KR101453302B1 - Novel Lactobacillus acidophilus NS with probiotic activities and use thereof - Google Patents

Abstract

More particularly, the present invention relates to a novel functional strain selection, which is separated from infant feces and has a function of suppressing acid resistance, bile acid tolerance, intestinal adherence ability, cholesterol lowering and obesity, so that a food composition such as a weight control food, Food, pharmaceutical composition, and the like, which can be widely used.

Description

Novel Lactobacillus acidosis NS with probiotic activity and its use {Novel Lactobacillus acidophilus NS with probiotic activities and use thereof}

More particularly, the present invention relates to a novel functional strain selection, which is separated from infant feces and has a function of suppressing acid resistance, bile acid tolerance, intestinal adherence ability, cholesterol lowering and obesity, so that a food composition such as a weight control food, Food, pharmaceutical composition, and the like, which can be widely used.

These microorganisms are called probiotics. Lactobacillus, which is represented by Lactobacillus or Bifidobacterium, accounts for most of the probiotics. In Korea, it has been recognized as a representative health functional food having function of formal action.

Lactic acid bacteria are bacteria that make lactic acid, which is usually called lactic acid bacteria, and it is widely distributed in human and animal digestive tracts, milk, yogurt, cheese, kimchi, plants and soil. It is a beneficial symbiosis that has a close relationship indirectly. In recent years, studies on lactic acid bacteria have been actively carried out, and their application range has been broadened not only as general foods but also as health foods and medicines. Lactic acid bacteria belong here streptomycin Cocos (Streptococcus), A Peddie Oh Caucus (Pediococcus), A kind Kono Stock (Leuconostoc) genus Lactobacillus (Lactobacillus) in, to Spokane Lactobacillus (Sporolactobacillus) in the Bifidobacterium (Bifidobacterium ). These fungi are gram-positive bacteria, anaerobically or organically anaerobic, and live in the intestines of animals. It is known that it plays an important role in maintenance of intestinal health by decomposing nutrients and fibrin which are ingested by animals and using it as an energy source and by producing lactic acid and antibiotics to suppress the development of harmful bacteria in the intestines.

In this case, there are various selection criteria for the lactic acid bacteria to be used in foods or medicines, and it is necessary to confirm acid resistance, bile acid resistance, cholesterol lowering effect, antimicrobial effect and the like. Only lactic acid bacteria having such effects are required to normalize intestinal flora, (lactose intolerance), lowering of blood cholesterol level, anticancer effect and strengthening of immune system.

Globally, the population of obesity is rapidly increasing, and the proportion of suffering complications is increasing. Especially when the cholesterol content is increased by obesity, the risk of heart disease, cerebrovascular disorder, hypertension and the like is increased. The risk of these diseases is rising significantly in Korea and is closely related to the eating habits of Koreans. Cholesterol is divided into LDL (low density lipoprotein) - cholesterol and HDL (high density lipoprotein) - cholesterol. LDL cholesterol has a radius of about 20-25 nm (10-9 m), the outermost is a phospholipid monolayer, and has a protein called apoB-100. And it has cholesterol and fatty acid inside. The main role is to carry fatty acids and cholesterol to cells. In addition, most of the blood cholesterol is present in the form of LDL cholesterol, which migrates through the low-density lipoprotein receptor. When the intracellular cholesterol concentration is increased, the rate of LDL-receptor synthesis decreases and cholesterol absorption from the blood into the cells slows down. Therefore, the blood cholesterol concentration is increased, leading to other complications.

In general, many drugs such as statins and niacin are used as therapeutic agents for lowering cholesterol. Lovastatin, provastatin, and simvastatin, statin-based compactin derivatives, are among the most commonly used drugs worldwide. These drugs inhibit the activity of HMG-CoA Reductase (3-hydroxy-methyl-glutamyl coenzyme A reductase), a cholesterol biosynthesis regulator. As a result, the amount of LDL-receptor expression is increased and the intracellular level of LDL is increased, thereby lowering the blood cholesterol level.

Despite these cholesterol-lowering functions, these drugs have side effects such as hepatotoxicity, myalgia, gastrointestinal disorders, and carcinogenicity. Therefore, rather than relying on pharmacotherapy, diuretic therapy is being performed in a direction that expects to be effective. However, dieting is not an easy method, and patients with high cholesterol levels need a different approach.

For this reason, a number of other efficacies have been researched besides the suitability of GRAS grade lactic acid bacteria. Representative studies have been extensively applied to treatment of skin diseases such as cholesterol lowering, immunity, and atopy. In particular, studies on cholesterol have shown that the efficacy has been studied extensively, but the mechanism is unknown. It is known that cholesterol is adsorbed to the ingested lactobacillus, and that two mechanisms are known to reduce the content of the bile in the body and to decompose the bile acid salt through the bile salt hydrolase produced by lactic acid bacteria.

The primary goal of the present invention is to select a bacterium having excellent basic properties as probiotics such as acid resistance, bile acid and intestinal adherence, and to identify cholesterol-lowering effects of cholesterol and ultimately obesity inhibition Thus completing the present invention.

It is an object of the present invention to provide a method of isolating a live strain from an infant (within 2 months of age) feces and having an acid resistance, a bile acid resistance, a long-term adherence, a cholesterol lowering and an obesity inhibiting effect, The purpose of the invention is to select a new strain.

The above object of the present invention is to provide a process for isolating lactobacillus of the genus Lactobacillus from infant feces, having acid resistance, bile acid tolerance, long-term adherence, cholesterol lowering and obesity inhibiting effect.

Hereinafter, the present invention will be described in detail.

The present inventors isolated and cultured new lactic acid bacteria from infant feces (within 2 months of age) using the MRS medium and finally identified them by morphological and biochemical characterization (see Table 1 and Table 2). As a result, it was confirmed that the lactic acid bacteria isolated in the present invention were Lactobacillus acidophilus .

The present inventors named the lactic acid bacterium strain of the present invention having the above characteristics as Lactobacillus acidophilus NS and deposited it on November 14, 2012 at the Korean Society for Microbiological Protection (accession number: KCCM 11322P). It was named Lactobacillus aspirophilus NS in the following description.

The lactic acid bacteria according to the present invention have the following physiological activities for functionality as probiotics.

First, the lactic acid bacteria of the present invention show acid resistance (see Table 3).

Specifically, the lactic acid bacterium of the present invention exhibited excellent survival rate at pH 2.5 to pH 3.5, indicating acid resistance. Therefore, when ingested orally, it can survive in poor conditions such as stomach, and maintain its functionality after intramuscular fixation.

Second, the lactic acid bacteria of the present invention exhibit bile acid resistance (see Table 4).

Specifically, bile is a type of surfactant secreted from the gallbladder into the duodenum, and is composed mainly of cholic acid, deoxycholic acid, and chendeoxycholic acid. Therefore, most of the lactic acid bacteria are not able to exert their function because the survival rate is lowered by the suck acid. However, the lactic acid bacterium of the present invention is resistant to bile acid because it has a tolerance to grow in a medium containing 0.5% of bile acid than other lactic acid bacteria.

Third, the lactic acid bacterium of the present invention shows ability to adhere epithelial cells (see Table 5).

Specifically, when lactic acid bacteria are ingested, viability is also important for realizing the function, but the ability to adhere to intestinal epithelial cells to form microfilaria is important. In order to prevent colonization and other pathogenic microorganisms from being trapped, the ability to bind intestines is essential. In order to select strains with excellent adhesion ability, HT-29 cells formed with monolayer were tested for adhesion, and it was confirmed that the ability to adhere the cells was excellent.

Fourth, the lactic acid bacteria of the present invention exhibit a cholesterol-lowering effect (see FIG. 2).

Specifically, the lactic acid bacterium of the present invention was cultured for a certain period of time after inoculation with a strain of MRS broth supplemented with cholesterol-lecithin micelles, and it was confirmed that the lactic acid bacteria had a reduction effect by cholesterol adsorption.

For culturing the lactic acid bacteria of the present invention, a medium composed of carbon source, nitrogen source, vitamins and minerals is used. For example, MRS medium is used and cultured under usual lactobacillus culture conditions, for example, at 30 to 45 ° C for 10 to 40 hours . More preferably at 37 째 C for about 18 hours.

The lactic acid bacterium according to the present invention can be cultured under anaerobic conditions, preferably in a stationary culture or under a condition of 5 to 15% of carbon dioxide.

The lactic acid bacterium of the present invention can also be used as a composition for antioxidation, a composition for dressing, and a composition for live bacteria. In particular, the lactic acid bacterium of the present invention can be used as a feed composition, a composition for food addition, and other fermented products.

As described above, the present invention is separated from infant feces and has a function of suppressing acid resistance, bile acid tolerance, intestinal adherence ability, cholesterol lowering and obesity, and is widely used as food composition such as weight control food, Lt; RTI ID = 0.0 > probiotics < / RTI >

Figure 1 shows the results of 16s rRNA sequencing analysis of Lactobacillus aspirin pills NS.
Figure 2 relates to the cholesterol adsorption effect of Lactobacillus acidophilus NS.
Figure 3 relates to changes in body weight following high fat diet and Lactobacillus aspirin pills NS administration.
Figure 4 relates to changes in adipocyte size following high fat diet and Lactobacillus aspirin pills NS administration.
Figure 5 relates to changes in total cholesterol levels following high fat diet and Lactobacillus aspirin pills NS administration.
Figure 6 relates to changes in low density lipid cholesterol levels following high fat diet and Lactobacillus aspirin pills NS administration.
Figure 7 relates to a comparison of total cholesterol levels detected in the feces system and in the feces following administration of Lactobacillus aspirin pills NS.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

[Example 1] Isolation and identification of Lactobacillus asidophilus NS

Lactic acid bacteria were isolated using infant feces (within 2 months of age) as a sample. Specifically, the sample was streaked on a MRS agar medium (Difco, USA) containing 0.004% of bromocresol purple and stained with an anaerobic state (carbon dioxide 5-15%) at 37 Lt; 0 > C for 48 hours. Among the cultivated strains, yellow colonies were screened for Gram stain, catalase and oxydase test, gas formation and sugar availability, and the sequence analysis was performed by Bionics. The results were confirmed by NCBI blast search. Respectively.

The characteristics of Lactobacillus acidophilus NS strain isolated by the above method and selected by the selection criteria in the following examples are shown in Tables 1 and 2 below.

Morphological and Physiological Characteristics of Lactobacillus asidophilus NS 1. Morphological properties (morphology when grown on MRS agar medium)  1) the shape of the cell Bacillus (bar)  2) Polymorphism of the cell (polymorphism) none  3) Motility none  4) Apoforming none 2. Physiological properties  1) Gram staining positivity  2) catalase voice  3) Oxidase voice  4) Growth temperature and time 37 ° C, 24 to 48 hours  5) Oxygen demand Organized anaerobic

Analysis of sugar availability of Lactobacillus ashidophilus NS Per type Lactobacillus acidophilus NS Glycerol - Erythritol - D-Arabinose - L-Arabinose - Ribose - D-Xylose - L-Xylose - β-Methyl-xyloside - Galactose + D-Glutose + L-Glutose + D-Mannose + L-Sorbose - Rhamnose - Dulcitol - Inositol + Mannitol - Sorbitol - alpha -Methyl-D-mannoside - alpha -Methyl-D-glucoside - N-acetyl glucosamine + Amygdaline + Arbutine - Esculin + Salicine - Cellobiose - Maltose + Lactose + Melibiose + Sacchaose + Trehalose - Inuline - Melezitose + D-Raffinose + Amidon + Glycogen - Xylitol - β-Gentiobiose + D-turanose + D-Lyxose - D-Tagatose - D-fucose - L-fucose + D-arabitol - L-Arabitol - Gluconate - 2-ceto-gluconate - 5-ceto-gluconate -

On the other hand, in order to preserve all the isolated and identified lactic acid bacteria, the cell pellet was supplemented with skimmed milk (10%) and lactose (2%) by 2 passages in MRS medium and centrifuged %) And yeast extract (0.3%) were prepared and mixed, and 1 mL of the medium was dispensed into a glass bottle and lyophilized.

[Example 2] Evaluation of acid resistance

The strain selected from the lactic acid bacteria selected in Example 1 was used for the experiment in the second subculture for 18 hours in MRS broth. 0.05 M sodium phosphate solution was prepared and pepsin (Sigma Co., USA) was added to the MRS broth adjusted to pH 2.0 and 2.5 with HCl to 1,000 units / mL. Inoculated with 0.05 M sodium phosphate buffer and incubated at 37 ° C for 2 hours, the number of colonies was counted to evaluate the acid resistance, so that the initial number of bacteria was about 10 ⁷ cfu / ml. The results of the acid resistance test of Lactobacillus ashidophilus NS are shown in Table 3 below.

Acid resistance test of Lactobacillus ashidophilus NS pH Viable cell count (cfu / ml) Initial pH (pH 7) 1.92 x 10 ⁷ After pH 2.5 treatment, 1.09 × 10 ⁶ After pH 2.0 treatment, 3.8 × 10 ⁴

[Example 3] Evaluation of bile acidity in bile

Lactobacillus aspirophilus NS suspension was inoculated on the MRS medium containing filtered filtrate of oxgall (Difco, USA) to an oxgall content of 0.5% (w / v), and cultured at 37 ° C for 0 hours and 24 hours. . The bile acid tolerance results of Lactobacillus ashidophilus NS are shown in Table 4. As can be seen from the results, it can be confirmed that the tolerance to bile acid is excellent enough to grow at a concentration of 0.5% oxgall.

Bile bile acid test of Lactobacillus ashidophilus NS Number of viable cells after 0.5% oxgall treatment (cfu / ml) Initial number of bacteria 2.06 × 10 ⁶ Initial number of bacteria 2 2.31 × 10 ⁶ Average initial bacterial count 2.19 × 10 ⁶ 1 time after 24 hours 4.9 × 10 ⁶ Twice after 24 hours 4.0 × 10 ⁶ Average after 24 hours 4.45 × 10 ⁶

[Example 4] Evaluation of long-term adhesion

Monolayer HT-29 cells were washed 5 times with PBS buffer and 0.5 ml of RPMI 1640 medium without antibiotics was added. Lactobacillus acidophilus NS was suspended in RPMI at a concentration of about 10 ⁹ CFU / ml, then inoculated on well-palates and cultured at 37 ° C for 2 hours. After cultivation was completed, the cells were washed three times with PBS buffer for 3 minutes at 200 rpm to confirm the ability to remove unadhered lactic acid bacteria and to adhere to them. After washing with 0.2% trypsin-EDTA, Cells were removed and viable cell counts were evaluated in MRS medium. As a result of the experiment, it was confirmed that more than 5.35 × 10 ⁷ bacteria survived after 2 hours, so that when Lactobacillus acidophilus NS was inoculated into the intestinal epithelium, .

Experimental study on intestinal adhesiveness of Lactobacillus acidophilus NS division Viable cell count (cfu / ml) 0 times 1 time 1.23 × 10 ¹⁰ 0 hours 2 times 1.56 × 10 ¹⁰ 0 hour average 1.39 × 10 ¹⁰ 1 time after 2 hours 6.2 × 10 ⁷ 2 hours later 2 times 4.5 × 10 ⁷ Average after 2 hours 5.35 × 10 ⁷

[Example 5] Cholesterol lowering effect

Lactobacillus acidophilus NS was inoculated with MRS broth (0.045% polyoxyethanyl cholesteryl sebacate, 0.05% cysteine), anaerobically cultured for 24 hours, centrifuged (12,000 × g, 4 ° C, 10 minutes) And the mixture was reacted in a water bath at 60 ° C for 10 minutes. After cooling, add 5 mL of hexane, mix well, add 3 mL of distilled water, and allow to stand for 15 minutes at room temperature. Then, 2.5 mL of the hexane layer (upper layer) was transferred to a new test tube, and the mixture was evaporated at 60 ° C with nitrogen gas. Then, 4 mL of o-phthalaldehyde reagent (0.5 mL of o-phthalaldehyde / glacial acetic acid 1 mL) After incubation for 2 minutes, 2 ml of concentrated sulfuric acid was added, and the reaction was continued for 10 minutes. The absorbance at 550 nm was measured and the cholesterol lowering effect was calculated by Equation (1).

&Quot; (1) "

The results are shown in Fig. 2, and it was confirmed that the cholesterol-absorbing activity was similar to that of the control group ( L. acidophilus ATCC 43121, Korea Univ. Based on the results of the above experiment, the experiment was conducted through animal experiments.

[Example 6] Evaluation of cholesterol lowering and obesity inhibiting activity by animal experiments

All animal experiments conducted in the present invention were approved by the Ethics Committee of the Animal Care Committee of Chonnam National University (CNU IACUC-YB-2012-40). (C57BL / 6) mice (19 g, n = 50, Chonnam National University experimental animal room) were maintained at 25 ± 1 ℃ for 7 weeks. standard rodent chow).

Lactobacillus acidophilus NS was daily diluted in a sterile diluent at a level of 1 × 10 ⁸ with high fat diet (45% caloric fat) and high fat diets for 10 weeks in an experimental group. The animals were weighed once a week. As shown in Fig. 3, the rats fed the high fat diet had a significantly higher body weight than the control rats. In the rats fed the lactic acid bacteria with the high fat diet, The weight gain was significantly inhibited (P <0.05) compared to rats fed only diet.

To further examine the efficacy of weight control, we analyzed changes in the size of adipocytes in epididymal adipose tissue. As shown in FIG. 4, in the case of the rats fed the lactic acid bacteria with the high fat diet, the fat cells were slightly increased compared with the control, but the fat cells were reduced to a significant level (P <0.05) as compared with the rats fed only with the high fat diet I could.

In addition, the total cholesterol content and LDL-cholesterol content were significantly lower in the group administered with Lactobacillus acidophilus NS than in the case of the high-fat diet alone, and the cholesterol-lowering effect was confirmed 5 and 6).

In addition, cholesterol detected in the feces was measured to confirm reduction of the cholesterol-lowering action by adsorption (Fig. 7). As a result of the experiment, it was confirmed that the amount of cholesterol detected was significantly (P <0.01) higher than that of the high fat diet alone. This suggests that when the Lactobacillus acidophilus NS Lactobacillus is simultaneously administered, the cholesterol is absorbed through the feces and released, thereby lowering the cholesterol content in the body.

[Example 7] Production of lactic acid bacteria preparation

Lactobacillus acidophilus NS of the present invention was prepared by using the lactic acid bacterium powder prepared in Example 1, and a lactic acid bacterial preparation such as a health functional food and a dressing agent using lactic acid bacteria was prepared. For this purpose, 20% by weight of dry powder of Lactobacillus acidophilus NS was mixed with 20% by weight of anhydrous crystalline glucose, 7% by weight of fructose, 3% by weight of fruit powder, 20% by weight of dietary fiber, 15% by weight of powdery cream, and 12% by weight of fruit powder. Thereafter, they were packed in the form of a stick or a capsule in a predetermined amount. The lactic acid bacterial preparation thus prepared maintained a viable cell count of 5 × 10 ⁸ cfu / g or more.

[Example 8] Preparation of weight control food

Lactobacillus acidophilus NS of the present invention was prepared by using the lactic acid bacterium powder prepared in Example 1. To this end, 30 to 40% by weight of isolated soybean protein, 10 to 20% by weight of fructose, 5 to 15% by weight of cocoa powder, 5 to 15% by weight of cereal mix and 5 to 15% by weight of cocoa powder were added to 2% by weight of dry powder of Lactobacillus acidophilus NS 5 to 15% by weight of lacto-oligosaccharide, 1 to 5% by weight of dietary fiber, 0.1 to 0.5% by weight of vitamin mineral mix, and 0.1 to 1% by weight of fruit flavor powder. Lactobacillus ashidophilus NS in the weight control food thus prepared maintained a viable cell count of 5 × 10 ⁸ cfu / g or more.

Korea Microorganism Conservation Center (overseas) KCCM11322P 20121114

Claims (5)

Acidic, bile acid, and adherent ability,
Cholesterol lowering and obesity inhibitory action,
Lactobacillus acidophilus NS (KCCM 11322P), which has reduced fat cell size and inhibits obesity. The method according to claim 1,
The Lactobacillus acidophilus NS is Lactobacillus acidophilus NS, which reduces total cholesterol and low density lipid cholesterol through cholesterol absorption. delete A composition for reducing blood cholesterol and inhibiting obesity comprising the Lactobacillus acidophilus NS (KCCM 11322P) of claim 1 as an active ingredient. A composition for controlling weight loss of blood cholesterol and obesity comprising Lactobacillus acidophilus NS (KCCM 11322P) according to claim 1 as an active ingredient.

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