KR20200071983A - Probiotics-microcapsule in which resveratrol is accumulated in cells and a method for producing the same - Google Patents

Abstract

The present invention relates to a microorganism preparation having resveratrol accumulated in cells and a manufacturing method thereof. The microorganism preparation according to the present invention provides the microorganism preparation capable of solving disadvantages of existing drugs (low solubility, low stability, unwanted toxicity, poor cell membrane permeability, etc.), and used microorganism absorbs resveratrol in a microbial culture medium, traces the same to the cells, and then acts as a delivery carrier, thereby being able to be safely delivered to the intestine.

Description

{Probiotics-microcapsule in which resveratrol is accumulated in cells and a method for producing the same}

The present invention relates to a microbial agent having resveratrol accumulated in cells and a method for manufacturing the same.

Lactobacillus is one of the most beneficial microorganisms available to humans, and refers to bacteria that produce lactic acid by decomposing carbohydrates such as glucose or lactose during fermentation. In 1858, the identity of lactic acid bacteria was discovered by French microbiologist Louis Pasteur, and Russian immunologist Ilya Mechinikov advocated "Fertilization by fermented milk" in the later years after receiving the Nobel Prize for Physiology of Medicine in 1908. A full-scale study has started since claiming that it is in, so far, about 300 lactic acid bacteria have been found. Lactic acid bacteria live in the intestines of most mammals, including humans. In particular, in the intestine of the human body, there are more than 100 trillion bacteria in 100 species, including beneficial and harmful bacteria. It is known to act as a bacterium that inhibits the growth of harmful bacteria and reduces the production of ammonia or carcinogens due to abnormal fermentation, as it induces acidification of the intestine by making lactic acid as a metabolite.

In addition, the physiological activity of lactic acid bacteria mainly suppresses the proliferation of harmful bacteria in the intestine by producing organic acids and lowers the pH in the intestine, maintains normal intestinal flora, and produces β-glucosidase to produce useful glycosides for the human body It is known to have a function of converting to a non-glycoside that is easily absorbed.

Resveratrol is a secondary metabolite contained in crops and plants, and is known to have antioxidant, anti-cancer, cardiovascular, and diabetes effects. In particular, naturally occurring molecules found in grapes and red wines have been studied in major fields in recent years. Resveratrol exists in three major structures, trans-resveratrol, cis-resveratrol, and resveratrol-piceid. However, it is known that resveratrol has various effects, but is difficult to absorb in the body due to low bioavaliability. Under these backgrounds, the present inventors have confirmed that the existing problems can be improved by using microorganisms or microbes containing them as raw materials using microorganisms, and have completed the present invention.

Republic of Korea Registered Patent No. 10-0442205 Republic of Korea Registered Patent No. 10-1825667

Accordingly, an object of the present invention is to provide a microbial agent comprising a microorganism or a crushed product of which resveratrol is accumulated.

In addition, another object of the present invention is to provide a method for producing a microbial agent with accumulated resveratrol.

In addition, another object of the present invention provides a composition comprising the microbial agent prepared above.

In one embodiment, the present invention provides a microbial agent comprising resveratrol-accumulating microorganisms or debris thereof.

Further, the microorganism is Streptococcus Thermo filler's (Streptococcus thermophillus), bifidobacteria bacteria (Bifidobacteria), Lactobacillus basil (Lactobacilli), Lactococcus (Lactococcus), flow Pocono Stokes (Leuconostocs), yeast (Yeast) or Aspergillus spread Lee ( Aspergilli ).

In addition, the resveratrol can be separated from one or more of grapes, audi, peanuts, mulberry leaves.

In one embodiment of the present invention, a microorganism is cultured in a medium containing resveratrol or reacted with a resveratrol-containing reactant to provide a method for producing a microbial agent comprising accumulating resveratrol in the microorganism.

Here, the microorganism is Streptococcus Thermo filler's (Streptococcus thermophillus), bifidobacteria bacteria (Bifidobacteria), Lactobacillus basil (Lactobacilli), Lactococcus (Lactococcus), low konoseu totseu (Leuconostocs), yeast (Yeast) or Aspergillus spread Lee ( Aspergilli ).

Here, the culture is characterized in that it comprises the step of first culturing the microorganism for the activation of the microorganism, and the second step of culturing the cultured microorganism in a medium containing resveratrol.

In one embodiment of the present invention provides a composition containing the microbial agent.

According to the present invention as described above, it is possible to accumulate and transfer resveratrol to a microorganism in its natural state without genetic manipulation of the microorganism, and then release it into the body, and improve the low human utilization rate of the existing resveratrol to be used stably and efficiently.

The effects according to the present invention in the above are not limited to the above, and other effects of the present invention are clearly understood by those skilled in the art to which the present invention pertains through the examples and claims described below. Will be understandable.

Figure 1 shows the mechanism of drug absorption of the microbial agent according to the present invention.
Figure 2 shows the resveratrol content by culture conditions (conditions 1 to 6) of S. thermophillus according to the present invention.

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

The present invention relates to a microbial agent having resveratrol accumulated in cells and a method for manufacturing the same.

The microbial preparation according to the present invention provides a microbial preparation capable of solving the disadvantages of the existing drugs (low solubility, low stability, unwanted toxicity, poor cell membrane permeability, etc.). The microorganism used in the preparation according to the present invention absorbs resveratrol in the microbial culture medium and traces it to cells, and then acts as a delivery carrier and safely delivers it to the intestine.

Unlike conventional techniques using genetically engineered microorganisms, the microorganisms used in the microbial preparations according to the present invention are used in the form of live bacteria, accumulate drugs in the microorganisms, and then deliver them to the large intestine, which is present in the mucosal layer (laminar propria mucosae) Through absorption and phagocytosis by resident macrophages, the drug is released into the body.

As illustrated in FIG. 1, uptake of drugs by microorganisms may be caused by active transport, carrier-mediated transport, and diffusion. Specifically, when administered orally, the microbial agent is phagocytosed/lysed by macrophages/dendritic cells in the large intestine, whereby the pharmacologically active substance can be delivered to the blood, or the microbial agent is absorbed by the large intestinal epithelial cells. Afterwards, the microorganisms are lysed in the cells and the pharmacologically active substance can be absorbed into the body by diffusion (probiotic carrier and active transport). These mechanisms cannot be identified as one by using different methods depending on the structural properties of the pharmacologically active substance, but in the end, the microbial agent according to the present invention is absorbed by macrophages (uptake) and passes through the intestinal cell membrane, resulting in poor cell membrane permeability. It can increase (active transport efficacy), after which the microbes are destroyed by the phagocytosis of macrophages, and the drug exerts the effect of being released into the body.

The pharmacologically active substances absorbed by the microorganisms used in the present invention are accumulated in the cells without being used for the physiological activity of the microorganisms, and are stable until the microorganisms are destroyed by various methods (physical crushing and phagocytosis, etc.) Is preserved. The amount of drug absorbed by microorganisms is in the order of tens to hundreds of micrograms, which is significantly lower than the hundreds of milligrams of drugs ingested. On the other hand, according to the pharmacokinetics showing the efficacy of the drug in the patient's body, the efficacy of the drug is sufficient if the drug is from several hundred nanograms to several micrograms, so delivery of the drug using the microbial agent of the present invention is excessive drug. Preventing abuse can lower unwanted side effects and toxicity.

Accordingly, according to the present invention, resveratrol can be accumulated in a microorganism and then delivered to the body in a natural state without genetic manipulation of the microorganism, and then released into the body, and can be used stably and efficiently by improving the low human utilization rate of the existing resveratrol.

In more detail, the present invention provides a microbial agent comprising a microorganism in which resveratrol is accumulated or a fragment thereof.

In the present invention, the microbial agent is provided as a probiotic-microcapsule (PM), which is a microbial agent in which resveratrol is accumulated, and prevents the destruction of resveratrol in the digestive system and safely passes into the body by acting as a capsule of lactic acid bacteria. I can do it.

In the present invention, resveratrol in lactic acid bacteria by probiotics-microcapsules is actively transferred to lamina propria or lymp node by macrophages (tissue resident macro phage) present in intestinal cells and released into blood vessels, thereby bioavailability. This becomes higher. In the present invention, the microbial agent is released into the body through resveratrol in the microorganism through absorption and phagocytosis by macrophages present in intestinal cells.

The microorganism used in the present invention is, for example, the microorganism is selected from the group consisting of lactic acid bacteria, Lactococcus, Corynebacterium GRAS (Generally recognized as safe) microorganism, Bifidus, yeast, Bacillus, Aspergillus and Clostridium It can be one or more. Preferably Streptococcus Thermo filler's (Streptococcus thermophillus), bifidobacteria bacteria (Bifidobacteria), Lactobacillus basil (Lactobacilli), Lactococcus (Lactococcus), flow Pocono Stokes (Leuconostocs), yeast (Yeast) or Aspergillus spread Li (Aspergilli) Any one or more may be used, and more preferably, Streptococcus thermophillus may be used.

In addition, in the present invention, resveratrol may be separated from one or more of grapes, audi, peanuts, and mulberry leaves. Here, the method for separating resveratrol may use a commonly known extraction method or fractionation method.

The lysate used herein refers to a cell lysate obtained by crushing the microorganism by a chemical method or a physical method of treating the microorganism with detergent, for example, bead mills, presses of microorganisms , Using a sonicator (Sonicator) or a microfluidizer (Microfluidizer) can be obtained crushed.

In another embodiment of the present invention, microorganisms can be cultured in a medium containing resveratrol to accumulate resveratrol components in the microorganisms, and specifically, microorganisms are cultured in a medium containing resveratrol or reacted with a reactant containing resveratrol. And accumulating resveratrol in the microorganism.

Here, the culture may include the step of first culturing the microorganism for activation of the microorganism, and the step of culturing the cultured microorganism in the medium containing resveratrol, specifically, the microorganism is first cultured in MRS medium. Thereafter, the cultured cells were transferred to MRS medium containing resveratrol, followed by secondary culture and washed. The cultured cells can be used directly after washing or by crushing the cells using a microfluidizer.

Since the method for manufacturing a microbial agent includes the same structure in which resveratrol accumulates in the microorganism, the above-described description applies equally to the method for preparing the microbial agent.

Further, as the medium used for cultivation, a known medium used for culturing microorganisms can be used, and any medium can be selected depending on the type of microorganism. For example, M9 minimal medium, LB (Luria-Bertani) medium, MRS (deMan Rogosa Sharpe) solid or broth medium, APT (All Purpose with Tween) medium, BHI (Brain Heart Infusion) medium or YPD (yeast extract-peptone Dextrose) solid or liquid media can be used. The medium may be used as a primary or secondary medium when culturing the microorganism of the present invention, and the secondary medium is characterized in that the resveratrol is further included.

The resveratrol contained in the secondary medium is not limited thereto, but may be included at a concentration of 0.01 to 20 g/L, more preferably, at a concentration of 0.01 to 10 g/L, and the concentration is cultured. Depending on the type of microorganism and the composition of the culture medium, it can be appropriately adjusted according to the skill level of those skilled in the art.

The microorganism cultured in the present invention has a body content of resveratrol of 0.1 to 2.0 ug/mg and is suitable as a microbial agent containing resveratrol.

In one embodiment of the present invention can be used as a composition containing the microbial agent. The composition according to the present invention may be, for example, a composition for antioxidant, a dressing composition, and a composition for probiotics including a microbial agent.

The composition according to the invention may further comprise excipients or carriers. Tablets, troches, capsules, elixirs, syrups, powders, suspensions, or granules by mixing with commonly used carriers and fragrances ( granules) and the like. As the carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, etc. can be used. The administration method may be oral, parenteral or applied, and the dosage may be appropriately selected according to the absorbency, inactivation rate and excretion rate of the active ingredient in the body, the age, sex, and condition of the recipient. Preferably, it can be administered at an effective dose of 10 mg to 1,000 mg per day, and the number of bacteria in the composition may be 10 ⁸ to 10 ¹² CFU/day.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only intended to materialize the contents of the present invention, and the present invention will not be limited thereby.

<Example 1> Preparation of resveratrol-containing Streptococcus thermophilus (S. thermophillus)

1. Culture conditions

S. thermophillus (OD 1.0) stored in a stock state in a cryogenic freezer was melted at room temperature, inoculated into a broth containing lasveratrol, and cultured at 36.5°C. The entire cultured S. thermophillus was inoculated into MRS broth to which resveratrol was added and cultured secondary.

2. Sample preparation

After lactic acid bacteria culture solution, lactic acid bacteria washing solution washing 1-3, lactic acid bacteria lysate, or resveratrol used in the experiment was fractionated using EtOAC and BuOH, the fractions were concentrated under reduced pressure to obtain fractions, and analysis was performed on the BuOH fraction containing resveratrol. Did. Each resveratrol was dissolved in MeOH and prepared and analyzed at a concentration of 100 ppm. Resveratrol, washing 1-3, lactic acid bacteria culture solution, lactic acid bacteria crushed BuOH fraction was prepared and analyzed at 2000 ppm concentration.

The analysis conditions are as follows:

-Instrument: Agilent Technologies 6410 Triple Quad (LC-MS/MS)

-Column: C18 (2.1 mm X 150 mm, 2.7 μm)

-Solvent: A; 0.1% Formic acid in Water, B; 0.1% Formic acid in Acetonitrile

-Flow rate: 0.2 ml/min

[Table 1] Concentration gradient

MS condition:

-Ionization Mode -ESI, sim mode

-Gas temp. 350℃

-Capillary volt. 4000 V

-Nebμlizer 40 psig

-Fragmentor 190 V

<Example 2> Resveratrol detection by LC-MS/MS

Streptococcus thermophilus lysates that absorbed resveratrol were analyzed using LC-MS/MS. At this time, experiments were conducted under six conditions according to the concentration of resveratrol contained in the medium, and the resveratrol content in the cultured microorganisms was confirmed as shown in FIG. 2. That is, as a result of analyzing resveratrol, it was confirmed that the content of resveratrol in the microorganism was changed according to the culture conditions.

<Example 3> Cell scale of resveratrol using yeast

According to the above conditions, in order to confirm whether the accumulation amount of resveratrol can be increased in the same manner in other strains, yeast Saccharomyces cerevisiae was added to broth containing rasveratrol. After inoculation and primary culture, the cultured yeast was further cultured by inoculating the secondary culture medium containing resveratrol. After incubation, this was centrifuged and washed to obtain yeast pellets.

The washed Saccharomyces cerevisiae was crushed three times using a microfluidizer, and the non-glycoside resveratrol compound was analyzed through LC/MS-MS. As a result, non-glycosylated resveratrol was detected in the bacterial lysate. The amount of non-glycoside in Saccharomyces cerevisiae before crushing was about 45 ng, and upon crushing, 1,222 ng of non-glycoside resveratrol was detected.

This means that even in the case of yeast, it absorbs a substance with pharmacological activity and contains it in cells, and it shows that microorganisms that can be used in this development are not limited to lactic acid bacteria.

<Example 4> Cell scale of resveratrol using Bifidobacterium

Bifidobacterium (Bifidobacterium) was inoculated into a broth containing rasveratrol, followed by primary culture, and then cultured in a medium containing resveratrol in the same manner as in Examples 2 and 3. After incubation, in order to separate the Bifidobacterium, the process of centrifugation and washing was repeated two or more times and the supernatant was removed to obtain a Bifidobacterium pellet.

The washed Bifidobacterium was crushed using a microfluidizer, and the non-glycoside resveratrol compound was analyzed through LC/MS-MS. As a result, non-glycosylated resveratrol was detected in the bacterial lysate. This means that even in the case of Bifidobacterium, it means that the pharmacologically active substance can be absorbed and contained in cells, and the microorganisms that can be used in this development are not limited to lactic acid bacteria.

As described above, specific embodiments of the present invention have been described in detail, but those skilled in the art who understand the spirit of the present invention may add other components within the scope of the same spirit, change, delete, etc., and degenerate other inventions. However, other embodiments included within the scope of the inventive concept may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims, which will be described later, rather than the detailed description above, and all the changed or modified forms derived from the meaning and scope of the claims and their equivalent concepts are included in the scope of the present invention. It should be interpreted as.

Claims (9)

Resveratrol (Resveratrol) is a microbial agent containing a microorganism or a fragment thereof accumulated. According to claim 1,
Wherein the microorganism is Streptococcus Thermo filler's (Streptococcus thermophillus), bifidobacteria bacteria (Bifidobacteria), Lactobacillus Bashile (Lactobacilli), Lactococcus (Lactococcus), flow Pocono Stokes (Leuconostocs), yeast (Yeast) or Aspergillus spread Li (Aspergilli) Microbial preparation, characterized in that any one or more. According to claim 1,
The resveratrol is a microbial agent characterized in that it is separated from one or more of grapes, audi, peanuts, mulberry leaves. A composition containing the microbial agent of claim 1. Method for producing a microorganism having accumulated resveratrol in the body, including culturing in a medium containing resveratrol. The method of claim 5, wherein the medium containing resveratrol has a resveratrol content of 0.01 to 20 g/L. The method of claim 5,
Wherein the microorganism is Streptococcus Thermo filler's (Streptococcus thermophillus), bifidobacteria bacteria (Bifidobacteria), Lactobacillus basil (Lactobacilli), Lactococcus (Lactococcus), low konoseu totseu (Leuconostocs), yeast (Yeast) or Aspergillus spread Li (Aspergilli) Method for producing a microorganism, characterized in that any one or more. The method of claim 5,
The microorganism is a method for producing a microorganism, characterized in that the body content of resveratrol is 0.1 to 2.0 ug/mg. The method of claim 5, wherein the microorganism is a method for producing microorganisms, characterized in that resveratrol contained in the medium is absorbed by macrophages present in intestinal cells or released into the body through phagocytosis.

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