JP2922013B2 - Novel bifidobacteria with acid and oxygen tolerance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel acid- and oxygen-tolerant Bifidobacterium breve.
bacterium breve ). The Bifidobacterium breve of the present invention is useful as a starter for fermented milk because it has good acid resistance and oxygen resistance.

[0002]

2. Description of the Related Art It is generally said that bifidobacteria are deeply involved in human health, from infants to elderly people. At present, pharmaceuticals and foods utilizing bifidobacteria are very large, and are particularly used in dairy products such as fermented milk (yogurt). However, few of these products maintain a high viable count. This is because bifidobacteria require anaerobic conditions for their survival and have low pH as fermented milk.
In the H range, it is difficult to survive, and nutritional requirements are complicated, and it is necessary to add a growth promoting substance such as yeast extract.

[0003] From such a point, studies have been conducted on acid-resistant strains and oxygen-resistant strains of Bifidobacterium. For Bifidobacterium bifidum , the presence of acid-resistant mutant strains and fermented milk produced by the strains (Japanese Patent Publication No. Sho 56-42250), a method for producing acid milk using an acid-resistant strain (Japanese Patent Laid-Open No. 61-20548).
No. 1), Bifidobacterium breve (B. bre)
Regarding ve), the presence of an oxygen-tolerant mutant strain, a method for producing fermented milk using the strain (Japanese Patent Publication No. 59-53031), and a method for producing acid milk using an acid-resistant strain (Japanese Patent Application Laid-Open No. Sho 61)
No. 2052051) Regarding Bifidobacterium longum (B. longum), the presence of an acid-resistant mutant strain and a method for producing fermented milk using the strain (JP-A-59-538)
No. 29), the presence of a hydrogen peroxide-resistant mutant, and a culture composition using the strain (Japanese Patent Application Laid-Open No. 61-185182) have been known so far. Bifidobacterium breve (B. breve) is recommended for infants and infants, and Bifidobacterium longum (B. longum) is recommended for infants and adults. (Chemistry and Biology Vol. 21, No. 1, pages 8-9).

[0004]

In view of this situation in the use of bifidobacteria, the present inventors have developed lactic acid bacteria which can survive with a high viable cell count in dairy products such as fermented milk, in other words, high acid resistance. Lactic acid bacteria showing sex and oxygen tolerance were searched.
As a result, a lactic acid bacterium having such properties was found from the feces of breastfeeding infants, and it was found that this lactic acid bacterium could be used as a starter for fermented milk, thus completing the present invention.

[0005] That is, an object of the present invention is to provide a novel lactic acid bacterium which has high acid resistance and oxygen resistance and can be used as a starter for fermented milk.

[0006]

SUMMARY OF THE INVENTION The present invention relates to Bifidobacterium breve which exhibits high acid and oxygen tolerance.

[0007] The present invention monaural, in order to obtain a strain having a strong acid resistance among Bifidobacterium breve,
A search using a variety of samples revealed that among the strains isolated from the stool of healthy breastfeeding infants, those with excellent aerobic growth were selected and selected, and treated several times with a low pH acetate buffer. Then, the strain Bifidobacterium breve SBR3212 having the highest viability among them was isolated. This strain exhibits the mycological properties of Bifidobacterium breve, but is novel in that it has higher acid resistance and oxygen resistance.
Deposited as No. 5.

[0008] The acid resistance of the present invention is as follows.
When the cells were suspended in a sterilized acetate buffer solution of No. 3 and stored at 5 ° C. for 7 days, they survived at least, and (2) the cells were cultured in reduced skim milk and quenched when the pH reached 4.1. 7 at ℃
It shows at least survival even if stored for days. Further, (3) oxygen tolerance indicates that the cells at least proliferate when cultured in stirred reduced skim milk for 48 hours.

The novel strain of the present invention can be obtained by inoculating and culturing Bifidobacterium into a milk medium consisting of whole milk, skim milk or reduced milk, or a synthetic or semi-synthetic medium containing lactose or glucose as a main component. It is possible to obtain a lactic acid bacterium starter containing bacteria and having high survivability.

When fermented milk such as drink yogurt and acidic milk drink is produced using this lactic acid bacteria starter, fermented milk having a high viable cell count can be obtained.

Next, the method for separating Bifidobacterium breve having high acid resistance and oxygen resistance according to the present invention will be described in detail. Separation was carried out from feces of eight breastfeeding infants a few months after birth according to the method of Mitsuoka (1980, published by Sobunsha Co., Ltd., Toshimitsu Mitsuoka, “World of Intestinal Bacteria”, pp. 53-92) to obtain 25 strains of Bifidobacterium. Bifidobacterium
The breve was thirteen strains.

The 13 strains were first screened for oxygen-resistant strains. That is, this strain is cultured in Briggs liver broth (Tomohito Mitsuoka: Clinical Laboratory, 18 , pp. 1163-1172 (1974)), collected and washed to prepare a bacterial cell solution twice as concentrated as the stock solution. did. This bacterial cell solution was added to 0.5% yeast extract and 12% reduced skim milk (18 ×
2% inoculated in a 180 mm test tube), stirred vigorously for 10 seconds, and cultured at 37 ° C. for 24 hours. The curd formation, lactic acidity and pH of the culture were measured, and at least those strains showing curd formation in the culture were defined as oxygen-resistant strains. Two oxygen-tolerant strains were obtained from 13 strains.

Next, a screening test for acid-tolerant strains was performed using the two oxygen-tolerant strains. That is, the oxygen-tolerant strain was cultured in Briggs River Broth, collected and washed, to prepare a bacterial cell solution having a concentration twice that of the stock solution. This bacterial cell solution was added to a 1/100 M acetate buffer (pH 18) (18 × 18).
0% test tube) and stored at 5 ° C. for 3 days.
Anaerobic culture was performed at 37 ° C. for 72 hours on an L-plate agar medium (manufactured by Nissui Pharmaceutical). The colonies that appeared by such culture were cultured in Briggs River Broth at 37 ° C. for 24 hours. Using the culture solution thus obtained, the preparation of the bacterial cell fluid, anaerobic culturing, and appearance of the colonies were repeated three times by culturing with Briggs River Broth, and the colonies formed on the BL plate agar medium in the final operation were repeated. Was used as acid-resistant bacteria. As a result, acid-fast bacteria 1
Got the strain. This strain was designated as Bifidobacterium breve SBR3212 strain and deposited with the Japan Institute of Fine Technology. The accession number of this bacterium was Microtechnological Laboratory No. 11915.

The bacteriological properties of the acid- and oxygen-tolerant strain of the present invention are as follows. 1. Taxonomic properties (1) Bacterial form (observation by light microscope) When anaerobic cultivation was carried out using a BL agar plate medium and a steel wool method at 37 ° C. for 48 to 72 hours. Size: 0.5 ± 0.3 × 1. 1 ± 0.5 μm Shape: Shows club-like or branched fungi (2) Gram stainability Positive or weakly positive when cultured under the same conditions as in (1) above (3) Colony morphology above (1) The morphology of the colony when cultured under the same conditions as above is as follows: Shape: Circular Ridge: Conical or convex Circumference Perimeter: Smooth Size: 1-3 mm Color Tone: White brown or reddish brown Table Surface: smooth and glossy (4) Sporulation: negative (5) Gas production: negative (6) Motility: negative (7) Catalase activity: negative (8) Milk coagulation: positive (9) Gelatin liquefaction: negative (10) Nitrate reduction: Negative (11) Indole production: Negative (12) Hydrogen sulfide production: Negative (13) Molar ratio of acetic acid / L (+) lactic acid: 1.7 ± 0.3

(14) Sugar fermentability The sugar was fermented according to the method of Mitsuoka [Tomota Mitsuoka: Clinical Examination, 18 , 1163-1172 (1974)]. Also, strains belonging to Bifidobacterium breve ATCC 15700 and J.
The same test was conducted for CM7016. Table 1 shows the results.

[0016]

[Table 1]

From the above properties, the SBR3212 of the present invention
Is Bergey's Manual of Systematic bacteriology (Will
iams & Wilkins, 1986) and the "World of Intestinal Bacteria" (Tomokazu Mitsuoka, Monobunsha, 1980) with reference to the classification criteria of Bifidobacterium breve.
ve). 2. Further, the SBR3212 strain of the present invention was subjected to a detailed comparison test of mycological properties with conventional Bifidobacterium breve. That is, SBR3212,
The test was performed by the following method using Bifidobacterium breve of ATCC15700, JCM7016, and four strains of isolate A from a commercial product.

[1] Comparative test of acid resistance (low pH buffer) (1) Method After culturing at 37 ° C. for 24 hours in Briggs River Broth, washing and collecting cells. The collected cells are made up to 4 times the concentration of the original bacterial solution with sterile physiological saline (containing 0.1% of L-cysteine hydrochloride and 0.1% of sodium thioglycolate). This bacterial solution was buffered at each pH (17 × 100 mm
5% dispensed into a Falcon tube)
And finally 3.8, 4.0, 4.3, 4.6,
Adjust to a pH of 5 steps of 5.0, store at 5 ° C,
The number of viable bacteria was measured over time. As the buffer, an acetate buffer of 1/100 mol of acetic acid / Na acetate was used. The number of viable bacteria was determined by the method of Mitsuoka [Clinical Inspection, 18 , 1163-1172 (1971).
According to 4)], the measurement was carried out by anaerobic culturing at 37 ° C. for 72 hours using a steel wool method using a BL plate agar medium to which no blood was added.

(2) Results The results are shown in Table 2. As is clear from Table 2, SBR32
No. 12 had higher survival than the other three strains at any pH, and the difference was remarkable especially in a low pH range of pH 3.8 to 4.3. In other words, SBR3212 is
37.69% at pH 4.3 when stored for days,
The other three strains show p.10% survival at pH 4.0 and 0.31% at pH 3.8.
At H4.3 or less, the survival rate was less than 1%.

[0020]

[Table 2]

[2] Comparative test of acid resistance (reduced skim milk) From the above test results, except for isolate A having poor survival, SBR
The test was carried out by the following method using Bifidobacterium breve of three strains, 3212, ATCC15700 and JCM7016. (1) Method 12% reduced skim milk containing 0.5% yeast extract (Difco) was sterilized at 115 ° C for 20 minutes, and 2% inoculated at 37 ° C.
After culturing for 18 hours, a starter was prepared. Next, 0.3
10% reduced skim milk containing 18% yeast extract (18 × 180 mm
10 ml dispensed into a test tube) is sterilized at 115 ° C for 20 minutes.
The starter was inoculated at 2% and cultured at 37 ° C.
When the culture reaches pH 4.6 and 4.1, quench and 5 ° C.
And the number of viable cells was measured over time.

(2) Results The results are shown in Table 3. From Table 3, SBR3212 is pH
At 4.6, the survival rate was almost the same as that of the other two strains on the seventh day. However, at pH 4.1, on the 7th day, 8.
The other two strains were less than 0.001%, while the survival rate was as high as 21%.

[0023]

[Table 3]

[3] Aerobic growth test SBR3212, ATCC15700, JCM7016
Using three strains of Bifidobacterium breve,
The test was performed by the following method. (1) Contents Each of the above strains was used as a starter and cultured at 37 ° C. for 18 hours in a 12% reduced skim milk (sterilized at 115 ° C. for 20 minutes) medium containing 0.5% yeast extract. 150 ml of 16% reduced skim milk was placed in a 300 ml Erlenmeyer flask, covered with a cotton stopper, sterilized at 115 ° C. for 20 minutes, and then stirred and cooled to 37 ° C. Thereafter, the above starter was inoculated at 2%, dispersed by stirring, and cultured at 37 ° C. to measure the number of viable bacteria, lactic acidity, and pH over time. In addition, the growth rate (%) = viable cell count at each time / initial viable cell count × 100 was calculated.

(2) Results The results are shown in Table 4. As is clear from Table 4, SBR32
In No. 12, the growth rate was 1000% or more after 24 hours of culture, and was maintained at 500% after 48 hours. on the other hand,
The other two strains did not show a growth rate of about 400% after 24 hours and 50% or less after 48 hours.

[0026]

[Table 4]

[0027]

The acid- and oxygen-tolerant strains of Bifidobacterium known to date include acid- and oxygen-tolerant strains of Bifidobacterium bifidum, oxygen-tolerant strains and acid-tolerant strains of Bifidobacterium breve. And acid-resistant or hydrogen peroxide-resistant strains of Bifidobacterium longum. However, a strain of Bifidobacterium breve having both acid resistance and oxygen tolerance characteristics was not known. Therefore, the present inventors have selected a strain excellent in aerobic growth among those isolated from the stool of a healthy breastfeeding infant, and further, a strain having the highest growth potential among those treated with a low pH buffer. SBR3212 was separated.

Further, a test for ascertaining the properties of the isolate SBR3212 was performed. The results were (1) identified as a strain belonging to Bifidobacterium breve from a taxonomic property test. (2) From a comparative test of acid resistance (low pH buffer solution), pH3.
At any pH of 8, 4.0, 4.3, 4.6, 5.0, the other three strains of B. breve (ATCC 15700,
JCM7016, which has higher viability than the commercial product isolate A),
In particular, when stored at 5 ° C. for 10 days, it shows a survival rate of 37.69% at pH 4.3, 7.10% at pH 4.0, and 0.31% at pH 3.8, whereas the other 3 The strain is p
At H4.3 or less, the survival rate was less than 1%. (3) In a comparative test of acid resistance (reduced skim milk), a test was performed except for a commercially available isolate A having poor survival from the control. From this test, at pH 4.6, there was almost no difference from the other two strains at 5 ° C. for 7 days. However, at pH 4.1, the other two strains showed a survival rate of less than 0.001% while showing a survival rate of 8.21% on the seventh day, showing a remarkable difference. (4) In the aerobic growth test, the growth rate became 1000% or more after 24 hours of culture, and was maintained at 500% after 48 hours. On the other hand, the other two strains did not show a growth rate of about 400% after 24 hours and 50% or less after 48 hours. From this, it is considered that there is growth in a reduced skim milk medium containing a large amount of dissolved oxygen. It became.

As described above, the SBR 3212 has (2)
In the low pH buffer and reduced skim milk culture of (3),
The survival rate is remarkably different from that of other B. breve, and it has excellent acid resistance. In addition, even in the aerobic growth of (4), the growth rate is different from the growth rate of other Bifidobacterium breve, and it has oxygen tolerance. This indicates that SBR3212 has excellent acid resistance and oxygen resistance, and is an excellent novel strain not described in the conventional literature.

In addition, the viable cell count during storage of a culture using this strain or a processed product thereof can be processed into food and drink in a wide pH range with a small decrease. For use in beverages and the like, a high viable cell count can be maintained. In addition, it can be used as an intestinal medicine. Furthermore, it can be added to feed.

[0031]

Example 1 Bifidobacterium breve SBR3212 starter and Lactobacillus casei which had been cultured in a stirred and cooled medium after sterilizing 18% reduced skim milk were sterilized.
Each starter was inoculated in an amount of 1% and cultured at 37 ° C. for 18 hours under aerobic conditions. 5000 ml of this culture
And syrup 5000 ml containing 800 g of sucrose were mixed and homogenized with a homogenizer to obtain fermented milk. Lactic acidity of the product immediately after production 0.80%, pH 4.30, viable bacterial count of Bifidobacterium breve SBR3212 2.0 × 10
⁹ / ml, viable bacterial count of Lactobacillus casei 6.5 x 1
0 ⁸ / ml, and the number of viable bacteria of Bifidobacterium breve SBR3212 was 5.3 × 10 ⁷ / m 7 days after storage.
1, viable cell count of Lactobacillus casei 5.8 × 10 ⁸ /
ml, Bifidobacterium breve SBR3
The survival rate of 212 was 2.7%.

[0032]

Example 2 After sterilizing 18% reduced skim milk, 2% of a previously cultured Bifidobacterium breve SBR3212 starter was inoculated into a stirred and cooled medium, and
The cells were cultured under aerobic conditions at 7 ° C. for 48 hours. Similarly, Lactobacillus casei starter was cultured at 37 ° C. for 48 hours. After culture, Bifidobacterium breve SBR
Syrup 50 containing 2500 ml of 3212 culture, 2500 ml of Lactobacillus casei culture and 800 g of sucrose
Then, the mixture was mixed with 00 ml, and after adding an acidulant, the mixture was homogenized with a homogenizer to obtain fermented milk. Lactic acidity of freshly manufactured product 0.7
At 3%, pH 4.54, the viable cell count of Bifidobacterium breve SBR3212 was 1.2 × 10 ⁸ / ml and the viable cell count of Lactobacillus casei was 7.8 × 10 ⁸ / ml. Bifidobacterium breve SB
The viable cell count of R3212 was 1.1 × 10 ⁷ / ml, the viable cell count of Lactobacillus casei was 6.3 × 10 ⁸ / ml, and the survival rate of Bifidobacterium breve SBR3212 was 9.2%. .

[0033]

Example 3 Polypeptone 40 g, meat extract 20
g, yeast extract 40g, lactose 120g, KH ₂ PO _{4 2}
0 g, and K ₂ HPO ₄ 4g, of 4000ml containing sodium L- ascorbate 2g media were filter sterilized. Then, 200 ml of a seed culture of Bifidobacterium breve SBR3212 previously cultured at 37 ° C. for 18 hours in a medium having the same composition was inoculated into the above-mentioned culture solution.
The cells were cultured at 7 ° C for 18 hours. The number of viable bacteria after culture was 3.2 ×
It was 10 ⁹ / ml. Then, the cells were collected by a centrifugal separator, resuspended in sterilized physiological saline at the same amount as the medium at 95 ° C. for 30 minutes, and centrifuged again to collect the cells. The obtained cells were suspended in 1000 ml of a dispersion containing 100 g of skim milk powder sterilized at 95 ° C. for 30 minutes and 10 g of sodium glutamate, and freeze-dried. 113 g of the obtained powder,
The number of viable bacteria was 1.0 × 10 ¹¹ / g.

────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Kiyotaka Shiraiwa 1-6-6 Shinooka, Komaki-shi, Aichi Prefecture Prefectural Shinooka Residential Building No. 501 501 (72) Inventor Shigeyuki Mitsuhashi 4-3-47 Nishiharacho, Tanashi-shi, Tokyo Nishihara Green Heights 7-302 (56) References JP-A-61-205481 (JP, A) JP-A-53-109961 (JP, A) JP-B-56-42250 (JP, B1) (58) Fields investigated (Int.Cl. ⁶ , DB name) A23C 1/00-23/00 C12N 1/20

Claims (1)

(57) [Claims] 1. Bifidobacterium breve S having high acid resistance and oxygen tolerance
BR3212 (Microbial Lab. No. 11915).