U.S. patent application number 09/485875 was filed with the patent office on 2002-03-14 for immunity enhancing lactic acid bacteria.
Invention is credited to GILL, HARSHARNJIT S., GOPAL, PRAMOD K., SMART, JOHN B..
Application Number | 20020031503 09/485875 |
Document ID | / |
Family ID | 25645584 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020031503 |
Kind Code |
A1 |
GILL, HARSHARNJIT S. ; et
al. |
March 14, 2002 |
IMMUNITY ENHANCING LACTIC ACID BACTERIA
Abstract
Novel bacteria Lactobacillus rhamnosus HN001 and HN 067,
Lactobacillus acidophilus HN017, and Bifidobacterium lactis HN019
are claimed. Each strain provides immune enhancing effects when
ingested.
Inventors: |
GILL, HARSHARNJIT S.;
(PALMERSTON NORTH, NZ) ; SMART, JOHN B.;
(ASHHURST, NZ) ; GOPAL, PRAMOD K.; (PALMERSTON
NORTH, NZ) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
25645584 |
Appl. No.: |
09/485875 |
Filed: |
February 16, 2000 |
PCT Filed: |
August 18, 1998 |
PCT NO: |
PCT/NZ98/00122 |
Current U.S.
Class: |
424/93.45 |
Current CPC
Class: |
A23C 9/1234 20130101;
C12R 2001/01 20210501; C12R 2001/23 20210501; A61K 39/39 20130101;
A61P 37/00 20180101; C12R 2001/225 20210501; A61P 37/04 20180101;
C12N 1/205 20210501; A23L 29/065 20160801; A61K 35/747 20130101;
Y10S 435/854 20130101 |
Class at
Publication: |
424/93.45 |
International
Class: |
A01N 063/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 21, 1997 |
AU |
P0 8699 |
Aug 28, 1998 |
AU |
PP 3225 |
Claims
1. A biologically pure culture of either L. rhamnosus HN001 AGAL
deposit number NM97/09514 dated Aug. 18, 1997 or L. rhamnosus HN067
AGAL deposit number NM97/01925 dated Feb. 11, 1998.
2. A biologically pure culture of L. rhamnosus HN001 AGAL deposit
number NM97/09514 dated Aug. 18, 1997.
3. A biologically pure culture of L. rhamnosus HN067 AGAL deposit
number NM97/01925.
4. A composition of a biologically pure culture of any one of L.
rhamnosus HN001 as claimed in claim 2, L. rhamnosus HN067 as
claimed in claim 3, B. lactis HN019 AGAL deposit number NM97/09513
dated Aug. 18, 1997 or L. acidophilus HN017 AGAL deposit number
NM97/09515 dated Aug. 18, 1997 in an immunostimulating
concentration, with a physiologically acceptable excipient or
diluent.
5. A composition as claimed in claim 4 containing any two or more
of said strains.
6. A composition as claimed in claim 4 or 5 wherein said
physiologically acceptable excipient or diluent is a food.
7. A composition as claimed in claim 6 wherein said food is any one
of cultured milk, yoghurt, cheese, milk drink or milk powder.
8. A composition as claimed in claim 4 or 5 which is a
pharmaceutical composition and wherein said excipient or diluent is
pharmacologically acceptable excipient or diluent.
9. Immunity enhancing physiologically acceptable biologically pure
strains of homologues or mutants of any one of the strains: L.
acidophilus HN017, L. rhamnosus HN001, B. lactis HN019, or L.
rhamnosus HN067.
10. A method of enhancing natural and acquired immunity which
comprises administering to a mammal a biologically pure cultures as
claimed in any one of claims 1-3 and 9 at an immunostimulating
dosage rate.
11. A method as claimed in claim 10 wherein biologically pure
cultures of two or three of the above-defined strains are
present.
12. A method of enhancing natural and acquired immunity which
comprises administering to a mammal a composition as claimed in any
one of claims 4 to 8.
13. A method as claimed in claim 10 wherein said physiologically
acceptable excipient or diluent is a food.
14. A method as claimed in claim 10 wherein said food is cultured
milk, yoghurt, cheese, milk drink or milk powder.
Description
TECHNICAL FIELD
[0001] This invention relates to novel strains of lactic acid
bacteria and their use in enhancing immunity.
BACKGROUND ART
[0002] The consumption of products containing lactic acid bacteria
(LAB) is associated with a range of health benefits including
enhancement of immunity. There are thousands of strains of lactic
acid bacteria but only some strains exhibit health-promoting
properties. The ability of these bacteria to tolerate acids and
bile salts, adhere to mucosal epithelial cells, and to survive
passage through the gastrointestinal tract is considered an
important criterion for selection of health-promoting strains. Only
a few strains of lactic acid bacteria with proven health benefits
have been identified to date.
[0003] Strains of LAB showing good adhesion to the cells of the
mucosal epithelium of the small intestine thereby lending
themselves to therapeutic applications are known from New Zealand
Patent 248057. The micro-organisms described in this patent enhance
both natural immunity (phagocyte function) and acquired immunity
(antibody responses and lymphocyte proliferation responses).
[0004] It is desirable to have other LAB bacteria that enhance a
broad spectrum of immune responses including phagocyte
function.
[0005] It is an object of this invention to go some way towards
achieving these desiderata or at least to offer the public a useful
choice of immune enhancing lactic acid bacteria.
DISCLOSURE OF THE INVENTION
[0006] Accordingly, in one aspect the invention may be said broadly
to consist of a biologically pure culture of Lactobacillus
rhamnosus HN001, AGAL deposit number NM97/09514 dated Aug. 18,
1997.
[0007] In another aspect the invention may be said broadly to
consist of a biologically pure culture of Lactobacillus rhamnosus
HN067, AGAL deposit number NM97/01925 dated Feb. 17, 1998.
[0008] In another aspect the invention may be said broadly to
consist of a composition of a biologically pure culture of any one
of Lactobacillus acidophilus HN017, AGAL deposit number NM97/09515
dated Aug. 18, 1997, Lactobacillus rhamnosus HN001, Lactobacillus
rhamnosus HN067 or Bifidobacterium lactis HN019, AGAL deposit
number NM97/09513 dated Aug. 18, 1997 in an immunostimulating
concentration, with a physiologically acceptable excipient or
diluent.
[0009] In one embodiment said composition contains any two or more
of said strains.
[0010] Preferably said physiologically acceptable excipient or
diluent is a food.
[0011] Preferably said food is any one of cultured milk, yoghurt,
cheese, milk drink or milk powder.
[0012] Alternatively said composition is a pharmaceutical
composition and said excipient or diluent is pharmacologically
acceptable excipient or diluent.
[0013] Immunity enhancing, physiologically acceptable, biologically
pure strains of homologues or mutants of any one of the
strains:
[0014] Lactobacillus acidophilus HN017,
[0015] Lactobacillus rhamnosus HN001,
[0016] Bifidobacterium lactis HN019, or
[0017] Lactobacillus rhamnosus HN067.
[0018] In another embodiment the invention may be said broadly to
consist of a method of enhancing natural and acquired immunity
which comprises administering to a mammal any one of the above
biologically pure cultures at an immunostimulating dosage rate.
[0019] In another embodiment substantially biologically pure
cultures of two or three of the above-defined strains are
present.
[0020] Preferably said culture is administered in the form of a
composition with a physiologically acceptable excipient or
diluent.
[0021] Preferably said physiologically acceptable excipient or
diluent is a food.
[0022] Preferably said food is cultured milk, yoghurt, cheese, milk
drink or milk powder.
[0023] This invention may also be said broadly to consist in the
parts, elements and features referred to or indicated in the
specification of the application, individually or collectively, and
any or all combinations of any two or more of said parts, elements
or features, and where specific integers are mentioned herein which
have known equivalents in the art to which this invention relates,
such known equivalents are deemed to be incorporated herein as if
individually set forth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the effect of supplementation of mice with
product fermented with L. rhamnosus HN001 or unfermented product
containing L. rhamnosus HN001 on phagocyte activity of peripheral
blood leukocytes as described in example 5. BALB/c nice were fed on
milk based diets containing 10.sup.9 cfu (per day) L. rhamnosus
HN001 in either fermented or unfermented product for 14 days.
Phagocytic activity of peripheral blood leukocytes was determined
using flow cytometry and fluoroscein isothiocyanate-labelled
Escherichia coli. Values are mean.+-.standard error. Significant
differences (ANOVA, the SAS program) from the control:
**P<0.0001.
[0025] FIG. 2 shows the effect of supplementation of mice with live
L. rhamnosus HN001 or heat killed L. rhamnosus HN001 on phagocytic
activity of peripheral blood leukocytes as described in example 7.
BALB/c mice were fed on milk based diets and orally administered
10.sup.9 cfu (per day) of either live or heat killed L. rhamnosus
HN001 for 14 days. Phagocytic activity of peripheral blood
leukocytes and peritoneal macrophages were determined using flow
cytometry and fluoroscein isothiocyanate--labelled Escherichia
coli. Values are mean.+-.standard error. Significant differences
(ANOVA, the SAS program) from the control, **P<0.0001.
[0026] FIG. 3 shows the effect of supplementation of mice with L.
rhamnosus HN001 or B. lactis HN019 on bacteria translocation in
mice challenged with S. typhimurium as described in example 8.
Unsupplemented and B. lactis HN019, or L. rhamnosus HN001
supplemented BALB/c mice were orally challenged with S. typhimurium
following continuous daily supplementation. Six days after
challenge mice were humanely killed and their livers and spleens
were harvested for monitoring bacterial translocation. Tissue
suspensions from the harvested organs were then cultured on
MacConkey agar plates for 24-48 hr prior to enumeration. Values are
mean.+-.standard error. Significant differences (ANOVA, the SAS
program) from the control: *P<0.05.
[0027] FIG. 4 shows the effect of supplementation of mice with L.
rhamnosus HN001 or B. lactis HN019 on the phagocytic activity of
peripheral blood leukocytes from mice challenged with S.
typhimurium as described in example 8. Unsupplemented and B. lactis
HN019, or L. rhamnosus HN001 supplemented BALB/c mice were orally
challenged with S. typhimurium following continuous daily
supplementation. Phagocytic activity of peripheral blood leukocytes
was determined six days after challenge using flow cytometry and
fluoroscein isothiocyanate-labelled Escherichia coli. Values are
mean.+-.standard error. Values (mean.+-.standard error) with
different superscripts are significantly different (ANOVA, the SAS
program): P<0.01.
[0028] FIG. 5 shows the effect of supplementation of mice with L.
rhamnosus HN001 or B. lactis HN019 on the proliferative responses
of spleen lymphocytes from mice challenged with S. typhimurium as
described in example 8. Unsupplemented and B. lactis HN019, or L.
rhamnosus HN001 supplemented BALB/c mice were orally challenged
with S. typhimurium following continuous daily supplementation. Six
days after challenge the proliferative responses of spleen
lymphocytes were measured colourimetrically following the
incorporation of 5-bromo-2'-deoxyuridine for the final 16 hrs of
the 96 hr incubation. Values (mean.+-.standard error) with
different superscripts are significantly different (ANOVA, the SAS
program): P<0.01).
MODES OF CARRYING OUT THE INVENTION
[0029] Freeze dried cultures of the four bacterial strains have
been deposited at the Australian Government Analytical Laboratories
(AGAL), The New South Wales Regional Laboratory, 1 Suakin Street,
Pymble, NSW 2073, Australia. Details of the deposits are:
1 Strain Number Date L. acidophilus HN017 NM97/09515 August 18 1997
L. rhamnosus HN001 NM97/09514 August 18 1997 B. lactis HN019
NM97/09513 August 18 1997 L. rhamnosus HN067 NM97/01925 February 11
1998
[0030] The four strains identified above have been found to enhance
a broad spectrum of immune responses including both natural and
acquired immune responses.
EXAMPLE 1
Morphology and General Properties
[0031] RAPD analysis, 16S rRNA sequencing and SDS-PAGE analyses
were used to confirm taxonomical characterisation of strains. It
was also found that L. acidophilus HN017 was genetically different
from L. acidophilus (LC1) of New Zealand Patent No.248057.
[0032] RAPD analysis, 16S rRNA sequencing and SDS-PAGE analyses
were used to confirm taxonomical characterisation of L. rhamnosus
HN067; species-specific primers used for characterisation of L.
rhamnosus HN067 at molecular level included Pr I (forward)
5-CAGACTGAAAGTCTGACGG-3 and Pha II (reverse)
5-GCGATGCGAATTTCTATTATT-3.
[0033] The morphology and sugar fermentation properties of this
strain are detailed in Tables 1 and2.
2TABLE 1 Morphology and other characteristics L. acidophilus L.
rhamnosus B. lactis L. rhamnosus HN017 HN001 HN019 HN067 Short to
medium Short to medium Microaerophilic to Short to medium rods with
rounded rods with square anaerobic rods with rods with square ends,
generally ends in chains, characteristic shapes ends in chains,
occurring singly generally 0.7 .times. such as middle generally 0.7
.times. 1.1 .times. or in pairs or short 1.1 .times. 2.0-4.0 .mu.m,
enlarged cells, `V` or 2.0 to 4.0 .mu.m, chains, when when grown in
palisade arrangement when grown in grown in MRS MRS broth. of cells
when grown MRS broth. broth. Gram positive, on TPY agar slabs. Gram
positive, Gram positive, non-mobile, non- In MR5 broth with
catalase negative, non-spore spore forming, 0.05% cysteine
non-mobile, non forming, catalase catalase negative hydrochloride,
they spore-forming, negative facultative form middle-enlarged
facultative facultatively anaerobic rods cells and club shaped
anaerobic rods anaerobic rods with optimum (spatulated with optimum
with optimum growth extremities) cells, growth growth temperature
of Gram positive, non- temperature of temperature of 37 .+-.
1.degree. C. and motile and non-spore 37 .+-. 1.degree. C. and 37
.+-. 1.degree. C. and optimum pH of forming, catalase optimum pH of
6.0 optimum pH of 6.0-6.5. These negative rods with to 6.5. These
are 6.0-6.5. These are facultatively optimum growth facultatively
are obligately heterofermentative temperature of heterofermentative
homofermentative bacteria and no 37 .+-. 1.degree. C. and optimum
bacteria and no bacteria and no gas produced from pH of 6.0-7.0.
gas produced from gas is produced glucose. Fructose-6-phosphate
glucose. from glucose. phospho-ketolase positive.
[0034]
3TABLE 2 Carbohydrate fermentation pattern of selected
Lactobacillus and Bifidobacterium strains S1. No. Name of the
bacterium Score* 1 L. acidophilus HN 017 5755546 2 L. rhamnosus
HN001 5757177 3 B. lactis HN019 1051622 4 L. rhamnosus HN067
5757175 API 50 CH sugar fermentation kit was used to determine the
sugar fermentation pattern. *The scores are based on scores of 22
prominent sugars (Bergey's manual)
EXAMPLE 2
Adhesion to Intestinal Cells
[0035] The ability of probiotic strains to adhere to human
intestinal epithelial cells (HT-29 and CaCo-2) was assessed in
vitro using differentiated cell-lines. Monolayers of HT-29 and
CaCo-2 cells were grown on cover slips and placed in multi-well
dishes. 10.sup.8 cfu/ml of LAB in 1 ml of spent culture supenatant
was then added to cell layers along with 1 ml of DMEM medium and
incubated for 1 hr at 37.degree. C. in 10% CO.sub.2-90% air.
Monolayers were washed 4 times with PBS, fixed in methanol, Gram
strained and the number of bacteria adhering to epithelial cells
determined microscopically. On average, 20 fields were counted and
the results are summarised in Table 3.
4TABLE 3 Adherence to HT-29 and CaCo-2 cell lines* STRAIN HT-29
CaCo-2 L. acidophilus HN 017 98 .+-. 17 171 .+-. 16 L. rhamnosus HN
001 161 .+-. 18 218 .+-. 35 B. lactis HN 019 188 .+-. 27 194 .+-.
25 *Number (mean .+-. SEM) of bacteria/100 epithelial cells
EXAMPLE 3
Enhancement of Natural and Acquired Immunity
[0036] The immunoenhancing effects of the three strains L.
rhamnosus HN001, L. acidophilus HN017 and B. lactis HN019 were
examined by determining phagocyte (blood leukocytes and peritoneal
macrophage) function, and quantifying concentrations of specific
antibodies to protein antigens used for mimicking responses to
vaccines in mice.
[0037] The following experimental protocol was used:
[0038] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were
used.
[0039] 2. Mice were randomly allocated to different treatment
groups (Table 4)
[0040] 3. Mice were fed L. acidophilus HN017, L. rhamnosus HN001 or
B. lactis HN019 (10.sup.9 cfu/day) in 50 .mu.l skim milk for 10
days. Control mice received 50 .mu.l of skim milk powder only.
[0041] 4. All mice received skim milk powder based diet throughout
the experiment.
[0042] Blood leukocytes and macrophages from mice receiving L.
acidophilus HN017, L. rhamnosus HN001 or B. lactis HN019 showed
significantly greater phagocytic capacity compared with cells from
control mice (Table 4). The production of oxygen radicals
(oxidative burst) by leukocytes from probiotic fed mice was also
higher than the control mice (data not shown).
5TABLE 4 The effect of dietary L. acidophilus HN017, L. rhamnosus
HN001 and B. lactis HN019 on phagocyte function in mice % Blood %
Peritoneal leukocytes with macrophage with Treatment phagocytic
activity phagocyte activity Control 14.33 .+-. 0.87 66.1 .+-. 3.5
L. acidophilus HN017 22.7 .+-. 1.21** 79.0 .+-. 1.0** L. rhamnosus
HN001 24.84 .+-. 0.93** 80.5 .+-. 1.8** B. lactis HN019 23.19 .+-.
0.95** 77.4 .+-. 2.6* BALB/c mice were orally administered with
10.sup.9 cfu (per day) L. acidophilus HN017, L. rhamnosus HN001 or
B. lactis HN019 for 10 days. Phagocytic activity of blood
leukocytes and peritoneal macrophages was determined using flow
cytometry and fluorescein isothiocyanate-labelled Escherichia coli.
Values are mean .+-. standard error. Significant differences
(Students t test) from the control: *P < 0.05, **P <
0.01.
[0043] The concentration of specific IgG antibodies in the sera and
in the intestinal washings of mice receiving L. acidophilus HN017,
L. rhamnosus HN001 or B. lactis HN019 was also greater than those
of control mice (Table 5).
6TABLE 5 The effect of dietary L. acidophilus HN017, L rhamnosus
HN001 and B. lactis HN019 on serum and mucosal antibody responses
Serum antibody Mucosal antibody response response Treatment
(units/ml) (units/ml) Control 80.2 .+-. 6.0 1350 .+-. 96.0 L.
acidophilus HN017 134.6 .+-. 25.2* 1548 .+-. 270.0 L. rhamnosus
HN001 118.5 .+-. 12.5** 1512 .+-. 198.0 B. lactis HN019 158.1 .+-.
51.6*** 1548 .+-. 234.0 BALB/c mice were orally administered with
10.sup.9 cfu (per day) L. acidophilus HN017, L. rhamnosus HN001 or
B. lactis HN019 for 10 days. Mice were immunised with cholera toxin
(an antigen used to mimic enteric infection) on days 0 and 7. The
concentration of specific antibodies in serum and intestinal
secretions were measured using ELISA on day 10. Values represent #
mean .+-. standard error. Significant differences (Students t test)
from control: *P = 0.08; **P < 0.05; ***P < 0.01.
EXAMPLE 4
Immunostimulating Effects Following Supplementation with LAB for
Four Weeks
[0044] The immunostimulating effects of L. acidophilus HN017, L.
rhamnosus HN001, and B. lactis HN019 were assessed in mice using
the following experimental protocol:
[0045] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were
used.
[0046] 2. Mice were randomly allocated (18/group) to different
treatment groups.
[0047] 3. After acclimatisation (for 7 days), mice were given
10.sup.9 cfu (per day) L. acidophilus HN017, L. rhamnosus HN001, or
B. lactis HN019, in 50 .mu.l skim milk, for 28 days (from day 0 to
day 28). Control mice received 50 .mu.l skim milk (without any
micro-organisms) only.
[0048] 4. Mice were offered a skim milk powder based-diet and water
ad libitum, throughout the experiment.
[0049] 5. Immunostimulating effects were assessed by monitoring
phagocytic activity of blood leukocytes and peritoneal macrophages,
NK-cell activity of splenic lymphocytes, lymphocyte proliferation
(spleen cells) responses to a T-cell mitogen, ConA (an indicator of
cell-mediated immunity) and antibody responses to Tetanus
vaccine.
[0050] As seen in Table 6, leukocytes (neutrophils, monocytes and
macrophages) from mice receiving L. acidophilus HN017, L. rhamnosus
HN001, or B. lactis HN019 exhibited significantly greater
phagocytic activity (an indicator of natural immunity) than
leukocytes from control mice.
7TABLE 6 The effect of dietary L. acidophilus HN017, L. rhamnosus
HN001, and B. lactis HN019 in mice % Blood leukocytes with %
Peritoneal macrophages Treatment phagocytic activity with
phagocytic activity Control 15.5 72.67 L. acidophilus HN017 29.4**
82.2* L. rhamnosus HN001 24.2** 82.8** B. lactis HN019 31.1**
83.0** Mice (18/group) were given 10.sup.9 cfu (per day) L.
acidophilus HN017, L. rhamnosus HN001, or B. lactis HN019 in 50
.mu.l skim milk for 28 days. Phagocytic activity of blood
leukocytes/peritoneal macrophages was determined on day 28 using
flow cytometry and fluorescein isothiocyanate-labelled E. coli.
Values are least square means. Significant differences (the SAS
analysis): *P < 0.002, **P < 0.0005.
[0051] Consumption of L. acidophilus HN017, L. rhamnosus HN00 1, or
B. lactis HN019 for 28 days also resulted in an increase in the
NK-cell activity, lymphocyte proliferation responses to ConA and
antibody responses to Tetanus vaccine. For all these indicators of
immunocompetence, mice receiving L. acidophilus HN017, L. rhamnosus
HN001, or B. lactis HN019 had higher responses than those of
control mice (Table 7).
[0052] Together these results show that supplementation for
extended periods with L. acidophilus HN017, L. rhamnosus HN001, or
B. lactis HN019 is able to induce a sustained enhancement in
several aspects of natural and acquired immunity.
8TABLE 7 The effect of dietary L. acidophilus HN017, L. rhamnosus
HN001, and B. lactis HN019 on NK cell activity and lymphocyte
proliferation responses to ConA and antibody responses to Tetanus
vaccine. Lymphocyte Antibody NK cell proliferation responses to
ConA activity to ConA Tetanus vaccine Treatment (%) (absorbance)
(units/ml) Control 8.8 1.4 .+-. 0.125 402.5 .+-. 41.4 L.
acidophilus HN017 9.9 1.6 .+-. 0.44 923.9 .+-. 116.0* L. rhamnosus
HN001 11.5 1.8 .+-. 0.1* 711.5 .+-. 127.2* B. lactis HN019 10.5 1.7
.+-. 0.5* 844.6 .+-. 134.7* Mice (18/group) were given 10.sup.9 cfu
(per day) L. acidophilus HN017, L. rhamnosus HN001, or B. lactis
HN019 in 50 .mu.l skim milk for 28 days (i.e. from days 0 to 28).
NK-cell activity of splenic lymphocytes was determined on day 28
using flow cytometry and D275-labelled Yac-1 cells. Lymphocyte
proliferation responses of splenic lymphocytes to ConA were
assessed on day 28 using a commercial cell proliferation kit
(Boebringer Mannheim, Germany). For antibody responses, #mice were
immunised with Tetanus vaccine (50 .mu.l/dose, CSL, Australia) on
days 7 and 21. The concentration of specific antibodies were
determined using an ELISA; antigen supplied by the vaccine
manufacturers (CSL, Australia) was used for coating plates. Values
are least square means of 18 mice. Significant differences (the SAS
analysis): *P < 0.05.
EXAMPLE 5
Enhancement of Natural and Acquired Immunity Using Fermented versus
Unfermented Products
[0053] The aim was to assess the immunoenhancing efficacy of
yoghurt made (fermented) using the probiotic strain L. rhamnosus
HN001 compared to unfermented product containing L. rhamnosus
HN001. The immunoenhancing effects were examined by determining the
phagocyte function (peripheral blood leukocytes and peritoneal
macrophages) and lymphocyte proliferative responses to a B-cell
mitogen (LPS).
[0054] The following experimental protocol was used:
[0055] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were
used.
[0056] 2. Mice were randomly allocated to different treatment
groups.
[0057] 3. Control mice received a whole milk powder-based diet
throughout the experiment.
[0058] 4. Test mice received 2.5 g yoghurt made using L. rhamnosus
HN001 (10.sup.9 cfu/day) or 2.5 g whole milk containing L.
rhamnosus HN001 (10.sup.9 cfu/day) per day as well as a whole milk
powder based diet for 14 days.
[0059] Results
[0060] Mice receiving yoghurt made with L. rhamnosus HN001 or whole
milk containing L. rhamnosus HN001 displayed a significantly higher
level of phagocytic activity of peripheral blood leukocytes than
was observed in mice receiving the control diet (FIG. 1). This
increase was seen irrespective of whether the L. rhamnosus HN001
was delivered in the yoghurt (fermented with L. rhamnosus HN001) or
unfermented product containing L. rhamnosus HN001. There was no
difference in the level of phagocytic activity between mice
receiving the fermented yoghurt made using L. rhamnosus (HN001)
compared to unfermented WMP product containing L. rhamnosus
(HN001).
[0061] Both the unfermented and L. rhamnosus HN001 fermented
product fed mice showed higher lymphocyte proliferative responses
to LPS than the control mice (Table 8). There was no significant
difference in the response between mice receiving unfermented
product containing L. rhamnosus HN001 and mice receiving product
fermented with L. rhamnosus HN001.
9TABLE 8 The effect of fermented and unfermented L. rhamnosus HN001
on lymphocyte proliferative responses in mice Lymphocyte
proliferation to Treatment LPS (absorbance) Control (WMP) 0.4699
.+-. 0.028 WMP Fermented with L. rhamnosus HN001 0.5361 .+-. 0.028
Unfermented WMP with L. rhamnosus 0.5518 .+-. 0.028* HN001 BALB/c
mice were fed on milk based diets containing 10.sup.9 cfu (per day)
L. rhamnosis HN001 in either unfermented product or yoghurt made
with L. rhamnosus HN001 (fermented product) for 14 days. Control
mice received milk-based diet without any LAB. Proliferative
responses were measured colourimetrically following the
incorporation of 5-brome-2'-deoxyuridine for the final # 16 hrs of
the 96 hr incubation. Values are means .+-. standard error.
Significant differences (Students t test) from the control: *P =
0.05.
[0062] Together these results suggest that supplementation with L.
rhamnosus HN001 enhances a range of immune functions including
phagocytic activity and lymphocyte cell proliferation. L. rhamnosus
HN001 presented in either fermented or unfermented product is
effective at eliciting enhancement of immune function, with
fermented product giving a greater response for some functions and
unfermented being superior in others.
EXAMPLE 6
Enhancement of Natural and Acquired Immunity by L. rhamnosus
HN067
[0063] Experiment 1
[0064] The immunoenhancing effects of L. rhamnosus HN067 were
examined by monitoring phagocytic capacity of peripheral blood
leukocytes and peritoneal macrophages (indicator of non-specific
immunity), and quantifying concentrations of specific antibodies to
an immunisation antigen, cholera toxin (used for mimicking
responses to enteric vaccines) in mice.
[0065] The following experimental protocol was used:
[0066] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were
used. They were fed on a skim milk-based diet throughout the
experiment.
[0067] 2. Mice in the test group (n=6) were orally administered L.
rhamnosus HN067 (10.sup.9 cfu/day) in 50 .mu.l skim milk for 10
days. Control mice (n=6) received 50 .mu.l of skim milk powder
(without any LAB) only.
[0068] Results
[0069] Blood leukocytes and peritoneal macrophages from mice
receiving L. rhamnosus HN067 showed significantly greater
phagocytic activity (enhanced phagocyte function) compared with
cells from control mice. The results are set out in Table 9
below.
10TABLE 9 The effect of dietary L. rhamnosus HN067 on phagocyte
function % Peritoneal % Blood leukocytes with macrophages with
Treatment phagocytic activity phagocytic activity Control 13.1 .+-.
1.5 76.4 .+-. 1.9 L. rhamnosus HN067 23.7 .+-. 1.5** 87.2 .+-. 1.9*
BALB/c mice (6/group) were fed on milk-based diet with or without
oral administration of L. rhamnosus HN067 (10.sup.9 cfu/day) for 10
days. Phagocytic activity of blood leukocytes and peritoneal
macrophages were determined using flow cytometry and fluorescein
isothiocyanate-labelled E. coli. Values represent least square mean
.+-. standard error LSM. Significant differences # (the SAS
program) from the control: *P = 0.0005, **P = 0.0001.
[0070] The concentration of specific antibodies to cholera toxin,
an antigen used for oral immunisation, in the sera and in the
intestinal washings of mice receiving L. rhamnosus HN067 was also
significantly greater than those of control mice (Table 10).
11TABLE 10 The effect of dietary supplementation with L. rhamnosus
HN067 on serum and mucosal antibody responses to cholera toxin
Serum Mucosal antibody anitbody response response Treatment
(units/ml) (units/ml) Control 63.1 .+-. 43.2 1969.7 .+-. 279.5 L.
rhamnosus HN067 246.5 .+-. 43.2** 2995.5 .+-. 465.2* BALB/c mice
were fed on milk-based diet with or without L. rhamnosus HN067
(10.sup.9 cfu/day) for 10 days. Mice were immunised orally with
cholera toxin (10 .mu.g/dose), an antigen used to mimic enteric
infection, on days 0 and 7. Antibody levels in serum and intestinal
secretions were measured using ELISA on day 10. Values represent
least square mean .+-. standard error LSM. Significant differences
(the SAS program) from control: *P = 0.02; **P = 0.0039
[0071] Experiment 2
[0072] The immunostimulating effects of L. rhamnosus HN067 were
assessed in mice using the following experimental protocol:
[0073] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were
used. They were offered skim milk powder based diet and water ad
libitum, throughout the experiment.
[0074] 2. After acclimatisation for 7 days, mice in group 1 (n=20)
were orally administered with 10.sup.9 cfu (per day) L. rhamnosus
(HN067) in 50 .mu.l skim milk (group 1 n=20) for 14 days. Control
nice (group 2, n=20) received skim milk without any
microorganisms.
[0075] 3. Immunostimulating effects were assessed by monitoring
phagocytic activity of blood leukocytes and peritoneal macrophages,
and spleen lymphocyte proliferation responses to
phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) (T and
B-cell mitogens respectively).
[0076] Results
[0077] Blood leukocytes and peritoneal macrophages from mice
receiving L. rhamnosus HN067 exhibited significantly greater
phagocytic activity (an indicator of natural immunity) than
leukocytes and macrophages from control mice (Table 11).
12TABLE 11 The effect of dietary L. rhamnosus HN067 on phagocyte
function in mice % Blood % Peritoneal Leukocytes with macrophages
Treatment phagocytic activity with phagocytic activity Control 13.7
.+-. 0.07 64.6 .+-. 2.1 L. rhamnosus HN067 22.5 .+-. 0.07** 75.8
.+-. 1.7* BALB/c mice were fed on milk-based diet with or without
oral administration of L. rhamnosus HN067 (10.sup.9 cfu/day) for 14
days. Phagocytic activity of blood leukocytes/peritoneal
macrophages were determined on day 14 using flow cytometry and
fluorescein isothiocyanate-labelled E. coli. Values represent least
square mean .+-. standard error LSM. Significant differences (the
SAS program): *P = 0.002, **P = 0.0001.
[0078] Mice receiving L. rhamnosus HN067 for 14 days also displayed
higher lymphocyte proliferation responses to PHA and LPS compared
with control mice (Table 12).
13TABLE 12 The effect of L. rhamnosus HN067 supplementation on
lymphocyte proliferation responses to PHA and LPS ConA Lymphocyte
Lymphocyte Treatment proliferation to PHA proliferation to LPS
Control 1.18 .+-. 0.08 0.99 .+-. 0.07 L. rhamnosus HN067 1.37 .+-.
0.07* 1.24 .+-. 0.06** BALB/c mice were fed on milk-based diet with
or without oral administration of L. rhamnosus HN067 (10.sup.9
cfu/day) for 14 days. Lymphocyte proliferation responses of spleen
cells to PHA and LPS were assessed on day 14 using a commercial
cell proliferation kit (Boehringer Mannheim, Germany). Values
represent least square mean .+-. standard error LSM. Significant
differences (the SAS program): *P < 0.08, **P < 0.01.
[0079] In summary, mice receiving L. rhamnosus HN067 displayed
significant enhancement of a range of host immune responses
including leukocyte phagocytic function, antibody responses to oral
immunisation, and lymphocyte proliferation responses to T and
B-cell mitogens. Blood leukocytes (neutrophils and monocytes) and
macrophages are major effectors of natural immunity and play a
major role in protection against microbial infections. A
correlation between in vitro lymphocyte proliferation responses to
mitogens (T- and B-cell mitogens) and immunocompetence of an
individual is also well documented. Therefore, these results
suggest that supplementation with L. rhamnosus HN067 is able to
enhance several aspects of natural and acquired immunity.
EXAMPLE 7
Enhancement of Natural and Acquired Immunity Using Live and Heat
Killed L. rhamnosus HN001
[0080] The aim of the present study was to investigate the
immunoenhancing effects of the probiotic strain L. rhamnosus HN001
when presented in either the live or heat killed form. The effect
on immune function was assessed by determining phagocytic activity
of peripheral blood leukocytes. The effect of live and heat killed
L. rhamnosus HN001 on humoral immunity was investigated by
immunising mice with cholera toxin, and measuring the
concentrations of specific antibodies produced.
[0081] The following experimental protocol was used:
[0082] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were
used.
[0083] 2. Mice were randomly allocated to different treatment
groups.
[0084] 3. Control mice received a skim milk powder based diet
throughout the experiment.
[0085] 4. Test mice receive either 10.sup.9 cfu/day of live L.
rhamnosus HN001 or to cfu/day heat killed L. rhamnosus HN001 per
day as well as a skim milk powder-based diet for 14 days.
[0086] 5. Mice were orally immunised with cholera toxin on day 0
and day 7 of feeding.
[0087] Results
[0088] L. rhamnosus HN001 feeding significantly enhanced the level
of phagocytic activity of peripheral blood leukocytes compared to
mice receiving the control diet (FIG. 2). This increase was seen
irrespective of whether the L. rhamnosus HN001 was delivered in the
live or heat killed form. There was no difference in the level of
phagocytic activity between the mice receiving live L. rhamnosus
HN001 compared to heat killed L. rhamnosus HN001.
[0089] Feeding of both live and dead L. rhamnosus HN001 induced an
increase in both serum and mucosal antibody responses compared to
the control mice. However, the level of response was significantly
greater in the mice fed the live L. rhamnosus HN001 (Table 13).
14TABLE 13 The effect of live and heat killed L. rhamnosus HN001 on
serum and mucosal antibody responses to Cholera Toxin in mice Serum
antibody Mucosal antibody Treatment response (units/ml) response
(units/ml) Control 88.69 .+-. 18.52 708.6 .+-. 146.9 Live L.
rhamnosus HN001 214.89 .+-. 62.33* 2054.5 .+-. 285.8*** Heat Killed
L. rhamnosus 174.89 .+-. 44.78 1533.6 .+-. 319.3 HN001 BALB/c mice
were fed on milk-based diets and orally administered 10.sup.9 cfu
(per day) L. rhamnosus HN001 in either live or heat killed form for
14 days. Control mice received no LAB. Mice were orally immunised
with Cholera Toxin on days 0 and 7. Antibody responses (serum and
intestinal secretions) were measured using an ELISA on day 14.
Values are mean .+-. standard error. Significant differences
(Students t test) from the control: *P = 0.05, ***P = 0.0005.
[0090] These results suggest that both live and heat killed L.
rhamnosus HN001 are able to enhance aspects of natural and acquired
immunity in mice.
EXAMPLE 8
Anti-infection Properties of B. lactis HN019 and L. rhamnosus
HN001
[0091] The aims of the current study were to:
[0092] 1. Assess the protection efficacy of B. lactis HN019 and L.
rhamnosus HN001 against the gastrointestinal pathogen Salmonella
typhimurium.
[0093] 2. Determine the role of immunostimulation induced by B.
lactis HN019 and L. rhamnosus HN001 in protection against S.
typhimurium infection in mice.
[0094] Anti-infection properties were assessed by measurement of
bacterial translocation to the liver and spleen. The
immunoenhancing effects were examined by determining the phagocyte
function (peripheral blood leukocytes and peritoneal macrophages)
and lymphocyte proliferative responses to a T-cell mitogen
(PHA).
[0095] The following experimental protocol was used:
[0096] 1. Six-to-seven week old BALB/c mice, weighing 20-30 g were
used.
[0097] 2. Mice were randomly allocated to 4 difference treatment
groups and were individually housed.
[0098] 3. All mice received a skim milk powder based diet
throughout the experiment
[0099] 4. Test mice commenced daily feeding of B. lactis HN019 or
L. rhamnosus HN001 (10.sup.9 cfu/day) 7 days prior to challenge,
and continued for the duration of the trial.
[0100] 5. Mice administered with B. lactis HN019 or L. rhamnosus
HN001 and a control group (no LAB) were orally challenged with
Salmonella typhimurium (ATCC 1772) 8.times.10.sup.5 cfu/day for 5
days starting on day 7.
[0101] 6. An uninfected control group did not receive S.
typhimurium challenge.
[0102] 7. On day 6 after challenge mice were used for the
measurement of bacterial translocation to the liver and spleen, and
for immune function assessment.
[0103] Results
[0104] Both the B. lactis HN019 and L. rhamnosus HN001 supplemented
mice showed significantly lower levels of bacterial translocation
into the liver and spleen than the S. typhimurium alone fed mice
(FIG. 3).
[0105] Challenge infection resulted in a significant suppression of
phagocyte function (FIG. 4); the phagocytic activity of control
mice challenged with S. typhimurium was significantly lower than
that of the uninfected mice. However, infection with S. typhimurium
had no effect on the phagocytic ability of peripheral blood
leukocytes of mice supplemented with B. lactis HN019 or L.
rhamnosus HN001. This was shown by similar levels of phagocytic
activity in mice supplemented with B. lactis HN019 or L. rhamnosus
HN001 and challenged with S. typhimurium and the normal uninfected
control mice.
[0106] Both the B. lactis HN019 and L. rhamnosus HN001 supplemented
mice showed higher lymphocyte proliferative responses to PHA than
the S. typhimurium challenged control (FIG. 5). There was no
significant difference in the response between mice receiving B.
lactis HN019 or L. rhamnosus HN001 and the uninfected control
mice.
[0107] Together these results suggest that supplementation with B.
lactis HN019 or L. rhamnosus HN001 is able to confer protection
against enteric pathogens such as Salmonella typhimurium. Enhanced
resistance to infection is accompanied by an increase in immune
performance.
* * * * *