771. Results are expressed as means and
standard deviations and the statistically significant differences
were established applying the Mann-Whitney U Test (p<0.05).
Panels B-E. The number of fatty hepatocytes in a histology section
of liver tissue stained with hematoxylin and eosin, in accordance
with the degree of accumulation of fat in the cell, is shown in
ascending order (0-3). Panel B: SD; Panel C: SD+B. uniformis CECT
7771; Panel D: HFD; Panel E: HFD+.beta.. uniformis CECT 7771.
[0126] FIG. 3 shows the effect of administering the B. uniformis
CECT 7771 strain (5.times.10.sup.8 cfu/day) to obese C57BL/6 mice
(n=6/group) for 7 weeks on adipocyte development, classified by
size intervals. Panel A: SD, control animals with a standard diet,
SD+B, control animals with a standard diet+B. uniformis CECT 7771,
HFD, high-fat diet, HFD+B, with a high-fat diet+B. uniformis CECT
7771. Results are expressed as means and standard deviations and
the statistically significant differences were established applying
the Mann-Whitney U Test (p<0.05). Panels B-E. The size of the
adipocytes in a histology section of epididymal tissue stained with
hematoxylin and eosin is shown. Panel B: SD; Panel C: SD+B.
uniformis CECT 7771; Panel D: HFD; Panel E: HFD+.beta.. uniformis
CECT 7771.
[0127] FIG. 4 shows the effect of administering the B. uniformis
CECT 7771 strain (5.times.10.sup.8 cfu/day) to obese C57BL/6 mice
(n=6/group) for 7 weeks on the number of fat micelles accumulated
in the enterocytes in histology sections stained with hematoxylin
and eosin. SD, control animals with a standard diet, SD+B, control
animals with a standard diet+B. uniformis CECT 7771, HFD, high-fat
diet, HFD+B, with a high-fat diet+B. uniformis CECT 7771. Results
are expressed as means and standard deviations. Statistically
significant differences were established applying the Mann-Whitney
U Test (p<0.05).
[0128] FIG. 5. Panel A shows the effect of administering the B.
uniformis CECT 7771 strain (5.times.10.sup.8 cfu/day) to obese
C57BL/6 mice (n=6/group) for 7 weeks on the role of macrophages
stimulated with lipopolysaccharide (LPS) in the synthesis of
inflammatory cytokines (TNF-.alpha.) and anti-inflammatory
cytokines (IL-10). Panel B shows the effect of administering the
strain in the respiratory burst in macrophage phagocytosis. SD,
control animals with a standard diet, SD+B, control animals with a
standard diet+B. uniformis CECT 7771, HFD, high-fat diet, HFD+B,
with a high-fat diet+B. uniformis CECT 7771. Results are expressed
as means and standard deviations. Statistically significant
differences were established applying the Mann-Whitney U Test
(p<0.05).
[0129] FIG. 6. A. Shows the effect of administering the B.
uniformis CECT 7771 strain (5.times.10.sup.8 cfu/day) to obese
C57BL/6 mice (n=6/group) for 7 weeks on the function of dendritic
cells stimulated with lipopolysaccharide (LPS) in the synthesis of
inflammatory cytokines (TNF-a) and anti-inflammatory cytokines
(IL-10). B. Shows the effect of administration of the strain on the
interaction between T cells and dendritic cells and their
proliferation ability. CD4 +T lymphocytes (TL) were incubated with
mature dendritic cells (DC) from different experimental groups of
obese C57BL/6 mice (n=6/group) that were administered the strain
(10.sup.8 cfu/day) for 7 weeks. The cell ratio (TL/DC) in the
mixture was 1:1, 1:2 and 1:4. SD, control animals with a standard
diet, SD+B, control animals with a standard diet+B. uniformis CECT
7771, HFD, high-fat diet, HFD+B, with a high-fat diet+B. uniformis
CECT 7771. Results are expressed as means and standard deviations.
Statistically significant differences were established applying the
Mann-Whitney U Test (p<0.05).
[0130] FIG. 7. A. Shows the effect of administering the B.
uniformis CECT 7771 strain (10.sup.8 cfu/day) to obese C57BL/6 mice
(n=6/group) and controls for 7 weeks on the ability of intestinal
microbiota (faeces) to stimulate the synthesis of inflammatory
cytokines (TNF-a) and anti-inflammatory cytokines (IL-10) in
macrophages (A) or dendritic cells (B) of control mice. SD, control
animals with a standard diet, SD+B, control animals with a standard
diet+B. uniformis CECT 7771, HFD, high-fat diet, HFD+B, with a
high-fat diet+B. uniformis CECT 7771. Results are expressed as
means and standard deviations. Statistically significant
differences were established applying the Mann-Whitney U Test
(p<0.05).
EXAMPLES
[0131] The invention is illustrated below by tests conducted by the
inventors. The following specific examples provided herein serve to
illustrate the nature of the present invention. These examples are
included solely for illustrative purposes and must be interpreted
as limitations to the invention claimed herein. Therefore, the
described examples are not intended to limit the scope thereof.
Example 1
Isolation and Identification of the B. uniformis CECT 7771
Strain.
[0132] We proceeded to the isolation of strains of the genus
Bacteroides from faeces of healthy infants who had not been
subjected to treatments with antibiotics for at least the month
prior to sampling. The samples were kept at 4.degree. C. and
analysed within two hours after collection. Two grammes of each
were diluted in 10 mM phosphate buffer containing a concentration
of 130 mM NaCl (PBS) and homogenised in a Stomacher 400 Lab Blender
(Seward Medical, London, UK) for 3 minutes and were diluted in
peptone water. Aliquots of 0.1 ml of various decimal dilutions were
inoculated onto Schaedler agar (Scharlau, Barcelona, Spain)
supplemented with kanamycin (100 mg/L), vancomycin (7.5 mg/L) and
vitamin K (0.5 mg/L), at 37.degree. C. under anaerobic conditions.
After incubation for 48 h at 37.degree. C. under anaerobic
conditions (AnaeroGen, Oxoid, UK), isolated colonies were selected
and their morphology was confirmed under Gram staining. The
identity of the isolates was confirmed by sequencing of the 16S
ribosomal RNA gene from total DNA. The sequenced fragment was
amplified using primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3': SEQ ID NO:
2) and 1401 r (5'-CGGTGTGTACAAGACCC-3': SEQ ID NO: 3) and purified
using the GFX.TM. PCR commercial system (Amershan, Bioscience, UK).
For the sequencing, primers 530F (5'-GTGCCAGCAGCCGCGG-3': SEQ ID
NO: 4) and U-968f (5'-AACGCGAAGAACCTTAC-3': SEQ ID NO: 5) were also
used, in accordance with the procedures described by other authors
(Gerhard et al., 2001. Appl. Environ. Microbio/, 67: 504-513;
Satokari et ai, 2001. Appl. Environ. Microbiol. 67, 504-513; Favier
of ai, 2002. Appl. Environ. Microbio!, 68: 219-22). Sequencing was
performed using an ABI 3700 {Applied Biosystem, Foster City,
Calif.) automatic DNA sequencer.
[0133] Sequence 1,335 kb of the 16S ribosomal RNA gene of the CECT
7771 strain is SEQ ID NO: 1. The search for more closely related
sequences was performed in the GenBank database using the BLAST
(Altschul et al algorithm, J. Mol 1990 Biol, 215: 403-410)
algorithm.
[0134] Upon comparing SEQ ID NO: 1 with the most similar sequences,
an identity of 99% was obtained with respect to other strains of
the species B. uniformis (GeneBank access no. AB0501). These
results indicate that the strain of the present invention may very
likely belong to said species.
[0135] The strain of the invention was molecularly typed using RAPD
analysis using the M13 primer (5'-GAGGGTGGCGGTTCT-3': SEQ ID NO: 6)
and according to the previously described methodology (Antonie Van
Leeuwenhoek, 2010, 98(1):85-92). The profiles of the randomly
amplified DNA fragments showed that the strain object of the
invention (.beta.. uniformis CECT 7771) is different from other
strains of the same species.
Example 2
Selection of the B. uniformis CECT 7771 Strain in Accordance With
its Capability to Modulate, In Vitro, the Macrophage Response
Related to Chronic Low-Grade Inflammation Associated With Obesity
and Acccording to the Ability to Modify Lipid Accumulation and use
of Glucose By Hepatocytes
2.1. Evaluation of the Effect of Bacterial Strains on
Macrophages
[0136] Bacterial strains and culture conditions. The following
strains of the genus Bacteroides were used: B. uniformis CAY1 (CECT
7771), B. uniformis CBD2, B. distasonis CAY3, B. fragilis SX3, B.
fivegoldi SX2, B. dorei SS1, B. ovatus SV2, B. thetaiotaomicron
SAC4 and B. caccae SV3. The strains were inoculated into 10 ml of
Brain Heart broth (BH; Schartau Chemie S.A., Barcelona, Spain)
containing 0.05% cysteine (BH), at 1% with 24 hours of culture were
incubated for 22 h at 37.degree. C. in anaerobiosis. (AnaeroGen;
Oxoid, Basingstoke, UK). The cells were collected by centrifugation
(6,000 g, 15 minutes), washed twice in PBS (10 mM sodium phosphate,
130 mM sodium chloride, pH 7.4), and re-suspended in PBS containing
20% glycerol. Aliquots of these suspensions were frozen with liquid
nitrogen and stored at -80.degree. C. The number of viable cells
after the freezing-thawing cycle was determined by counting on
Schaedler Agar agar plates (Schartau, Barcelona, Spain)
supplemented with kanamycin (100 mg/L), vancomycin (7.5 mg/L) and
vitamin K (0.5 mg/L). Viability was over 90% in all cases. Each
aliquot was used for a single assay. In order to evaluate the
effects of dead bacteria, some of the aliquots were cold-activated
(three freeze-thaw cycles -20.degree. C.) and heat inactivated (30
minutes at 80.degree. C.). The pH values of the supernatants
obtained were adjusted to 7.2 with NaOH and sterilised by
filtration (0.22-.mu.m pore size, Millipore, Bedford, Mass.) to
eliminate the possible presence of viable cells. In order to
evaluate the effects of other metabolites and compounds secreted
into the culture medium, aliquots of the cell-free supernatants
were conserved at -80.degree. C. until use thereof. Likewise, the
effect of incorporating probiotics into the culture medium by
replacing part of the glucose of the BH medium with inulin (Inulin
L-Light and Co LTD, Colnbrook, UK) was evaluated, its final
concentrations being 0.5 g/l and 1.5 g/l, respectively. Under these
conditions, the cells and supernatants were obtained from each
strain and were used to perform the same macrophage and hepatocyte
stimulation assays.
[0137] Macrophage culture and stimulation. Cells of the RAW 264.7
murine macrophage cell line were grown in Dulbecco 's Modified
Eagle Medium (DMEM, Sigma, USA), supplemented with 10% of fetal
bovine serum (Gibco, Barcelona, Spain), streptomycin (100 .mu.g /ml
, Sigma) and penicillin (100 U/ml, Sigma). In order to conduct the
stimulation experiments, cells were incubated at a concentration of
10.sup.5 ppm in Polystyrene Flat Bottom Plates with 24 Wells
(Corning, Madrid, Spain) at 37.degree. C., at 5% of CO.sub.2.
Suspensions of live and dead bacteria equivalent to
1.times.10.sup.6 colony-forming units (cfu)/ml and supernatant
volumes of 30 .mu.l were used as a stimulus. Lipopolysaccharide
(LPS) purified from Salmonella enterica serotype Typhimurium (Sigma
Chemical Co., Madrid, Spain) at a concentration of 1 .mu.g/ml was
used as a positive control. Cytokine production in non-stimulated
cells was tested as a negative control. Each type of stimulus was
tested in triplicate in two independent experiments. The culture
supernatants were collected by centrifugation, fractioned and
stored in aliquots at -20.degree. C. until the detection of
cytokines and chemokines. Determination of cytokines and
chemokines. The concentrations of cytokines (TNF-.alpha. and IL-10)
of the supernatants of the macrophage cultures was measured using
ELISA kits (BD Biosciences, San Diego, Calif.) following the
company's instructions.
TABLE-US-00001 TABLE 1 Example the effect of stimulation with
viable cells of different species and strains of the genus
Bacteroides in the synthesis of pro-inflammatory and
anti-inflammatory cytokines by macrophages. Cytokine concentration
Bacteroides strains TNF-.alpha. (pg/ml) IL-10 (pg/ml)
TNF-.alpha./IL-10 DEMEN (control) 491.2 (112.1).sup.a 97.2
(10.8).sup.a 5.00 (0.60).sup.a,b' LPS (1 mg/ml) 1,425.4
(77.6).sup.b 162.3 (37.6).sup.a 9.04 (1.61).sup.b,a' B. dorei SS1
3,765.5 (150.0).sup.b,,a' 215.8 (12.5).sup.b,b' 17.53
(1.71).sup.b,b' B. ovatus SV2 4,515.7 (211.3).sup.b,b' 271.5
(8.1).sup.b,b' 16.62 (0.28).sup.b,b' B. distasonis CAY3 4,462.4
(173.9).sup.b,b' 215.8 (9.7).sup.b,b' 20.74 (1.74).sup.b,b' B.
uniformis CECT 7771 2,998.4 (50.4).sup.b,a' 341.3 (13.5).sup.b,a'
9.91 (3.86).sup.b,a' B. uniformis CBD2 2,640.5 (80.2).sup.b,a'
105.4 (10.5).sup.a,b' 31.00 (16.55).sup.b,b' B. thetaiotaomicron
SAC4 2,931.2 (464.5).sup.b,a' 109.2 (3.0).sup.a,,b' 26.95
(5.01).sup.b,b' B. fragilis SX3 6,657.3 (278.3).sup.b,b' 81.2
(14.6).sup.a,a',b'' 219.17 (41.39).sup.b,b' B. caccae SV3 11,622.0
(818.3).sup.b,b' 171.7 (12.9).sup.b,b' 67.69 (0.32).sup.b,b' B.
finegoldii SX2 6,535.8 (62.2).sup.b,b' 83.5 (17.4).sup.a,b' 80.75
(16.07).sup.b,b' *Results are expressed as means and their standard
deviation (sd, values n parentheses). Statistically significant
differences were established applying Tukey's Test at a value of P
< 0.050. Different letters in the same column indicate
significant differences between the means in relation to the
control value (a-b) or to the value of the cells stimulated with B.
uniformis CECT 7771 (a'-b'). The data corresponding to the strain
of the invention are underlined.
[0138] The strain object of the invention is selected from among
others of the same genus due to being one of the strains that
induced the lowest concentrations of pro-inflammatory molecules
(TNF-.alpha.) involved in the state of chronic inflammation
associated with obesity that causes resistance to the effects of
insulin and leptin (Table 1). The strain of the invention was also
selected for inducing the synthesis of the highest concentration of
the anti-inflammatory and regulatory IL-10 cytokine by macrophages,
which can help reduce inflammation in the context of obesity (Table
1). Other strains of the same species as B. uniformis CBD2 induced
a significantly higher proportion of the TNF-.alpha./IL-10 factor
object by the patent (CECT 7771), indicating that the
pro-inflammatory/anti-inflammatory cytokine balance induced by the
latter is more favorable than that induced by the other strains.
The immunological properties of the selected bacteria are not
common to all the intestinal bacteria of the same genus
(Bacteroides) or species (.beta.. uniformis) and, therefore, make
it particularly suitable for use in the treatment and prevention of
excess weight, obesity and metabolic alterations, associated or
not, and related to inflammation.
2.2. Evaluation of the Effect of the Bacterial Strains in
Hepatocytes
[0139] Bacterial strains and culture conditions. The following
strains of the genus Bacteroides are used: B. uniformis CAY1 (CECT
7771), B. uniformis CBD2, B. distasonis CAY3, B. fragilis SX3, B.
fivegoldi SX2, B. dorei SS1 and B. ovatus SV2. The strains were
grown in BH broth containing 0.05% cysteine and the cell
suspensions and culture supernatants were collected and stored
until use as indicated in section 2.1.
[0140] Cultivation of HepG2 cells. Cultures of liver-derived human
cells belonging to the HepG2 cell line, widely used as a hepatic
model, were used. The cells were cultivated in DMEM supplemented
with fetal calf serum (10%, v/v) (FBS), penicillin (100 units/rip
and streptomycin (100 .mu.g ml). Cultures were maintained
(37.degree. C.) in a humidified 5% CO.sub.2 atmosphere with medium
change every 48 h until reaching 70-80% of convergence, at which
time they were used for the studies. Prior to its addition to the
cell culture, a mixture of oleic acid (18: 1TO9, Sigma-Aldrich)
(AO) was prepared and albumin (BSA) (A2153, Sigma-Aldrich), under
aseptic conditions. An aliquot (5 g) of BSA dissolved in Dulbecco's
modified medium (protein-free) (DMEM), used for cell culture,
tempered to 40.degree. C. The pattern of oleic acid was added to
this solution, drop-wise and under constant stirring, a final
concentration of 0.8 M. Based on the convergence cultures, in order
to carry out and standardise the results of the various studies,
the cells were re-suspended in DMEM and seeded in multiple-well
plates (.times.24) at a density of 1.times.10.sup.5 cells/well. The
cells were incubated (37.degree. C./5% CO2) under these conditions
for 24 hours. After this period, the cultures were washed
(.times.2) with buffered saline solution (PBS) and 1 ml of DMEM
(not supplemented with FBS) was added, at a concentration of 2 mM
of the AO/BSA mixture in the presence or absence of cell
suspensions (10.sup.8 colony-forming units/rip of the different
bacterial strains indicated in the preceding section. The cultures
were returned to the incubator for an additional 24 hours. Control
cultures incubated with DMEM (not supplemented with FCS) but
without AO cultures were included in all the studies.
[0141] Effect of various species and strains of Bacteroides in the
accumulation of triglycerides and cholesterol in HepG2 cultures.
The quantification of the total concentration (nmol/L) of
triglycerides (TG) and cholesterol (CHOL) in HepG2 cultures exposed
(24 hours) to the AO/BSA mixture (2 mM in DMEM) in the presence or
not of the cell suspensions of the different bacterial strains
described was carried out using commercial enzymatic kits
(Triglycerides and Cholesterol Liquid, Quimica Analitica Aplicada,
S.A., Spain). Quantification of TG and CHOL was conducted in cell
homogenates with a solution (300 .mu.i) of PBS (pH 7)/0.1%
Triton-X100, using the pattern provided in the corresponding
commercial kit.
[0142] The strain object of the invention was selected from among
others of the same genus and of the same species for their ability
to reduce triglyceride and cholesterol accumulation in hepatocytes
(FIG. 1). The B. uniformis CECT 7771 strain reduced the
accumulation of triglycerides in comparison with other strains of
different species such as B. dorei, B. fivegoldi, B. fragilis and
B. ovatus and of the same species such as B. uniformis CBD2 (FIG.
1A). The B. uniformis CECT 7771 strain also reduced the
accumulation of cholesterol in comparison with other strains of
different species such as B. distasonis, B. dorei, B. fivegoldi, B.
fragilis and B. ovatus (FIG. 1B).
[0143] Effect of Various Species and Strains of Bacteroides on
Glucose Use and Insulin Resistance
[0144] The influence of different bacterial strains on insulin
resistance induced by treatment with oleic acid was evaluated by
incubating (4 hours) HepG2 cultures exposed for 24 hours to 2 mM of
AO/BSA in DMEM with a glucose solution (100 .mu.g/mL) supplemented
with insulin (10 ng/mL) in the presence or absence of different
bacterial suspensions. The potential uptake of glucose by the
bacteria was considered by incubating these means without addition
to cell cultures. The influence of the bacterial suspensions on
insulin resistance was evaluated by quantifying glucose uptake and
intracellular concentration thereof in hepatocytes using a
commercial enzymatic kit (Glucose Liquid, Quimica Analitica
Aplicada, S.A., Spain). In FIG. 1C it can be observed that
hepatocytes exposed to oleic acid have a reduced ability to use
glucose even in the presence of insulin with respect to the
controls. However, the strain of the invention (.beta.. uniformis
CECT 7771) improved the ability of hepatocytes to use glucose and,
thus, their insulin sensitivity to a greater degree than other
strains of the same genus and species, due to which it was
considered the best candidate for the applications object of the
patent.
Example 3
Effect of Administration of the B. uniformis CECT 7771 Strain on
Biometric and Bionchemical Parameters, Absorption of Lipids in the
Intestine and Histology of Adipose Tissue and Liver
3.1. Animal Model of Obesity and Sampling.
[0145] Adult male C57BL-6 mice (6-8 weeks; Harlan Laboratorios)
were used. The animals were kept under controlled temperature
(23.degree. C.), with a 12 hour light/dark cycle in an atmosphere
with 40%-50% relative humidity.
[0146] Obesity was induced by feeding the mice a high-fat diet
(HFD) which provided 60% of energy in the form of lipids (60/Fat,
Harlan Laboratorios) at the expense of a reduction in
carbohydrates, for 7 weeks, while the non-obese mice were fed a
conventional diet that provided 12.4% of energy as lipids. The mice
had free access to water and diet. Weight was monitored weekly. The
experiments were conducted in accordance with the rules of the
Animal Ethics Committee.
[0147] The animals were randomly divided into four groups
(n=6/group): (1) controls that were fed a standard diet (SD),
controls that were administered the strain of the invention
(SD+strain), obese mice on being fed a high-fat diet (HFD) and
obese mice that were administered the strain CECT 7771
(HFD+strain). The strain was administered at a daily dose of
5.times.10.sup.8 cfu/day by intragastric catheter orally for 7
weeks. The bacterium is administered in the form of a nutritional
composition comprising 10% skimmed milk supplemented with the
bacteria at the indicated concentration of 5.times.10.sup.8 cfu per
each 100 .mu.l of composition. The nutritional composition was
administered to the SD-fed control groups and HFD-fed obese mice
without the bacteria as a placebo. After the treatment periods, the
animals were anaesthetised and sacrificed by cervical dislocation
and samples of adipose (epididymal) and hepatic tissue and blood
was drawn by cardiac puncture for analysis as described below.
3.2. Effects on the Liver and Adipose Tissue.
[0148] Samples of adipose (epididymal) and hepatic tissue were
washed with saline solution and fixed in 10% buffered formalin,
embedded in paraffin, cut into 4-5 .mu.m sections and stained with
hematoxylin eosin. The severity of the steatosis (lipid
accumulation in the liver) was determined by analysing ten fields
of each section set with bright-field microscope (Olympus),
according to the following scale: grade 1 (no steatosis); grade 2,
when hepatocyte fat occupied less than 33% of the cell; grade 3,
when hepatocyte fat occupied between 34%-66% of the cell; grade 4,
when hepatocyte fat occupied over 66% of the cell. Adipocyte size
was measured by image analysis using NIS-Elements BR 2.3 software,
evaluating at least 100 cells per experimental group and tissue
type.
[0149] The results indicate that the strain object of the invention
reduces the size of adipocytes in epididymal tissue, whose increase
(hypertrophy) at certain stages of life (childhood and adolescence)
favors the development of excess weight and obesity in adulthood is
associated with a positive imbalance between energy intake and
energy expenditure (Macia et al., 2006. Genes Nutr., 1: 189-212).
By contrast, the reduction in adipocyte size is related to the
decreased resistance to insulin and glucose concentrations (Varady
et al., 2009. Metabolism 58: 1096-101). In particular,
administration of the CECT 7771 strain to obese animals gives rise
to an increase in small adipocytes (<2000 .mu.m.sup.2), while in
obese animals which have not been administered the strain recorded
an increase in all large adipocytes (2000-7000 .mu.m.sup.2) and a
reduction in small adipocytes (<2000 .mu.m.sup.2) (Example 3,
FIG. 3). Histology sections of adipose tissue also show these
effects.
[0150] The increased size of the adipocytes is also related to the
increase in the uptake of fatty acids by the liver, giving rise to
hepatic steatosis and complications, so that the strain can also
help to avoid or ameliorate these alterations. Therefore, the B.
uniformis CECT 7771 strain reduces the size of adipocytes, i.e. it
is useful in the treatment of alterations in the development of
this type of cells leading to hypertrophy thereof which, maintained
over time, can lead to excess weight and obesity, in addition to
other diseases not necessarily associated with obesity.
[0151] The strain of the invention reduces the accumulation of fat
in the liver (steatosis) associated with the intake of high-fat
diets, obesity and various diseases such as non-alcoholic hepatitis
(Musso et al., 2010. Hepatology 52:
[0152] 79-104). Specifically, the strain produces a decrease in the
number of grade 2 and 3 hepatocytes, with maximum fat content, and
an increase in the number of grade 0 and 1 hepatocytes, with lower
fat content; however, in obese animals that were not administered
the strain, proportion of hepatocyte type is reversed,
predominating those with maximum fat content. In control animals,
the administration of the strain produces an increase in grade 0
hepatocytes (lean) and a reduction in the number of grade 1 and 2
hepatocytes. (Example 3, FIG. 2). Thus it is demonstrated that
administration of the strain reduces overall fat accumulation in
the liver, diet-induced or otherwise.
3.3. Effects on Biometric and Biochemical Parameters.
[0153] Total body weight was monitored weekly and final weight gain
was determined with respect to initial weight. Additionally, the
weight of adipose tissue (epididymal and perirenal) per 100 g body
weight was estimated after sacrifice. The glucose, triglyceride and
cholesterol levels were determined in serum samples obtained from
peripheral blood after sacrifice using colorimetric methods
(Quimica Clinica Aplicada, S.A., Amposta, Spain) and insulin
samples obtained using ELISA (BD Bioscience, San Diego, Calif.,
USA). Furthermore, the cholesterol and triglyceride levels were
determined in the lipids extracted from the liver after sacrifice
using the same methodology. In order to evaluate the postprandial
glycemic response, at 6 weeks of treatment and after fasting for 4
hours, the mice were also administered an oral dose of glucose (2
g/kg) and blood samples were taken at different times (15, 30, 60,
90 and 120 min), whereupon the changes in glucose concentration was
determined using test strips (Glucosa strips; Ascensia Esyfill,
Bayer, Tarrytown, N.Y., USA) and a glucometer (Ascensia VIGOR,
Bayer Tarrytown, N.Y., USA).
[0154] As shown in Table 2, administration of the strain of the
patent, B. uniformis CECT 7771, to obese animals reduces their
weight gain significantly after 7 weeks of intervention, indicating
that it is effective in the prevention and treatment of excess
weight and obesity.
TABLE-US-00002 TABLE 2 Biometric and metabolic parameters in mice
fed a high-fat or standard diet, supplemented or not with the B.
uniformis CECT 7771 strain. Experimental Groups SD HFD SD + B HFD +
B Parametro Mean sd Mean sd Mean sd Mean sd Biometric parameters
Weight gain (%) 24.21 3.34 36.19 1.55 23.61 3.17 30.33 0.92 Adipose
tissue (g)/100 g 0.06 0.04 0.15 0.03 0.03 0.02 0.14 0.04 Body
weight Serum parameters Cholesterol (mg/dl) 120.00 13.67 176.02
14.91 128.22 11.91 143.97 17.29 Tryglicerides (mg/dl) 130.31 11.56
156.99 27.47 129.77 13.94 118.21 10.04 Glucose (mg/dl) 219.81 26.41
229.47 13.83 372.41 13.50 233.52 30.62 Insulin (.mu.g/l) 0.57 0.47
1.59 0.09 0.69 0.05 0.92 0.14 Leptin (ng/ml) 8.07 1.12 18.28 4.28
6.80 1.23 12.98 3.24 Hepatic lipids Cholesterol (mg/g) 29.94 4.08
35.51 4.35 29.22 6.32 27.48 6.39 Tryglicerides (mg/g) 22.93 13.03
45.99 11.53 31.36 4.76 34.17 9.51 Analisis estadistico Value P
Value P Value P Parameter HFD vs SD SD + B vs SD HFD + B vs HFD
Biometric parameters Weight gain (%) 0.007* 0.890 0.005* Adipose
tissue (g)/100 g body weight 0.016* 0.150 0.423 Serum parameters
Cholesterol (mg/dl) <0.001* 0.222 0.003* Tryglicerides (mg/dl)
0.041* 0.937 0.004* Glucose (mg/dl) 0.001* 0.584 0.002* Insulin
(.mu.g/l) 0.018* 0.892 0.018* Leptin (ng/ml) <0.001* 0.048*
0.014* Hepatic lipids Cholesterol (mg/g) 0.029* 0.801 0.024*
Tryglicerides (mg/g) <0.001* 0.142 0.039* SD: standard diet
group (control) (n = 6) SD + B: group with DS and supplemented
orally with 5.0 .times. 10.sup.8 CFU/day of B uniformis CECT 7771
(n = 6), HFD: group with high-fat diet (n = 6), HFD + B: HFD group
and supplemented orally with 5.0 .times. 10.sup.8 CFU/day of B
uniformis for 7 weeks (n = 6). The biochemical parameters were
determined in plasma after the intervention. .sup.aTotal weight
gain was calculated at the end of the intervention and expressed in
relative values with respect to the initial weight of each mouse.
.sup.bThe relative weight of adipose tissue, including epididymal
and perirenal tissue, per each 100 g of body weight was calculated
after the intervention. The values of all the parameters are
expressed as means and their standard deviations. *The statistical
analysis was performed applying ANOVA and, subsequently, Tukey's
Test for normally distributed data or the Mann-Whitney U Test for
those without normal distribution. Significant differences were
established at a value of P < 0.050.
[0155] As shown in Table 2, the strain of the invention
administered in vivo also regulates glucose metabolism, reducing
its concentration in peripheral blood in obese animals; for
example, high levels of serum glucose 485.9 mg/dl detected in obese
mice tend to become normalised after administering the strain
object of the invention, achieving significantly lower levels of
233.5 mg/dl (P=0.002), in proportion to the reduction of insulin
levels (1.593 versus 0,920 .mu.g/l; P=0.018). The increase in
plasma glucose concentration is indicative of an alteration in
their metabolism and in insulin synthesis or response and can be
positively regulated by the strain object of the invention,
reducing the risk of developing insulin resistance and diabetes and
enhancng treatment. Furthermore, the HOMA (Homeostasis Model
Assessment) index was determined, which enables the estimation of
insulin resistance (a high index indicates low insulin sensitivity)
sensitivity) and the function of pancreatic beta cells. This index
was estimated based on fasting glucose and insulin levels using the
following equation HOMA=Insulin (.mu.g/l).times.Glucose
(mg/dl)/405. The HOMA index in obese subjects was 1, 91 1, while in
obese subjects treated with the strain it was 0.530, thereby
detecting a significant reduction that indicates the positive
effect of the strain on improving insulin sensitivity. Moreover,
the strain of the invention reduces postprandial glycemic response
after an oral dose of glucose, reducing the area under the glucose
curve in obese subjects, which also indicates an improvement in
glucose metabolism and insulin sensitivity. As shown in Table 2,
the strain object of the invention also reduces the hyperleptinemia
characteristic of diet-induced obesity, indicating an improvement
in its function in the regulation of lipid and glucose
metabolism.
[0156] The strain of the invention administered in vivo regulates
lipid metabolism, reducing, in particular, the concentration of
triglycerides and cholesterol in peripheral blood in obese animals.
Thus, the elevated serum triglyceride levels detected in obese mice
were significantly reduced upon administering the strain object of
the invention from 156.99 to 118.21 mg/dl (P=0.004) assuming a
reduction of 25%. Elevated concentrations of serum cholesterol
detected in obese mice were also significantly reduced upon
administering the strain of the invention from 176.02 to 143.97
mg/dl (P=0.003) assuming a reduction of 18%. Moreover, the strain
of the invention significantly reduces the accumulation of
cholesterol and triglycerides in the liver that may contribute to
hepatic steatosis.
3.4. Effects of the Administration of the B. uniformis CECT 7771
Strain on the Absorption of Dietary Lipids in the Intestine.
[0157] Following sacrifice, intestinal tissue samples were taken
and washed with saline solution and fixed in 10% buffered formalin,
embedded in paraffin, and cut into 4-5 .mu.m sections which were
stained with hematoxylin and eosin. The number of chylomicrons or
enterocyte fat micelles was determined by counting ten fields in
each section fixed with a bright-field microscope (Olympus) and
expressed in number of chylomicrons per enterocyte.
[0158] As can be seen in FIG. 4, the strain of the invention
reduces the number of fat micelles or chylomicra formed in the
enterocytes by more than 50%. These results are consistent with
those of Table 2, demonstrating that the strain of the invention
reduced blood triglyceride levels.
Example 4
Effect of Administering the B. uniformis 7771 CECT Strain on the
Function of Immune System Cellls; on Immunological Parameters in
Pweipheral Tissues; and on the Composition of Intestinal Microbiota
and its Inflammatory Properties
4.1. Preparation of Cultures of the Strain Object of the
Invention
[0159] The B. uniformis CECT 7771 strain was grown in Brain Heart
broth (Schariab, S.L., Barcelona, Spain) supplemented with 0.05%
(w/v) cysteine at 37.degree. C. under anaerobic conditions
(AnaeroGen; Oxoid, Basingstoke, UK) for 22 hours. The cells were
collected by centrifugation (6,000 g for 15 minutes), washed with a
phosphate-buffered saline solution (PBS, 10 mM sodium phosphate,
130 mM sodium chloride, pH 7.4), and re-suspended and administered
as a nutritional composition composed of 10% skimmed milk and the
bacterial strain at a concentration of 5.times.10.sup.8 cfu of the
CECT 7771 strain per 100 .mu.l of composition, similarly to the
nutrient composition described in example 3. Aliquots of these
suspensions were frozen with liquid nitrogen and stored at
-80.degree. C. until use. The viability of the bacteria was tested
by counting on Schaedler Agar agar plates (Scharlau, Barcelona,
Spain) supplemented with kanamycin (100 mg/L), vancomycin (7.5
mg/L) and vitamin K (0.5 mg/L) after 48 hours of incubation and was
approximately 90%. Each aliquot was thawed only once.
4.2. Obesity Animal Model
[0160] The same mice were described in example 3 and the same
experimental groups were used, two of which were administered the
strain object of the invention as a nutritional composition,
followingthe same pattern, and the placebo controls (nutritional
composition without the strain). At the end of the treatment
period, the animals were anaesthetised and sacrificed by cervical
dislocation and various biological samples taken: adipose tissue
and pancreas to determine immunological parameters (cytokines),
faeces to determine the effect on the composition of the microbiota
and immunocompetent cells (macrophages, dendritic cells and T
cells) obtained as described below, to evaluate the effect of the
intervention on the immune responses of these cells.
4.3. Evaluation of the Effect of Administering the B. uniformis
CECT 7771 Strain on Macrophage Function in Obese and Normal-Weight
Mice.
[0161] In order to demonstrate the effect of administering the CECT
7771 strain on improving the response of innate immune system
cells, macrophages were aseptically obtained from each experimental
group of mice by injecting, via the intraperitoneal route,
Dulbecco's Modified Eagles Medium (DMEM) solution (Sigma.TM.-St.
Louis, Mo. AJSA) supplemented with 10% inactivated fetal bovine
serum at 56.degree. C. for 30 minutes (Gibco, Barcelona, Spain),
100 .mu.g /ml streptomycin and 100 U/ml penicillin (Sigma Chemical
Co.). The macrophages obtained from each group of mice were
adjusted to a concentration of 1.times.10.sup.5 cells/ml in DMEM
and, after incubating for 1 hour at 37.degree. C. in a 5% CO.sub.2
atmosphere, the wells were washed with serum-free DMEM to remove
non-adherent cells. The adherent cells were incubated for 24 hours
and, at the end of this period, were stimulated with 1 .mu.g/ml LPS
from Salmonella enterica serotype Typhimurium (Sigma Chemical Co.,
Madrid, Spain) to assess their response to a bacterial component of
potential pathogens. Additionally, control mice cells were
stimulated with faeces (diluted 1/9 in PBS) for each experimental
group of mice to determine their inflammatory potential. In
parallel, unstimulated macrophages were assessed to determine the
basal cytokine production. After stimulation, the supernatants were
collected and the concentrations of these cytokines were
determined: TNF-.alpha. and IL-10 by ELISA (Ready SET Go! Kit, BD
Bioscience, San Diego, Calif., USA).
[0162] The results obtained indicate that the strain of the
invention improves the functioning of innate immune system cells
such as macrophages when administered in vivo to normal-weight and
obese subjects, increasing their ability to respond to infectious
agents, antigens or allergens. In particular, the administration of
the strain to animal models of obesity induced by a high-fat diet
improves, inter alia, the role of macrophages in phagocytosis and
cytokine synthesis (FIG. 5). The administration of the strain
increases the respiratory burst of peritoneal macrophages in
response to a stimulus or foreign allergen (pathogen), enhancing
phagocytic capacity and, therefore, immunological defences in obese
and normal-weight subjects (FIG. 5). This capacity is significantly
decreased in obese animals compared to non-obese controls (FIG. 5).
Earlier studies also show that the respiratory burst of phagocytic
cells responsible for the elimination of pathogens is also altered
in subjects with diabetes (Marhoffer et al., 1992. Diabetes Care,
15(2): 256-60). Additionally, the cultivation test with peritoneal
macrophages extracted from obese animals and controls and in vitro
stimulation thereof with lipopolysaccharide (LPS) of a pathogen,
demonstrate that administration of the strain object of the
invention improves the synthesis of cytokines responsible for
stopping a possible infection such as TNF-.alpha. in obese animals
(FIG. 5). This macrophage function is also reduced as a result of
diet-induced obesity.
4.4. Evaluation of the Effect of Administering the B. uniformis
CECT 7771 Strain on the Function of Dendritic Cells and T Cells of
Obese and Normal-Weight Control Mice.
[0163] In order to demonstrate the effect of administering the
strain of the invention on the ability of dendritic cells to
stimulate T lymphocyte response and thus, the adaptive immune
response, the ability of mature dendritic cells to induce the
proliferative response of CD4+ T lymphocytes in a mixed lymphocyte
reaction. The assay was performed by comparing the responses of
dendritic cells extracted from obese and control mice which were
administered or not the strain object of the invention as described
above.
[0164] The dendritic cells were generated from mouse tibia and
femoral bone marrow. The tibias and femurs of each mouse were
extracted and the surrounding tissue was removed aseptically. After
cutting the ends, the bone marrow was extracted by flushing with
PBS using a syringe and needle of 0.45 mm in diameter. The obtained
cells were washed once with PBS and aliquots of 10.sup.6 cells
diluted in RPMI, supplemented with antibiotics (100 IU/ml
penicillin and 100 .mu.g/ml streptomycin), 10% FBS and 20 ng/ml of
mouse GM-CSF, and seeded in 100 mm bottles. On the third day, 10 ml
of culture medium were added and, on the seventh day, the medium
was replaced with fresh medium. On the eighth day, the non-adherent
cells were harvested by gentle pipetting. The cells were washed
with PBS and re-suspended in culture medium without GM-CSF. The
dendritic cells were activated by adding LPS (100 ng/ml) for 24
hours before performing the Mixed Lymphocyte Reaction. Mature
dendritic cells were used to stimulate CD4+T lymphocytes. CD4+T
lymphocytes were isolated from spleens of 7-8 week-old C57BL/6
mice. After being removed, the spleens were suspended in PBS with
FBS and passed through a nylon mesh, the cell suspension obtained
was washed once and re-suspended in lysis buffer for 5 minutes.
After washing twice with PBS, CD4+ T cells were immunomagnetically
separated by positive selection using CD4 (L3T4) MicroBeads
(Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the
manufacturer's instructions.
[0165] In order to perform the Mixed Lymphocyte Reaction, aliquots
of dendritic cells were distributed in 96-well plates in triplicate
to stimulate, in each case, 1.times.10.sup.5 CD4+ T lymphocytes in
the following ratios (CD4+ T lymphocytes/dendritic cells): 1:1,
1:2, 1:4 in 100 .mu.l of culture medium and incubated at 37.degree.
C. for 72 hours in 5% CO.sub.2 atmosphere. Dendritic cells and CD4+
T lymphocytes with and without ConA (5 .mu.g/ml; Sigma), used as
mitogen, were used as controls. Lymphocyte proliferation was
determined using an ELISA kit (BrdU-colorimetric assay, Roche
Diagnostics, Germany) and quantified by measuring absorbance at 440
nm.
[0166] In dendritic cell cultures from each experimental group of
mice, their ability to synthesise cytokines when stimulated with
LPS, as an example of pathogenic stimulation, was also assessed;
and in dendritic cell cultures of control mice the potentially
inflammatory effect of faeces from each experimental group of mice
was determined by measuring cytokine synthesis, as indicated in the
case of macrophage cultures. The strain object of the invention has
also been shown to enhance the function of dendritic cells and T
cells when administered in vivo. Dendritic cells extracted from
obese mice that had been administered the strain, incubated in the
presence of T cells in various proportions (1:1, 1:2 and 1:4),
increase their proliferation and activation ability, properties
which are diminished in obese animals which have not been
administered the strain (FIG. 6). The improved functioning of the
dendritic cells in the obese animals to which the strain was
administered is also evident because, after stimulation with LPS in
vitro, they are capable of inducing increased secretion of
cytokines involved in the response to pathogens (for example
TNF-.alpha.) (FIG. 6). The administration of the strain object of
the patent also increases the capacity of LPS-stimulated dendritic
cells to produce the anti-inflammatory cytokine IL10, which helps
to regulate inflammation processes, avoiding chronic inflammation.
The effects on the described dendritic cells for the strain object
of the invention are also significant in normal-weight animals.
These properties make the strain object of the patent ideal, as the
functionality of dendritic cells and T cells is altered in obesity
and related diseases such as diabetes, not always associated with
obesity. In particular, dendritic cells exhibit functional
alterations associated with weight gain, characterised by their
reduced capacity to present antigens and stimulate allogeneic T
cells (Macia et al., 2006. J Immunol., 177(9): 5997-6006; Verwaerde
et al., 2006. Scand J Immunol., 64(5): 457-66). The
pro-inflammatory properties of naive T cells are increased in
response to a stimulus (mitogen or antigen), and can contribute to
low-grade chronic inflammatory condition associated with obesity;
and, by contrast, T cells previously exposed to antigens present a
defect in proliferation and preferably secrete Th2-type cytokines.
This explains the high incidence of infections in obese subjects
and the lack of response to vaccination and infection mediated by
memory T cells (Karlsson et al., 2010. J Immunol, 184: 3127-33). T
cell function is also deficient in diabetics, showing reduced
capacity to proliferate in response to a stimulus and to synthesise
IL2 (Chang y Shaio. 1995. Diabetes Res Clin Pract, 28(2):
137-46).
4.5 Effect of Administering the B. uniformis CECT 7771 Strain on
Inflammation of Peripheral Tissues.
[0167] In order to determine the effect of the strain object of the
patent on inflammation in peripheral tissues associated with
obesity and related diseases (for example, diabetes), the cytokine
concentration in adipose tissue and pancreas was determined after
homogenization with a polytron, by ELISA. Obesity increases the
concentration of TNF-.alpha. and reduces IL-10 in adipose tissue.
However, in obese subjects the strain object of the patent reduces
the concentration of TNF-.alpha. and increases the synthesis of the
anti-inflammatory cytokine IL-10 in adipose tissue, reducing
inflammation. TNF-.alpha. synthesis is increased in obesity and
other diseases and contributes to the development of insulin and
leptin resistance in tissues, inhibiting its anorectic effects
(reducing feelings of hunger) and its role in the regulation of
body weight and lipid and glucose metabolism (Example 4, Table 3).
Furthermore, in obese subjects, the strain object of the patent
reduces the concentration of TNF-.alpha. in the pancreas, which can
improve the function of this organ in the regulation of glucose
metabolism (Example 4, Table 3).
TABLE-US-00003 TABLE 3 Cytokine concentration in adipose tissue and
pancreas of mice fed a high-fat or standard diet, supplemented or
not with the B. uniformis CECT 7771 strain. *Experimental groups
**Student t test SD HFD HFD + B Value-P Value P Cytokine
concentration SD vs HFD vs Tissue (Mean .+-. sd pg/ml) HFD HFD + B
Adipose TNF-.alpha. 1098.1 .+-. 208.5 3075.7 .+-. 282.8 1628.6 .+-.
407.8 <0.001 0.001 IL-10 32089.5 .+-. 2936.5 6578.8 .+-. 890.3
11178.0 .+-. 1013.5 <0.001 0.005 Pancreas TNF-.alpha. 8698.7
.+-. 822.5 10693.6 .+-. 1481.1 2780.3 .+-. 360.6 0.260 0.001 IL-10
21894.9 .+-. 1952.3 11131.7 .+-. 2704.3 10037.9 .+-. 759.8 0.005
0.700 *SD: standard-diet group (control) (n = 6); HFD: high-fat
diet group (n = 6), HFD + B: HFD group and supplemented orally with
5.0 .times. 10.sup.8 CFU/day of B uniformis CECT 7771, for 7 weeks
(n = 6). The concentration of cytokines in different tissues was
determined by ELISA after sacrifice. **Significant differences
established applying ANOVA and the Student t test for comparisons
between two means at a value of P < 0.050.
4.6. Evaluation of the Effect of Administering the B. uniformis
Strain CECT 7771 Strain on the Composition of the Intestinal
Microbiota and Inflammatory Properties.
[0168] In order to evaluate the effect of the CECT 7771 strain on
the composition of the microbiota, stool samples were collected at
the end of the intervention from the different experimental groups
of mice, a dilution of 1:10 (w/v) in PBS (pH 7.2) was prepared and,
after homogenisation, DNA was extracted using the QIAamp DNA stool
Mini kit commercial system (Qiagen, Hilden, Germany).
Quantification of the concentration of each bacterial group was
performed by real-time PCR using the ABI PRISM 7000-PCR Sequence
Detection System (Applied Biosystems, UK). The reaction mixture was
composed of 25 SYBR.RTM. Green PCR Master Mix (SuperArray
Bioscience Corporation, USA), 1 of each primer at a concentration
of 0.25 .mu.M and 1 .mu.l of DNA. The concentrations of each
bacterial group were determined using the Ct values obtained for
each case study. Standard curves were built using plasmid dilutions
in which the group-specific PCR-amplified fragment of each
bacterial group had been cloned. The results were expressed in
number of copies of the 16S rRNA gene per gramme of faeces.
[0169] The results show that the B. uniformis CECT 7771 strain
partially restores the composition of the intestinal microbiota,
normalising the alterations associated with excess weight and/or
obesity and the inflammatory effect caused by these alterations
(FIG. 7), as well as the changes associated with other pathological
conditions associated not only to excess weight and/or obesity. The
administration of the strain of the invention to a model of obesity
increases the number of Bacteroides spp. and of the group C.
coccoides and reduces the number Bifidobacterium spp. These changes
in the microbiota composition also result in a reduction of the
pro-inflammatory properties thereof. Both in macrophages and
dendritic cells, the microbiota of obese animals that are
administered the strain induces decreased synthesis of
pro-inflammatory cytokines, such as TNF-.alpha., with respect to
obese animals that are not administered the strain (Example 4, FIG.
7). Alterations in the intestinal microbiota are considered one of
the possible inflammatory stimuli that cause weight gain, insulin
resistance, obesity and diabetes (Cani y Delzenne 2009. Curr Opin
Pharmacol., 9(6): 737-43). Furthermore, these alterations cause
other types of pathological conditions. The CECT 7771 strain also
induces changes in the microbiota of lean animals, for example,
increasing the concentration of Bifidobacterium spp. and reducing
their ability to induce TNF-.alpha. in macrophages and, hence,
cause inflammation.
TABLE-US-00004 TABLE 4 Example of the effect of administering the
CECT 7771 strain on the composition of the intestinal microbiota of
obese and normal-weight animals. *Experimental groups SD HFD SD + B
HFD + B .sup.aMedian .sup.aMedian .sup.aMedian .sup.aMedian
Bacterial group (IQR) (IQR) Valor-p.sup.b (IQR) Valor-p.sup.c (IQR)
Valor-p.sup.d Total bacteria 10.8 10.5 0.092 11.4 0.010* 11.0 0.629
(10.6-11.1) (10.3-10.8) (11.3-11.6) (10.7-11.2) Lactobacillus group
9.9 9.4 0.040* 9.6 0.470 9.7 0.936 (9.4-10.5) (9.2-9.5) (9.3-9.8)
(9.5-10.1) Bacteroides spp. 8.4 8.7 0.674 9.3 0.004* 9.0 0.016*
(8.3-8.6) (8.3-9.0) (9.1-9.5) (8.8-9.3) Bifidobacterium spp. 7.1
6.0 0.004* 8.1 0.013* 7.5 0.004* (6.8-7.2) (5.9-6.3) (7.9-8.3)
(7.0-7.7) C. leptum group 8.4 7.6 0.004* 9.6 0.004* 8.5 0.936
(8.3-8.6) (7.5-7.7) (9.4-9.8) (8.1-8.7) C. coccoides group 9.1 8.4
0.016* 9.9 0.054 9.6 0.036* (8.6-9.3) (8.2-8.5) (9.4-10.0)
(9.4-9.7) Enterobacteriaceae 7.3 8.1 0.019* 8.1 0.052 7.9 0.029*
(7.2-7.7) (7.8-8.2) (7.6-8.2) (7.5-8.0) *SD: standard-diet and
placebo group (control) (n = 6); SD + B: standard-diet group and a
daily dose of 5.0 .times. 10.sup.8 CFU/day of B. uniformis CECT
7771 (n = 6); HFD: high-fat diet and placebo group (n = 6), HFD +
B: high-fat diet group and a daily dose of 5.0 .times. 10.sup.8
CFU/day of B. uniformis CECT 7771 (n = 6). The treatment was
maintained for 7 weeks and the placebo or the bacteria were
administered daily by gavage. .sup.aResults are expressed as the
median (interquartile range) of the number of copies of the 16S
rRNA gene amplified with group-specific primers specific for each
bacterial group per gramme of faeces. "Significant differences
between the SD and HFD groups. .sup.cSignificant differences
between the SD and SD + B groups. .sup.dSignificant differences
between the HFD and HFD + B* groups. Significant differences were
established at P < 0.050 values, applying the Mann-Whitney U
Test.
Sequence CWU 1
1
611335DNABacteroides uniformismisc_featureGene sequence of the
ribosomal RNA16S from Bacteroides uniformis CECT 7771 1agtcgagggg
cagcatgaac ttagcttgct aagtttgatg gcgaccggcg cacgggtgag 60taacacgtat
ccaacctgcc gatgactcgg ggatagcctt tcgaaagaaa gattaatacc
120cgatggcata gttcttccgc atggtagaac tattaaagaa tttcggtcat
cgatggggat 180gcgttccatt aggttgttgg cggggtaacg gcccaccaag
ccttcgatgg ataggggttc 240tgagaggaag gtcccccaca ttggaactga
gacacggtcc aaactcctac gggaggcagc 300agtgaggaat attggtcaat
ggacgagagt ctgaaccagc caagtagcgt gaaggatgac 360tgccctatgg
gttgtaaact tcttttatac gggaataaag tgaggcacgy gtgccttttt
420gtatgtaccg tatgaataag gatcggctaa ctccgtgcca gcagccgcgg
taatacggag 480gatccgagcg ttatccggat ttattgggtt taaagggagc
gtaggcggac gcttaagtca 540gttgtgaagt ttgcggctca accgtaaaat
tgcagttgat actgggtgtc ttgagtacag 600tagaggcagg cgcaattcgt
ggtgtagcgg tgaaatgctt acatatcacg aagaactccg 660attgcgaagg
cagcttgctg gactgtaact gacgctgatg ctcgaaagtg tgggtatcaa
720acaggattag ataccctggt agtccacaca gtaaacgatg aatactcgct
gtttgcgata 780tacagtaagc ggccaagcga aagcgttaag tattccacct
ggggagtacg ccggcaacgg 840tgaaactcaa aggaattgac gggggcccgc
acaagcggag gaacatgtgg tttaattcga 900tgatacgcga ggaaccttac
ccgggcttga attgcaactg aatgatgtgg agacatgtca 960gccgcaaggc
agttgtgaag gtgctgcatg gttgtcgtca gctcgtgccg tgaggtgtcg
1020gcttaagtgc cataacgagc gcaacccttg tcgatagtta ccatcaggtt
atgctgggga 1080ctctgtcgag actgccgtcg tgagatgtga ggaaggtggg
gatgacrtca aatcascacg 1140gsccttacrt ccggggctac acacgtgtta
caatgggggg tacagaaggc agctacacgg 1200cgacgtgatg ctaatcccta
aagcctctct cagttcggat tggagtctgc aacccgactc 1260catgaagctg
gattcgctag taatcgcgca tcagccacgg cgcggtgaat acgttcccgg
1320gtcttgtaca caccg 1335220DNAArtificial SequenceSequence of
primer 27F 2agagtttgat cctggctcag 20317DNAArtificial
SequenceSequence of primer 1401r 3cggtgtgtac aagaccc
17416DNAArtificial SequenceSequence of primer 530f 4gtgccagcag
ccgcgg 16517DNAArtificial SequenceSequence of primer U-968f
5aacgcgaaga accttac 17615DNAArtificial SequenceSequence of primer
M13 6gagggtggcg gttct 15
* * * * *