KR20100135612A - Method for production of conjugated linolenic acid using bifidobacterium breve lmc520 strain - Google Patents

Abstract

PURPOSE: A method for producing conjugated linoleic acid(CLN) using Bifidus sp. strain is provided to naturally CLN and to enhance functionality of milk product. CONSTITUTION: A conjugated linoleic acid(CLN) is produced by culturing Bifidobacterium breve LMC520 (deposit number: KCTC 10455BP) in a medium containing linoleic acid. The linoleic acid is alpha-linoleic acid containing non-conjugated double bond at cis-9, cis-12, and cis-15. A fermented milk containing CLN is produced by culturing Bifidobacterium breve LMC520 in a milk or skim milk containing linoleic acid.

Description

Method for production of conjugated linolenic acid using Bifidobacterium breve LMC520 strain}

The present invention relates to a method for producing conjugated linolenic acid (CLN) using a bifidus strain, and more particularly, by culturing the Bifidobacterium breve LMC520 strain, a lactic acid bacterium isolated from the human body, in a medium containing linolenic acid, The present invention relates to a method for producing conjugated linolenic acid using Bifidobacterium breve LMC520 strain which is converted to conjugated linolenic acid (CLN).

Probiotic is defined as "a living microbial additive that has a beneficial effect by enhancing the balance of intestinal microflora in the host." These probiotics are getting more and more attention in terms of nutrition as scientific evidences about their benefits, functionality, and traits are promoted in health promotion.

Among these probiotics, Bifidobacteria have unique human benefits, such as mineral utilization, enhanced vitamin synthesis, enhanced immunity, reduced gastrointestinal disorders and cancer. In addition, some Bifidobacterium breves have been reported to produce conjugated linoleic acid (CLA), a functional fatty acid.

Recently, however, several unique pharmacological effects have been found in CLA as well as in conjugated linolenic acid (CLN). CLN is a positional isomer and geometric isomer of unsaturated fatty acids with three conjugated double bonds, which are produced a lot in some plant seed oils. Funninic acid (cis-9, trans-11, cis-13-CLN) is present in about 72% pomegranate seed oil, and α-eleostearic acid (cis-9, trans-11, trans-13-CLN) 60% and 70% of bitter gourd and tung seed oils, respectively. In addition, the catalpa seed oil contains about 31% of catalic acid (trans-9, trans-11, cis-13-CLN), and the pot marigold seed oil contains carrenic acid (trans-8, trans-10). Cis-12-CLN). There is considerable research showing that a mixture of α-linolenic acid (α-LNA) or CLN isomers produced by alkali isomerization of seed oil has some pharmacological function in anticancer activity and body fat reduction. In addition, Bitter God seed oil enriched with purified α-Eleostearic acid and α-Eleostearic acid showed anticancer activity in vitro and other studies showed that the feeding of pomegranate seed oil containing purinic acid in rat fat tissue cells Has been reported.

CLN is known to be abundant in plant seed oil, but its effectiveness is very limited. Thus, CLN, which is used as only a few nutritional supplements, is produced through organic synthesis, but has not been commercialized yet. In addition, CLN contained in organically synthesized CLN and plant seed oil has a variety of unidentified isomers and requires the selection of safe isomers for use in pharmaceutical or health foods.

The present invention devised to solve the problems of the prior art relates to a method for producing conjugated linolenic acid (CLN), and more specifically, Bifidobacterium breve which is a lactic acid bacterium isolated from the human body in a medium containing linolenic acid. Conjugated linolenic acid using Bifidobacterium breve LMC520 strain which can enhance the functionality of dairy products by enhancing conjugated linolenic acid when used as starter in dairy products by culturing LMC520 strains naturally It is an object of the present invention to provide a method for producing the same.

In one embodiment, the present invention provides a method for producing conjugated linolenic acid by culturing Bifidobacterium breve LMC520 strain (KCTC 10455BP) in a medium comprising linolenic acid.

In the present invention, the linolenic acid is an unsaturated fatty acid having three double bonds and a carboxylic acid, and is a colorless, odorless liquid present as a glycerin ester in vegetable dry oil. The linolenic acid is largely divided into α-linolenic acid and γ-linolenic acid. α-linolenic acid is called (Z, Z, Z) -9,12,15-octadecatrienoic acid, which is an essential fatty acid that is not synthesized in the body and should be consumed as a food. It is found in vegetable oils such as walnut oil. In addition, γ-linolenic acid is called (Z, Z, Z) -6,9,12-octadecatenoic acid, and is naturally contained in evening primrose, blackcurrant seed oil, borage oil, etc. It is known to be essential for in vivo synthesis of prostaglandin, which is effective in lowering blood cholesterol levels.

In the present invention, all linolenic acid such as α-linolenic acid or γ-linolenic acid may be used as the linolenic acid, but it is preferable to use α-linolenic acid having a non-conjugated double bond in the cis-9, cis-12, cis-15 positions. Most preferred.

In addition, in the present invention, conjugated linolenic acid (CLN) refers to a series of positional and conformational isomers of all linolenic acids having conjugated double bonds in cis or trans configuration. These CLNs take a cis or trans configuration at positions 9, 11, 15, 9, 11, 13 or 8, 10, 12 and have conjugated double bonds at these positions.

In one embodiment of the present invention, using linolenic acid α- linolenic acid to cis-9, trans-11, cis-15-CLN containing a conjugated double bond in the cis-9, trans-11, cis-15 position Switch. Its structure can be represented by the following formula (1) or (2).

As another aspect, the present invention provides a method for producing a fermented milk containing CLN by culturing the Bifidobacterium breve LMC520 strain (KCTC 10455BP) in milk or skim milk containing linolenic acid.

In the present invention, the fermented milk has a meaning of general fermented milk, and means fermented crude milk or dairy products with lactic acid bacteria, yeast, etc. Can be. The types of fermented milk are classified into liquid fermented milk and rich fermented milk, which are classified into liquid fermented milk in the case of 3.0% or more and rich fermented milk in the case of more than 3.0% according to the non-fat solid content. In addition, rich fermented milk is subdivided into a staple-type yogurt made by putting fruit and a drink-type yogurt made by drinking juice.

As another aspect, the present invention provides a fermented milk containing CLN prepared by culturing Bifidobacterium breve LMC520 strain (KCTC 10455BP) in milk or skim milk containing linolenic acid.

Therefore, the method of producing CLN using the Bifidobacterium breve LMC520 strain of the present invention has the effect of naturally producing CLN by using the Bifidobacterium breve LMC520 strain, which is isolated from the human body.

In addition, the production method of CLN using the Bifidobacterium breve LMC520 strain of the present invention by producing a CLN naturally using the Bifidobacterium breve LMC520 strain, which is isolated from the human body, as a starter in dairy products In use, it has the effect of promoting the functionality of dairy products by promoting CLN.

The terms or words used in this specification and claims are not to be construed as being limited to their ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to best describe their invention. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that the present invention.

Therefore, the embodiments described in the specification and the drawings shown in the drawings are only the most preferred embodiment of the present invention and do not represent all of the technical idea of the present invention, various modifications that can be replaced at the time of the present application It should be understood that there may be equivalents and variations.

Example 1 Microbial Production of CLN

In this example, the Bifidobacterium breve LMC520 strain (KCTC 10455BP) having the highest CLN production activity among the screened Bifidobacteria was used.

For the production of CLN, the strain was 0.05% L − in a Belco tube (18 × 150 mm; Bellco, Vineland, NJ, USA) covered with septum stopper (Bellco) and aluminum seal (Bellco). MRS medium containing cysteine-HCl (mMRS; Sigma) was subcultured twice for 18 hours at 37 ° C. under CO ₂ without O ₂ . The activated culture is transferred to new mMRS and then cultured under different substrate concentration conditions, i.e., α-linolenic acid (α-LNA) concentration conditions of 1, 2, 4, 6, 8 and 10 mM, respectively. It was. Growth rate was evaluated by measuring optical density (OD) using a microplate reader (BIO-RAD, Hercules, CA, USA) at 600 nm.

Example 2 Fatty Acid Analysis of Strain Cultures

In order to extract fatty acids from the strain culture of Example 1 in which CLN was produced, 1 ml of the culture to which heptadecanoic acid (C _{17: 0} ) was added as an internal standard (IS) material was added to 12 ml of chloroform /. Extracted with methanol (1: 1, v / v). The bottom layer of the extract was vigorously mixed with 2 ml of 0.88% KCl solution and then evaporated to nitrogen until dry.

The extracted lipids were ethyl esterified with 2% H ₂ SO ₄ in 10 ml of ethanol at 80 ° C. for 60 minutes. After addition of 8 ml of saturated NaCl solution and 4 ml of n-hexane, fatty acid ethyl ester was obtained in the n-hexane layer and 7890A gas chromatography using flame ionization detector (Agilent Technologies) Total fatty acids including CLN isomers were analyzed by chromatography (GC).

The fatty acid ethyl ester was a Supelcoax-10 fused silica capillary column, 100 m × 0.25 mm id, 0.2 μm film thickness at 1.2 mL / min helium airflow; Supelco, Inc., Bellefonte, PA , USA). The temperature of the oven was increased from 190 ° C. to 240 ° C. at a rate of 4 ° C./min. The temperature of the injector and detector was both 260 ° C. 1 μl of sample was injected into the column in split mode (50: 1).

Peaks of each CLN isomer and other fatty acids were identified and quantified by comparing the retention time and peak area of each fatty acid standard. Internal standards were added as internal reference materials prior to extraction to determine the recovery of fatty acids in each sample.

The spectral results analyzed by the GC are shown in FIGS. 1A and 1B.

Figure 1a shows the spectral results before addition and incubation of the α-LNA to the strain culture, the rightmost peak was the peak of the α-LNA.

Figure 1b shows the spectral results after the production of CLN by culturing by adding α-LNA to the Bifidobacterium breve LMC520 strain (KCTC 10455BP) culture according to the present invention. Three hours after CLN production began, some α-LNA remained because the conversion was not maximum yet, and the rightmost peak was the CLN peak.

After the GC measurement, the obtained fatty acid ethyl ester was analyzed for molecular weight through GC / MS.

In the analysis, a DB-23 column (60 m × 0.25 mm id, 0.25 μm) was used as the helium stream at 1.0 mL / min. It was increased from 150 ° C. to 200 ° C., then from 200 ° C. to 230 ° C. at a rate of 2 ° C./min and held at 230 ° C. for 5 minutes. The injector temperature was 250 ° C. and the detection temperature (Aux temp.) Was 280 ° C. The split ratio was 2: 1.

2 shows the spectral results of the CLN produced from the Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention by GC / MS.

In Figure 2, peak 1 is 9, 11, 15-Octadecatrienoic acid ethyl ester (linolenic acid ethyl ester, C _{18: 3} , Mw 306, 9, 11 , 15-CLN), peaks 2 and 3 are 9, 12-octadecadienoic acid ethyl ester (linoleic acid ethyl ester, C _{18: 2).} , Mw 308), peak 4 corresponds to the peak of 9-Octadecenoic acid ethyl ester (ethyl oleate, C _{18: 1} , Mw 310), 5 Burn peak corresponds to the peak of Octadecanoic acid ethyl ester (Stearic acid ethyl ester, C _{18: 0} , Mw 312), peak 6 is Hexadecanoic acid ethyl ester Corresponds to the peak of (C _{16: 0} , Mw 284).

Therefore, as can be seen from the results of FIG. 2, CLN produced from the Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention, 9, 11, 15-CLN isomers of the CLN isomers are the main Isomers (> 90%) and no other CLN isomers were produced.

Example 3 Structural Analysis of Fatty Acids Isolated from Strain Cultures

In order to clarify the structure of the fatty acid obtained from the strain culture of Example 1, ¹ H-NMR, ¹³ C-NMR, gCOSY and gHMBC with a nuclear magnetic resonance spectrometer (400 MHz NMR, Inova AS400, Varian, CA, USA) Measured. CDCl ₃ was used as the NMR solvent. The results are shown in Table 1 below.

number δ _H ^a δ _C ^b gHMBC ^c One - 179.9 2 2.32 (t, J = 7.2 Hz, 2H) 34.1 H2↔C3 ( J ₂ ), H2↔C1 ( J ₂ ) 3 1.61 (tt, J = 7.2, 7.2 Hz, 2H) 24.7 H3↔C1 ( J ₃ ), H3↔C2 ( J ₂ ) 4
1.20-1.38 (m, 8H) 29.1 5 29.2 6 29.7 7 31.7 8a 2.13 (tdd, J = 7.2, 14.8, 10.8 Hz, 1H) 27.7 H8↔C10 ( J ₃ ) 8b 2.14 (tdd, J = 7.2, 6.8, 10.8 Hz, 1H) 9 5.33 (ddd, J = 7.2, 14.8, 10.8 Hz, 1H) 133.8 H9↔C11 ( J ₃ ), H9↔C8 ( J ₂ ),
H9↔C10 ( J ₂ ) 10 5.92 (dd, J = 10.8, 10.8 Hz, 1H) 128.5 H10↔C12 ( J ₃ ), H10↔C8 ( J ₃ ),
H10↔C11 ( J ₂ ), H10↔C9 ( J ₂ ) 11 6.29 (dd, J = 14.8, 10.8 Hz, 1H) 125.8 H11↔C13 ( J ₃ ), H11↔C9 ( J ₃ ),
H11↔C12 ( J ₂ ), H11↔C10 ( J ₂ ) 12 5.64 (dt, J = 14.8, 7.2 Hz, 1H) 130.1 H12↔C14 ( J ₃ ), H12↔C10 ( J ₃ ),
H12↔C13 ( J ₂ ), H12↔C11 ( J ₂ ) 13 2.12 (td, J = 7.2, 7.2 Hz, 2H) 33.0 H13↔C11 ( J ₃ ), H13↔C15 ( J ₃ ) 14 2.03 (td, J = 7.2, 7.2 Hz, 2H) 27.1 H14↔C12 ( J ₃ ), H14↔C16 ( J ₃ ) 15 5.29 (td, J = 7.2, 10.8 Hz, 1H) 128.1 H15↔C17 ( J ₃ ), H15↔C13 ( J ₃ ) 16 5.38 (td, J = 7.2, 10.8 Hz, 1H) 132.0 H16↔C18 ( J ₃ ), H16↔C14 ( J ₃ ) 17 1.24 (qt, J = 7.6, 7.2 Hz, 2H) 22.7 H17↔C15 ( J ₃ ), H17↔C18 ( J ₂ ) 18 0.86 (t, J = 7.6 Hz, 3H) 14.2 H18↔C17 ( J ₂ ), H18↔C16 ( J ₃ )

^{A 1} H-NMR (400MHz)

^{b 13} C-NMR (100 MHz)

^{c 1} H- ¹³ C gradient heteronuclear multiple bonding connectivity: mainly, J ₃ correlation

Analysis of ¹ H-NMR and ¹³ C-NMR data indicated that the fatty acid was an unsaturated fatty acid and contained various fatty acids. However, the structure of fatty acids was primarily confirmed through the signal of the major constituents.

¹ H-NMR spectrum analysis, six olefinic methine (methine) Proton _{[δ H 6.29 (dd, J} = 14.8, 10.8Hz, H-11), δ H 5.92 (dd, J = 10.8, 10.8Hz, H- 10), δ _H 5.64 (dt, J = 14.8, 7.2 Hz, H-12), δ _H 5.38 (td, J = 7.2, 10.8 Hz, H-16), δ _H 5.33 (ddd, J = 6.8, 14.8 , 10.8 Hz, H-9) and δ _H 5.29 (td, J = 7.2, 10.8 Hz, H-15)], 8 allyl methylene protons [δ _H 2.32 (t, J = 7.2 Hz, H-2), δ _H 2.14 (tdd, J = 7.2, 14.8, 10.8 Hz, H-8b), δ _H 2.13 (tdd, J = 7.2, 14.8, 10.8 Hz, H-8a), δ _H 2.12 (td, J = 7.2, 7.2 Hz, H-13), δ _H 2.03 (td, J = 7.2, 7.2 Hz, H-14)], 12 methylene protons (δ _H 1.61 (tt, J = 7.2, 7.2 Hz, H-3), δ _H 1.20-1.38 (m, H3 ~ H7, H17)], and δ _H 0.86 (t, J = 7.6 Hz, H-18), the terminal methyl of fatty acids was observed. From this, it was confirmed that the measurement compound is a fatty acid including three olefin groups.

^{The 13} C-NMR spectra are carbonyl carbon signals at δ _C 179.9 (C-1), six olefin methane carbon signals [δ _C 133.8 (C-9), δ _C 132.0 (C-16), δ _C 130.1 (C -12), δ _C 128.5 (C-10), δ _C 128.1 (C-15) and δ _C 125.8 (C-11)], 10 methylene carbon signals [δ _C 34.1 (C-2), δ _C 33.0 (C-13), δ _C 31.7 (C-7), δ _C 29.7 (C-6), δ _C 29.2 (C-5), δ _C 29.1 (C-4), δ _C 27.7 (C-8) , δ _C 27.1 (C-14), δ _C 24.7 (C-3) and δ _C 22.7 (C-17)], and δ _C 14.2 (C-18). In total, these 18 carbon signals were shown to be unsaturated octadecanoic acid comprising three olefin groups.

In addition, the positions of the three olefin groups were accurately identified by the gCOSY and gHMBC spectra. In the gCOSY spectrum, the olefin methine proton signal H-9 (δ _H 5.33) is H-10 with a J ₃ correlation peak with H-8a (δ _H 2.13) / 8b (δ _H 2.14) and a chemical constant of 10.8 Hz. J ₃ correlation peak with δ _H 5.92), indicating that the olefin methine proton exhibits a ( Z ) -configuration, ie a cis configuration. H-10 showed a J ₃ correlation peak with H-11 (δ _H 6.29) with a chemical constant of 10.8 Hz, indicating that H-10 represents the ( Z ) -array, or cis-array. there was. And H-11 (δ _H 6.29) showed a J ₃ correlation peak with H-12 (δ _H 5.64) with a chemical constant of 14.8 Hz, from which H-11 was ( E ) -arranged It could be seen that. Thus, two olefin groups were connected in order like ZZE. H-16 (δ _H 5.38) showed a J ₃ correlation peak with H-15 (δ _H 5.29) linked to a chemical constant of 10.8 Hz, from which H-16 resolved the ( Z ) -array, the cis array. It can be seen that. The final structures of the fatty acids comprising the positions of the three olefin groups were confirmed by gCOSY and gHMBC. As a result, the fatty acids produced by the Bifidobacterium breve LMC520 strain according to the present invention were cis-9, trans-11, cis-15. It was identified as 9Z, 11E, 15Z-octadeca-9, 11, 15-trienoic acid which is -CLN (see Formula 1 and Formula 2).

Example 4 Investigation of the growth rate of Bifidobacterium breve LMC520 strain and the amount of CLN produced therefrom

Cultures of the Bifidobacterium breve LMC520 strain (2%, v / v) were inoculated in mMRS containing 2 mM α-LNA. And it was incubated for 48 hours at 37 ℃ under anaerobic conditions.

Production of CLN produced from Bifidobacterium breve LMC520 strain by measuring the optical density (OD) with a microplate reader (BIO-RAD, Hercules, CA, USA) at 600 nm in cultured as described above And growth rate was confirmed.

Figure 3 shows a graph of the yield and growth rate of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention (where a, b, c and d represent different values, p <0.05).

In FIG. 3, CLN production increased significantly 6 hours after initiation of the middle of the logarithmic growth phase, reaching a maximum level in the initial stationary growth phase. CLN production decreased slightly after 24 hours of incubation. In addition, as the CLN is gradually produced it was confirmed that the pH in the culture decreases.

Meanwhile, the incubation of Bifidobacterium breve LMC520 strain (2%, v / v) in mMRS containing 2mM LA, the growth rate of the strain with time while incubating for 48 hours at 37 ℃ in anaerobic conditions And CLA production were investigated.

The results are shown in FIG. As can be seen from FIG. 4, CLA production increased significantly with growth, where a, b and c represent different values.

As can be seen from the results of Figures 3 and 4, the CLA production according to the prior art significantly increased with the growth of the strain, CLN production according to the present invention is the logarithmic growth phase (0 o'clock) From 6 o'clock).

Example 5 Investigation of Production and Conversion Rate of CLN Produced from Bifidobacterium breve LMC520 Strain According to α-LNA Concentration

The Bifidobacterium breve LMC520 strain (KCTC 10455BP) was incubated in mMRS containing different concentrations of α-LNA (1 mM to 10 mM). The culture was carried out for 24 hours at 37 ℃ in anaerobic conditions.

Figure 5 shows a graph of the yield and conversion of CLN produced from Bifidobacterium breve LMC520 strain according to the present invention at different α-LNA concentrations (a, b, c, d and e described in each graph). Indicates different values, <0.05).

In FIG. 5, the production of CLN produced by culturing the Bifidobacterium breve LMC520 strain in a medium containing 1 mM of α-LNA was about 0.2 mg / ml, and was produced when cultured in a medium containing 8 mM added thereto. The yield of about 1.4mg / ㎖ was found to increase about 7 times.

In addition, when the Bifidobacterium breve LMC520 strain was cultured in a medium supplemented with α-LNA, the conversion rate at which the culture was converted to CLN was almost 100%, and even growth reached its maximum level. CLN was previously obtained with high yield. The conversion to CLN was maintained at 90% or more until 8 mM of α-LNA was added.

Meanwhile, Bifidobacterium breve LMC520 strain (KCTC 10455BP) was inoculated in mMRS containing different concentrations of LA (1 mM to 8 mM), incubated for 24 hours at 37 ° C. under anaerobic conditions, and then each concentration of LA The production and conversion rate of CLA produced from the above strain was investigated.

The results are shown in FIG. As can be seen from FIG. 6, the optimal LA concentration in producing CLA was 1 mM (a, b, c, d and e described in each graph represent different values).

As can be seen through the results of FIGS. 5 and 6, the optimal concentration of LA in producing CLA is 1 mM, whereas the optimal concentration of α-LNA in CLN production according to the present invention was 2 mM.

In addition, the CLN production according to the present invention was 30% higher than the conventional CLA production at 2 mM LA, and the conversion was higher. These results indicate that the Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention has more isomerase compared to conventional CLA production.

Example 6 CLN Production of Bifidobacterium breve LMC520 Strains According to pH

pH is an important factor not only in the growth activity of bacteria in CLN production but also in the activity of CLN producing enzyme.

By varying the pH from 3.5 to 9.0, the yield of CLN produced by the Bifidobacterium breve LMC520 strain (KCTC 10455BP) at each pH condition was investigated. The culture was carried out for 24 hours at 37 ℃ in anaerobic conditions. The results are shown in FIG.

On the other hand, while varying the pH from 4.0 to 10.0, the CLA production amount produced by the Bifidobacterium breve LMC250 strain (KCTC 10455BP) under each pH condition was investigated, and the results are shown in FIG. 8 (a, b described in the graph). , c, d, e, f, g, h, i and j represent different values).

pH 5 and pH 5.5 are the optimum range of activity of most starter strains in dairy production, and it can be seen from FIG. 7 that the conversion rate to CLN is quite high. In addition, the conversion rate to CLN was considerably high even under basic conditions, and it was found that the conversion rate to CLN was relatively higher than the conversion rate to CLA even under acidic conditions by comparing FIGS. 7 and 8.

Overall, CLN production capacity using Bifidobacterium breve LMC520 strain was higher than CLA production capacity at the same pH condition, and it was found that pH was not significantly affected.

Example 7: CLN conversion of Bifidobacterium breve LMC520 strain according to oxygen conditions

Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention was cultured in aerobic or anaerobic conditions, respectively, and the amount of CLN produced therefrom was compared. Cultivation was performed at 37 ° C. for 48 hours.

The results are shown in FIG. As can be seen from Figure 9, the conversion was higher in anaerobic conditions up to 6 hours, the conversion was similar in aerobic and anaerobic conditions from 12 hours.

In general, Bifidobacterium strains are inhibited in activity under aerobic conditions, but the results of FIG. 9 indicate that Bifidobacterium breve LMC520 strain (KCTC 10455BP) is not significantly affected by the CLN conversion. there was. Therefore, it was found that when the industrial production of CLN-containing dairy products using Bifidobacterium breve LMC520 strain can produce CLN-containing dairy products with or without oxygen.

Although the present invention has been shown and described with reference to the preferred embodiments as described above, it is not limited to the above embodiments and those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

The present invention provides a method for producing CLN microbially by naturally producing CLN using Bifidobacterium breve LMC520 strain, as well as using the strain as a starter of dairy products to enhance CLN in dairy products. It is an invention useful in the food industry because it can provide dairy products with increased functionality.

Figure 1a is the result of analysis by GC before adding and culturing α-LNA in the strain culture according to the present invention.

1B is a result of analyzing the GN when CLN was produced after culturing by adding α-LNA to the Bifidobacterium breve LMC520 strain (KCTC 10455BP) culture according to the present invention.

Figure 2 shows the results of analyzing the CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention by GC / MS.

3 is a graph showing the growth rate of Bifidobacterium breve LMC520 strain (KCTC 10455BP) and the amount of CLN produced therefrom according to incubation time.

4 is a graph showing the growth rate of Bifidobacterium breve LMC520 strain (KCTC 10455BP) and the amount of CLA produced therefrom.

5 is a graph showing the yield and conversion rate of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention at different α-LNA concentrations.

Figure 6 is a graph of the yield and conversion of CLA produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) at different LA concentrations.

Figure 7 is a graph of the production of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention at each pH.

FIG. 8 is a graph of the yield of CLA produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) at each pH.

Figure 9 is a graph of the production of CLN produced from Bifidobacterium breve LMC520 strain (KCTC 10455BP) according to the present invention in aerobic or anaerobic conditions.

Claims (7)

A method for producing conjugated linolenic acid (CLN) by culturing Bifidobacterium breve LMC520 strain (Accession Number: KCTC 10455BP) in a medium comprising linolenic acid. The method of claim 1, Wherein said linolenic acid is an α-linolenic acid comprising a non-conjugated double bond in cis-9, cis-12, cis-15 positions. The method of claim 1, Wherein the conjugated linolenic acid comprises a conjugated double bond in cis-9, trans-11, cis-15 positions. A method for preparing fermented milk containing conjugated linolenic acid (CLN) by culturing Bifidobacterium breve LMC520 strain (Accession Number: KCTC 10455BP) in milk or skim milk containing linolenic acid. The method of claim 4, wherein The linolenic acid is α-linolenic acid containing a non-conjugated double bond in the cis-9, cis-12, cis-15 position, a method for producing a fermented milk containing conjugated linolenic acid. The method of claim 4, wherein The conjugated linolenic acid is a method for producing a fermented milk containing conjugated linolenic acid, including a conjugated double bond in the cis-9, trans-11, cis-15 position. A fermented milk prepared according to the method for producing the fermented milk of any one of claims 4 to 6, containing conjugated linolenic acid.

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