KR101544180B1 - Novel Lactobacillus plantarum strains having high-productivity of conjugated linoleic acid, and probiotics and conjugated linoleic acid enhanced food prepared by using the strains - Google Patents

Abstract

In the present invention, new Lactobacillus plantai strain S48 and P1201 strains excellent in the productivity of conjugated linoleic acid and excellent in acid resistance and bile acid resistance are provided, and probiotics and conjugated linoleic acid-rich foods produced using these strains are provided .
The novel Lactobacillus plantarum S48 strain and P1201 strain of the present invention are excellent in CLA productivity and can be used for production of CLA-enhanced foods. In addition, the strains of the present invention have acid resistance and bile acid resistance, and thus are highly utilizable as probiotics. The strains according to the present invention and foods made from the cultures of the present invention can be used as functional foods that exhibit a synergistic effect on weight loss, cholesterol lowering, hyperlipidemia improvement, and atherosclerosis relief by increasing the content of CLA.

Description

[0001] The present invention relates to a conjugate linolenic acid high-productivity lactobacillus plantarum strain, a conjugate linolenic acid strains having high conjugate linoleic acid, and probiotics and conjugated linoleic acid enhanced food preparations using the strains}

The present invention relates to novel Lactobacillus plantarum strains having excellent productivity of conjugated linoleic acid, and to a food product in which a probiotic agent and a conjugated linoleic acid produced using the same are increased. More particularly, the present invention relates to a novel Lactobacillus plantaitus S48 strain and P1201 strain excellent in the productivity of conjugated linoleic acid and excellent in acid resistance and bile acid resistance, probiotics prepared using these strains and foods having increased conjugated linoleic acid .

Modern people are exposed to excessive stress and negative environment of health, high protein and high cholesterol foods have been exposed to many foods and various diseases have increased rapidly. Especially, diabetes, hypertension, hyperlipidemia, atherosclerosis and dementia, which are various metabolic syndrome diseases caused by obesity, are increasing as a result of an increase in the annual expenditure of medical expenses.

Conjugated Linoleic Acid (CLA) is an isomer of linoleic acid and has a conjugated double bond structure. It has anticancer activity, reduction of body fat, prevention and alleviation of arteriosclerosis, blood total- and LDL-cholesterol and TG (triglyceride; (PARK YK et al., 1997 Lipids 32: 853-858; McCarty MF 2000. Medical hypotheses 55: 187-188), which are known to lower triglyceride levels and to exhibit various physiological activities such as diabetic therapeutic effects.

Linoleic acid has cis - type double bonds at 9 and 12 carbons, whereas double bond positions of conjugated linoleic acid are 9 and 11, 10 and 12, 11 and 13, and double bond type cis - cis , cis - trans , trans - cis , trans - trans and so on, the existence of twelve conjugated linoleic acid isomers is known.

Conjugated linoleic acid (CLA) is present in very low concentrations in meat derived from rumen, and because of the use of skim milk in fermented foods of lactic acid bacteria, especially yogurt. In addition, there is no conjugated linoleic acid (CLA) in soy-related foods such as soy milk.

The conjugated linoleic acid (CLA) is chemically synthesized or produced by microorganisms. When chemically synthesized, expensive equipments are required, or the process takes too much time, and there is a possibility that all the isomers of conjugated linoleic acid (CLA) are produced, which is inefficient. By micro-organisms is cis -9, trans -11 conjugated linoleic acid (CLA) that is mainly produced, in consideration of the time and cost of the separation process of the produced CLA, it was added to the cells of the microorganism itself capable of producing a directly CLA CLA (I. E., Patent No. 1201184). ≪ / RTI >

Probiotics are microorganisms that help balance intestinal microbes, microorganisms with antimicrobial or enzymatic activity, and products they produce (Fuller, R. J Appl. Bacteriol. 66 (5): 365-378 , 1989). In addition, the probiotic agent is defined as a single or complex strain of live bacteria which is supplied in the form of a dried cell or fermentation product to a human or an animal, and which has a good effect by improving intestinal microflora of a human or animal host. Probiotic agents should survive on the way to non-pathogenic, non-toxic, intestinal, with human intestinal tract as a habitat. Furthermore, it should be susceptible to antibiotics used to maintain viability and activity and to prevent infection prior to consumption in the food product, and should not have plasmids resistant to antibiotics. In addition, resistance to acids, enzymes, and bile in the intestinal environment should be established (Mishra, C. et al., Asia Pacific J Clin Nutr 5: 20-24, 1996).

However, the CLA-producing strains identified so far have not shown a low CLA productivity, remain in the process of passing through the stomach after ingestion, and have little role as probiotics. Therefore, an invention for a strain that can be used as a probiotic agent due to its high survival ability and activity in the body, together with excellent CLA production ability, still remains as a problem.

Soybean is a source of excellent vegetable protein (35 ~ 45%) and fat (10 ~ 15%) and is consumed especially in Korean food such as tofu, soybean paste, soy milk and soybean oil. As a result of research on physiologically active substances such as saponin, isoflavone, and lecithin as well as nutritional aspects of soybeans, attention has been paid to health functional effects such as anticancer, anti-arteriosclerosis, Particularly, since more than 50% of the soybean fat is linoleic acid, there has been a demand for development of a probiotic agent capable of efficiently converting the linoleic acid into CLA.

As a result of continuing the study to meet the demand in the prior art, the present inventors have found that a novel Lactobacillus plant ( Lactobacillus ( Lactobacillus )) having a very high productivity of conjugated linoleic acid (CLA) plantarum ) was isolated and identified, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a novel Lactobacillus plantarum strain having excellent acid-fastness and bile acid resistance with excellent CLA productivity.

It is still another object of the present invention to provide a prophylactic agent comprising the strain of the present invention or a culture thereof as an active ingredient.

It is also an object of the present invention to provide a method for producing a food having an increased content of CLA using the strain of the present invention or a culture thereof and a food having an increased content of CLA prepared therefrom.

In order to achieve the above object, the present invention provides novel Lactobacillus plantai S48 and P1201 strains excellent in acid resistance, bile acid resistance and CLA production.

The present inventors isolated various lactic acid bacterial species from fermented foods (kimchi, salted fish, fermented soy sauce, fermented soy sauce, and fermented soybeans) and selected strains that produce CLA in the MRS liquid medium supplemented with linoleic acid. By isolating and identifying the strains which can be used as probiotic agent and excellent CLA productivity through the bile acid tolerance test, the strains according to the present invention were identified. The strains according to the present invention were named 'Lactobacillus plantai S48' and 'Lactobacillus plantai P1201' respectively and deposited on August 13, 2013 at the National Institute of Agricultural Science and Technology (KACC) : KACC 91849P and KACC 91848P).

Identification of Lactobacillus plantarum S48 and P1201 strains was performed using a plate culture medium and a Gram stain (morphological observation) (FIGS. 1A to F and FIG. 2), and physiological / biochemical characterization was performed using an API CHB50 kit was used, and cell wall fatty acid analysis (MIDI analysis) and 16S rDNA sequencing of the strain were confirmed. It was confirmed that the strains of the present invention based on the 16S rRNA base sequence, Lactobacillus plantai S48 and P1201 belong to Lactobacillus sp. Strains (Fig. 3).

The strains according to the present invention, Lactobacillus plantai S48 and P1201, are more excellent in CLA producibility than the conventional strains (Table 1).

According to another object of the present invention, there is provided a probiotics comprising the strain of the present invention, lactobacillus plantai S48, P1201 or a culture thereof as an active ingredient.

The probiotics can be supplied in various forms such as dried cell form or fermented composition. Since the probiotics according to the present invention are selected from safe fermented foods, the strains can be used as a non-toxic probiotics because they are secured. Therefore, the probiotics according to the present invention can be used for the edible use of animals including humans, and specifically for food.

According to another object of the present invention, there is provided a method for producing a food product in which the content of CLA is increased using the strain of the present invention, Lactobacillus plantaitum S48, P1201 or a culture thereof.

The method for producing a food having an increased content of CLA according to the present invention comprises the step of adding a strain according to the present invention and fermenting at 20 to 40 ° C for 12 hours or more.

The amount of the strain added can be in the range of 1.0% (v / v) to 10.0% (v / v).

In the present invention, the food may be a soybean product (soymilk, tofu, soybean meal, soybean stack, soybean cake), dairy product (milk, cheese), fermented milk (liquid yogurt, yogurt yogurt), fermented food (kimchi, But are not limited thereto.

In the production process of the present invention, it may include a step of treating the food material with an enzyme of cellulase, protease and / or esterase before fermentation to promote the production of free-linoleic acid. The enzyme throughput can be easily determined by a person skilled in the art depending on the material of the food. Preferably, the throughput of each enzyme is between 1 and 10 units, and the production amount is not significantly affected by more than 10 units. By such an enzyme treatment, CLA productivity can be increased by 1.5 to 2 times.

In addition, in the method for producing foods in which the CLA content of the present invention is increased, vegetable oil having a high content of linoleic acid can be added before fermentation.

Vegetable oils with high linoleic acid content are preferably safflower seed oil (77.0%), evening primrose oil (75.0%) and gucci mulberry seed oil (68.4%).

In the production method of the present invention, the addition amount of the vegetable oil is 0.1 to 5.0% (v / v), preferably 1 to 2% (v / v).

As an example of the production method of the present invention,

The soybean was soaked and pulverized to prepare a bean pulverized liquid,

The soybean pulverizing solution was subjected to a thickening treatment,

1 to 10 units of an enzyme of a cellulase, a protease and / or an esterase is added to the soybean pulverized solution to prepare a soybean hydrolyzate,

The present invention provides a method for producing a soybean product in which the strain of the present invention is inoculated to a soybean hydrolyzate and fermented at 20 to 40 캜 for 12 hours or more to thereby increase the content of CLA.

It is preferable to perform the thickening treatment at 100 to 120 DEG C for 30 to 60 minutes.

Enzyme treatment is performed by adding 1 ~ 10 units and treating at 30 ~ 40 ℃ for more than 12 hours.

Further, in the method for producing a soybean product in which the content of CLA of the present invention is increased, vegetable oil having a high content of linoleic acid can be added after the planting.

According to still another object of the present invention, there is provided a food having an increased content of CLA prepared using the strain of the present invention, Lactobacillus plantai S48, P1201 or a culture thereof. In the present invention, the types of foods having increased CLA content include soybean products (soybean milk, tofu, soybean meal, soybean snack, soybean cake), dairy products (milk and cheese), fermented milk (liquid yogurt, yogurt yogurt), fermented foods Pastries, pickles), fermented beverages, and the like.

The novel Lactobacillus plantarum S48 strain and P1201 strain of the present invention are excellent in CLA productivity and can be used for production of CLA-enhanced foods. In addition, the strains of the present invention have acid resistance and bile acid resistance, and thus are highly utilizable as probiotics.

The strains according to the present invention and foods made from the cultures of the present invention can be used as functional foods that exhibit a synergistic effect on weight loss, cholesterol lowering, hyperlipidemia improvement, and atherosclerosis relief by increasing the content of CLA.

The method of producing a CLA-containing food of the present invention can further increase the productivity of CLA compared to the conventional method by using the strain of the present invention having high CLA productivity and adding an enzyme treatment.

FIG. 1 is a photograph showing clone morphology in 16 strains of MRS flat plate culture selected in the present invention (A: S48, B: S52, C: S56, D: S64, E: S65, F: P1201, , H: K24, I: D56, J: D58, K: 13, L: 14, M: 15, N: 29, O: 41, P: 104).
2 is a Gram stain image of 16 strains selected in the present invention (A: S48, B: S52, C: S56, D: S64, E: S65, F: P1201, G: K23, H: D56, J: D58, K13, L14, M15, N19, O41, P104).
FIG. 3 is a flow diagram showing the homology relationship between Lactobacillus plantai S48 and P1201 strains in the genus Lactobacillus according to the present invention.
4 is a graph showing the degree of bacterial growth and CLA productivity of Lactobacillus platelets S48 and P1201 according to the concentration of linoleic acid (4a: degree of bacterial growth of Lactobacillus plantai S48, 4b: degree of bacterial growth of Lactobacillus plantarium S48 CLA productivity, 4c: degree of bacterial growth of Lactobacillus plantai P1201, and 4d: CLA productivity of Lactobacillus plantai P1201).
FIG. 5 is a graph showing the degree of bacterial growth and CLA productivity of Lactobacillus platelets S48 and P1201 according to the concentration of added safflower seed oil (5a: degree of bacterial growth of Lactobacillus plantai S48, 5b: 5c: CLA productivity of Lactobacillus plantarium S48, and 5d: CLA productivity of Lactobacillus plantai P1201).
FIG. 6 is a graph showing that the productivity of CLA is increased during the production of soybean products by adding the safflower seed oil according to the present invention (6a: CLA productivity in the soybean hydrolyzate of Lactobacillus plantai S48, 6b : CLA Productivity in Daphne Soybean Hydrolyzate of Lactobacillus plantai S48, 6c: CLA Productivity in Always Chan Hydrolyzate of Lactobacillus Plantai S48, 6d: Daphne Soybean Hydrolyzate of Lactobacillus Plantai S48 at 48 hours 6e: CLA productivity in the soybean hydrolyzate of Lactobacillus plantai P1201, 6f: CLA productivity in the soybean hydrolyzate of Lactobacillus plantai P1201, 6g: Lactobacillus plantai P1201, CLA productivity in hydrolyzate, 6h: GC-chromatogram at 48 hrs. Of Lactobacillus plantai P1201).

The present invention will be described in more detail by the following examples. These examples are provided for illustrating the present invention, and the scope of the present invention should not be limited thereby.

Example

Example 1 Isolation of Strain

10 m1 samples were taken from fermented beverages of Kimchi, Makgeolli, Soybean, Pancreas and Sanayasia (sugar extracted) and put into 90 ml of physiological saline, diluted to 10 ⁴ to 10 ⁸ by 10-step dilution, and then 0.1 ml of diluted solution was added to Lactobacilli MRS medium (DIFICO). The cells were plated on Lactobacilli MRS medium with sterilized physiological saline (0.5% w / v) to give 30 ~ 150 colony, and then cultured for 72 hours in a constant temperature incubator maintained at 30 ~ 35 ° C. Each strain was subcultured twice in MRS medium and then inoculated 1% in 20 ml test tube. Bovine serum albumin was added to 5 ml of MRS liquid medium. 0.02% (100 μg / ml) of mixed linoleic acid emulsion (Sigma-Arich Co.) was added and cultured for 48 hours.

The fatty acid was extracted by the usual fatty acid pretreatment method and analyzed by gas chromatograph (GC), and the concentrations of linoleic acid (LA) and conjugated linoleic acid (1: 1) CLA) standard curve to compare the amount of CLA produced, and 16 strains producing CLA were selected.

Clonal morphology of 16 selected strains on MRS plate culture medium is shown in Fig. 1, and Gram staining photograph is shown in Fig.

Example 2. Measurement of CLA Productivity of a Strain

The CLA productivity of the final 16 strains selected in Example 1 was measured.

Specifically, 0.02% (100 μg / ml) of linoleic acid emulsion was added to each of the MRS liquid medium and the 8% skim milk medium, and each strain of Example 1 was inoculated at 2.5% and cultured at 37 ° C. for 48 hours. Were measured.

For CLA production, 2 ml of each culture was taken, and 2 ml of chloroform: methanol (1: 1) was added to extract the fatty acid. The extracted fat was filtered through the saponification and methylation process and analyzed by fatty acid analysis The results are shown in Table 1. The results are shown in Table 1. < tb > < TABLE >

Strain Log cfu / ml Concentration (g / ml)
MRS
Skim milk MRS Skim milk c9t11 t10C12 Total amount c9t11 t10C12 Total amount S48 9.48 9.23 17.32 13.41 30.73 21.48 17.11 38.59 S52 9.54 9.57 8.23 6.01 14.24 10.24 8.89 19.13 S56 9.43 9.33 9.42 6.97 16.39 12.33 10.68 23.01 S64 9.72 9.64 11.22 8.06 19.28 15.33 12.86 28.19 S65 9.30 9.18 15.42 10.26 25.68 15.65 13.01 28.66 P1201 9.52 9.46 17.79 13.83 31.62 22.35 17.86 40.21 K23 9.31 9.26 14.06 10.11 24.17 16.37 13.42 29.79 K24 9.60 9.42 8.66 6.46 15.12 12.34 10.50 22.84 D56 9.54 9.58 12.33 10.83 23.16 15.93 12.30 28.23 D58 9.46 9.39 13.46 11.28 24.74 16.11 12.74 28.85 13 9.62 9.55 4.44 3.02 7.46 7.98 5.88 13.86 14 8.51 8.44 10.11 6.98 17.09 11.42 5.89 17.31 15 9.36 9.28 6.23 4.86 11.09 9.24 6.59 15.83 29 9.14 9.07 5.24 3.51 8.75 8.72 5.51 14.23 41 9.44 9.32 8.01 6.18 14.19 11.06 7.22 18.28 104 9.31 9.16 10.37 8.04 18.41 13.85 10.44 24.29

As shown in the above table, strains S48 and P1201 produced 38.59 μg / ml and 40.21 μg / ml CLA, respectively, while simultaneously producing cis- 9, trans- 11 and trans- 10 and cis- Was significantly higher than that of other strains.

Example 3. Probiotic insecticidal test of strain

Resistance to acid, artificial stomach acid and bile acid was determined in order to determine whether the 16 strains selected in Example 1 could be used as a probiotic agent.

The acid resistance was measured by incubating the strains at 37 ° C for 48 hours in a MRS liquid medium (Lactobacilli MRS Broth; DIFCO), adding 10 ⁶ CFU to the new MRS liquid medium adjusted to pH 2.0, 2.5 and 3.0 respectively with 3M hydrochloric acid / ml. The diluted cells were incubated at 37 DEG C for 4 hours. After incubation, the cells were continuously diluted with phosphate buffer (0.1 M, pH 6.2) to neutralize the acidity of the culture medium, plated on MRS plate medium (Lactobacilli MRS Agar; DIFCO), and cultured at 30 ° C for 48 hours. Were measured. Survival rates were determined from the initial bacterial concentration and Lactobacillus sp . And the ratio of colonies of bacteria was calculated. The results are shown in Table 2.

Strain Survival rate (%) pH 2.0 pH 2.5 pH 3.0 Incubation time Incubation time Incubation time 2 hr 4 hr 2 hr 4 hr 2 hr 4 hr S48 48.69 33.11 91.26 63.25 110.49 108.62 S52 22.32 13.87 80.51 52.31 99.62 98.21 S56 24.85 12.33 78.92 50.34 101.30 97.14 S64 20.12 13.85 66.74 47.45 84.60 78.80 S65 22.47 11.52 64.53 45.21 86.46 82.63 P1201 46.84 34.60 84.63 58.14 100.39 99.34 K23 22.28 11.44 77.24 50.46 98.72 98.46 K24 23.65 10.52 79.23 51.33 97.06 91.44 D56 18.44 6.52 63.88 42.31 84.94 80.75 D58 24.18 12.85 81.69 53.65 97.39 92.44 13 15.33 5.21 70.11 42.52 110.52 90.79 14 36.52 25.22 88.57 41.40 97.41 86.66 15 12.23 6.34 62.58 33.85 94.78 90.93 29 9.54 3.45 36.52 22.11 63.05 57.44 41 16.34 10.55 40.85 30.78 74.66 60.36 104 9.87 2.54 48.96 31.55 68.39 58.42

The artificial gastric juice resistance was measured by adding 1% pepsin to the MRSB liquid medium and adjusting the pH to 2.0, 2.5 and 3.0 respectively. The test was carried out in the same manner as the acid resistance measurement described above, and the results are shown in Table 3.

Strain Survival rate (%) pH 2.0 pH 2.5 pH 3.0 Incubation time Incubation time Incubation time 2 hr 4 hr 2 hr 4 hr 2 hr 4 hr S48 28.11 18.69 88.52 59.57 98.82 90.31 S52 17.64 10.45 59.66 44.21 97.60 87.78 S56 19.55 9.99 48.22 30.33 101.39 94.93 S64 18.33 10.42 46.62 27.99 84.33 78.31 S65 25.52 14.21 72.33 49.41 101.89 100.13 P1201 26.85 15.32 92.56 62.22 112.55 102.79 K23 19.42 9.21 60.45 42.25 82.74 79.41 K24 23.76 13.87 74.98 47.32 106.87 95.99 D56 25.66 13.54 80.45 53.47 105.35 98.13 D58 24.89 14.77 84.22 55.67 106.41 99.03 13 10.56 2.35 40.98 24.87 78.00 55.64 14 18.54 9.65 48.76 30.21 80.44 56.45 15 10.23 2.98 42.77 23.58 64.55 40.13 29 5.12 1.11 46.88 17.93 53.67 47.55 41 8.34 2.12 20.55 10.87 44.49 30.54 104 8.80 1.54 22.09 11.06 38.25 28.88

The measurement of the biliary properties was carried out as follows. 15 ml (same as 10 ⁶ CFU / ml) of the cells cultured in the MRS liquid medium for 48 hours at 30 ° C were treated with MRS containing oxgall bile at different concentrations (1, 2, 3, 4% w / v) Spotted on a plate culture medium, and then cultured at 30 DEG C for 5 days. The minimum inhibitory concentration (MIC) of bile acid for the strains was determined as the minimum concentration that inhibited bacterial growth in the spot by spotting by visual inspection. The criteria for visual examination were +++ for 15 mm directly, ++ for 10 mm, + for 8 mm, and - for 0 mm. The visual inspection results are shown in Table 4.

Strain
Bile acid concentration (%) One 2 3 4 S48 +++ +++ +++ ++ S52 +++ +++ +++ ++ S56 +++ +++ +++ +++ S64 +++ +++ ++ ++ S65 +++ +++ +++ ++ P1201 +++ +++ +++ ++ K23 +++ +++ ++ ++ K24 +++ +++ ++ ++ D56 +++ +++ +++ ++ D58 +++ +++ +++ ++ 13 ++ + w w 14 + w w w 15 ++ + w w 29 +++ ++ ++ + 41 + w w w 104 +++ +++ ++ +

As can be seen from the above tables, the strains according to the present invention were most resistant to acid and artificial gastric acid among the strains selected for Lactobacillus platelets S48 and P1201, and about 50% at 2 hours even at pH 2.0 , And the survival rate of bile acid was 4%. Therefore, it can be seen that the strains according to the present invention, Lactobacillus plantai S48 and P1201 strains can be sufficiently used as a probiotic agent.

Example 4. Identification of Lactobacillus plantai S48 and P1201 strains

Lactobacillus platelets S48 and P1201, which had excellent CLA productivity and excellent acid resistance, artificial gastric juice resistance and bile acid tolerance, were selected and the morphological, physiological, biochemical and molecular genetic characteristics Respectively.

The Lactobacillus plantarum S48 strain was Gram positive bacteria. As a result of analysis of cell wall fatty acid as a bacterium, the major fatty acids C16: 0 and C18: 1 w9c were 31.52% and 23.86%, respectively. Lactobacillus plantai P1201 was a Gram-positive bacterium. As a result of amino acid analysis of the cell wall fatty acids (bacterium), the major fatty acids, C16: 0 and C18: 1 w9c, accounted for about 55.31% (Table 5).

Types of fatty acids Furtherance(%) Strain S48 P1201 C10: 0 0.06 - C12: 0 0.15 0.17 C14: 0 4.68 2.67 C15: 0 ISO 0.19 - C16: 0 31.52 37.81 17: 1 w8c - 0.09 C18: 1 w9c 23.86 17.50 C18: 1 w7c 4.84 8.22 C18: 0 2.23 2.41 C19: 0 ISO 1.39 0.92 19: 1 ISO - 0.23 19: 0 CYCLO w8c - 7.45 Sum Feature 3 2.82 2.90 Sum Feature 7 28.25 19.62 Summed feature 3 - C15: 0 ISO 20H / C16: 1w7c
Summed feature 7 - C19: 0 CYCLO w10c / C19w6

The API CHB50 kit, a method for testing the glycosyltransferase (BioMerieux, France), the Lactobacillus plantarum S48 strain was found to be able to magnetize 24 carbon sources including Ribose and to produce lactic acid bacteria of the lactobacillus genus The growth temperature was 10 ~ 40 ℃ and the growth pH was 4.5 ~ 11.0.

The Lactobacillus plantai P1201 strain was able to magnetize 25 carbon sources including L-Arabinose and was assumed to be a lactic acid-anaerobic Lactobacillus sp. Strain. The growth temperature was 10 to 40 占 폚, The growth pH was 4.5 ~ 10.0 (Table 6).

No reaction No

reaction Strain Strain S48 P1201 S48 P1201 0 Control - - 25 Esculine + + One Glycerol - - 26 Salicine + + 2 Ertythritol - - 27 Cellobiose + + 3 D-Arabinose - - 28 Maltose + + 4 L-Arabinose - + 29 Lactose + + 5 Ribose + + 30 Melibiose + + 6 D-Xylose - - 31 Saccharose + + 7 L-Xylose - - 32 Trehalose + + 8 Adonitol - - 33 Inuline + + 9 beta-methyl-xyloside - - 34 Melezitose + + 10 Galactose + + 35 D-Raffionse - - 11 D-Glucose + + 36 Amidon - - 12 D-Fructose + + 37 Glycogene - - 13 D-Mannose + + 38 Xylitol - - 14 L-sorbose - - 39 β-Gentiobiose + + 15 Rhamnose - - 40 D-Turanose + + 16 Dulcitol - - 41 D-Lyxose - - 17 Inositol - - 42 D-Tagatose - - 18 Mannitol + + 43 D-Fucose - - 19 Sorbitol + + 44 L-Fucose - - 20 alpha -Methyl-D-mannoside + - 45 D-arabitol - - 21 alpha -Methyl-D-glucoside - - 46 L-Arabitol - - 22 N- Acetyl glucosamine + + 47 Gluconate + + 23 Amygdaline + + 48 2-ceto-gluconate - - 24 Arbutine + + 49 5-ceto-gluconate - -

Sequence analysis of 16S rRNA as a molecular genetic method revealed that both strains showed 99% high homology with Lactobacillus plantarum strain through BLASTN (nucleotidenucleotide BLAST) database (Fig. 3 ).

The identified strains were named Lactobacillus plantai S48 and P1201, respectively, based on morphological, physiological and molecular genetic characteristics.

Example 5 CLA Productivity of Lactobacillus plantarium S48 and P1201 according to Linoleic Acid Concentration

After addition of 250, 500 and 1000 μg / ml of linoleic acid emulsion to 8% skim milk liquid medium, Lactobacillus platelets S48 and P1201 were inoculated respectively and samples were collected at culture time 0 hr, 12 hr, 24 hr and 48 hr And the degree of bacterial growth and the degree of CLA production were measured. The results are shown in Figs. 4A to 4D.

Growth of Lactobacillus plattarium S48 was maximal at 9.35 to 9.64 log cfu / ml at 24 hours at the initial 6.0 log cfu / ml level, and slightly decreased after 48 hours (Fig. 4a). The degree of growth of the strain was similar regardless of the concentration of linoleic acid.

CLA production in the case of culture at 48 hours linoleic acid 250 μg / ml was added cis -9, trans -11 is 25.89 μg / ml and trans -10, cis -12 is of 19.08 μg / ml, linoleic acid, 500 μg / ml was added to produce If cis -9, tran s-11 was 48.22 μg / ml and trans -10, cis -12 was generated 41.67 μg / ml, linoleic acid for 1000 μg / ml was added cis -9, trans -11 was 51.02 μg / ml And trans- 10 and cis- 12 were 42.64 μg / ml (FIG. 4b), indicating that the productivity of CLA increased depending on the concentration of linoleic acid.

The growth of Lactobacillus plantai P1201 was maximal at 9.41 to 9.56 log cfu / ml at 24 hours at the initial 6.0 log cfu / ml level and slightly decreased after 48 hours (Fig. 4c). The degree of growth of the strain was similar regardless of the concentration of linoleic acid.

The amount of CLA produced by cis-9, trans-11, and cis- 12 was 250 μg / ml, 27.89 μg / ml and trans- 10 and 20.47 μg / If cis -9, trans -11 was 48.54 g / ml and trans -10, cis -12 in the case of, it was produced 41.97 μg / ml linoleic acid, 1000 μg / ml was added cis -9, trans -11 and is 50.99 μg / ml trans- 10 and cis- 12 were produced at 41.03 μg / ml (FIG. 4d), indicating that the productivity of CLA was increased depending on the concentration of linoleic acid.

Example  6. Safflower oil  Depending on concentration Lactobacillus Pantalum S48  And P1201 of CLA  productivity

Lactobacillus platelets S48 and P1201 were inoculated by adding 0.5%, 1.0% and 2.0% of Safflower oil emulsion to 8% skim milk powder liquid medium, respectively, and incubated at 0 hr, 12 hr, 24 hr and 48 hr Samples were collected and the degree of bacterial growth and the degree of CLA production were measured. The results are shown in Figs. 5A to 5D.

The degree of growth of the strain was increased from about 6.0 log cfu / mL at the initial stage to about 10.0 log cfu / mL at the stage (Figs. 5A and 5B). The degree of growth of the strain was similar regardless of the concentration of safflower oil.

As a result of CLA productivity, the CLA production increased with increasing the amount of oil added to sour milk, and Lactobacillus plantai S48 ( cis- 9, trans- 11, 57.03 μg / ml and trans- 10, cis- 12 It is Lactobacillus Planta volume P1201 (cis -9, trans -11 is 62.39 μg / ml, and trans -10, cis -12 exhibited a slightly higher CLA productivity is 50.22 μg / ml) (Fig than 45.02 μg / ml) 5c and 5d).

Example  7. Lactobacillus Pantalum S48  And P1201 Using CLA Bean product manufacturing

100 g of purified water was added to 10 g of each of Daewon bean, Daeungbong bean, Soybean bean, Soybean bean powder, Soybean bean powder, and soybean powder (after soaking for 6 hours and drying at 50 to 55 ° C for 30 minutes at 100 ° C for 2 to 3 days) And sterilized at 120 DEG C for 15 minutes.

Sterilized soybean and soy sauce soybean compositions were divided into two groups, one group was used as it is, and the other group was added with cellulase, protease and esterase (Sigma-Aldrich) And treated at 37 ° C for 24 hours to prepare soybean hydrolyzate.

The resulting soybean composition and soybean hydrolyzate were inoculated with 2.5% (2.5 ml) of Lactobacillus plantai S48 and Lactobacillus plantai P1201, respectively, and cultured at 37 ° C for 48 hours. The amount of CLA produced was measured, The results are shown in Table 7.

Soybean variety CLA production (g / ml) Sweet potato powder Increased soybean powder c9t11 t10C12 Total amount c9t11 t10C12 Total amount Lactobacillus platarium S48 Daewon bean
Composition 18.11 13.02 31.13 21.76 16.32 38.08 Daesung bean
Composition 22.54 15.98 38.52 25.34 18.24 43.58 Always a cold
Composition 23.18 16.23 39.41 26.78 19.55 46.33 Daewon bean
Hydrolyzate 38.53 29.02 67.55 40.53 31.50 72.03 Daesung bean
Hydrolyzate 44.67 37.34 82.01 48.12 39.23 87.35 Always a cold
Hydrolyzate 46.14 38.45 84.59 49.88 40.21 90.09 Lactobacillus plantai P1201 Daewon bean
Composition 20.35 15.26 35.61 23.61 17.71 41.32 Daesung bean
Composition 24.62 18.22 42.84 28.42 19.89 48.31 Always a cold
Composition 26.55 19.22 45.77 30.11 22.58 52.69 Daewon bean
Hydrolyzate 38.21 30.33 68.54 42.28 33.54 75.82 Daesung bean
Hydrolyzate 46.64 37.87 84.51 50.89 41.87 92.76 Always a cold
Hydrolyzate 47.22 39.01 86.23 52.99 43.50 96.49

Compared with the soybean composition, which had no increase or enzyme treatment as a whole, the soybean hydrolyzate treated with increased and enzyme-treated soybean showed high CLA productivity. When the soybean powder was used, it was judged that the gel was formed from the culture for 24 hours, and the smooth contact of the cells did not occur and the CLA production was small.

Example  8. Safflower oil  By addition CLA  Manufacture of soybean food with further increased content

100 ml of purified water was added to 10 g of the soybean powder as in Example 7, sterilized at 120 ° C for 15 minutes, added with 0%, 0.5% and 1.0% of safflower seed oil, and then the cellulase, protease and Esterase was added to the final 10 units, respectively, and the mixture was treated at 37 ° C for 24 hours to prepare soybean hydrolyzate of oil added with safflower seed oil. The resulting soybean hydrolyzate was inoculated with 2.5% (2.5 ml) of Lactobacillus plantai S48 and Lactobacillus plantai P1201, respectively, and cultured at 37 ° C for 48 hours. The degree of bacterial growth was measured and shown in Table 8 (Lactobacillus Pantotarium S48) and Table 9 (Lactobacillus plantai P1201), and the degree of CLA production was measured and shown in Figs. 6a to 6h.

Soybean variety
sample Fermentation time (hr) 0 12 24 48
Daewon bean SSPHB 5.57 7.42 9.31 9.43 SSPHB + 0.5% SF 5.33 7.38 9.22 9.41 SSPHB + 1.0% SF 5.46 7.4 9.27 9.44
Daesung bean SSPHB 5.36 7.33 9.34 9.45 SSPHB + 0.5% SF 5.34 7.36 9.33 9.4 SSPHB + 1.0% SF 5.4 7.38 9.3 9.43
Always a cold SSPHB 5.42 7.38 9.3 9.42 SSPHB + 0.5% SF 5.39 7.43 9.36 9.47 SSPHB + 1.0% SF 5.38 7.34 9.4 9.45 SSPHB: 10% Increase soybean powder hydrolyzate, SF: safflower seed oil
Strain: Lactobacillus plantarium S48

Soybean variety
sample Fermentation time (hr) 0 12 24 48
Daewon bean SSPHB 5.66 7.49 9.45 9.46 SSPHB + 0.5% SF 5.62 7.4 9.34 9.42 SSPHB + 1.0% SF 5.68 7.41 9.33 9.42
Daesung bean SSPHB 5.65 7.46 9.43 9.45 SSPHB + 0.5% SF 5.64 7.46 9.42 9.4 SSPHB + 1.0% SF 5.64 7.47 9.43 9.43
Always a cold SSPHB 5.66 7.48 9.36 9.42 SSPHB + 0.5% SF 5.69 7.43 9.35 9.41 SSPHB + 1.0% SF 5.64 7.44 9.32 9.42 SSPHB: 10% Increase soybean powder hydrolyzate, SF: safflower seed oil
Strain: Lactobacillus plantai P1201

In the soybean cultured with Lactobacillus plantarum S48, the growth of the strain was increased to 9.5 log cfu / mL after 48 hours at the initial level of about 5.4 log cfu / mL. (Table 8). The growth of the strain was increased to 9.5 log cfu / mL after 48 hours at the initial level of about 5.6 log cfu / mL. The growth rate of the strain was higher than that of the safflower seed oil (Table 9).

As can be seen from Figs. 6a to 6h, the content of CLA in soybean products increased in a concentration-dependent manner as the amount of safflower seed oil was increased. In the case of Lactobacillus plantarum S48 strain, the highest CLA content ( cis- 9, trans- 11, 77.66 μg / ml and trans- 10, cis -12 is showed a 57.71 μg / ml) (Fig. 6a ~ 6d), Lactobacillus Planta volume if the strain P1201 is also the highest CLA content in safflower oil, soy products with added 1.0% soy hydrolyzate Dafeng (cis - 9, 89.97 μg / ml for trans- 11 and 68.78 μg / ml for trans- 10 and cis- 12) (Figs. 6e to 6h).

National Institute of Agricultural Science KACC91849P 20130813 National Institute of Agricultural Science KACC91848P 20130813

Claims (14)

Lactobacillus plantarum S48 strain deposited with accession number KACC91849P,
A productivity of conjugated linoleic acid of 44.97 to 93.66 μg / ml at a linolenic acid concentration of 250 to 1000 μg / ml, and having enhanced acidity and bile bile acidity.
Lactobacillus plantarum P1201 deposited with accession number KACC91848P,
A productivity of conjugated linoleic acid of 44.97 to 93.66 μg / ml at a linolenic acid concentration of 250 to 1000 μg / ml, and having enhanced acidity and bile bile acidity.
A probiotics comprising the strain according to claim 1 or 2 or a culture thereof as an active ingredient.
The active agent according to claim 3, wherein the active agent is in the form of a dried cell or a fermented composition.
A food having increased conjugated linoleic acid content comprising the strain according to any one of claims 1 or 2 or a culture thereof.
The food according to claim 5, wherein the food is any one selected from the group consisting of soybean products, dairy products, fermented milk, fermented foods and fermented beverages.
A method for producing a food having an increased conjugated linoleic acid content,
Adding a strain according to claim 1 or 2 to a food material and fermenting the mixture at 20 to 40 DEG C for at least 12 hours,
Further comprising, prior to fermentation, treating the food material with at least one enzyme selected from the group consisting of cellulase, protease and esterase.
delete The method according to claim 7, wherein before fermentation, at least one vegetable oil selected from the group consisting of safflower seed oil, evening primrose oil, and Gucci mulberry seed oil is added.
A food having an increased content of conjugated linoleic acid produced by the method according to claim 7.
A method for producing a soybean product having an increased conjugated linoleic acid content,
The soybean was soaked and pulverized to prepare a bean pulverized liquid,
The soybean pulverizing solution was subjected to a thickening treatment,
1 to 10 units of at least one enzyme selected from the group consisting of cellulase, protease and esterase is added to the soybean pulverized liquid to prepare soybean hydrolyzate,
Which comprises fermenting soybean hydrolyzate at 20 to 40 DEG C for at least 12 hours by inoculating the strain according to claim 1 or 2.
12. The method according to claim 11, wherein, after the thickening treatment, at least one vegetable oil selected from the group consisting of safflower seed oil, evening primrose oil, and Gucci mackerel oil is added.
A soybean product having an increased content of conjugated linoleic acid produced by the method according to claim 11.
14. The soybean product according to claim 13, wherein the soybean product is processed into any one form selected from the group consisting of soybean milk, tofu, soybean meat, soybean snack, and soybean paste.

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