KR101471033B1 - Novel Strains of Weissella sp. F22 with Anti-Obesity Ability - Google Patents

Abstract

The present invention relates to strains of Weissella sp. F22 having anti-obesity ability. The strains of the present invention have excellent anti-obesity ability, show growth inhibition activity against food poisoning-inducing virus, and have bile tolerance and strong acid resistance. Also, the strains show acid production fermentation characteristic, thereby being used as strains for fermentation starter to produce fermented milk products.

Description

Novel Strains of Weissella sp. ≪ RTI ID = 0.0 > F22 with Anti-Obesity Ability}

The present invention relates to a novel Weissella sp. Strain F22, and more particularly to a novel Weissella sp. F22 which has an ability to inhibit obesity suitable for producing fermented milk.

Obesity means that the energy source such as carbohydrate or lipid is abnormally accumulated as a triglyceride in living tissue, especially subcutaneous adipose tissue or adipose tissue of organ organs, and the body weight is increased beyond the limit of skeletal system or physiological function. In particular, visceral adipose tissue accumulated around the intestines of the mesentery and the like is known to cause the abnormalities of carbohydrate metabolism and lipid metabolism, and there are cases in which lifestyle-related diseases such as cardiovascular disease, arteriosclerosis, hypertension, diabetes, . Treatment methods for obesity are diet - exercise, surgery and drug therapy. Dietary-exercise therapy is a method of treating low-calorie-low-fat meals and oxygen-consuming physical activity, which is perceived to be difficult to see because of the need to be patient, repetitive, and continuous. Surgery is a method of physically removing body fat through surgical operations, which is advantageous in that it can be effective in a short period of time. However, it is limited due to the necessity of surgery, lack of persistence of effect, and cost . Pharmacotherapy is a method to treat or prevent obesity by using drugs that induce appetite reduction or inhibit fat absorption. Recently, many studies for the development of obesity treatment drugs have been carried out. One of them is pancreatic lipase inhibitor. Pancreatic lipase acts as a key enzyme that breaks down triglyceride into 2-monoacylglycerol and fatty acid [Bitou, N., M. Nimomiya, T. Tsjita and H. Okuda. 1999. Screening of lipase inhibitors from marine algae. Lipids 34, 441-445]. Representative pancreatic lipolytic inhibitors are tetrahydrolipstatin (Orlistat), a derivative of lipstatin derived from Streptomyces toxitricini , which is known to be effective in inhibiting about 30% of the fat ingested [Drent, ML, et al., 1995. Orlistat RO 18-0647), a lipase inhibitor, in the treatment of human obesity: a multiple dose study. Int. J. Obes. 19, 221 -226.], And is currently also being marketed as a pharmaceutical product. Despite these benefits, however, tetrahydrolipstatin has been known to have side effects such as gastrointestinal disorders, hypersensitivity, biliary secretion, and lipid-soluble vitamin absorption inhibition. Recently, studies for the development of pancreatic lipolytic enzyme inhibitors from foods and natural products without side effects have been carried out.

Lactic acid bacteria refers to a bacterium that produces about 50% or more of lactic acid as a metabolite in a sugar that is commonly consumed. It is a food or a substance harmful to human body in human and animal intestines, such as an indole is used to conceptually generically refer to bacteria that do not produce substances such as indole, skatole, phenol, amine, and ammonia and prevent decay. Lactic acid bacteria have long been used in the manufacture of foods such as fermented milk, lactic acid bacteria beverages, fermented butter, and other dairy products, but lactic acid bacteria themselves have various pharmacological effects as well as a dressing effect and are therefore also used as materials for health foods and medicines . Korean Patent Laid-Open No. 10-2010-0109661 discloses a novel Lactobacillus carvettus strain having a blood cholesterol lowering effect and an obesity-inhibiting effect, and Korean Patent Publication No. 10-2012-0128260 discloses a novel Lactobacillus species Discloses a composition for treating or preventing obesity and obesity-related diseases including lactic acid bacteria, and Korean Patent Laid-Open No. 10-2010-0010015 discloses a lactobacillus Johnsonia strain having a blood cholesterol lowering and anti-obesity activity.

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.

Korean Patent Publication No. 10-2010-0109661 Korean Patent Publication No. 10-2012-0128260 Korean Patent Publication No. 10-2010-0010015

The present inventors have sought to find lactic acid bacteria having excellent ability to inhibit obesity. As a result, we succeeded in isolating and identifying Weissella sp. F22 strain having excellent ability to inhibit obesity from feces. The applicability of this strain to a fermentation product was confirmed experimentally and the present invention was completed.

Accordingly, an object of the present invention is to provide a novel lactic acid bacteria strain having excellent ability to inhibit obesity.

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 Weissella sp. F22 (accession number: KACC 91867P) excellent in the ability to inhibit obesity.

The Weissella sp. F22 strain of the present invention was isolated from feces 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 microorganism of the present invention was isolated from adult feces and was inoculated with modified MRS medium (see Table 1 of Example 1 below) diluted with peptone diluent and NaN ₃ in a planar state by 0.1 ml, For 48 hours. Each strain was purified from the transformed MRS medium and then transformed into yellow as a potential lactic acid bacterium. The selected strains were plated on the electrotransformed MRS medium and 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. In addition, it did not grow at 15 ° C, grew at 45 ° C, and did not produce ammonia from gas and arginine from glucose. When 49 sugar fermentation tests were carried out, acid was produced from 24 kinds such as glucose. (See Table 3 in the following examples)

The result obtained using the 16S rRNA gene sequence (Fig. 1) was finally identified as Weissella sp. Weissella sp. F22 was named as a strain having excellent ability to inhibit obesity in adult feces of the present invention and designated as Accession No. KACC91867P (National Institute of Agricultural Biotechnology, KACC) ≪ / RTI >

Microbial growth

The viable cell count and pH were measured, and the growth of the lactic acid bacteria of the present invention was tested according to the temperature. As a result, the optimum growth temperature was 40 캜.

Antibiotic resistance

The antimicrobial resistance test showed that the antimicrobial activity of the antibiotics in the presence of antibiotics was not affected by the antimicrobial resistance to polymyxin, streptomycin and vancomycin, (See Table 4 in the following Examples).

Enzyme activity

As a result of the enzyme activity test on the isolated lactic acid bacteria of the present invention, Weissella sp. F22 showed high enzyme activity in acid phosphatase.

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

When the lactic acid bacteria of the present invention were tested for growth resistance in the MRS medium, the tolerance to bile was measured to be slightly inhibited by 0.25 log difference between when the bile was not added and when the bile was added after 7 hours of culture, And that the tolerance was sufficient. As a result of the test for pH resistance, it was confirmed that even at pH 2, which is a strong acid, it is not influenced by growth and has a strong acid resistance, compared with the control pH of 6.4.

Antibacterial activity

In the antimicrobial activity test, Weissella sp. F22 of the present invention showed the highest inhibitory effect against Staphylococcus aureus (87.3%) and the inhibitory effect against Salmonella typhimurium (60.0%) against Salmonella typhimurium Respectively. On the other hand, there was no inhibition against Escherichia coli .

Obesity inhibitory activity

The Enterococcus faecalis of the present invention, The MD366 strain showed excellent pancreatic lipase inhibitory activity and showed excellent anti-adipogenic activity in the adipocyte differentiation activity test using 3T3-L1 adipocyte precursor cells.

According to another aspect of the present invention, there is provided a fermentation starter composition comprising a fermentation substrate inoculated with a strain of Weissella sp. F22 (Accession No .: KACC 91867P).

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 milk.

According to another aspect of the present invention, there is provided a fermentation product prepared by fermenting the above fermented starter composition, comprising a fermentation product containing a strain of Weissella sp. F22 (accession number: KACC 91867P) do.

According to yet another aspect of the present invention, the present invention provides a food composition comprising a fermentation product comprising the strain Weissella sp. F22 (Accession No. KACC 91867P).

The present invention relates to a strain of Weissella sp. F22 which is excellent in the ability to inhibit obesity. The strain of the present invention is excellent in the ability to inhibit obesity, has a growth inhibitory activity against food poisoning-causing bacteria, and has bile resistance and strong acid resistance. Further, since the skim milk exhibits acid-producing fermentation characteristics, it can be used as a fermentation starter strain for producing fermented milk products.

Fig. 1 shows the results of the phylogenetic tree of F22 isolates isolated from adult feces.
FIG. 2 shows the results of measuring the inhibitory activity of the lactic acid bacterium Weissella sp. F22 according to the present invention on adipocyte differentiation using 3T3-L1 adipose precursor cells.
FIG. 3 shows the results of measurement of growth rate at 34 ° C., 37 ° C. and 40 ° C. for 24 hours using 10% skimmed milk powder of the lactic acid bacterium Weissella sp. F22 of the present invention.
Fig. 4 shows the results of measurement of changes in pH in the medium for 24 hours at 34 ° C, 37 ° C and 40 ° C using 10% skim milk powder for the lactic acid bacterium Weissella sp. F22 of the present invention.
Fig. 5 shows the result of measuring the bile resistance of the lactic acid bacterium Weissella sp. F22 of the present invention.
6 shows the results of measurement of fermentation characteristics of the lactic acid bacterium Weissella sp. F22 of the present invention in skimmed 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

Crude milk and adult fecal samples used as samples were inoculated into modified MRS media (see Table 1) supplemented with Bromcresol purple and sodium azide (see Table 1) in a flatness condition of 0.3 ml, And cultured for 48 hours. 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: Obesity inhibitory ability of lactic acid bacteria

Among 1,272 strains isolated from crude oil and 269 strains isolated from feces, porcine pancreatic lipase was used to measure lipase activity inhibition, and anti-lipase activity ) Were 65% or more. The selected four strains were inoculated 1% in 10% skim milk solution, cultured at 37 ° C, lyophilized, and then added to a 3T3-L1 cell line at a concentration of 100 μg / ml. Adipogenic activity experiments were conducted to determine whether the cells induced differentiation and lipid accumulation (adipogenesis) of the captive. The results of this experiment are shown in Table 2 and FIG.

As shown in Table 2, it was confirmed that the isolated strain of lactic acid bacteria F22 was 68.03% in lipolysis inhibition rate and 87.02% in lipolysis rate.

Example 3: Identification of microorganisms

The taxonomic identification of microorganisms was performed according to the method of Hammes et al. [The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)]. 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 did not grow at 15 ℃ and grew at 45 ℃, and did not produce ammonia from gas and arginine from glucose. When 49 sugar fermentation tests were carried out, acid was produced from 24 kinds such as glucose. The result obtained using the 16S rRNA gene sequence (Fig. 1) was found to be Weissella sp. F22. Weissella sp. F22 was named as an isolate of obesity-inhibiting ability isolated from adult feces by the above procedure and was deposited on October 1, 2013 by KACC (Korea Research Institute of Bioscience and Biotechnology) KACC 91867P.

Table 3 below summarizes the physiological and biochemical characteristics of the novel lactic acid bacterium Weissella sp. F22 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 number of viable cells was inoculated to 150 ml of 10% skim milk powder, and 1 ml of a lactic acid bacterium was inoculated with 1 ml of a pipette. Each sample was incubated at 34 ° C, 37 ° C, and 40 ° C for 3 hours at intervals of 3 hours, BCP 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. 3 and FIG. As can be seen from Figs. 3 and 4, the optimum temperature for lactic acid bacteria growth was 40 占 폚.

Example 5: Antibiotic resistance test

The antimicrobial resistance test was carried out by using a tryptic soy broth (Difco, USA), and the growth inhibition was examined by a 2-fold dilution method to determine the lowest inhibitory concentration (MIC) . Table 4 below shows the results of measuring antibiotic susceptibility of Weissella sp. F22.

Antibiotic The lowest inhibitory concentration (占 퐂 / ml) Aminoglycoside system Amikacin 320 Gentamicin 20 Kanamycin 400 Neomycin 400 Streptomycin * 1600 beta -lactam system Penicillin-G * 5 Methacillin 80 Oxacillin 30 Ampicillin 320 Gram-positive spectrum Bashtrasin * 3.75 Rifampicin 30 Novobaiosin 30 Lincomycin 800 Gram-voice spectrum Polymycin B * 2400 Broad spectrum Chloramphenicol 40 Vancomycin * 1600

*: units / ml

As can be seen in Table 4 above, Weissella sp. F22 is more resistant to polymyxin, streptomycin and vancomycin than penicillin and bacitracin, but is more susceptible to penicillin and bacitracin than other antibiotics.

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 enzyme activity is shown in Table 5 as the value of 0-5 compared with the standard color trademark. Table 5 below shows the results of comparing enzyme activities of Weissella sp. F22.

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
0
One
2
2
0
2
0
4
2
2
One
0
One
One
0
0
One

*: 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 shown in Table 5 above, Weissella sp. F22 showed high enzyme activity in acid phosphatase. 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 from FIG. 5, after the incubation for 7 hours, the difference was 0.25 log between the addition and the absence of bile.

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 6 below shows the results of comparing Weissella sp. F22 resistance after 3 hours in a hydrochloric acid solution.

Culture time (hr) pH 2 pH 3 pH 4 pH 6.4 0 7.37 7.40 7.41 7.39 One 7.41 7.47 7.50 7.46 2 7.50 7.55 7.56 7.58 3 7.48 7.53 7.55 7.54

Unit: CFU per ㎖ (Log number)

As can be seen from Table 6, the strain of the present invention was not 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 performed according to Gilliland & Speck, J. Food Prot., 40 (12), 820, 1977, and the measurement results are shown in Table 7 below. Table 7 below shows the results of measuring the activity of Weissella sp. F22 food poisoning bacteria (*) in the MRS medium.

Food poisoning bacteria Growth Food poisoning bacteria ^a Weissella sp. F22 ^{*, a} % Inhibition CFU / ml pH CFU / ml pH  Escherichia coli 1.2 × 10 ⁸ 6.46 1.8 x 10 ⁸ 5.54 -  Salmonella typhimurium 2.0 × 10 ⁶ 6.48 8.0 × 10 ⁵ 5.50 60.0  Staphylococcus aureus 1.5 × 10 ⁸ 6.54 1.9 × 10 ⁷ 5.40 87.3

* Weissella sp. Initial number of F22: 3.3 X 10 < ⁶ > CFU / ml

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

As can be seen in Table 7, the most inhibitory effect was high as 87.3% with respect to the strain-way gelatinase SP (Weissella sp.) F22 is stearyl Pyrococcus fluorescein mouse (Staphylococcus aureus) of the invention, Salmonella tie blood head Titanium (Salmonella typhimurium ) was inhibited by 60.0%. On the other hand, there was no inhibition against Escherichia coli .

Example 10: Measurement of pH change for product application of Weissella sp. Strain F22

To confirm whether the strain of the present invention is suitable as a fermented milk starter, Weissella sp. F22 was used for the test while culturing in skimmed milk. The skim milk was inoculated with 1.0% each of Weissella sp. F22 and cultured at 37 ° C for 3 hours for 24 hours. The pH change was measured. The measurement results are shown in FIG. As can be seen from FIG. 6, fermentation was carried out in skimmed milk. Thus, the lactic acid bacteria of the present invention were found to be suitable as starter strains of fermented 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 KACC91867P 20131001

Claims (5)

Weissella sp. F22 (accession number: KACC 91867P) which is excellent in the ability to inhibit obesity.
The strain according to claim 1, wherein the strain Weissella sp. F22 (accession number: KACC 91867P) has a pancreatic lipase inhibitory activity and an anti-adipogenic activity Lt; / RTI >
A fermentation starting material composition comprising a fermentation substrate inoculated with a strain of Weissella sp. F22 (accession number: KACC 91867P) having excellent inhibitory activity against obesity.
A fermentation product obtained by fermenting the fermentation starting material composition of claim 3, wherein the fermentation product is a strain of Weissella sp. F22 (accession number: KACC 91867P).
A food composition comprising the fermented product of claim 4.

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