Mutant bifidobacteria strains with acid, bile salt and...

Details Mutant bifidobacteria strains with acid, bile salt and... Mutant bifidobacteria strains with acid, bile salt and...

1997-12-30

2001-10-23

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C426S061000, C426S071000, C435S243000, C435S244000, C435S822000

Reexamination Certificate

active

06306638

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to Bifidobacterium strains and to a method for culturing these strains.
BACKGROUND OF THE INVENTION
The presence of Bifidobacterium in an infant's intestine is typically an indication of a healthy intestinal bacterial flora. While types and sizes of Bifidobacterium populations vary with age, bifidobacteria are present throughout the life cycle of a human being.
Bifidobacteria have various physiological properties. For instance, these bacteria can inhibit putrefactive and pathogenic bacteria, maintain a normal intestinal bacterial population, inhibit production of toxic amines, synthesize vitamin B groups, and produce L-lactic acid.
Since bifidobacteria tend to be more anaerobic than other lactic acid-producing bacteria, the development of food products containing viable bifidobacteria has encountered problems such as difficulties in maintaining bacterial viability during storage of the food products. In addition, orally administered bacteria are under the stress caused by, e.g., gastric acid and bile salt encountered in the gastrointestinal environment.
Nearly a hundred different kinds of bifidobacteria-containing food products have been developed to date, yet none of the strains used in these products are tolerant to oxygen, gastric acid, and bile salt. Many strains disclosed in the prior art, for example, are acid- and oxygen-tolerant, yet few of them are bile salt-tolerant (see, e.g., Tables I and II).
According to some studies, Bifidobacterium spp. ATCC 15700, ATCC 15696, ATCC 15697, and ATCC 15707 have extremely low growing abilities when cultured in the presence of 0.3% glycocholate for 24 hours. Studies of acid-tolerant strains have been devoted largely to the bacterial viability in acidic dairy products (pH 4 to 4.8) over a period of storage under refrigerating conditions. Some studies carried out to test tolerance to gastric acid set pH values at about 3 to 3.5 and not at about 2, which occurs during secretion of gastric acid.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is to provide a gastric acid-tolerant strain of
Bifidobacterium longum
isolated from healthy infants' feces, and based on the isolated strain, to provide strains having multiple characteristics such as tolerance to gastric acid, bile salt and oxygen by means of artificial mutagenesis and screening.
Within the scope of the invention are isolated Bifidobacterium strains characterized by their ability to grow aerobically (e.g., in the open air) and their tolerance to hydrochloric acid at a pH of approximately 1.5 to 4.5 (e.g., 2.0), and its tolerance to oxgall at approximately 0.3% (weight/volume or “w/v”) in a culture medium. By “tolerance to hydrochloric acid at a pH of approximately 1.5 to 4.5” is meant that the strain has a survival rate of 10
−6
or higher (e.g., 10
−5
or 10
−4
) in a physiological saline buffer containing hydrochloric acid at about pH 1.5-4.5 (see, e.g., Tables III and IV, infra, and the accompanying text). By “tolerance to oxgall at approximately 0.3%” is meant that bacteria from the strain can grow (i.e., proliferate) in the presence of oxgall present at about 0.3%
TABLE I
Comparison of patented bifidobacteria strains with the invention strains
Isolation
Strains
Inventors
sources
Characteristics
Assignee
Reference
Bifidobacterium
Mutai et al.
Feces of healthy
1. Aerobic growth
Yakult
U.S. Pat. No. 4087559
bifidum
YIT-4002
infants fed with
without growth
Honsha
(1978)
(FERM-P 3371)
mother milk
promoting substance
*Growth up to 5.0 × 10
9
CFU/ml in the
presence of 6.5 ppm
oxygen over 24 hrs.
and up to 6.5 × 10
9
CFU/ml in the
presence of 0.1 ppm
oxygen over 24 hrs..
B. bifidum
YIT-
Mutai et al.
same as above
1. Oxygen tolerance:
Yakult
U.S. Pat. No. 4091117
4005
results of growth are
Honsha
(1978) JP
(FERM-P 3372)
the same as that of
second
anaerobic growth and
publication
can be up to 10
9
No. Sho-56-
CFU/ml.
42250
2. Acid tolerance: 4%
(1981)
survived after
storing at pH 4.2 for
7 days (similar
results obtained both
in milk and buffer
solution).
B. breve
YIT-4006
Mutai et al.
Same as above
1. Growth with
Yakult
U.S. Pat. No. 4187321
(FERM-P 3906)
tolerance against
Honsha
(1980) JP
oxygen in absence of
second
growth promoting
publication
substances and can be
No. Sho-59-
up to 10
9
CFU/ml
53031
after 24 hrs.
(1984)
B. breve
HW-
Ishikawa et al.
Feces of healthy
1. Acid tolerance
Midorina
JP first
107 (FERM 5774)
infant fed with
(gastric acid): pH
Co., Ltd.
publication
milk
3.5, 37° C./1 hr.,
No. Sho-57-
bacteria population
99190
decreased 3.89 log
(1982)
value (buffer
system).
B. longum
M-8201
Kawashima et
Feces of healthy
1. Acid tolerance:
Morinaga
JP second
(FERM 6548)
al.
infant fed with
11.8% survived after
Milk
publication
milk
storing at pH 4.6 for
Industry
No. Sho-59-
7 days (buffer
Co., Ltd.
53829
system); 53.6%
(1984)
survive after storing
at pH 4.8 for 7 days
(milk system).
B. breve
SBR 3212
Yoshino et al.
Feces of healthy
1. Acid tolerance: 8%
Snow
JP first
(FERM 11915)
infant born
survived after
brand
publication
several months
storing at pH 4.0 for
Milk Prod
No. Hei-4-
and fed with
7 days (milk system)
Co., Ltd.
320642
mother milk
and 23% (buffer
(1992)
system).
2. Oxygen tolerance:
growth increased 10-
fold after 24 hour
culturing and kept at
5-fold increase after
48 hrs..
B. infantis
Sozzi
1. Acid tolerance:
Nestec
U.S. Pat. No. 4870020
CNCM T-372
survivabilities after
S.A.
(1989)
B. bifidum
storing at pH 4.0 for
(Switzer-
JP first
CNCM I-373
7 days were 100%
land)
publication
B. breve
(I-372), 75% (I-373),
No. Sho-61-
CNCM I-374
and 70% (I-374); and
205481
after 40 days, 70%
(1986)
(I-372), 60% (I-373)
EP 86101202
and 14% (I-374) (milk
(1986)
system).
2. Oxygen tolerancing
substances with
growth promoting
substances
B. longum
Mura et al.
UV mutant of
B.
1. Acid tolerance:
Mei Ji
JP first
No. 1022
longum
ATCC
after storing at pH
Milk
publication
(FERM-P 8033)
15708
4.7 for 7 days,
Industry
No. Sho-61-
survivability of
Co., Ltd.
185182
original
B. longum
(1986)
ATCC 15708 is only 7%
of that of mutant
B. breve
Kawashima et
1. Acid tolerance:
Morinaga
JP second
M 7204 (FERM1324)
al.
0.02% survived after
Milk
publication
storing at pH 4.6 for
Industry
No. Sho-47-
7 days (buffer
Co., Ltd.
29995
system); 1.5%
(1972)
survived after
storing at pH 4.8 for
7 days (milk system)
B. longum
ATCC
Feces of healthy
1. Gastric acid
The present
55816
infant
tolerance: population
invention
decreased 3-4 log
value after treatment
at pH 2, 37° C./2 hr
(0.85% NaCl/0.01 N
HCl system)
2. Tolerance to bile
salt: growth up to
10
8
-10
9
CFU/ml in the
presence of 0.3%
oxgall for 24 hrs.
3. Oxygen tolerance:
growth up to 10
9
CFU/ml for 24 hrs.
without growth
promoting substances.
TABLE II
Comparison of Tolerant Strains Disclosed in the Literature with the Invention Strains
Isolation
Strains
sources
Characteristics
Research Unit
References
B. longum
TQB
Feces of mid-
1. Oxygen tolerance: growth
Ten-jin Light
Been Fa,
21-2-2
age men
increased 25% after thin layer
Industries
Sheu et al.
standing for 24 hrs.; and
College
(1994)
increased 25% after shaking for
60 hrs..
B. bifidum
1. Oxygen tolerance: growth up to
Wuu-Hann Light
Sheau Chau,
10
9
CFU/ml after non-anaerobic
Industries
Fuh et al.
culturing for 5-7 hrs. (milk
Research
(1990, 1992)
system).
Institute
2. Acid tolerance;
13.5% survived after storing at
pH 4.5 for 10 days (milk system).
B. bifidum
GSNE
Feces of
1. Oxygen tolerance: Growth
Yakult Honsha
Wumada
infant fed
increased 1.3 log value in the
Research
(1982)
with milk
presence of 6.2-6.4 ppm dissolved
Institute
oxygen for 24 hrs., and 1.8 log
value in the presence of 0 ppm
dissolved oxyyen for 24 hrs.
(milk system).
B. bifidum
1. Gastric acid tolerance:
Same as above
Mutai (1978)
B. breve
Population decreased 5.3 and 2.6
log values, respectively, after
culturing at pH 3, 37° C. for 3
hrs. (0.2% NaCl, 0.32% pepsin
system).
2. Bile salt tolerance: growth
rate in the presence of 135 ppm
bile salt were 67% and 45%,
respectively (culture medium
syste

Affiliated with

Also associated with

Rating

Mutant bifidobacteria strains with acid, bile salt and... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Mutant bifidobacteria strains with acid, bile salt and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant bifidobacteria strains with acid, bile salt and... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2563412