KR101995705B1 - A conjugated linoleic acid production method from linoleic acid by combination of Bifidobacterium breve LDTM8001(KCTC 18423P) and Bifidobacterium breve LDTM 8002(KCTC 18424P) - Google Patents

Abstract

The present invention relates to a conjugate for increasing the content of conjugated linolenic acid contained in dairy products so that dairy products produced in dairy cows can be recognized as functional foods having functions such as antioxidative effect, immunity enhancement effect, body fat reduction effect, Bifidobacterium breve, which produces linolenic acid LDTM8001 (KCTC 18423P) and Bifidobacterium breve (KCTC 18424P) and Bifidobacterium breve LDTM8001 (KCTC 18423P) and Bifidobacterium breve LDTM 8002 (KCTC 18424P) in combination to convert linoleic acid to conjugated linoleic acid Can be provided.
Bifidobacterium breve, which produces the conjugated linolenic acid according to the present invention The new strain of LDTM 8001 (KCTC 18423P) is characterized by being a Bifidobacterium strain isolated from the feces of infants with an age of 14 days or more but less than 100 days after birth.

Description

A method for converting linoleic acid into conjugated linoleic acid by a combination of Bifidobacterium breve LDTM8001 (KCTC 18423P) and Bifidobacterium breve LDTM 8002 (KCTC 18424P), and a method for producing a conjugated linoleic acid from linoleic acid by combination of Bifidobacterium breve LDTM8001 (KCTC 18423P) and Bifidobacterium breve LDTM 8002 (KCTC 18424P)}

TECHNICAL FIELD The present invention relates to a method for producing a conjugated linoleic acid, and more particularly, to a method for producing a conjugated linoleic acid-producing microorganism capable of producing a conjugated linoleic acid- Which is required to increase the content of the conjugated linoleic acid contained in the culture medium.

In general, conjugated linolenic acid (CLA) has been known to have functions such as anti-cancer effect, antioxidant effect, immunity enhancement effect, body fat reduction effect, cholesterol reduction, diabetes suppression and protein maintenance effect. The conjugated linoleic acid is an unsaturated fatty acid having 18 carbon atoms and having two double bonds, such as linoleic acid, which is an essential fatty acid. The linoleic acid has a single bond between carbon number 10 and carbon number 11, carbon number 11 and carbon number 12, and a double bond between carbon number 9 and carbon number 10 and between carbon number 12 and carbon number 13. Linoleic acid has two single bonds between two double bonds, whereas conjugated linoleic acid shifts the position of double bonds, forming a conjugated double bond with only one single bond between two double bonds.

The conjugated linoleic acid which forms a conjugated double bond may exist in a total of 8 types because there are cis type and trans type geometric isomers and positional isomers in which the position of double bond of linoleic acid is changed. Among them, there is a cis-shaped double bond between carbon number 9 and carbon number 10, a trans-type double bond between carbon number 11 and carbon number 12 (9cis, 11trans), and trans between carbon number 10 and carbon number 11 (10trans, 12cis) with a cis-double bond between carbon number 12 and carbon number 13 is known to have high physiological activity.

In general, trans fatty acids are single unsaturated fatty acids with only one double bond. The conjugated linoleic acid also contains a trans-type double bond, which is why the action in the body is wrong because it is co-conjugated. In conjugated linoleic acid, two pairs of double bonds are mutually influenced, resulting in a marked difference in physical and chemical properties from the independently existing double bonds. Therefore, conjugated linoleic acid has a different effect from linoleic acid, which is a non-conjugated double bond, as well as a trans fatty acid having only one double bond.

While these conjugated linoleic acids are found in a variety of foods, the main dietary source is ruminant tissue and milk. The conjugated linoleic acid is produced as an intermediate product of the hydrolysis of linoleic acid by Butyrivibrio fibrisolvens bacteria in the ruminant stomach, which is the digestive tract of the cow. Recently, There is also a possibility that it can be changed. Profiles of conjugated linoleic acid isomers found in ruminant tissues or dairy products (dairy products) vary, but are known to be particularly dominant, with cis-9 and trans-11 isomers accounting for more than 90%.

The conjugated linoleic acid is an alkaline isomerization method in which caustic soda (NaOH) is added to a safflower seed oil containing a large amount of linoleic acid and then reacted at a temperature as high as about 200 ° C, a method in which an anaerobic microorganism produces a linoleic acid isomerase It can be produced by an artificial method such as isomerization of linoleic acid which is a substrate. The content of conjugated linoleic acid present in milk is from about 2.5 mg / g to about 6.0 mg / g, which is 0.3% to 0.6% of the milk fat. However, the content of the conjugated linoleic acid is considered to be a functional food that can show the effect of conjugated linoleic acid.

Therefore, the development of an excellent conjugate linoleic acid-producing strain capable of enhancing the content of conjugated linoleic acid in milk produced by dairy cows is expected to greatly contribute to the revitalization of the dairy market and the economic profit of dairy farmers.

The present invention relates to a method of converting a linoleic acid into a conjugated linoleic acid in combination with Bifidobacterium breve LDTM8001 (KCTC 18423P) and Bifidobacterium breve LDTM 8002 (KCTC 18424P) to produce an antioxidative effect, The purpose of this study is to solve the problem of increasing the content of conjugated linoleic acid in dairy products to be recognized as a functional food having functions such as body fat reduction effect and body protein maintenance effect.

The present invention relates to Bifidobacterium breve LDTM8001 (KCTC 18423P) and Bifidobacterium breve LDTM 8002 (Bifidobacterium breve LDTM 8002) isolated from infant feces with no antibiotic resistance to ampicillin, gentamicin and streptomycin for more than 14 days and less than 100 days KCTC 18424P) are combined and cultivated to convert linoleic acid to conjugated linoleic acid.

Bifidobacterium breve LDTM8001 (KCTC 18423P) and Bifidobacterium breve LDTM 8002 (KCTC 18424P) according to the present invention can be cultivated in combination to convert linoleic acid to conjugated linoleic acid in an excellent manner.

The combination of these strains can support the production of meat-functional milk with more abundant conjugated linoleic acid compared to ordinary milk.

Figure 1 is a screening of Bifidobacterium isolated for the production of conjugated linoleic acid.
Fig. 2 is a graph showing the results of the measurement of the concentration of the Bifidobacterium buret LDTM8001 (KCTC 18423P) fresh strain by gas chromatography, the Bifidobacterium breve LDTM 8002 (KCTC 18424P) strain, LA-5 strain alone or in combination.
Fig. 3 shows the distribution pattern of the Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain of Bifidobacterium strain according to the present invention.
Fig. 4 is a graph showing the effect of the Bifidobacterium breve strain LDTM 8002 (KCTC 18424P) shows the distribution pattern of the new strain.
FIG. 5 shows a standard curve of a new strain of Bifidobacterium breve LDTM8001 (KCTC 18423P) according to the present invention.
FIG. 6 shows the results of measuring the acid tolerance of the new strain of Bifidobacterium breve LDTM8001 (KCTC 18423P) according to the present invention measured in MRS broth.
FIG. 7 shows the results of measuring the tolerance of the new strain of Bifidobacterium breve LDTM8001 (KCTC 18423P) according to the present invention.

The present invention relates to a conjugate linoleic acid-producing strain,

(KCTC 18423P), which is isolated from the feces of an infant with a birthday of 14 days or less but less than 100 days after birth. Hereinafter, the Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain of the present invention, Bifidobacterium breve LDTM 8002 (KCTC 18424P) The process of selecting a new strain will be described in detail. First, the Bifidobacterium strain according to the present invention is characterized by being a Bifidobacterium strain isolated from the feces of infants and young children of 14 days or more after birth but less than 100 days after birth.

As a method for obtaining such strains, the inventors of the present invention used healthy infant feces of 14 days or more but less than 100 days. All fecal samples were kept refrigerated immediately after collection and transferred to the laboratory to conduct experiments for the isolation of Bifidobacterium strains within 12 hours after discharge from the infant's body. The experimental procedure is as follows. First, the feces are uniformly mixed and then diluted to 10 ⁶ to 10 ⁸ CFU / ml using an anaerobic microbial dilution. Thereafter, 100 μl of a sample diluted on a pre-prepared TOS agar plate is plated and cultured for 37 to 48 hours using an anaerobic incubator. Then, the isolated bifido bacterium is enriched with two or more subcultures and cultured at 37 ° C for 18 to 24 hours using Modified MRS broth. Then, the selected strains were subcultured in MRS broth, centrifuged to recover the cells, mixed with glycerol-containing MRS broth, and each ml was dispensed and stored frozen at -70 ° C .

Hereinafter, the present invention will be described in more detail by way of examples, with reference to the Bifidobacterium breve LDTM8001 (KCTC 18423P) fresh strain, Bifidobacterium breve Newer strains of LDTM 8002 (KCTC 18424P) will be described in more detail.

Example 1. Bifidobacterium breve LDTM8001 (KCTC 18423P) New strain, Bifidobacterium breve Identification of a new strain of LDTM 8002 (KCTC 18424P)

The isolated Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve To identify the new strain of LDTM 8002 (KCTC 18424P), 16s rRNA sequencing was commissioned to Macrogen, a sequencing company, and the results are shown in Tables 1 and 2.

designation

Sequencing results
LDTM 8001
Bifidobacterium breve ACS-071-V Sch8b,

complete genome
LDTM 8002
Bifidobacterium breve S27,

complete genome

LDTM 8001-F GGGGGGCAGGTATCGGAATATTGGGGCGTAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGACCGTAGAGATACGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGTGCGAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCCGTCTAAGGTGAGGCTCGTGATTGGACTAATCAAGGGGGGGGGCCCGCCAACATATAAAAACCGG


LDTM 8001-R CCCCTCTTTTCGCTCTCAGCGTCAGTAACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCGAAAGGTACACTCAACACAAAAATGCCTTGCTCCCTAACAAAAGAGGTTTACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGAAGCCATGGTGGGCCGTTACCCCGCCATCAAGCTGATAGGACGCGACCCCATCCCATGCCGCAAAGGCTTTCCCAACACACCATGCGGTGTGATGGAGCATCCGGCATTACCACCCGTT

LDTM 8002-F GGGGGGTACTTATCCGGAATTATTGGGCGTAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGACCGTAGAGATACGGTTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGTGCGAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCCGTCTAAGGTGAGGCTCGTGATGGACTATACGGAGGGG



LDTM 8002-R GCGCTCTTTTCGCTCCTCAGCGTCAGTAACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAACGCTTGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCGAAAGGTACACTCAACACAAAAATGCCTTGCTCCCTAACAAAAGAGGTTTACAACCCGAAGGCCTCCATCCCTCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGAAGCCATGGTGGGCCGTTACCCCGCCATCAAGCTGATAGGACGCGACCCCATCCCATGCCGCAAAGGCTTTCCCAACACACCATGCGGTGTGATGGAGCATCCGGCATTACCACCCGTTTCCAGGAGCTATTCCGGTGCATGGGGCAGGTCGGTCACGCATTACTCACCCGTTCGCCACTCTCACCACCAGGCAAAGCCCGATGGATCCCGTTCGACTTGCAATGTGTTAAGCACGCCGCCAGCGTTCATCCTGACGGGAAAAAACATAAAAAAAAGGGCCCCCGAAAA

Example 2: Spectrophotometric assay

Firstly, using a spectrophotometer, the above-mentioned Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve It was confirmed whether or not the new strain of LDTM 8002 (KCTC 18424P) produced conjugated linoleic acid, and the new strain of Bifidobacterium breve LDTM8001 (KCTC 18423P), Bifidobacterium breve LDTM 8002 (KCTC 18424P) Screening of the optimal medium composition for the production of conjugated linoleic acid by the new strain. In this experiment, the Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve LDTM 8002 (KCTC 18424P) The experiment was conducted including LA-5, a strain other than the new strain. The Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve The new strain of LDTM 8002 (KCTC 18424P) and the LA-5 were cultured in a modified MRS broth containing 2% (w / v) Tween 80 and 0.1% and 0.2% (w / v) linoleic acid 1% (v / v), and then cultured in an anaerobic chamber at 37 ° C for 24 hours. Then, the cultured culture was centrifuged at 4,000 g for 10 minutes, and 1 ml of chloroform and 2 ml of methanol were mixed with 10 ml of the supernatant liquid except for the pellet, followed by vortexing for 5 minutes. Respectively. At this time, 100 μl of a chloroform layer extracted with fatty acid and 900 μl of methanol were mixed, and the absorbance was measured at 233 nm using a spectrophotometer.

As a result, as shown in Fig. 1, a new strain of Bifidobacterium breve LDTM8001 (KCTC 18423P), Bifidobacterium breve All of the new strains of LDTM 8002 (KCTC 18424P) were identified as producing conjugated linoleic acid, and it was confirmed that the conjugate linoleic acid content of the mMRS broth containing 0.2% LA was higher than that of mMRS broth containing 0.1% LA I could.

Example 3. Fatty acid analysis

The Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve Inoculated LDTM 8002 (KCTC 18424P) fresh strain and LA-5 to a total inoculum of 1% (v / v) in modified MRS broth containing 0.2% linoleic acid and 2% (w / v) Tween 80 , Mixed culture was carried out under seven conditions, and cultured in an anaerobic chamber at 37 ° C for 24 hours. 1, 2, 2, 2, and 4, respectively. The strains were grown in a medium containing 0.2% LA, 2% tween 80 in mMRS medium, 2 LDTM8001, 3 LDTM8002, 4 LA-5, 5 LDTM8001 + LDTM8002, 7, LDTM8002 + LA-5, and 8, LDTM8001 + LDTM8002 + LA5, respectively.

1 ml of the culture was placed in a tube and 100 μl of C13: 0 stock dissolved in methanol at a ratio of 100: 1 was added. Then, 700 μl of 10 N KOH and 5.3 ml of methanol were mixed, Lt; / RTI > for 90 minutes. After 90 min, 580 μl of 24N H ₂ SO ₄ was added and reacted at 55 ° C for 90 minutes. After 90 minutes, 2 ml of hexane was added and vortexed for 5 minutes, followed by centrifugation at 3,000 rpm for 10 minutes. The centrifuged supernatant was then transferred to a GC vial and analyzed using GC.

As a result, as shown in Fig. 2, the Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve The new strain of LDTM 8002 (KCTC 18424P) was found to convert linoleic acid to conjugated linoleic acid. As shown in the drawing, No. 2 of the experiment was inoculated with DTM8001 strain, No. 3 was inoculated with LDTM 8002, No. 4 was inoculated with LA-5, and No. 6 was inoculated with LDTM8001 + LA-5 , LDTM8002 + LA-5 strain, and LDTM8001 + LDTM8002 + LA5 strain, respectively. In comparison with these individual strains or combination strains, LDTM8001 + LDTM8002 strain 5 The conjugated linoleic acid CLA production was particularly effective when inoculated with the combination of the two strains. The CLA production capacity was significantly higher than that of using only the LDTM8002 strain No. 3 and only using the LDTM8001 strain No. 2, It is confirmed that it brings great synergy effect.

CLA LA One 11,732 238,737 2 13,487 257,928 3 45,739 284,886 4 11,250 246,102 5 92,879 255,713 6 13,275 268,209 7 13,232 272,337 8 12,936 268,240

1: 0.2% LA, 2% tween 80 in mMRS, 2: LDTM 8001, 3: LDTM 8002, 4: La-5, 5: LDTM 8001 + LDTM 8002, 6: LDTM 8001 + La- + La-5, 8: LDTM 8001 + LDTM 8002 + La-5

Example 4. Observation of morphological characteristics of bifidobacteria using optical microscope

In order to confirm the morphological characteristics of the strain, general optical microscope was used. The strain cultured anaerobically for 48 hours at 37 ° C in BL broth was plated on a slide glass and subjected to heat fixation for 1 minute. Then, the cells were stained with crystal violet for 1 minute and then washed with sterilized water. Thereafter, the cells were fixed with iodine for 30 seconds, decolored with ethyl alcohol at 95% concentration, stained with safranin for 1 minute, and observed under a general optical microscope. 8001 (KCTC 18423P) and the LDTM8002 (KCTC18424P) as shown in Fig. 4 were observed.

Example 5. Physiological characteristics on the ability to utilize Bifidobacterium carbohydrate

The Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve A new strain of LDTM 8002 (KCTC 18424P) and API 50 CHL were used to confirm the carbohydrate availability of the LA-5. The separated samples were incubated subculture twice in BL broth to increase viability, and then the cells were recovered by centrifugation at 15,000 × g for 3 minutes. The recovered cells were washed twice with 0.85% NaCl solution, and the turbidity was adjusted to 4 Macfarland. One ml of the suspension was added to 9 ml of BCP medium, and then 130 μl of each was added to each cuff. The completed kit was incubated for 48 hours at 37 ° C using an anaerobic incubator.

Example 6 Antibiotic resistance evaluation

The culture broth was diluted in BL broth to a Macfarland 1 to 2, an OD ₆₀₀ of 0.3, and then plated on a BL agar medium evenly with a sterile cotton swab. Thereafter, 1 μg / ml of ampicillin, 2 μg / ml of ampicillin, 4 μg / ml of ampicillin, 32 μg / ml of gentamicin, 64 μg / ml of gentamicin, 128 μg / ml of gentamicin, streptomycin ), 64 μg / ml of streptomycin, 128 μg / ml of streptomycin and 256 μg / ml of streptomycin, respectively.

As a result, as shown in Table 3, Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve The new strain of LDTM 8002 (KCTC 18424P) showed no antibiotic resistance to ampicillin, gentamicin, or streptomycin.

Ampicillin
(Ampicillin)

Gentamicin
(Gentamicin)
Streptomycin
(Streptomycine)
Con.
(占 퐂 / ml)
One

2
4
32
64
128
64
128
256
LDTM 8001

-
-
-

-
-
-
-
-
-
LDTM 8002

-
-
-
-
-
-
-
-
-

Example 7. Confirmation of selected lactic acid bacteria strain LDTM 8001 (KCTC 18423P) strain

The final selected strains were subcultured in BL broth, and then 1% (v / v) of the culture was inoculated (where) and incubated for 24 hours and measured at absorbance at 600 nm. As shown in FIG. 5, the growth curve and the number of bacteria were measured. The colony forming unit (CFU / ml) according to absorbance, A standard curve was calculated to estimate the value. Then, in order to confirm the growth of bacteria according to the pH change of the culture medium, each BL broth adjusted to a pH value of 2 to 6 was prepared, and 1% of the culture broth was inoculated and cultured for 24 hours. Respectively. As a result, as shown in FIG. 6, it was confirmed that the CFU value decreased as the pH was lowered. Then, the isolate culture broth was inoculated 1% in each broth supplemented with 1.25%, 2.5%, 5%, and 10% oxgall, cultured for 24 hours, diluted with 0.85% NaCl solution and counted. As a result, as shown in FIG. 7, when the bile broth is contained in the BL broth, the CFU value decreases and increases with the increase of the bile acid concentration. However, when the bile acid concentration reaches 10% Respectively.

Example 8. Statistical Analysis

The results of this study were analyzed by ANOVA using SPSS 20.0 (SPSS INC., Illinois, USA). The significance of the treatment was verified by Duncan's multiple range test and it was judged to be statistically significant when p <0.05 . All analyzes were repeated 8 times or more, and the results were expressed as mean ± standard deviation. The embodiments described above are provided by way of example for the purpose of enabling a person skilled in the art to sufficiently transfer the technical idea of the present invention to a person skilled in the art, But may be embodied in other forms without limitation.

Bifidobacterium breve LDTM8001 (KCTC 18423P) new strain, Bifidobacterium breve New strain of LDTM 8002 (KCTC 18424P) was deposited with the Korea Biotechnology Research Institute.

Korea Biotechnology Research Institute KCTC18423P 20151109 Korea Biotechnology Research Institute KCTC18424P 20151109

Claims (4)

Bifidobacterium breve LDTM8001 (KCTC 18423P) and Bifidobacterium breve LDTM 8002 (KCTC 18424P) isolated from the infant feces of 14 days or more and less than 100 days after birth were cultured in combination, and linoleic acid was conjugated Linoleic acid.
(a) No antibiotic resistance to ampicillin, gentamycin, streptomycin. delete delete delete

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