Bifidobacterium strains having high IgA induction potential

The present invention is to provide an easy, rapid method for screening a great amount of substances having IgA induction potential. The IgA induction potential herein mentioned means the potential to activate and enhance the activity of IgA production cells to produce secretory-type IgA in response to antigen. The present invention is to provide bacterial strains of genus Bifidobacterium obtained by the method for screening substances having IgA induction potential. More specifically, the present invention is to provide Bifidobacterium longum YIT 4062, Bifidobacterium breve YIT 4063 and Bifidobacterium breve YIT 4064. The three strains obtained by the present invention have high IgA induction potential.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method capable of easy screening of a 
substance having production potential of secretory-type IgA (so called IgA 
induction potential) which inhibits the binding of microorganisms and 
allergens to tunica mucosa. 
The present invention also relates to a bacterial strain of genus 
Bifidobacterium, which is screened by the method and which exhibits strong 
IgA induction potential. 
2. Description of the Prior Art 
Immunoglobulin, being antibody or protein having a structural and 
functional relation with antibody, is classified in terms of functional 
and structural properties into five different classes i.e. IgG, IgM, IgA, 
IgD and IgE. 
IgA among them comprises two subclasses i.e. IgA1 and IgA2. In IgA1, the L 
chain (light chain) is covalently bonded with the H chain (heavy chain) 
thereof, while in IgA2 the L chain is bound in S--S bond with each other 
instead of binding to the H chain. As for the composition ratio regarding 
IgA1 and IgA2, IgA1 reaches 90% of total serum IgA, and IgA2 reaches 60% 
of total secretory-type IgA. 
The production sites of IgA exists in submucosal plasma cells such as 
gut-tunica propria and the like, sialaden and mammary gland. In human 
gut-tunica propria, the number of IgA production cells is far greater than 
that of IgG production cells and the ratio between them is about 20:1 in 
remarkable contrast of the ratio of 1:3 (IgA:IgG) in lympho nodes and 
spleen. IgA in secreted mucus is formed in dimer having a J-chain 
component and has a secretory component attached thereto which can be 
observed only in small amount in serum IgA. This component is attached to 
a dimeric IgA molecule while it is secreted from plasma cells beneath the 
tunica mucosa of gut and airway into mucus. 
Secretory-type IgA in secreted mucus inhibits the binding of highly 
pathogenic microorganisms and allergens to tunica mucosa. Thus, 
secretory-type IgA binds food components functioning as allergens to 
inhibit the absorption thereof through gastric wall, in addition to 
prevention of infection of pathogenic microorganisms. 
The preventive mechanism of antibody is now illustrated in the following 
inhibitive mechanism of absorption through gastric wall. Antibody directly 
binds to the surface of microorganisms in order to prevent infection of 
exotoxin secreted from pathogenic microorganisms. That is, the antibody 
may directly bind to the microorganisms to exert a variety of effects. 
Alternatively, there has been also observed some substances having the 
potential to process antigen effectively after nonspecific stimulation of 
antibody production cells, and such property is often called 
built-in-adjuvanticity. 
It is known, for example, that cholera toxin to be produced by Vibrio 
cholerae, a causative toxin for diarrhea, may affect tunica mucosa 
intestini tenuis to modify ionic permeability thereof, so that the toxin 
may induce intestinum tenuis to discharge a great amount of water and 
electrolytes to lead to the occurrence of diarrhea. It is also known that 
the cholera toxin B subunit obtained by detoxifying the principle 
component of the toxin exhibits immunological response such that it 
permeates into tunica mucosa to facilitate the production of IgA antibody 
(so-called IgA induction potential), namely, the cholera toxin B subunit 
functions as adjuvant. 
Ninety-nine percent of enterobacterium flora in breast-feeding babies 
within several weeks after birth is occupied by bacterial strains of genus 
Bifidobacterium as an enterobacterium which cannot exert any pathogenicity 
to humans and animals. The above fact has indicated that the bacterial 
strains may have certain roles in biophylaxis. 
Bifidobacterium longum, one of the genus Bifidobacterium, is known to 
increase total IgA in feces if administered orally. 
However, the induction potential of secretory-type IgA has not been 
comparatively studied yet and an easy method for the investigation 
therefor has never been established, either. 
It has not been completely elucidated yet how effective the enterobacterium 
may be. It has not been identified specifically whether there may be any 
difference in IgA induction potential among a variety of bacterial strains 
of genus Bifidobacterium known not to have any pathogenicity to humans and 
animals. 
SUMMARY OF THE INVENTION 
The objective of the present invention is to establish an easy method using 
IgA production cells for screening substances having IgA induction 
potential. Another objective of the present invention is to define any 
difference in IgA induction potential among bacterial strains of genus 
Bifidobacterium to screen a strain having strong IgA induction potential. 
The present invention may clearly identify the difference in IgA induction 
potential among the bacterial strains and realize the screening of a 
strain having strong IgA induction potential. 
Regarding the first invention, the method for screening substances having 
IgA induction potential, comprises culturing aseptically cells of Peyer's 
patch as one of the gut-associated lymphoid tissue containing a great 
amount of IgA production cells, adding the solution of a subjective 
substance or a suspension thereof to the culture medium for culture for a 
given period, measuring IgA secreted from the IgA production cells in the 
culture medium after culturing for a given period, and selecting a 
substance having IgA induction potential based on a ratio of the produced 
IgA in a group with addition of a subjective substance to that in a group 
without the substance. 
Regarding to the second invention, the bacterial strain of genus 
Bifidobacterium having IgA induction potential, is one obtained by the 
method for screening substances having IgA induction potential according 
to the first invention described above. 
In accordance with the first invention, a substance having IgA induction 
potential may be screened, based on a ratio of the IgA produced in a group 
with addition of a subjective substance to that in a group without the 
subjective substance after aseptically culturing cells containing a great 
amount of IgA production cells. The screening procedure is easy to 
perform, and may further accomplish, for a short period, screening for a 
great number of kinds of substances having IgA induction potential. 
Furthermore, the bacterial strain according to the second invention is one 
of genus Bifidobacterium obtained by using the screening method for 
substances having IgA induction potential according to the first 
invention. Accordingly, the IgA induction potential of genus 
Bifidobacterium which is known not to show any pathogenicity to humans and 
animals, exerts an adjuvant activity i.e. an ability to stimulate 
non-specifically antibody production cells to process antigen more 
effectively, together with the initial IgA induction potential due to 
foreign body cognition (the antigenicity of the bacterial strain). 
The bacterial strain of genus Bifidobacterium having IgA induction 
potential, obtained by the present invention, has an action to activate 
and enhance the activity of the IgA production cells to produce 
secretory-type IgA in response to antigen. More particularly, the present 
invention discloses three bacterial strains of genus Bifidobacterium, 
YIT4062, YIT4063 and YIT4064. 
Each of the bacterial strains, YIT4062, YIT4063 and YIT4064, has 
outstandingly stronger IgA induction potential, compared with the known 
bacterial strains of genus Bifidobacterium deposited in public 
organizations; the ratio of the IgA produced by the group with addition of 
a subjective substance to that by the group without the substance (namely, 
the increment) is 12 or more. Each strain may stimulate the potential of 
IgA production cells strongly. 
The bacterial strains, YIT4062, YIT4063 and YIT4064, have been already 
deposited in the Agency of Industrial Science and Technology, Fermentation 
Research Institute as FERM BP-2822, FERM BP-2823 and FERM BP-2824, 
respectively. 
In accordance with the first invention, a substance having IgA induction 
potential may be screened, based on a ratio of the IgA produced in a group 
with addition of a subjective substance to that in a group without the 
substance after aseptically culturing cells containing a great amount of 
IgA production cells of Peyer's patch. The screening procedure is easy to 
perform, and may further accomplish rapid screening for a great number of 
kinds of substances having IgA induction potential. 
Furthermore, the bacterial strain according to the second invention is one 
of genus Bifidobacterium obtained by using the screening method for 
substances having IgA induction potential according to the first 
invention. Accordingly, the IgA induction potential of genus 
Bifidobacterium which is known not to show any pathogenicity to humans and 
animals, exhibits an adjuvant activity i.e. an ability to stimulate 
non-specifically antibody production cells to process antigen more 
effectively, together with the initial IgA induction potential due to 
foreign body cognition (the antigenicity of the bacterial strain). 
The bacterial strain of genus Bifidobacterium having IgA induction 
potential, obtained by the present invention, has an action to activate 
and enhance the activity of the IgA production cells to produce 
secretory-type IgA in response to antigen. 
For the reasons described above, such strain may be widely used as nutrient 
agents and mixing compositions for so-called healthy foods and functional 
foods, besides prophylactic agents for enteral infection and incidence of 
allergy.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is to provide the finding that the three bacterial 
strains originating from 120 Bifidobacterium strains isolated from feces 
of newborn babies, babies, infants, adults and elderly, induce IgA 
production activity of Peyer's patch cells more strongly than the known 
Bifidobacterium strains. 
The known bacteria now used are those described on the catalogs of the 
American Type Culture Collection (ATCC) and the Japan Collection of 
Microorganisms (JCM). 
The screening procedure of the bacterial strains of genus Bifidobacterium, 
having strong IgA induction potential, according to the present invention, 
is now explained in examples. 
The identification technique of the bacterial strains used in experiments, 
and the preparation and experimental methods therefor are as follows; 
1) The identification of isolated bacteria 
Bacteria isolated are identified according to the sugar-fermentation test 
(see Anaerobe Laboratory Manual, 4th ed., ed. by L. V. Holdeman et. al. 
1977). 
2) The preparation of bacteria 
Each Bifidobacterium strain isolated from feces of newborn babies, babies, 
infants, adults and elderly, in total 120 strains, is inoculated on the 
GAM medium and cultured under anaerobic condition at 37.degree. C. for 48 
hours. These bacteria are washed in phosphate buffer two times and 
subjected to heating at 100.degree. C. for 30 min for use. 
3) Isolation of Peyer's patch cells 
Peyer's patch is aseptically taken out from mouse intestine and placed in a 
solution of Dispase (1.5 mg Dispase/ml Joklik-modified MEM), and then 
stirred at 37.degree. C. for 30 to 40 minutes to recover single cells 
separated into the solution. Such procedure is repeated 4 or 5 times 
before centrifuge and washing, to obtain Peyer's patch. 
EXAMPLE 1 
The isolated Bifidobacterium strains (120 strains) obtained after repeating 
three times the procedures for separation into single colonies, were 
examined of their IgA induction potential. IgA production was determined 
as follows; 
Cells of Peyer's patch (5.times.10.sup.5) and each of a variety of 
bacterial strains in suspension at a concentration of OD.sub.660 =0.275 
were placed in each well of a 96-well microtiter plate and cultured under 
5% CO.sub.2 /air at 37.degree. C. in the Eagle medium containing 5% fetal 
calf serum [Eagle MEM (manufactured by Nissui Pharmaceuticals, Co.Ltd) 9.4 
g/l, 200 mM glutamine 10 ml/l (2 mM), MEM nonessential amino acid 
(.times.100GIBCO) 10 ml/l, 100 mM sodium pyruvate (manufactured by Sigma, 
Co. Ltd.) 10 ml/l (1 mM), pH7.2, adjusted with 1N NaOH]. 
During the culture, there was continued every day the division per well of 
0.02 ml of the nutrient mixture [MEM essential amino acid (.times.50GIBCO) 
5 ml, MEM nonessential amino acid (.times.100GIBCO) 2.5 ml, 200 mM 
glutamine 2.5 ml, Dextrose 500 mg, Eagle MEM (--NaHCO.sub.3) (manufactured 
by Nissui Pharmaceuticals, Co. Ltd) 35 ml, adjusted at pH 7.2 with 1N NaOH 
before addition of 7.5 ml of 7.5% NaHCO.sub.3 ]. IgA secreted into the 
supernatant of the culture medium was measured by ELISA (enzyme-linked 
immunosorbent assay) over time for 7 days. 
ELISA was carried out as follows. As a buffer solution, sodium carbonate 
buffer (pH 9.6) was used. One hundred microliter of goat anti-mouse IgA 
(manufactured by Cappel Co. Ltd.) was added to an immuno-plate well and 
was reacted at 4.degree. C. overnight for adsorption. After washing with a 
washing solution (0.05% Triton X-100, PBS) three times, sodium carbonate 
buffer containing 1% bovine serum albumin (BSA) was added and reacted at 
37.degree. C. for 1.5 hours, in order to cover the unabsorbed part of the 
well. Again, after washing with the washing solution, 90 microliter of 
each diluted sample of the supernatant from the culture medium was added 
to each of 96 wells and left to stand at 37.degree. C. for 1.5 hours. 
After washing, 100 microliter of peroxidase-bound goat anti-mouse IgA 
(manufactured by Cappel Co. Ltd.) was added to each well and left to stand 
at 37.degree. C. for 1.5 hours. After washing again with the washing 
solution, 100 microliter of a substrate solution, which was produced just 
before use by adding 10 microliter of aqueous hydrogen peroxide solution 
to the solution of 20 mg of 0-phenylenediamine dissolved in 50 ml of 
citrate buffer, pH 7.2, was added and reacted at 37.degree. C. for 10 
minutes, immediately followed by addition of 50 microliter of 2.5M sulfate 
per well, in order to terminate the reaction. The absorbance of each well 
at OD.sub.492 nm was measured by using a Titertek Multiscan manufactured 
by Flow Laboratory Inc. 
As standard, mouse IgA (myelona) (manufactured by ICN Immuno-Biological, 
Co. Ltd.) was used. The value of IgA was represented in the unit 
microgram/ml supernatant of culture medium. 
The increment of IgA was determined by the following formula; 
##EQU1## 
As a result, there could be detected three bacterial strains (YIT 4062, 
4063 and 4064) having stronger IgA induction potential (index value of 12 
or more) than the 7 kinds of known bacterial strains, as is shown in Table 
1. 
TABLE 1 
______________________________________ 
IgA Induction Potential of Isolated Bacterial Strains 
and Known Strains 
Bacterial strains IgA (.mu.g/ml) 
Increment 
______________________________________ 
Isolated bacterial strains 
YIT4062 3.34 12.9 
.sup. 4063 4.87 18.7 
.sup. 4064 6.89 26.5 
Known strains 
B. Breve 
YIT4014 (ATCC15700) 
2.90 11.2 
YIT4015 (ATCC15698) 
1.64 6.3 
YIT4049 (ATCC15701) 
1.58 6.1 
YIT4006 2.81 10.8 
B. longum 
YIT4021 (ATCC15707) 
0.73 2.8 
YIT4037 (ATCC15708) 
0.94 3.6 
B. animalis 
YIT4044 (JCM1190) 
0.65 2.5 
No addition of bacteria 
0.26 1.0 
(only cells of Peyer's patch) 
______________________________________ 
EXAMPLE 2 
The three strains having strong IgA induction potential were identified by 
the sugar-fermentation test and the DNA homology test. The results of the 
sugar-fermentation tests are shown in Table 2. YIT 4062 was identified to 
be Bifidobacterium longum (B. longum); YIT 4063, 4064 were identified to 
be Bifidobacterium breve (B. breve). 
TABLE 2 
__________________________________________________________________________ 
Identification Test (Sugar Fermentation Test) 
Isolated 
Known strains bacterial strains 
B. breve ss 
B. breve ss 
Sugar breve parvulorum 
B. longum 
B. animalis 
YIT 4064 
YIT 4063 
YIT 4062 
__________________________________________________________________________ 
Arabinose 
- - + + - - + 
Xylose 
- - + + - - + 
Ribose 
+ + + + + + + 
Glucose 
+ + + + + + + 
Mannose 
+ + .+-. .+-. + + .+-. 
Fructose 
+ + + + + + + 
Galactose 
+ + + + + + + 
Sucrose 
+ + + + + - + 
Maltose 
+ + + + + + + 
Cellobiose 
+ + - - + + - 
Lactose 
+ + + + + + + 
Trehflose 
.+-. - - - - - - 
Raffinose 
+ + + + + + + 
Melezitose 
.+-. .+-. + - - - + 
Dextrin 
.+-. + .+-. .+-. + + - 
Starch 
.+-. + - - - - - 
Glycogen 
.+-. + - - - - - 
Inulin 
- .+-. - - - - - 
Mannitol 
+ - - - + - - 
Sorbitol 
+ - - - + - - 
Inositol 
- - - - - - - 
Esculin 
+ + - - + + - 
Salicin 
+ + - - + + - 
Amygdalin 
+ + - - - + - 
B. breve 
B. breve 
B. longum 
__________________________________________________________________________ 
As is described above, the fact that the three bacterial strains according 
to the present invention were identified as Bifidobacterium longum or 
Bifidobacteirum breve (H. Yasui, A. Mike and M. Ohwaki, 1989. J. Dairy 
Sci. 72:30-35) and that the strains are capable of enhancing IgA 
production potential of the cells of Peyer's patch indicates that the 
strains may act as adjuvant. 
As has been described above, the first invention may provide an easy, rapid 
method for screening a great amount of substances having IgA induction 
potential. The method may be used in near future for screening other 
substances or bacterial strains, having strong IgA induction potential, 
other than the three strains. 
Due to the fact that the Bifidobacterium strains of the second invention, 
detected by the present first invention, have been known not to show any 
pathogenicity for humans and animals, the IgA induction potential thereof 
may be considered to be the one as adjuvant actions, not as antigenicity 
as foreign bodies of themselves. 
The above experiments were carried out using the thermally killed three 
bacterial strains of the present invention. They proposed the evidence 
that the strains are capable of enhancing IgA production potential of the 
cells of Peyer's patch. Thus, they indicates that there may exist in the 
cell walls thereof some substances capable of activating IgA induction 
potential. Because these indicate that similar effects may be obtained by 
using live such bacteria, the strains may be prepared in pharmaceuticals 
in powders, drink or tablets, and may be used as sources for functional 
foods. 
These three strains enhance IgA production potential of the cells of 
Peyer's patch as a principal tissue in the gastric mucosal immune system. 
The oral administration thereof may enhance the production potentials of 
IgA and intraluminally secreting-type IgA of the cells of Peyer's patch. 
Additionally, the bacterial strains of the present invention may be 
expected to enhance strongly IgA production in mucosal layers other than 
Peyer's patch, so that they may be effective against prophylaxis of 
enteral infection and inhibition of allergy absorption. Furthermore, it 
may be suggested that they will exhibit satisfactory effects to prevent 
cold and allergic rhinitis. 
These bacteria may be administered orally, but they may not cause any 
problems on safety. In addition, they may be handled quite easily. 
The thermally killed microorganisms may be satisfactorily used as effective 
components, which provides easy preparation of the microorganisms into 
pharmaceuticals, and simple storage and use thereof. 
The killed bacteria may be thus used in a wide range such as mixing 
components for nutrient agents, so-called healthy foods and functional 
foods, besides prophylactic drugs for enteral infection and allergy 
episodes.