KR101548640B1 - Novel Strains of Lactobacillus plantarum JA71 with Folic Acid Producing Ability - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum JA71 strain having excellent folate productivity. The strain of the present invention is excellent in folic acid production ability, has a growth inhibitory activity against food poisoning causing bacteria, and has a bile resistance and a strong acid resistance. In addition, since it exhibits excellent acid-producing fermentation characteristics in soymilk, it can be used as a fermentation starter strain for producing a fermented soybean milk product.

Description

Novel Strains of Lactobacillus plantarum JA71 with Folic Acid Producing Ability ", which is a novel Lactobacillus plantarum JA71 strain having excellent folate-

The present invention relates to a novel Lactobacillus plantarum JA71 strain, and more particularly to a novel Lactobacillus plantarum JA71 having folate production ability suitable for producing fermented soybean milk.

It is a collective term for pteroylglutamic acid and derivatives with similar activities, derived from the word folium in Latin. Folic acid is composed of pteridine, para-aminobenzoic acid (PABA), and one or more glutamate molecules (glutamate). If there is only one glutamic acid molecule, it is called folic acid. The main form of folate present in foods and in the body is the reduced form of tetrahydrofolate, which is a pteroylpolyglutamate in which about 1-6 glutamates are additionally linked with various types of single carbon units. On the other hand, folic acid supplementation and fortified folate in food are included as relatively stable folate.

Folic acid is converted to basic tetrahydrofolic acid (THFA), a basic coenzyme form in cells. There are five such active THFAs, and this form of THFA serves as a cofactor for single-carbon transfer reactions in nucleic acid metabolism. In order to synthesize thymidylate necessary for DNA synthesis, folic acid is necessary. Therefore, when folic acid is insufficient, it is difficult for cell division to occur normally, and for purine and pyrimidine synthesis, DNA synthesis and repair . In addition, folic acid is needed in amino acid metabolism. Histidine degradation, metabolism of forminino-glutamic acid, an intermediate metabolite of histidine metabolism, interconversion of serine to glycine, and synthesis of methionine. In particular, methionine synthesized from homocysteine is converted to S-adenosylmethionine (S-adenosylmethionine), which is necessary for synthesis of neurotransmitters in the brain, and acts as an important material for providing methyl groups in various metabolic processes required in the body.

Folic acid is very likely to become deficient in folate during this period because it is highly required in infants, growing, pregnant, and lactating periods, where cell division is frequent. When folate intake is insufficient, serum folate concentration decreases, erythrocyte folate concentration decreases, and homocysteine concentration increases. In addition, there is a huge change in the composition of the cells where bone marrow and cell division occur rapidly, and anemia appears. Anemia that occurs when folate deficiency is anemia due to giant anemia is accompanied by weakness, fatigue, instability, and heart palpitations. In addition, folate deficiency affects gastrointestinal mucosa, which causes cell division very quickly, and causes gastrointestinal disturbances.

It has been reported that insufficient folate at the early pregnancy may cause birth defects in the neural tube due to obstruction of the neural tube formation in the fetus and lower birth defects in the neural tube damage by supplementing folic acid to women with these risk factors. Recently, it has been reported that not only neural tube damage but also other abnormal births such as lachrymia and Down syndrome are related to maternal folate deficiency. Also, a large amount of folic acid is recommended for patients with inflammatory bowel disease. It is therefore desirable to provide a natural source, such as probiotics, that can supply folic acid to the body continuously.

Korean Patent Publication No. 2007-0048776 discloses Bifidobacterium bacteria that produce folic acid and the use of this strain, and Korean Patent No. 10-0882278 discloses a method of fermenting multiple strains of Kimchi lactic acid bacteria and milk lactic acid bacteria Korean Patent No. 10-0882279 discloses that folic acid is produced when fermented with a complex of a plant Kimchi lactic acid bacterium and an animal milk lactic acid bacterium in a soybean containing extract of herbal medicine However, it is not reported that the lactobacillus strain as a single strain produces folic acid.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korea Patent Publication No. 2007-0048776 Korean Patent No. 10-0882278 Korean Patent No. 10-0882279

The present inventors have studied to find lactic acid bacteria having excellent folic acid production ability. As a result, Lactobacillus plantarum JA71 strain having excellent folate productivity was successfully isolated from the fermented seafood, and the applicability of the strain to the fermentation product was confirmed experimentally, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a novel Lactobacillus plantarum JA71 strain having excellent folate-producing ability.

Another object of the present invention is to provide a fermentation starting material composition comprising the strain.

It is still another object of the present invention to provide a fermented product prepared by fermenting the fermentation starting material composition.

It is still another object of the present invention to provide a food composition comprising the fermented product.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a strain of Lactobacillus plantarum JA71 (accession number: KACC 91876P) excellent in folic acid production ability.

The strain L. plantarum JA71 of the present invention was isolated from the fermented fish as lactic acid bacteria and screened and identified according to the method described in the following Examples. The identification of the microorganism of the present invention is based on the morphological, physiological and biochemical characteristics of the microorganism.

Microbial Isolation and Identification

The novel lactic acid bacteria of the present invention were inoculated on a modified MRS medium (see Table 1 in Example 1) diluted with a peptone diluent and NaN ₃ in a planar manner by 0.1 ml each at 37 ° C After 48 hours of incubation, each strain was purified from transformed MRS medium and then transformed into yellow as a potential lactic acid bacteria. The selected strains were plated on the modified MRS medium and then cultured aerobically to isolate pure strains. As a result of the identification of pure isolates, the strain was Gram positive bacillus and showed good growth regardless of presence or absence of oxygen and negative for catalase and motility. Also, it was grown at 15 ° C and 45 ° C, and it was confirmed that it belonged to Lactobacillus genus because it did not produce ammonia from gas and arginine from glucose. On the other hand, 49 kinds of sugar fermentation tests were conducted to confirm the species names. As a result, an acid was produced from 25 kinds of sugars such as glucose (see Table 3 in the following examples) The result obtained using the Gene Sequence (Fig. 3) was concordant with that of Lactobacillus plantarum ( L. plantarum ). The strains having excellent folic acid production ability, which were separated from the fermented fish paste of the present invention, ( Lactobacillus plantarum ) JA71, and deposited with Accession No. KACC 91876P on October 8, 2013 to the Korean Society for Agricultural Microbiology and Resources (KACC), Agricultural Biotechnology Research Institute.

Microbial growth

The viable cell count and pH were measured, and the growth of the lactic acid bacteria of the present invention was tested. As a result, the optimum growth temperature was 37 ° C.

Antibiotic resistance

The antimicrobial resistance test was performed to determine the minimum inhibitory concentration (MIC) of antibiotics in the presence of antibiotics. As a result, the antimicrobial resistance to neomycin, vancomycin and polymycin was found to be higher than that of bacitracin and chloramphenicol (See Table 5 below).

Enzyme activity

As a result of the enzyme activity test for the lactic acid bacteria isolated according to the present invention, the strain L. plantarum JA71 of the present invention was found to inhibit the enzymatic activity of valine arylamidase,? -Galactosidase and? -Glucosidase Activity. In the case of β-glucuronidase, a carcinogenic enzyme that converts benzopyrene to a carcinogenic substance, there is no enzyme activity and its safety can be confirmed.

Biliary and pH tolerance

The growth of lactic acid bacteria according to the present invention in the MRS medium was tested and the resistance to bile was not significantly affected until 4 hours of culture. When the bile was not added and added for 7 hours, 0.8 log , But it was found to be resistant to bile. As a result of the test for pH resistance, it was confirmed that even at pH 2, which is a strong acid, it is hardly influenced by growth and has a strong acid resistance compared with the control pH of 6.4.

Antibacterial activity

In the antibacterial test, the strain L. plantarum JA71 of the present invention showed 96.7% inhibition against Salmonella typhimurium and 76.8% against Staphylococcus aureus , Respectively. And 60.6% for Escherichia coli , which was slightly less inhibitory than other food poisoning bacteria.

According to another aspect of the present invention, there is provided a fermentation starting material composition comprising a fermentation substrate inoculated with a strain of Lactobacillus plantarum JA71 (Accession No: KACC 91876P).

The lactic acid bacteria of the present invention can be used as a starter strain of a fermentation starting material.

According to one embodiment of the present invention, in the fermentation starting material composition of the present invention, the fermentation substrate is soy milk.

According to another aspect of the present invention, there is provided a fermentation product prepared by fermenting the above fermentation starting material composition, comprising a fermentation product containing a strain of Lactobacillus plantarum JA71 (accession number: KACC 91876P) do.

According to another aspect of the present invention, there is provided a food composition comprising a fermentation product comprising the strain Lactobacillus plantarum JA71 (Accession No: KACC 91876P).

The present invention relates to a Lactobacillus plantarum JA71 strain having excellent folate productivity. The strain of the present invention is excellent in folic acid production ability, has a growth inhibitory activity against food poisoning causing bacteria, and has a bile resistance and a strong acid resistance. In addition, since it exhibits excellent acid-producing fermentation characteristics in soymilk, it can be used as a fermentation starter strain for producing fermented soybean milk.

FIG. 1 shows the chromatogram of folate standard measured by HPLC.
Fig. 2 shows the results of folate chromatogram of soymilk fermented by JA71 strain measured by HPLC.
Fig. 3 shows the results of the lineage count of JA71 strain isolated from the fermented seafood.
4 shows the results of measurement of the growth rate at 34 ° C, 37 ° C and 40 ° C for 24 hours using the MRS liquid medium for the lactic acid bacterium L. plantarum JA71 strain of the present invention.
FIG. 5 shows the results of measurement of the pH change in the medium for 24 hours at 34 ° C, 37 ° C and 40 ° C using the MRS liquid medium for the lactic acid bacterium L. plantarum JA71 strain of the present invention.
Figure 6 shows the results of measurement of bile tolerance of the lactic acid bacterium L. plantarum JA71 strain of the present invention.
FIG. 7 shows the results of measurement of fermentation characteristics of the lactic acid bacterium L. plantarum JA71 strain of the present invention in soybean milk.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Isolation of microorganisms

The fermented seafood was sampled and inoculated on a modified MRS medium (see Table 1) in which Bromcresol purple and sodium azide were added (refer to Table 1) in a flatness condition of 0.3 ml and cultured at 37 ° C for 48 hours Respectively. Then, each of the herbicides was separated pure from the above-mentioned modified MRS medium, and the yellow-transformed herb was selected as a potential lactic acid bacterium. The selected strains were plated on the modified MRS medium three times and then cultured aerobically to isolate pure strains. Table 1 below shows the composition of the modified MRS medium used for microbial isolation.

 ingredient Component Ratio (g / l)  Proteose Peptone # 3  10.0 Beef extract  10.0 Yeast extract 5.0 Lactose 20.0 Twin 80 1.0 Ammonium citrate 2.0 Sodium acetate 5.0 Magnesium sulfate 0.1 Manganese sulfate  0.05 Dipotassium phosphate 2.0 Sodium azide 0.25 Bromocresol purple 0.04

Example 2: Folic acid production ability lactic acid bacteria

600 strains isolated from 24 kinds of fermented seaweed and 13 kinds of Chungkukjang were inoculated 1% in MRS liquid medium and cultured for 24 hours. Folic acid content was measured by the following HPLC analysis conditions (Table 2, Fig. 1) These excellent strains are shown in Table 3 and Fig.

As shown in Table 3, the isolated strain of lactic acid bacteria JA71 was found to have a folate-producing ability of 9.03 μg / ml after culturing for 24 hours.

Example 3: Identification of microorganisms

The taxonomic identification of the microorganisms was carried out according to the method of Hammes et al. [The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)] and the results are shown in Table 4 below. All of these strains were Gram - positive, Bacillus, well grown regardless of presence or absence of oxygen, and catalase and motility were negative. In addition, it was grown at 15 ° C and 45 ° C, and ammonia was not generated from glucose and arginine, and thus it was confirmed to be a bacterium belonging to the genus of Lactobacillus . In order to identify the species, 49 kinds of sugar fermentation tests were carried out using API 50CHL kit (API bioMerieux, France). Acid was produced from 25 species such as glucose, The result obtained using the rRNA gene sequence (Fig. 3) was found to be identical with that of Lactobacillus plantarum . Lactobacillus plantarum JA71 was named as a strain having excellent folic acid production ability which was separated from the fermented seafood in the above process and was named as JA71 by the Korean Agricultural Microbiological Resource Center (KACC) of the Agricultural Biotechnology Research Institute on Oct. 8, KACC 91876P. Table 4 below summarizes the physiological and biochemical characteristics of the novel lactic acid bacterium L. plantarum JA71 of the present invention.

Gram reaction
Cell morphology
Spore formation
motility
Aerobic growth
Anaerobic growth
Catalase reaction
Grow at 15 ℃
Grow at 45 ° C
Gas production from glucose
Ammonia production from arginine +
rod
-
-
+
+
-
+
+
-
- Party ability Glycerol
Erythritol
D-arabinose
L-arabinose
Ribos
Glucose
Fructose
Mannos
Sorbos
Rams North
Dissytol
Inositol
Mannitol
Sorbitol
alpha -methyl-D-mannose
alpha -methyl-D-glucoside
N-acetylglucosamine
Amigalline
Arbutin
Esculin
Salincin
Cellobiose
Maltose
Lactose
Melibiose -
-
-
+
+
+
+
+
-
-
-
-
+
+
+
-
+
+
+
+
+
+
+
+
+ D-Cirrus
L-Cirrus
Adonitol
beta -methyl-D-thiazole
Galactose
Sucrose
Trehalose
Inulin
Melitos
Raffinos
Starch
Glycogen
Silica
Gentiobios
D-Turanos
D-lysos
D-tagatose
D-Fucose
L-Fucose
D-arabitol
L-arabitol
Gluconate
2-keto-gluconate
5-keto-gluconate
-
-
-
-
+
+
+
-
+
+
-
-
-
+
+
-
-
-
-
-
-
+
-
-

Example 4: Microbial growth test

The growth of lactic acid bacteria was measured by measuring viable cell count and pH. The viable cell counts were obtained by inoculating 1 ml of lactic acid bacteria into 150 ml of MRS liquid medium and incubating at 34 ° C, 37 ° C, and 40 ° C for 3 hours at 24 hours. The samples were diluted in 0.1% Plate count agar was poured on a plate, hardened, incubated at 35 ° C for 48 hours and counted by pH and temperature. The measurement results are shown in Fig. 4 and Fig. As can be seen from Figs. 4 and 5, the optimum temperature for lactic acid bacteria growth was 37 占 폚.

Example 5: Antibiotic resistance test

The antimicrobial resistance test was carried out using a tryptic soy broth (Difco, USA), and the lowest inhibitory concentration (MIC) value was determined by observing the growth by a 2-fold dilution method. The results are shown in Table 5 . Table 5 below shows the results of measuring antibiotic susceptibility of L. plantarum JA71.

Antibiotic The lowest inhibitory concentration (占 퐂 / ml) Aminoglycoside system Amikacin 160 Gentamicin 640 Kanamycin 1600 Neomycin 3200 Streptomycin * 1600 beta -lactam system Penicillin-G * 160 Methacillin 640 Oxacillin 120 Ampicillin 320 Gram-positive spectrum Bashtrasin * 30 Rifampicin 480 Novobaiosin 240 Lincomycin 100 Gram-voice spectrum Polymycin B * 2400 Broad spectrum Chloramphenicol 80 Vancomycin * 3200

*: units / ml

As can be seen in Table 5, L. plantarum JA71 was more resistant to neomycin, vancomycin and polymacine than to other antibiotics, while it was more susceptible to bacitracin and chloramphenicol.

Example 6: Enzyme activity test

The enzyme activity test was performed by diluting a strain cultured in MRS liquid medium at 37 ° C for 18 hours with physiological saline to prepare a sample of 10 ⁵ to 10 ⁶ cfu / ml, and then using API ZYM kit (API bioMerieux, France) Incubated at 37 ° C for 5 hours, and then subjected to enzyme reaction. The enzymatic activity was expressed as 0-5 in comparison with the standard color trademark, and the results are shown in Table 6. Table 6 below shows the results of comparing enzyme activities of L. plantarum JA71.

enzyme Enzyme activity Alkaline phosphatase
Esterase (C4)
Esterase lipase (C8)
Lipase (C14)
Leucine arylamidase
Valine arylamidase
Cysteine aryl amidase
Trypsin
Chymotrypsin
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase
? -galactosidase
? -galactosidase
β-glucuronidase
alpha -glucosidase
? -glucosidase
N-acetyl- beta -glucosamine amine
? -mannosidase
? -fucosidase One
One
One
One
4
5
2
0
0
One
3
One
5
0
2
5
4
0
0

*: Standard color is specified in steps from 0 to 5, 0 is negative and 5 is maximum intensity. 1 shows 5 nano moles, 2 has 10 nano moles, 3 has 20 nano moles, 4 has 30 nano moles, and 5 has an enzyme activity of 40 nano moles or more.

As can be seen from Table 6, L. plantarum JA71 showed high enzyme activity in valine arylamidase,? -Galactosidase and? -Glucosidase. In the case of β-glucuronidase, a carcinogenic enzyme that converts benzopyrene to a carcinogenic substance, there is no enzyme activity and its safety can be confirmed.

Example 7: Bile tolerance test

The bile tolerance test was performed according to the method of Gilliland and Walker (Gilliland & Walker, J. Dairy Sci., 73, 905, 1990), and the measurement results are shown in FIG. As can be seen in FIG. 6, there was little effect until 4 hours of incubation. After incubation for 7 hours, the difference was 0.8 log difference between when no bile was added and when added, .

Example 8: pH resistance test

The pH tolerance test was carried out according to the method of Clark et al. (Clark et al., Cultured Dairy Products J., 28 (4), 11, 1993), and the measurement results are shown in Table 6. Table 7 below shows the results of comparing the tolerance of L. plantarum JA71 after 3 hours in hydrochloric acid solution.

Culture time (hr) pH 2 pH 3 pH 4 pH 6.4 0 8.43 8.43 8.39 8.37 One 8.48 8.49 8.51 8.50 2 8.53 8.50 8.50 8.58 3 8.55 8.59 8.58 8.58

Unit: CFU per ㎖ (Log number)

As can be seen from Table 7, the strain of the present invention has very strong acid resistance because it is hardly affected by growth even at pH 2, which is a strong acid, compared to the control pH of 6.4.

Example 9: Antimicrobial activity test

The antimicrobial activity test was carried out according to Gilliland & Speck, J. Food Prot., 40 (12), 820, 1977. The measurement results are shown in Table 8 below. Table 8 below shows the results of measurement of L. plant arum JA71 inhibition (*) activity on the MRS medium.

* Initial number of L. plant arum JA71: 5.0 X 10 ⁶ CFU / ml

^a Measurement after incubation at 37 ° C for 6 hours

As can be seen in Table 8, the strain L. plantarum JA71 of the present invention showed 96.7% inhibition against Salmonella typhimurium , and the inhibitory effect against Staphylococcus aureus Staphylococcus aureus ) showed 76.8% inhibition. And 60.6% for Escherichia coli , which was slightly less inhibitory than other food poisoning bacteria.

Example 10: Lactobacillus plantarum ( L. plant arum) Measurement of pH change for application of strain JA71

In order to confirm whether the strain of the present invention is suitable as a fermented milk starter, L. plantarum JA71 was used in the test while culturing in soybean milk. L. plantarum JA71 was inoculated to soybean milk at a concentration of 1.0%, and the pH was changed after incubation at 37 캜 for 3 hours for 24 hours. The measurement results are shown in FIG. As can be seen from FIG. 7, fermentation was carried out in soybean milk, and thus the lactic acid bacteria of the present invention were found to be used as a starter strain of fermented soybean milk.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Agricultural Biotechnology Research Institute KACC91876P 20131008

Claims (4)

Lactobacillus plantarum JA71 (accession number: KACC 91876P), which has antifungal activity against Salmonella typhimurium and Staphylococcus aureus , and which has excellent folic acid production ability.
Fermented with Lactobacillus plantarum JA71 (accession number: KACC 91876P), which has excellent antifungal activity against Salmonella typhimurium and Staphylococcus aureus , A fermentation starter composition comprising a substrate.
A fermentation product obtained by fermenting the fermentation starting material composition of claim 2, wherein the fermentation product comprises a strain of Lactobacillus plantarum JA71 (accession number: KACC 91876P).
A food composition comprising the fermented product of claim 3.

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