KR100858288B1 - Novel strains of lactobacillus zeae rmk354 having ace inhibitory activity - Google Patents

Abstract

A novel strain of Lactobacillus zeae RMK354 is provided to improve milk coagulation activity and ACE(angiotensin converting enzyme) inhibitory activity as compared to conventional lactic acid bacteria, so that the strain is suitable for production of functional fermented milk. A novel strain of Lactobacillus zeae RMK354(KACC 91314P) showing improved milk coagulation activity and ACE inhibitory activity is isolated from raw milk by culturing the raw milk in modified MRS(Modified Rogosa SL) medium, isolating yellow colonies as lactic acid bacteria, selecting skim milk-coagulating strains, and selecting a strain showing excellent ACE inhibitory activity from the selected skim milk-coagulating strains, shows optimal temperature of 40 deg. C and needs 10 hours until a pH reaches 4.3 to 4.4 which is optimal pH of fermented milk.

Description

Novel Strains of Lactobacillus zeae RMK354 having ACE inhibitory activity}

1 is a graph showing the growth when inoculated with Lactobacillus Zia RMK354 10% reduced skim milk.

Figure 2 is a graph showing the pH change when inoculated with Lactobacillus Zia RMK354 10% reduced skim milk.

Figure 3 is a graph showing the growth of Lactobacillus Baia RMK354 in MRS medium containing L-cysteine with or without bile addition.

The present invention relates to a novel Lactobacillus Zea RMK354 strain, and more particularly, to Lactobacillus zeae ( Lactobacillus zeae ) RMK354, a novel lactic acid bacterium having the ability to inhibit lactate and ACE suitable for fermented milk production.

      In Korea, about 20% of adults have high blood pressure, 20% of which are severe hypertension, and the remaining 80% are known as moderate hypertension. According to the report of the labor union of the Korea Health Insurance Corporation, as a result of analyzing the health insurance medical records for one year from 02. 9 to 03. 9, there were 3,554 thousand patients with hypertension. Persons) were in their 60s, with 28.6% of the total.They were in their 50s (23.5%), over 70s (22.8%), 40s (16.9%), and 30s (6%). As this increase, hypertension was found to be more common.

Essential hypertension, which accounts for most of hypertension, a representative disease of modern adult diseases, is very important for the regulation of blood pressure by the renin-angiotensin system. In other words, angiotensinogen undergoes specific degradation of renin to produce angiotensin I, which in turn is angiotensin, which is vascular contracted by angiotensin converting enzyme (ACE). To generate II. ACE also breaks down and inactivates bradykinin with vascular relaxation, resulting in elevated blood pressure. As ACE is involved in the increase of blood pressure, the inhibition of ACE is essential for the drop of blood pressure, and the importance of ACE inhibitors in the prevention and treatment of hypertension has been highlighted as ACE inhibitors have been suggested to be developed as antihypertensive agents.

In 1977, captopril (2-D-mercaptopropanoyl-L-proline), a potent inhibitor of ACE, was developed, and several ACE inhibitors such as Enalapril and Benazepril were commercialized to treat high blood pressure. Although it is used, there are many side effects such as dry cough, anorexia, palate, rash, and white blood cell reduction (水 島 裕 & 宮本 昭 正, 505, 1996).

In the food industry, ACE inhibitors isolated from enzymatic degradation of food proteins as well as milk fermentation by lactic acid bacteria are emerging as functional foods. Currently, biopeptides released during milk fermentation by starter lactic acid bacteria are of considerable interest. Smacchi and Gubbetti (2000) describe the selection strains Lactobacillus helveticus , Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis subsp. Cremoris produced antihypertensive peptides. Calpis made of Lactobacillus helveticus and Saccharomyces cerevisiae contained antihypertensive biopeptides and showed inhibition of ACE and decreased blood pressure in vivo (Smacchi & Gubbetti, Food Microbiol. 17, 129, 2000).

Lactic acid bacteria, the starter of milk fermentation, can synthesize cell surface proteases, hydrolyze milk proteins and release some peptides from the medium (Gilbert et al, J. Dairy. Res. 64, 561, 1997). Since peptides are not all used for bacterial growth, large amounts of peptides accumulate during fermentation (Juillard et al, J. Bacteriol. 177, 3472, 1995). Some dairy products fermented with L. helveticus as starters have demonstrated antihypertensive activity in vivo (Sipola et al, J. Dairy Res. 69, 103, 2002).

In Korea, no significant progress has been made except for the study of antihypertensives of natural materials, but in June 2005, fermented milk that lowers blood pressure is being released from Namyang Dairyin under the product name Project 102-80. The main fermentation strain is a mixed strain of S. thermophilus and L. bulgaricus introduced by Wiseby, Germany, and it is used for fermentation. It is supplied and used and additionally, erythritol, fiber sol and natural substance extracts (RGP-HC90, ONC-129, YQ2) are added.

In addition, by making products using domestically developed lactic acid bacteria, it is expected to increase the import substitution effect of products and lactic acid bacteria and further international competitiveness to be exported to foreign countries.

Conventional lactobacillus starters are Gram-positive, rod-shaped or spherical, homo-lactic acid-fermenting bacteria, which are currently used as starters for fermented milk, but use spawns imported from foreign countries, and are isolated from crude milk and have excellent antihypertensive function. There was no case developed.

The present invention has been proposed to solve the problems of the prior art as described above, an object of the present invention is to provide a novel lactic acid bacteria Lactobacillus Giae RMK354 isolated from crude oil.

The present invention for achieving the above object is excellent strain Lactobacillus zeae milk coagulation and ACE inhibitory ability ( Lactobacillus zeae ) RMK354 (KACC 91314P).

Hereinafter, the present invention will be described in more detail.

The novel microorganisms of the present invention were isolated from crude oil and screened and identified according to the method of Examples described below. Identification of microorganisms was made based on their morphological, physiological and biochemical properties. The novel lactic acid bacteria isolated from crude oil was found to be suitable for fermented milk production with excellent lactic acid and ACE inhibitory ability, thus completing the present invention.

After culturing in modified MRS medium in crude oil, the inventors separated each colony purely and selected the colonies that turned yellow as potential lactic acid bacteria. The selected strains were plated in electro-modified MRS medium, and then separated by pure aerobic culture. As a result of identifying the purely isolated strains, the strains were Gram-positive bacillus, well grown with or without oxygen, and negative for catalase and motility. In addition, it was grown at 15 ℃ and 45 ℃, it did not generate ammonia from gas and alginic acid from glucose (glucose) was confirmed to belong to Lactobacillus ( Lactobacillus ).

On the other hand, 49 kinds of sugar fermentation tests were carried out to confirm the species name, acid was generated from 20 species such as lactose, inputted into the ATB identification system, and the results obtained by using the 16S rRNA gene sequence. Was matched and turned out to be Lactobacillus.

Therefore, the present invention is Lactobacillus zeae strains excellent in skim milk coagulation rate and ACE inhibitory ability among the skim milk coagulation strains isolated from crude milk Named RMK354.

As a result of testing the growth of lactic acid bacteria by measuring the number of viable cells and pH, the optimum temperature is 40 ℃, it takes 10 hours to reach the pH condition of pH 4.3-4.4. In addition, by measuring the growth inhibitory concentration (MIC) value through the antibiotic resistance test, the resistance to polymycin B and vancomycin compared to other antibiotics, while penicillin-G, bacitracin and novobio High sensitivity to God. As a result of enzyme activity test, enzyme activity was high in esterase and leucine arylamidase.

In addition, β-glucuronidase, a carcinogenic enzyme that converts benzopyrene to carcinogenic substances, has been shown to have no enzymatic activity, indicating safety. Bile resistance was measured by testing the growth of lactic acid bacteria in MRS medium. The time required to increase the OD value by 0.3 was 7.3 hours without bile and 8 hours with bile. It appears to be resistant to bile.

In addition, the test of the pH resistance shows that the acid resistance is hardly affected even at pH 2 which is a strong acid compared to the control pH of 6.4. In the antimicrobial test, it has no inhibitory activity against Escherichia coli and Staphylococcus aureus , but shows 60.0% inhibition against Salmonella typhimurium . As described above, the Lactobacillus geranium of the present invention. It can be seen that RMK354 is a suitable lactic acid bacterium for the production of fermented milk.

Lactobacillus geranium of the present invention RMK354 was deposited on May 4, 2007 with the accession number KACC 91314P to the Korea Institute of Agricultural Biotechnology (KACC).

Hereinafter, in the present invention, the Lactobacillus jiae RMK354 for convenience L. zeae Called RMK354.

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, but the scope of the present invention is not limited to these embodiments.

Example 1 Isolation of Microorganisms

Crude oil used as a sample was collected from ranches in each country in Korea and inoculated in a modified MRS medium containing bromcresol purple and sodium azide (Table 1) by 0.3 ml each in a plan view method 37 Incubated for 48 hours at ℃. Then, each colony was purely separated in electro-modified MRS medium, and the colony that turned yellow was selected as a potential lactic acid bacterium. The selected strains were plated with platinum three times in electro-modified MRS medium and then aerobicly cultured and purified. Pure isolates were inoculated with 10% reduced skim milk (Difco Laboratories, USA) and incubated at 37 ° C. for 24 hours to determine clotting.

TABLE 1 Composition of Modified MRS Medium Used for Microbial Separation in the Present Invention

 ingredient Ingredient ratio (gram / liter)  Proteas 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 Bromcresol Purple 0.04

Example 2 Selection of ACE Inhibitory Active Lactic Acid Strains

1% of lactic acid bacteria and commercial lactic acid bacteria isolated above were incubated in 10% reduced skim milk until pH 4.4 was reached at 37 ° C., and the results of measuring the ACE inhibition rate with the whey obtained from each are shown in Table 2 below.

<Table 2> Non-solidified milk coagulation of stored lactic acid strains and isolated strains in commercial strains, Korea Food Research Institute and results of ACE inhibition after culture

Storage lactic acid bacteria source Skim milk coagulation reaction pH ACE inhibition rate (%) 24hr 48hr L. acidophilus * NCFM, Rhone-poulenc + + 4.4 55.4 ± 5.6 L. acidophilus * Culture System + + 4.4 53.1 ± 3.4 Streptococcus themophilus * Culture System + + 4.4 44.3 ± 5.4 L. acidophilus * NCFM, Rhone-poulenc + + 4.4 48.7 ± 2.1 L. lactis subsp . lactis ATCC 21053 + + 4.4 63.1 ± 3.4 L. cremoris KFRI 00349 + + 4.4 62.6 ± 3.3 L. delbrueckii subsp . lactis ATCC 7830, IFO 3376 + + 4.4 73.1 ± 7.6 L. bulgaricus ATCC 33409 + + 4.4 84.8 ± 3.4 L. amylophilus NRRL B-4437 + + 4.4 74.5 ± 4.6 L. reuteri KFRI 00661 + + 4.4 82.5 ± 6.3 Selected Lactic Acid Bacteria RMK354 + + 4.4 88.6 ± 1.8

Note) * Commercial Strains

As shown in Table 2, the isolated lactic acid bacteria RMK354 strain was selected as a strain having excellent ACE inhibitory activity as the ACE inhibition rate was similar or higher than that of other strains.

Example 3 Identification of Microorganisms

The taxonomy of microorganisms is described by Hammes et al. The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992), and the results are shown in Table 3. These strains were both Gram-positive and bacillus, well grown with or without oxygen, and catalase and motility were negative. In addition, it was grown at 15 ℃ and 45 ℃ belonged to the genus Lactobacillus (genus) because it does not produce gas from glucose (glucose), and ammonia from arginine (arginine). In order to determine the species, when 50 kinds of sugar fermentation tests were conducted using API 50CHL kit (API bioMerieux, France), acid was generated from 20 species such as lactose and inputted into the ATB identification system. The results obtained using the Gene Sequence were confirmed to be Lactobacillus strains. Lactobacillus zeae RMK354 was named among the skim milk coagulation strains isolated from crude oil with excellent skim milk coagulation rate and ACE inhibitory activity, and deposited on the May 4, 2007 to the Korea Agricultural Microbiological Resource Center (KACC). Deposited as 91314P.

Table 3 Physiological and biochemical properties of the novel microorganism L. zeae RMK354 of the present invention

 Gram Responsive Cell Form Sporulation Motility Aerobic Growth Anaerobic Growth Catalase Reaction Growing at 15 ° C Growing at 45 ° C Producing Gas from Glucose Forming Ammonia from Arginine + rod--+ +-+ +-- Party use ability  Glycerol Erititol D-Arabinose L-Arabinose Ribose Glucose Fructose Mannose Sorbose Rhamnose Dulcitol Inositol Mannitol Sorbitol α-Methyl-D-Mannose α-Methyl-D-Glucoside N Acetyl Glucoseamine Amigalin Arbutin Esculin Salicylic Cellobiose Maltose Lactose Meliviose ---+ + + + +-+--+---+ + + + + + + +-  D-Cyros L-Cyros Adonitol β-Methyl-D-Cyrroside Galactose Sucrose Trehalose Inulin Melezitose Raffinose Starch Glycogen Silitol Genthiobios D-Turanos D-Lysose D-Tagatos D- Fucose L-fucose D-arabitol L-arabitol Gluconate 2-keto-gluconate 5-keto-gluconate ----+ + +-+----+-----------

To determine whether it is suitable as a starter for fermented milk, L. zeae RMK354 was used in the test while incubated in 10% reduced skim milk.

Example 4 Growth Test of Microorganisms

The growth of lactic acid bacteria was tested by measuring the number of live bacteria and pH. The number of viable cells was inoculated with 150 ml of 10% skim milk in 1 drop of lactic acid bacteria with a pipette for 1 ml, and each sample incubated for 3 hours at 34, 37, and 40 ° C for 24 hours was diluted in 0.1% peptone solution. Poured from the agar plate and hardened and counted by incubation at 35 ℃ for 48 hours, the pH change was measured by temperature and time and the results are shown in Figures 1 and 2. As can be seen in Figures 1 and 2, the optimum temperature is 40 ℃, it took 10 hours to reach the pH pH 4.3-4.4 optimal fermented milk conditions.

Example 5 Antibiotic Resistance Test

In the antibiotic resistance test, tryptic soy broth (Difco, USA) was used to determine the minimum inhibitory concentration (MIC) value by observing growth by a 2-fold dilution method and the results are shown in Table 4. It was.

TABLE 4 Antibiotic susceptibility of L. zeae RMK354

Antibiotic Lowest inhibitory concentration (㎍ / ㎖) Aminoglycosides Amikacin 1.25 Gentamicin 1.25 Kanamycin 100 Neomycin 12.5 Streptomycin * 50 β-lactam meter Penicillin-G * 0.156 Methicillin 160 Oxacillin 7.5 Ampicillin 20 Gram-positive spectrum Bassitracin * 0.312 Rifampicin 30 Novobiocin 0.312 Lincomycin 20 Gram-Voice Spectrum Polymycin B * 1,200 Broad spectrum Chloramphenicol 10 Vancomycin * 1,600

 *: units / ml

As can be seen in Table 4, L. zeae RMK354 was more resistant to polymycin B and vancomycin than other antibiotics, while it was highly susceptible to penicillin-G, bacitracin and novobiocin.

Example 6 Enzyme Activity Test

Enzymatic activity test is 10 ⁵ to dilute the strains were cultured for 37 ℃, 18 hours in MRS broth in saline - and then was adjusted to 10 ⁶ cfu / ㎖ level of the sample, using the API ZYM kit (API bioMerieux, France) After incubating at 37 ° C. for 5 hours, enzyme reaction was performed. Enzyme activity is shown in Table 5 by comparing the standard color table with a value of 0-5.

TABLE 5 Comparison of Enzyme Activities of L. zeae RMK354

enzyme Enzyme activity Alkaline phosphatase esterase (C4) esterase lipase (C8) lipase (C14) leucine arylamidase valine arylamidase cystine arylamidase trypsin chymotrypsin acid phosphatase naphthol-AS-BI-phosphohydrolase α-galacto Cedarase β-galactosidase β-gluronidase α-glucosidase β-glucosidase N-acecyl-β-glucosaminenidase α-mannosidase β-fucosidase  0 3 2 0 3 0 0 0 0 0 1 0 1 0 0 0 0 0 0

*: Standard colors are specified in steps from 0 to 5, where 0 is negative and 5 is maximum intensity. 1 is 5 nanomoles (nanomoles), 2 is 10 nanomoles, 3 is 20 nanomoles, 4 is 30 nanomoles, 5 is 40 nanomoles or more enzyme activity.

As can be seen in Table 5, L. zeae RMK354 showed high enzymatic activity in esterase and leucine arylamidase. Β-glucuronidase, a carcinogenic enzyme that converts benzopyrene to carcinogenic substances, was found to have no enzymatic activity, indicating safety.

Example 7 Bile Resistance Test

Bile tolerance test was measured according to the method of Gilliland and Walker (Gilliland & Walker, J. Dairy Sci., 73, 905, 1990) is shown in FIG. As can be seen in Figure 3, the time required for the OD value to increase by 0.3 is 7.3 hours without bile and 8 hours when bile is added. appear.

Example 8 pH Resistance Test

The pH resistance test was measured according to Clark et al. (Clark et al., Cultured Dairy Products J., 28 (4), 11, 1993) and the results are shown in Table 6.

Table 6 Comparison of L. zeae RMK354 resistance after 3 hours in hydrochloric acid solution

                                               Unit: CFU / mL (Log number)

Incubation time (hr) pH 2 pH 3 pH 4 pH 6.4 0 7.79 7.82 7.83 7.83 One 7.70 7.72 7.79 7.86 2 7.64 7.71 7.78 7.88 3 7.51 7.67 7.74 7.89

As can be seen in Table 6, it was shown that there was little effect even at pH 2, which is a strong acid, compared to the control pH of 6.4.

Example 9 Antibacterial Activity Test

The antimicrobial activity was measured according to Gilliland & Speck, J. Food Prot., 40 (12), 820, 1977. The results are shown in Table 7.

TABLE 7 L. zeae in MRS medium RMK354 Suppression of Food Poisoning Bacteria (*)

Food poisoning bacteria Growth Food poisoning bacteria ^a L. zeae RMK354 ^a % Inhibition CFU / mL pH CFU / mL pH Esherichia coli 9.2 × 10 ⁶ 6.56 2.3 × 10 ⁸ 5.64 -  Salmonella typhimurium 1.45 × 10 ⁷ 6.55 5.8 × 10 ⁶ 5.67 60.0 Staphyloccous aureus 3.2 × 10 ⁴ 6.55 7.7 × 10 ⁸ 5.76 -

* L. zeae Initial number of RMK354: 3.8 × 10 ⁶ CFU / mL

^a measurement after 6 hours of incubation at 37 ° C

As can be seen in Table 7, L. zeae RMK354 had no inhibitory activity against Escherichia coli and Staphylococcus aureus , but showed 60.0% inhibition against Salmonella typhimurium .

Example 10 ACE Inhibition Activity Optimal Culture Conditions

end. Effect of Reduced Skim Milk and Inoculation on ACE Inhibitory Activity

After culturing for 15 hours at 37 ℃ for each reduced skim milk content and inoculum inoculation, the results of measuring the ACE inhibitory activity is shown in Table 8. When the reduced skimmed milk concentration was 10%, the addition amount of the bacteria showed the highest ACE inhibitory activity when 0.1% was added.

<Table 8> Effect of reduced skim milk content and bacterial inoculum on ACE inhibitory activity

                   Inoculation amount (%) Reduced skim milk (%) 0.1 0.5 One ACE inhibition rate (%) 8 62.0 ± 5.7 76.4 ± 2.1 80.9 ± 7.2 10 89.2 ± 3.3 88.9 ± 6.7 88.5 ± 2.3 12 88.9 ± 8.3 75.5 ± 3.1 80.9 ± 7.2

I. Effect of Incubation Temperature and Incubation Time on ACE Inhibitory Activity

After adding 0.1% of the inoculum inoculum to 10% of the reduced skim milk concentration, which is the optimum condition obtained above, and culturing at 24 hours for 3 hours at each culture temperature, the ACE inhibition rate is measured as shown in Table 9 below. As shown in Table 9, the ACE inhibitory activity was 89.2% for 15 hours at 37 ° C and 89.4% for 15 hours at 40 ° C.

 <Table 9> Effect of incubation temperature and incubation time on ACE inhibitory activity

                 Incubation temperature (℃) Incubation time (hours) 34 37 40 ACE inhibition rate (%) 0 33.9 55.1 51.4 3 34.8 69.1 52.7 6 36.9 73.2 53.7 9 45.6 76.4 73.2 12 54.8 88.2 77.5 15 56.6 89.2 89.4 18 48.0 83.5 53.0 24 39.0 73.8 38.0

As described above, the present invention is Lactobacillus zeae ( Lactobacillus zeae ) that is a lactic acid fermentation bacteria collected from crude oil Provides RMK354. Lactic acid bacteria of the present invention is superior to the lactic acid fermentation bacteria found in the conventional ACE inhibitory activity, it is suitable for producing fermented milk.

Claims (1)

Lactobacillus zeae strain with excellent milk coagulation and ACE inhibitory activity RMK354 (KACC 91314P).

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