KR102420141B1 - Synbiotics lactobacillus manufacturing method for improved female immune activity and stability using NK cell culture medium composition - Google Patents

Abstract

Disclosed is a method for producing new biotics lactic acid bacteria with improved female immune activity and stability using an NK cell culture medium composition. When producing new probiotics lactic acid bacteria, an NK cell culture medium is used, which can show not only the effect of lactic acid bacteria but also the immune activation effect of NK cells. The method of the present invention includes the steps of: (a) inoculating and culturing NK cells in a culture medium; (b) recovering NK cells after the culturing is completed; (c) inoculating and culturing lactic acid bacteria in the culture medium from which the NK cells have been removed; and (d) freeze-drying a culture solution after the culturing of the lactic acid bacteria is completed, so as to make powder.

Description

Synbiotics lactobacillus manufacturing method for improved female immune activity and stability using NK cell culture medium composition}

The present invention relates to a method for producing a synbiotic lactic acid bacteria with improved female immune activity and stability using a NK cell culture medium composition, and more particularly, to the use of a NK cell culture medium when preparing a new biotic lactic acid bacteria. Rather, it relates to a method for producing synbiotic lactic acid bacteria with improved female immune activity and stability using an NK cell culture medium composition that can simultaneously exhibit the immunoactive effect of NK cells.

NK cells are known to play an important role in the initial body defense mechanism and tumor immunity of the human body. That is, NK cells can kill specific autologous cells, allogeneic cells, and even heterogeneous cancer cells without the process of acquiring immunity according to the expression of the Major Histocompatibility Complex (MHC). It is better able to kill non-target cells. Therefore, NK cells can effectively kill most cancer cells that do not express MHC, and in addition, they can kill virus-infected cells and bacteria such as Salmonella typhi. However, NK cells, which have an excellent effect on killing cancer cells and virus-infected cells, account for only 5 to 15% of peripheral blood lymphocytes even in normal humans, and especially in patients with severe cancer, the ratio is unlikely to decrease to less than 1%. Therefore, there is a limit in effectively attacking cancer cells without a separate amplification process through immunotherapy.

Immunotherapy involves extracting the most important immune cells for cancer treatment, such as natural killer cells (NK cells), dendritic cells (DC), B cells, and T cells, from the patient’s blood and then using various types of stimulants to fight cancer. It is a method of growing strong immune cells and then injecting them back into the patient. Since the patient's own blood is used, it has fewer side effects compared to conventional chemotherapy and the administration method is simple, so it is being actively studied in recent years. However, such immunotherapy has to be performed by a doctor with a medical license due to the nature of re-injecting cells collected from a patient, and the management is also very difficult and difficult, so there is a limit to the general people using it. In particular, in the case of the NK cells, culture is performed in exclusion for culture, but when injected into the human body, only the cells are selectively re-injected. The utilization rate of by-products is declining.

On the other hand, more than 400 types of microorganisms such as bacteroides, eubacteria, bifidobacteria, and lactobacilli inhabit the human intestine. In the intestine of a healthy person, beneficial and harmful bacteria, represented by lactic acid bacteria, live in a balanced (microflora) environment. As the intestinal flora is disturbed due to harmful environment, unsanitary food intake, and antibiotic use, lactic acid bacteria decrease and harmful bacteria such as E. coli and salmonella increase. will do

Probiotics are microorganisms that live in the gastrointestinal tract of humans or animals and have beneficial effects on humans or animals, and include lactobacilli, bifidobacteria, and enterococci.

The intestinal physiologically active functions of lactic acid bacteria include maintaining the balance of intestinal flora, inhibiting proliferation of harmful bacteria, preventing diarrhea, protecting intestinal epithelial cells, inhibiting absorption of toxic substances, and inhibiting carcinogenesis. In order for lactic acid bacteria to act as probiotics, they must bind to and proliferate in the intestinal mucosa and have antagonistic power against harmful bacteria.

In order for lactic acid bacteria to attach to the intestinal mucosa of the host, it must be attached through competition with the host's flora. Nurmi and Rantala were the first to introduce competitive exclusion of harmful microorganisms and reported that freshly hatched chicks were inoculated with the intestinal contents of chickens to reduce Salmonella infection. It was reported that the normal intestinal microbes of chickens adhered better to the adsorbed portion of the intestinal surface cells, produced fatty acids and other antimicrobial substances, and were advantageous in competition for nutrients.

Lactobacillus has the characteristic of adsorbing to the intestinal mucosa of the host, and this is known to be because lipoteichoic acid, polysaccharides, and proteins, which are components of the cell wall of lactic acid bacteria, are involved in adhesion to the intestinal mucosa.

The cell structure of lactic acid bacteria is composed of a cell membrane and a cell wall surrounding it. The cell wall functionally represents the shape of the lactic acid bacteria and surrounds the cell membrane to protect the lactic acid bacteria from the external environment. The four important components of the cell wall are peptidoglycan, teichoic acid, S-layer, and polysaccharide, and are involved in binding to the extracellular matrix (ECM) of the intestinal mucosa. ECM is a stable macromolecular tissue underlying epithelial and endothelial cells. Lactobacillus binds to dendritic cells (DC) in the intestinal mucosa and then transmits various cellular signals including intestinal function to prevent immune diseases.

Conventionally, these lactic acid bacteria have been cultured using a general medium, but by culturing lactic acid bacteria on a medium having specific components using the characteristics of lactic acid bacteria that absorb surrounding substances and grow, a method of culturing lactic acid bacteria having specific components or specific effects A lot of this is being developed.

Therefore, it is expected to develop a method for culturing lactic acid bacteria with improved immune activity and stability by utilizing the metabolites of NK cells, which are by-products generated during NK cell culture, the culture medium after culture, and the culture medium containing NK cells.

(0001) Republic of Korea Patent Publication No. 10-2021-0011977 (0002) Republic of Korea Patent Registration No. 10-2182076

The present invention has been devised to solve the above problems, and an object of the present invention is to provide a method for producing lactic acid bacteria with improved immune activity and stability in women by culturing specific lactic acid bacteria using a medium in which NK cells are cultured. .

The problems of the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention comprises the steps of (a) inoculating and culturing NK cells in a culture medium; (b) recovering NK cells after the culture is completed; (c) inoculating and culturing lactic acid bacteria in the culture medium from which the NK cells are removed; and (d) lyophilizing and powdering the culture medium after the culturing of the lactic acid bacteria is completed.

In one embodiment, the step (a) comprises the steps of: (i) preparing a basal medium by mixing IL2, IL12 and IL18 in a floating cell culture basal medium solution; (ii) mixing peripheral blood mononuclear cells isolated from blood into the basal medium; (iii) injecting an anti-CD56 antibody into the basal medium mixed with the peripheral blood mononuclear cells; (iv) injecting the anti-CD3 antibody into the basal medium in which the anti-CD56 antibody-infused peripheral blood mononuclear cells are mixed; and (v) adding a steroid to a basal medium mixed with peripheral blood mononuclear cells injected with the anti-CD3 antibody, wherein step (b) includes injecting the culture medium into a centrifuge; rotating the centrifuge into which the culture medium is injected at 1000 to 2000 rpm; and recovering the NK cells deposited on the bottom of the centrifuge, wherein step (c) includes: recovering the culture medium from which the NK cells are partially removed after the centrifugation is completed; mixing the culture medium with the MRS medium in a weight ratio of 1:1 to 3:1; inoculating lactic acid bacteria into the culture medium mixed with the MRS medium; Culturing the culture medium inoculated with the lactic acid bacteria at a temperature of 30 to 40 ° C for 1 to 5 days; And when the culture medium reaches pH 2 to 3, 1 to 5 parts by weight of the fisetin compound and 0.5 to 3 parts by weight of the damask rose extract are mixed with respect to 100 parts by weight of the culture solution, wherein the lactic acid bacteria are separated from the female genitalia. Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus reuteri (Lactobacillus reuteri) and Lactobacillus fermentum (Lactobacillus fermrntum) is a mixture, the lactic acid bacteria is Lactobacillus rhamnosus 0.01 to 0.5 compared to 100 parts by weight of the culture medium mixed with the MRS medium Inoculating parts by weight, 0.01 to 0.1 parts by weight of Lactobacillus reuteri, and 0.01 to 0.2 parts by weight of Lactobacillus fermentum, the step (d) comprises substituting an inert gas for the inside of the freeze dryer; Cooling the freeze dryer with the interior substituted with the inert gas to a temperature of -70 ~ -20 ℃; spraying the culture solution on which the culture is completed through a nozzle having a diameter of 0.1 to 2 mm; and removing moisture inside the freeze dryer by circulating the inert gas, wherein 2 to 8 parts by weight of dietary fiber, 2 to 5 parts by weight of fructooligosaccharide and water to 5 to 10 parts by weight of the freeze-dried lactic acid bacteria. preparing synbiotics by mixing 20 to 30 parts by weight; 80 to 100 parts by weight of the temperature-dependent oil is mixed with 20 to 50 parts by weight of the synbiotics, followed by emulsification; mixing 10 to 20 parts by weight of a coating agent obtained by mixing 200 to 500 parts by weight of water with respect to 100 parts by weight of the enteric cellulose compound with the emulsified synbiotics; and recovering the synbiotics on which the coating treatment has been completed.

According to an embodiment of the present invention, it is possible to provide lactic acid bacteria with improved immune activity and stability of women by culturing specific lactic acid bacteria using a medium in which NK cells are cultured.

In addition, the present invention can improve the in vivo utilization and engraftment rate of the lactic acid bacteria by mixing the lactic acid bacteria produced using the NK cell culture medium and the prebiotics serving as the food for the lactic acid bacteria to produce new biotics.

In addition, the present invention provides a method for producing synbiotics lactic acid bacteria that can greatly improve the amount of lactic acid bacteria engrafted in the intestine by coating the prepared synbiotics with an enteric film, thereby minimizing the death of lactic acid bacteria due to the attack of gastric acid. can provide

The effect according to the present invention is not limited by the contents exemplified above, and more various effects are included in the present invention.

1 shows a method for producing synbiotic lactic acid bacteria according to an embodiment of the present invention.

Hereinafter, various embodiments will be described in more detail with reference to the accompanying drawings. The embodiments described herein may be variously modified. Certain embodiments may be depicted in the drawings and described in detail in the detailed description. However, the specific embodiments disclosed in the accompanying drawings are only provided to facilitate understanding of various embodiments. Therefore, the technical idea is not limited by the specific embodiments disclosed in the accompanying drawings, and it should be understood to include all equivalents or substitutes included in the spirit and scope of the invention.

Terms including an ordinal number, such as first, second, etc., may be used to describe various elements, but these elements are not limited by the above-described terms. The above terminology is used only for the purpose of distinguishing one component from another component.

In the present specification, terms such as "comprises" or "have" are intended to designate that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, but one or more other features It is to be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof. When a component is referred to as being “connected” or “connected” to another component, it may be directly connected or connected to the other component, but it is understood that other components may exist in between. it should be On the other hand, when it is said that a certain element is "directly connected" or "directly connected" to another element, it should be understood that the other element does not exist in the middle.

Meanwhile, as used herein, a “module” or “unit” for a component performs at least one function or operation. And “module” or “unit” may perform a function or operation by hardware, software, or a combination of hardware and software. In addition, a plurality of “modules” or a plurality of “units” other than a “module” or “unit” that must be performed in specific hardware or are executed in at least one processor may be integrated into at least one module. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be abbreviated or omitted.

1 schematically shows a method for preparing synbiotic lactic acid bacteria of the present invention.

The present invention comprises the steps of (a) inoculating and culturing NK cells in a culture medium; (b) recovering NK cells after the culture is completed; (c) inoculating and culturing lactic acid bacteria in the culture medium from which the NK cells are removed; And (d) it relates to a synbiotic lactic acid bacteria production method with improved female immune activity and stability using an NK cell culture medium composition comprising the step of lyophilizing and powdering the culture medium after the culture of the lactic acid bacteria is completed.

The step (a) is a step of culturing NK cells in a culture medium, culturing NK cells using a culture medium, and proliferating NK cells, NK cell metabolites and NK cell components in the culture medium. to be.

To this end, step (a) comprises the steps of (i) preparing a basic medium by mixing IL2, IL12 and IL18 in a suspension cell culture basic medium solution; (ii) mixing peripheral blood mononuclear cells isolated from blood into the basal medium; (iii) injecting an anti-CD56 antibody into the basal medium mixed with the peripheral blood mononuclear cells; (iv) injecting the anti-CD3 antibody into the basal medium in which the anti-CD56 antibody-infused peripheral blood mononuclear cells are mixed; and (v) adding a steroid to the basal medium in which the anti-CD3 antibody-injected peripheral blood mononuclear cells are mixed.

The basal medium can be used by purchasing a commercially available basal medium for floating cell culture as a part on which the NK cells grow. In the case of such a basic medium for culturing suspended cells, since the culture efficiency per unit area is excellent compared to the agar medium used for experiments, it can be widely used as an industrial medium. In addition, IL2, IL12 and IL18 antibodies can be mixed and used in the basal medium for culturing the floating cells. In the case of IL2 used at this time, activation may be induced by directly stimulating NK cells, but it may be activated by stimulating th1 cells, and the activated th1 cells may then be activated by stimulating NK cells. In addition, in the case of IL12 and IL18, it is possible to directly stimulate and activate the NK cells.

When the preparation of the basal medium is completed as described above, peripheral blood mononuclear cells can be injected into the basal medium. The peripheral blood mononuclear cells used in this case may be peripheral blood mononuclear cells collected from a lactobacillus user, an NK cell user, or a donor, and preferably may be a peripheral blood mononuclear cell of a lactobacillus user or an NK cell user, but the peripheral blood mononuclear cells provided from another donor It is also possible to use blood mononuclear cells.

The peripheral blood mononuclear cells collected as described above may be mixed with the basal medium to activate NK cells. In addition, by injecting the anti-CD56 antibody into the basal medium, it is possible to further stimulate the NK cells, thereby maximizing the activation of the NK cells.

After the NK cells are activated, the anti-CD3 antibody may be injected into the basal medium. In the case of the anti-CD3 antibody, the T cells are stimulated. The T cells activated by the stimulation can be combined with the NK cell stimulating antibodies to activate the NK cells more strongly.

However, in the case of the anti-CD3 antibody, regulatory T cells can also be stimulated. The regulatory T cells have immunosuppressive activity and may interfere with the proliferation of the NK cells. Therefore, in order to suppress the regulatory T cells, it is preferable to inject the regulatory T cell inhibitory drug into the basal medium after the stimulation of NK cells and T cells as described above is completed.

In this case, it is preferable to use a steroid as the drug for inhibiting regulatory T cells, and the steroid may be a glucocorticoid type. Cortisol (Cortisol), cortisone (Cortisone), prednisolone (Prednisolone), prednisone (prednisone), methylprednisolone (Methylpredni soone), dexamethasone (Dexamethasone), betamethasone (Triamcinolone), cortisone (Triamcinolone) as an embodiment It may be one or more selected from the group consisting of acetate (Fludrocortisone acetate) and deoxycorticosterone acetate (deoxycorticosterone acetate).

After suppression of regulatory T cell activity by the steroid as described above, anti-CD56, an antibody for stimulating NK cells, may be additionally injected 1 to 5 times. In the case of the IL2, IL12 and IL18 antibodies, they are contained in excess in the basal medium and are responsible for the initial stimulation, but in the case of the anti-CD56 antibody, it is possible to continuously stimulate and activate NK cells. However, in the case of the anti-CD3 antibody for T cell stimulation, it is only required for initial stimulation, and there is no need for additional supply due to problems such as stimulation of regulatory T cells. Therefore, it is preferable to continuously activate the NK cells by additionally supplying the anti-CD56 antibody 1 to 5 times after the injection of the steroid. If the supply of the anti-CD56 antibody exceeds 5 times, further NK cell activation does not appear, so it is preferable to supply only up to 5 times.

After culturing of NK cells as described above is completed, NK cells can be isolated from the basal medium. Isolation of the NK cells may be performed using a centrifuge.

To this end, the step (b) includes injecting the culture medium into a centrifuge; rotating the centrifuge into which the culture medium is injected at 1000 to 2000 rpm; And it may include the step of recovering the NK cells precipitated on the bottom of the centrifuge.

In the case of the culture medium, a commercially available floating cell culture medium is used as the basal medium, and accordingly, the NK cells can be activated and cultured in a suspended state in the basal medium. Therefore, when using a centrifuge, it is possible to easily separate cultured NK cells. In this case, the centrifuge may use a centrifuge container, but in case of commercial production, the culture container may be installed in a centrifuge as it is, and the basal medium containing the NK cells is moved to a container suitable for centrifugation. It is also possible to separate In particular, since it can receive a strong centrifugal force during centrifugation, the centrifugation vessel can be manufactured in a cylindrical shape with a length longer than its width, and an NK cell collection unit is formed at the bottom to easily separate NK cells.

The centrifugation preferably uses a low-speed rotation, unlike the conventional centrifuge. In the case of a commercially available centrifuge, it can rotate at a maximum of 20,000 to 50,000 rpm, but in the case of the NK cells of the present invention, when subjected to a strong centrifugal force, the cells may be destroyed. Therefore, in the centrifugation of the present invention, it is possible to prevent the destruction of the cultured NK cells by rotating at 1000 ~ 2000rpm. However, when centrifuging at low speed as described above, some NK cells are separated in a suspended state in the medium. can be manufactured. When the centrifugation is rotated at a speed of less than 1000 rpm, the recovery rate of NK cells may be reduced due to low-speed rotation, and when rotating at a speed exceeding 2000 rpm, destruction of NK cells occurs, and NK cells in the synbiotic lactic acid bacteria of the present invention Cell content may drop.

The NK cells recovered as described above can be used for other purposes. For example, the immune system of the human body can be strengthened by directly injecting the NK cells into the human body, and a composition using the NK cells can also be prepared to facilitate the injection of the NK cells.

Therefore, in the case of the synbiotic lactic acid bacteria of the present invention, an additional product can be manufactured using the manufactured NK cells, so that the production of the synbiotic lactic acid bacteria can be more economical than the conventional method.

In the case of the NK cell culture medium isolated as described above, it may contain NK cell metabolites, NK cell body components, and non-separated NK cells. Therefore, when proliferating lactic acid bacteria using this, it is possible to produce lactic acid bacteria containing active ingredients.

The step (c) is a step of inoculating and culturing the lactic acid bacteria in the culture medium from which the NK cells are removed, and the centrifugation is completed to recover the culture medium from which the NK cells are partially removed; mixing the culture medium with the MRS medium in a weight ratio of 1:1 to 3:1; inoculating lactic acid bacteria into the culture medium mixed with the MRS medium; Culturing the culture medium inoculated with the lactic acid bacteria at a temperature of 30 to 40 ° C for 1 to 5 days; And when the culture medium reaches pH 2 to 3, it may include mixing 1 to 5 parts by weight of the fisetin compound, 0.5 to 3 parts by weight of the damask rose extract relative to 100 parts by weight of the culture solution.

In the case of the culture medium from which NK cells are separated using a centrifuge as in step (b), conventionally, NK cells are cultured or discarded again by recycling, but the efficiency of NK cell cultivation is low, and the cost increases when discarded. will do However, in the present invention, the components of the culture medium can be recycled by inoculating and culturing the lactic acid bacteria in the NK cell culture medium.

The lactic acid bacteria used at this time may be a mixture of Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus fermentum isolated from the female genitalia. In particular, the lactic acid bacteria corresponds to the lactic acid bacteria present in a woman's body, and when ingested, it can bring about normalization of the intestinal flora, and when applied to the genitals, it can also bring about improvement of the bacterial flora in the genital organs. In addition, since the culture medium of the NK cells contains NK cell metabolites, it may have antibacterial properties, and thus may serve to reduce harmful bacteria in the genital organs.

The lactic acid bacteria is preferably inoculated with 0.01 to 0.5 parts by weight of Lactobacillus rhamnosus, 0.01 to 0.1 parts by weight of Lactobacillus reuteri, and 0.01 to 0.2 parts by weight of Lactobacillus fermentum relative to 100 parts by weight of the culture medium mixed with the MRS medium. The lactic acid bacteria may exhibit an optimal engraftment rate and effect within the above range, and if outside the above range, the engraftment rate may drop or the effect may be reduced.

The MRS medium is a medium developed for culturing lactic acid bacteria, Lactobacillus, and is a medium named after the first letters of the developers deMan, Rogosa, and Sharpe. This MRS medium contains peptone, egg extract and yeast extract as sources of carbon, nitrogen, vitamins and amino acids, and may contain a certain amount of sodium acetate to inhibit the growth of microorganisms competing with Lactobacillus. A commonly used MRS medium is 10 parts by weight of peptone, 8 parts by weight of egg extract, 4 parts by weight of yeast extract, 20 parts by weight of glucose, 5 parts by weight of sodium acetate, 1 part by weight of surfactant, 2 parts by weight of ammonium citrate, 0.2 parts by weight of magnesium sulfate. It is mixed in a proportion of parts by weight and 0.05 parts by weight of manganese sulfate, and the pH can be measured as 6.2. In addition, Difco MRS medium using beef extract can be used instead of egg extract.

The MRS medium may be mixed with the NK cell culture medium and used. In the case of the NK cell culture medium, it contains various components for culturing NK cells, but it is only for culturing NK cells, and it is difficult to use as it is for culturing lactic acid bacteria. Therefore, it is preferable to culture the lactic acid bacteria by mixing the MRS medium used for culturing the lactic acid bacteria with the NK cell culture medium. At this time, the NK cell culture medium is preferably mixed with the MRS medium in a weight ratio of 1:1 to 3:1. If the mixing ratio is less than 1:1, that is, when a large amount of MRS medium is used compared to the NK cell culture medium, the content of the NK cell metabolites in the lactic acid bacteria may be lowered, and when it exceeds 3:1, relatively small It may be difficult to culture the lactic acid bacteria due to the MRS medium.

MRS medium is mixed with the NK cell culture medium as described above, and then the lactic acid bacteria are inoculated, and this can be cultured for 1 to 5 days at a temperature of 30-40 ° C. The lactic acid bacteria are lactic acid bacteria found in the human body, and optimal culture conditions can be formed under conditions similar to those of the human body. Therefore, it is possible to optimize the culture rate of the lactic acid bacteria by culturing at a similar temperature. If the culture temperature is less than 30 ° C or cultured for less than 1 day, the lactic acid bacteria may not be sufficiently cultured, and when cultured at a temperature exceeding 40 ° C, the lactic acid bacteria may be killed due to the high temperature. In addition, when cultured for more than 5 days, other bacteria in competition with the lactic acid bacteria may breed.

After the culture is completed as described above, when the culture medium reaches pH 2 to 3, 1 to 5 parts by weight of the fisetin compound and 0.5 to 3 parts by weight of the damask rose extract can be mixed with respect to 100 parts by weight of the culture solution. When culturing the lactic acid bacteria, the culture medium becomes acidic due to metabolites of the lactic acid bacteria. Therefore, by measuring the acidity of the culture medium, it is possible to confirm the culture degree of the lactic acid bacteria. In this case, if the acidity (pH) is less than 2, the lactic acid bacteria may be killed due to over-cultivation, and if the acidity exceeds 3, the lactic acid bacteria may not be sufficiently cultured.

After reaching the above acidity, the facetin compound and the damask rose extract may be mixed in the culture medium.

The fisetin compound is a compound having the structure of Formula 1 below and has anti-inflammatory and antibacterial effects.

[Formula 1]

The fisetin compound is a kind of flavonoid, and is represented by the molecular formula of "C15H10O6", and may be represented by the following Chemical Formula 1, and can be mainly found in the heartwood of Sumacaceae plants in nature. In addition, the damask rose extract is a material extracted from the damask rose, a sub shrub of the rose family Rosaceae, a dicotyledonous plant. The present invention may have excellent antibacterial activity by including the fisetin compound and the damask rose extract. For example, the fisetin compound and the damask rose extract may have excellent antifungal activity against Candida genus fungi, more specifically, Candida albicans. In addition, the composition is a gram-negative strain of Escherichia genus, more specifically Escherichia coli, and a gram-positive strain of the genus Staphylococcus, more specifically Staphylococcus aureus ( Staphylococcus aureus) may have excellent antibacterial activity. That is, it is possible to prevent or reduce diseases occurring in the vulva because it is possible to suppress fungal skin diseases that frequently occur in the vulva of women and also to reduce gram-negative bacteria.

After the culture of the lactic acid bacteria is completed as described above, the culture solution may be freeze-dried and powdered. In the case of a commonly used heat drying method or a reduced pressure drying method, since it is highly likely to destroy the lactic acid bacteria and NK cells contained in the culture medium, it can be dried while preserving the lactic acid bacteria and NK cells through the freeze-drying method.

At this time, the freeze-drying method comprises the steps of substituting an inert gas inside the freeze-drying machine; Cooling the freeze dryer with the interior substituted with the inert gas to a temperature of -70 ~ -20 ℃; spraying the culture solution on which the culture is completed through a nozzle having a diameter of 0.1 to 2 mm; and circulating the inert gas to remove moisture inside the freeze dryer.

The interior of the freeze dryer may be substituted with an inert gas. This can prevent aerobic microorganisms from propagating due to oxygen contained therein during the freeze-drying period, and can prevent oxidation of active ingredients due to contact with oxygen. In this case, the inert gas is not used for the aerobic microorganisms, and any gas capable of preventing oxidation may be used without limitation, but nitrogen, helium, neon, argon or xenon may be used, and nitrogen may be preferably used.

After replacing the inside of the freeze dryer with an inert gas as described above, the inside of the freeze dryer may be cooled to -70 ~ -20 °C. At this time, it is also possible to cool the inside of the freeze dryer using only a cooler, but the initial cooling is performed using liquid nitrogen, so that additional cooling and replacement with an inert gas may be performed at the same time. At this time, if the inside of the freeze dryer is less than -70 ℃, it may require a lot of cost to maintain the temperature, and if it has a temperature exceeding -20 ℃, it may not be sufficiently cooled, so that the drying efficiency may be reduced.

After the cooling is completed as described above, the culture solution in which the culture of the lactic acid bacteria is completed may be sprayed into the freeze dryer through a nozzle having a diameter of 0.1 to 2 mm. Through this spraying, the culture solution can be frozen while forming small particles inside the freeze dryer. Cooling and drying can be performed at a high speed. At this time, if the diameter of the nozzle is less than 0.1mm, the amount of spraying may be reduced and drying efficiency may be reduced. Not only that, but the size of the generated particles may increase, and thus the drying efficiency may decrease.

As described above, the dried lactic acid bacteria powder can be prepared with synbiotics to facilitate engraftment in the body.

As used herein, the term “synbiotics” refers to a mixture of lactic acid bacteria (probiotics) and food (prebiotics) of the lactic acid bacteria. .

The synbiotics may be prepared by mixing 2 to 8 parts by weight of dietary fiber, 2 to 5 parts by weight of fructooligosaccharide, and 20 to 30 parts by weight of water to 5 to 10 parts by weight of the freeze-dried lactic acid bacteria. Conventional synbiotics are prepared by simply mixing prebiotic powder with lactic acid bacteria powder, but in the present invention, it is preferable to mix with water to prepare a liquid because it undergoes an encapsulation process to be described later.

In this case, the dietary fiber and fructooligosaccharide are prebiotics, that is, substances used as food for the lactic acid bacteria, and can exert an optimal effect within the above range. In addition, in the case of the water, when the above range is included, it can be smoothly manufactured into a capsule, and when it is included below the above range, it is difficult to form a capsule. effectiveness may decrease.

The synbiotics prepared as described above may be encapsulated. In general, in the case of lactic acid bacteria, it is known that most of them are killed by gastric acid present in the stomach when taken orally. To prevent this, oral administration of antacids before ingestion of lactic acid bacteria or a method of mixing antacids with lactic acid bacteria have been developed, but this may cause indigestion and thus has been used only limitedly. In the present invention, by encapsulating the lactic acid bacteria and then performing an enteric coating on the surface, the microcapsules containing the lactic acid bacteria can reach the intestine, and can be dissolved in the intestine to release the lactic acid bacteria.

To this end, 80 to 100 parts by weight of the temperature-dependent oil is mixed with 20 to 50 parts by weight of the synbiotics, followed by emulsification; mixing 10 to 20 parts by weight of a coating agent obtained by mixing 200 to 500 parts by weight of water with respect to 100 parts by weight of the enteric cellulose compound with the emulsified synbiotics; and recovering the synbiotics on which the coating treatment has been completed.

The temperature-dependent oil may be any one of coconut oil, triglycerides, fatty acids, or a mixture thereof, and preferably, the temperature-dependent oil may be coconut oil. The temperature-dependent oil corresponds to a non-polar substance that is not mixed with the synbiotics. However, through the emulsification process as described above, the synbiotics can be dispersed in a certain size in the temperature-dependent oil. In this case, in the emulsification process, the synbiotics may be dispersed using an emulsifier, but in the present invention, since the emulsifier may have a bad effect on the human body, it is preferable to emulsify it through mechanical mixing. Through the mechanical mixing as described above, the synbiotic particles can be crushed and emulsified with the temperature-dependent oil. That is, a shape in which the temperature-dependent oil surrounds the outside of the synbiotics may be configured. When the synbiotics are mixed in less than 20 parts by weight, the film treatment efficiency may be reduced, and when the synbiotics are mixed in excess of 50 parts by weight, the emulsification process is not smooth, and synbiotics with large particles may be formed. .

After the emulsification treatment as described above is completed, 10 to 20 parts by weight of the coating agent mixed with 200 to 500 parts by weight of water relative to 100 parts by weight of the enteric cellulose compound may be mixed with the emulsified synbiotics. The enteric cellulose compound may include hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate phthalate (CAP), their respective derivatives, or mixtures thereof. The enteric cellulose compound does not dissolve in gastric acid, and is a compound having a property of being dissolved by an enzyme in the intestine. Therefore, when a film is formed on the surface of the synbiotics using the compatible cellulose compound, it will be possible to prepare microcapsules that pass through the stomach and are dissolved and released in the intestine.

In the case of the enteric cellulose compound, it is also possible to dissolve directly in the water, but it may be more easily dissolved when a pH adjusting agent is added to and dissolved in water. In this case, the pH adjusting agent used may be a basic material including sodium, preferably sodium hydrogen carbonate. 200 to 500 parts by weight of water can be mixed with respect to 100 parts by weight of the cellulose compound, and when the water is included in less than 200 parts by weight, the thickness of the film is not uniform, and when the water exceeds 500 parts by weight, the thickness of the film becomes thin It can be destroyed by stomach acid.

The coating agent formed as described above may be mixed with the emulsified synbiotics. At this time, since the synbiotics are emulsified in the temperature-dependent oil, the synbiotics-coating agent may form a double structure or the synbiotics-temperature-dependent oil-coating agent triple structure may be formed. The amount of the coating agent to be added is preferably 10 to 20 parts by weight, and when it contains less than 10 parts by weight, the formation of the film is not smooth. .

After the membrane-breaking treatment is completed as described above, the membrane-treated synbiotics are separated and dried to produce synbiotic lactic acid bacteria with improved female immune activity and stability using the NK cell culture medium composition.

The present invention can also provide a synbiotic lactic acid bacteria with improved female immune activity and stability using the NK cell culture medium composition prepared by the above method.

Hereinafter, preferred embodiments of the present invention will be described so that those of ordinary skill in the art can easily implement them with reference to the accompanying drawings. In addition, in the description of the present invention, when it is determined that a detailed description of a related known function or a known concept may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, certain features presented in the drawings are enlarged, reduced, or simplified for ease of description, and the drawings and their components are not necessarily drawn to scale. However, those skilled in the art will readily appreciate these details.

Example 1

2.2 mg of IL-2, 20 μg of IL12, and 125 μg of IL18 were mixed in the suspension cell culture basal medium, and finally 10 L was used as the basal medium.

300 ml of peripheral blood 1 day after blood collection from the donor was placed in a 500 ml conical tube and centrifuged. Thereafter, the autologous plasma of the upper layer was put into a heparin tube for treatment, and then placed in a new 500 ml conical tube and centrifuged again to separate the plasma components of the upper layer.

After that, add PBS to the blood tube from which plasma has been removed, adjust the volume to 300 ml, mix well, transfer to a tube containing Ficoll-Paque Plus, centrifuge at 800xg for 15 minutes, and then centrifuge the second layer, peripheral blood mononuclear cells. The buffy coat layer was separated and collected in a 500 ml conical tube, and the volume was adjusted to 500 ml with PBS and then mixed. Thereafter, the supernatant was discarded by centrifugation 2 to 3 times, and peripheral blood mononuclear cells were isolated. The number of peripheral blood mononuclear cells obtained at this time was about 10 x 10 ⁷ pieces.

The peripheral blood mononuclear cells collected by the above method were injected into 45 ml of the basal medium, and then 10 ml of the basal medium in which the anti-CD56 antibody was dissolved at a rate of 10 μg/ml was injected.

After the anti-CD56 antibody was injected, 10 ml of a basal medium in which the anti-CD3 antibody was dissolved at a rate of 8 μg/ml was injected.

After the injection of the anti-CD3 antibody was completed, Dexamethasone sodium phosphate as a steroid was dissolved in the basal medium at a concentration of 1ug/mL, and 50ml of this was added.

After the addition of the steroid was completed, 10 ml of the basal medium in which the anti-CD56 antibody was dissolved at a rate of 10 μg/ml was additionally injected twice.

After culturing of the NK cells as described above was completed, they were inserted into a centrifuge and centrifuged at 1500 rpm for 10 minutes to separate the settled NK cells and the NK cell culture solution.

The NK cell culture medium and the MRS medium were mixed in a weight ratio of 2:1 to prepare a culture solution, and then 0.2 parts by weight of Lactobacillus rhamnosus isolated from the female genitalia, 0.05 parts by weight of Lactobacillus reuteri And Lactobacillus fermentum (Lactobacillus fermrntum) was inoculated in a ratio of 0.1 parts by weight and lactic acid bacteria culture was performed at a temperature of 35 ℃ 3 days.

It was confirmed by using a pH meter that the lactic acid bacteria culture was completed (pH2.2), and 3 parts by weight of the facetin compound and 2 parts by weight of the damask rose extract were mixed in the culture medium in which the lactic acid bacteria culture was completed.

The lactic acid bacteria culture method was repeated several times to obtain 1 L of lactic acid bacteria culture solution.

By injecting liquid nitrogen into the freeze dryer, the inside of the dryer was lowered to -45° C. and the inside was replaced with a nitrogen atmosphere. The lactic acid group culture solution was sprayed through a nozzle having a diameter of 1 mm into the cooled and substituted lyophilizer with a nitrogen atmosphere, and lyophilization was performed by continuously circulating nitrogen therein to obtain freeze-dried lactic acid bacteria.

A synbiotic solution was prepared by mixing 8 g of the freeze-dried lactic acid bacteria, 5 g of dietary fiber, and 3 g of fructooligosaccharide in 25 g of water.

A coating agent was prepared by dissolving 10 g of sodium hydrogen carbonate in 100 g of water and then dissolving 30 g of hydroxypropyl methylcellulose acetate succinate.

The synbiotic solution was mixed with 100 g of coconut oil and then continuously mixed to emulsify, and 13 parts by weight of the coating agent was mixed to form a film on the surface of the emulsified synbiotic solution.

After the formation of the film was completed, coconut oil and the synbiotic lactic acid bacteria on which the film was formed were separated and dried.

Example 2

It was carried out in the same manner as in Example 1, except that lactic acid bacteria were cultured without using the NK cell culture medium.

Example 3

The same procedure was carried out in Example 1 except that the facetin compound and the Damask rose extract were not used.

Example 4

The same procedure was performed except that lactic acid bacteria culture was not performed in Example 1.

Example 5

The same procedure was performed except that in Example 1, dietary fiber and fructooligosaccharide were not included.

Example 6

The same procedure was performed except that the coating agent was not used in Example 1.

Example 7

In Example 1, the same procedure was performed except for using a formulation in which lyophilized lactic acid bacteria, dietary fiber, and fructooligosaccharide were mixed.

Test Example 1

An experiment for measuring the antibacterial activity of the synbiotic lactic acid bacteria prepared in Examples 1 to 7 was conducted.

Candida, Staphylococcus aureus, Enterococcus faecalis, 5X5mm size immersed in a solution in which the synbiotic lactic acid bacteria prepared in Examples 1 to 7 were dissolved at a ratio of 1g/L on an agar medium inoculated with Candida bacteria of paper was placed in the center. After 12 hours, the antibacterial properties were measured by measuring the diameter (cm) of the part where the bacteria were killed.

candida Staphylococcus aureus enterococci yeast fungus Example 1 4.8 3.2 3.6 4.3 Example 2 2.8 1.6 1.5 2.1 Example 3 3.9 2.9 2.3 2.6 Example 4 1.4 0.9 1.1 1.0 Example 5 3.1 2.9 2.8 2.4 Example 6 5.1 3.9 3.9 4.3 Example 7 6.8 6.4 6.1 6.6

As shown in the above example, Examples 6 and 7, which were not treated with a coating agent, were found to have a higher effect than that of Example 1. However, Example 2 without using NK cell culture medium, Example 3 without using facetin compound and Damask rose extract, Example 4 without using lactic acid bacteria, Example 5 without using dietary fiber and fructooligosaccharide was found to have a lower antibacterial activity than in Example 1.

Experimental Example 2

An experiment was conducted to test the effect of gastric acid. In order to simulate the effect of gastric acid, 30 vol% of hydrochloric acid was injected into the synbiotic lactic acid bacteria solution prepared in Experimental Example 1 at a ratio of 5 g/L and left for 1 hour. Then, it was neutralized using sodium hydrogen carbonate. Thereafter, the procedure was carried out in the same manner as in Experimental Example 1.

candida Staphylococcus aureus enterococci yeast fungus Example 1 4.5 2.9 3.1 4.1 Example 2 2.6 1.6 1.3 1.9 Example 3 3.1 2.1 2.0 2.0 Example 4 1.1 0.8 0.8 0.6 Example 5 2.9 2.6 2.4 2.0 Example 6 3.1 1.9 1.6 2.1 Example 7 1.7 1.6 1.1 1.4

As shown in Table 1, in the case of Examples 1 to 5 in which the film was formed, it was confirmed that the effect was not significantly lowered even by gastric acid, but Example 6 in which the film was not used and Example 7 in which the microcapsule was not used. In the case of , it was confirmed that the effect was dramatically reduced by gastric acid.

In the above, preferred embodiments of the present invention have been illustrated and described, but the present invention is not limited to the specific embodiments described above, and it is common in the technical field to which the present invention pertains without departing from the gist of the present invention as claimed in the claims. Various modifications are possible by those having the knowledge of, of course, these modifications should not be individually understood from the technical spirit or perspective of the present invention.

Claims (2)

(a) inoculating and culturing NK cells in an NK cell-only medium;
(b) recovering NK cells after the culture is completed;
(c) inoculating and culturing lactic acid bacteria in the NK cell-only medium from which the NK cells have been removed; and
(d) lyophilizing and powdering the culture solution after the culture of the lactic acid bacteria is completed;
In the method for producing synbiotics with improved antibacterial activity and stability against gastric acid using a NK cell culture medium composition comprising:
The step (a) is,
(i) preparing a basal medium by mixing IL2, IL12 and IL18 in a NK cell-only medium solution;
(ii) mixing peripheral blood mononuclear cells isolated from blood with the NK cell-only medium;
(iii) injecting the anti-CD56 antibody into the NK cell-only medium mixed with the peripheral blood mononuclear cells;
(iv) injecting the anti-CD3 antibody into the NK cell-only medium mixed with the peripheral blood mononuclear cells injected with the anti-CD56 antibody; and
(v) adding a steroid to an NK cell-only medium mixed with peripheral blood mononuclear cells injected with the anti-CD3 antibody;
includes,
Step (b) is,
injecting the NK cell-only medium into a centrifuge;
rotating the centrifuge into which the NK cell-only medium is injected at 1000 to 2000 rpm; and
recovering the NK cells precipitated on the bottom of the centrifuge;
includes,
Step (c) is,
Recovering the culture solution from which the centrifugation is completed and NK cells are removed;
mixing the culture medium with the MRS medium in a weight ratio of 2:1;
inoculating lactic acid bacteria into the culture medium mixed with the MRS medium;
Culturing the culture medium inoculated with the lactic acid bacteria at a temperature of 30 to 40 ° C for 1 to 5 days; and
When the culture medium reaches pH 2 to 3, mixing 1 to 5 parts by weight of the fisetin compound, 0.5 to 3 parts by weight of the damask rose extract compared to 100 parts by weight of the culture solution;
includes,
The lactic acid bacteria is a mixture of Lactobacillus rhamnosus, Lactobacillus reuteri and Lactobacillus fermentum isolated from the female genitalia,
The lactic acid bacteria are inoculated with 0.2 parts by weight of Lactobacillus rhamnosus, 0.05 parts by weight of Lactobacillus reuteri, and 0.1 parts by weight of Lactobacillus fermentum relative to 100 parts by weight of the culture medium mixed with the MRS medium,
Step (d) is,
substituting the inside of the freeze dryer with an inert gas;
Cooling the freeze dryer with the interior substituted with the inert gas to a temperature of -70 ~ -20 ℃;
spraying the culture solution on which the culture is completed through a nozzle having a diameter of 0.1 to 2 mm; and
removing moisture inside the freeze dryer by circulating the inert gas;
includes,
preparing synbiotics by mixing 2 to 8 parts by weight of dietary fiber, 2 to 5 parts by weight of fructooligosaccharide, and 20 to 30 parts by weight of water to 5 to 10 parts by weight of the freeze-dried lactic acid bacteria;
80 to 100 parts by weight of the temperature-dependent oil is mixed with 20 to 50 parts by weight of the synbiotics, followed by emulsification;
mixing 10 to 20 parts by weight of a coating agent obtained by mixing 200 to 500 parts by weight of water with respect to 100 parts by weight of the enteric cellulose compound with the emulsified synbiotics; and
recovering the synbiotics on which the coating treatment has been completed;
includes,
The synbiotics are synbiotics with improved antibacterial activity and gastric acid stability using a NK cell culture medium composition, characterized in that it has antibacterial activity against Candida, Staphylococcus aureus, Enterococcus faecalis and yeast. manufacturing method. delete

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