KR20220143231A - Manufacturing methods of heat-killed strain powder oriented from phellinus linteus, heat-killed strain powder and uses thereof - Google Patents

Abstract

The present invention relates to a manufacturing method of heat-killed strain powder, which includes the steps of: obtaining a concentrate by culturing L. fermentum VIMPP04 KCTC14499BP in a first medium and concentrating the same; obtaining a culture by culturing L. plantarum VIOAP03 KCTC14498BP in a second medium; and performing tyndallization and drying a mixed culture medium obtained by mixing the concentrate and the culture. The manufacturing method can manufacture heat-killed strain powder which can increase the number of bacteria by improving the growth of the strain, has an increased content of β-glucan, an effective substance capable of improving the hypersensitive immune response, and has excellent antioxidant effect.

Description

Method for producing dead cell powder of Sanghwang mushroom-derived strain, dead cell powder and its use

The present invention relates to a method for producing a dead cell powder of a strain derived from Sanghwang mushroom, a dead cell powder, and a use thereof, and more specifically, to obtain a concentrate by culturing L. Fermentum VIMPP04 KCTC14499BP in a first medium to obtain a concentrate, and L. Plantarum To obtain a culture by culturing VIOAP03 KCTC14498BP in the second medium, and then to a method for producing a dead cell powder through a tindil process and drying a mixed culture solution in which the concentrate and the culture solution are mixed.

Probiotics are live microorganisms that enter the body and give good health effects, and the World Health Organization (WHO) defines them as living microorganisms that confer health benefits to the host when administered in an appropriate amount. The definition of probiotics is changing little by little with the passage of time. In the 1980s, probiotics were defined as probiotics that have a beneficial effect on the host by improving the intestinal microbiota and refer to living microorganisms. was expanded to the dead cells and the components constituting the cells.

Probiotic dead cells having the opposite concept to probiotic live cells means a form in which live cells obtained through culture and fermentation and their metabolites are prevented from growing by heat treatment or the like. Products using these dead cells have superior stability compared to live products. In particular, since it has high stability and heat resistance to the external environment, it has the advantage of being easier to distribute and not difficult to store than probiotic live bacteria products. Patents related to products using dead cells have been previously known. Specifically, Korea Patent No. 1472190 contains Lactobacillus plantarum PMO 08 dead cells as an active ingredient as a composition for preventing, treating or improving constipation, and Korean Patent Application Publication No. 2020-0051953 discloses a composition for preventing, treating or improving constipation. , It contains Lactobacillus salivarius V133 dead cells as an active ingredient as a composition for improvement or treatment. However, in the prior patents, there is no description of a method for preparing the novel strains identified in the present invention as dead cell powder and using the same.

On the other hand, since all microorganisms have different properties, the components, types, and culture conditions of the medium for culturing the microorganisms are very important. In particular, since nutritional requirements vary depending on the type of microorganism or culture conditions, it is necessary to select and culture each appropriate medium. Accordingly, various media compositions and patents for methods of utilizing them have been known in the prior art. Specifically, Korean Patent No. 2222953 relates to a medium composition for high-yield culture of egg-culture anaerobic microorganisms, and an amino acid mixture of N-acetylhexosamine, L-aspartic acid and L-cysteine and cobalt as supplements to the medium composition. Calibacterium prausnitzii, including amines Faecalibacterium prausnitzii , Anaerostipes caccae , Akkermansia muciniphila , and the group consisting of Bifidobacterium longum Anaerobic microorganisms selected from were cultured in high yield to enable mass production. Korea Patent No. 2123581 relates to an optimal medium composition for promoting the growth of strains, and includes molasses, sucrose and peptone as the medium composition, and the accession number is Bacillus subtilis KCCM12433P. (Bacillus subtilis ) The growth of the SRCM102046 strain was increased to increase the production of the cells. However, the prior patents do not disclose a culture using a medium for culturing the novel strains identified in the present invention and a method for producing a dead cell powder therethrough.

Hypersensitivity immune response refers to an allergy. Specifically, when an allergen penetrates the human body, the allergen and IgE antibody interact to release an inflammatory substance, thereby causing inflammation in the tissue and ultimately causing allergic symptoms. In general, atopic dermatitis, rhinitis, asthma, etc. correspond to hypersensitive immune response. In order to improve these overactive immune spots, it is important to strengthen the immune system and balance the immune system. It is known that representative 'probiotics' products among immune enhancing substances generally use live lactic acid bacteria or dead lactic acid bacteria.

Accordingly, the present inventors have made intensive research efforts to overcome the problems of the prior art, and as a result, after culturing L. Fermentum VIMPP04 KCTC14499BP in the first medium, concentrating to obtain a concentrate, and culturing L. Plantarum VIOAP03 KCTC14498BP in the second medium to obtain a culture, and then, in the case of preparing a dead cell powder through a tindil process and drying a mixed culture solution in which the concentrate and the culture solution are mixed, the growth of the strain can be improved to increase the number of bacteria, and the hypersensitive immune response is improved It was confirmed that the content of β-glucan (β-gulcan), an effective substance that can be used, was significantly increased, and the present invention was completed.

US 10-1472190 B1 US 10-2020-0051953 A US 10-2222953 B1 US 10-2123581 B1

Therefore, the main object of the present invention is to increase the number of dead cells by improving the growth of the number of bacteria, and the content of β-glucan, an effective substance that can improve the hypersensitive immune response, is significantly increased. An object of the present invention is to provide a method for producing a cell powder.

Another object of the present invention is to provide a dead cell powder prepared through the method for producing the dead cell powder.

Another object of the present invention is to provide a food composition for preventing or improving a hypersensitive immune response using the dead cell powder.

Another object of the present invention is to provide a health functional food composition for preventing or improving hypersensitive immune response using the dead cell powder.

According to one aspect of the present invention, the present invention is a first step of culturing L. Fermentum VIMPP04 KCTC14499BP in a first medium to obtain a concentrate, and culturing L. Plantarum VIOAP03 KCTC14498BP in a second medium to obtain a culture a third step of mixing the concentrate obtained in the first step with the culture obtained in the second step to obtain a mixed culture solution, and drying the mixed culture solution obtained in the third step after the Tyndil process to provide a method for producing a dead cell powder comprising; a fourth step of obtaining a dead cell powder.

The present inventors confirmed that when each of the strains isolated from the Sangha mushroom was cultured in an optimal medium to prepare a dead cell powder, the number of bacteria was increased and the content of the active substance was significantly increased, and the present invention was completed.

In the production method of the dead cell powder of the present invention, the second medium is characterized in that it contains the extract derived from the Sangha mushroom, and the extract derived from the Sangha mushroom contains 10 to 30% by weight based on the total weight of the second medium composition. characterized.

According to an experimental example of the present invention, it was confirmed that L. Plantarum VIOAP03 KCTC14498BP among the strains isolated from Sanghwang mushroom had better growth in the medium containing the Sanghwang mushroom-derived extract than in the general medium that does not contain the Sanghwangmushroom-derived extract. And, a medium containing the extract derived from the Sangha mushroom was selected as an optimal medium for culturing L. Plantarum VIOAP03 KCTC14498BP (see Experimental Example 1 and Table 1).

According to an experimental example of the present invention, it was confirmed that L. Fermentum VIMPP04 KCTC14499BP among the strains isolated from the Sangha mushroom grew better in the general medium not containing the Sanghwang mushroom-derived extract than in the medium containing the Sanghwang mushroom-derived extract. And, a normal medium not containing the extract derived from the Sangha mushroom was selected as the optimal medium for culturing L. Fermentum VIMPP04 KCTC14499BP (see Experimental Example 1 and Table 1).

In the production method of the dead cell powder of the present invention, the first medium and the second medium are glucose (Glucose), yeast extract (Yeast Extract), soy peptone (Soy peptone), sodium acetate (Sodium Acetate), diphosphate Potassium (Dipotassium phosphate), sodium citrate (Sodium Citrate), Tween 80 (Tween 80), magnesium sulfate (Magnesium sulfate) and made of containing one or more components selected from the group consisting of manganese sulfate (Manganese sulfate), preferably is glucose 2 to 5.5 wt%, yeast extract 1 to 3 wt%, sodium acetate 0.5 to 1 wt%, potassium dibasic phosphate 0.01 to 0.1 wt%, sodium citrate 0.01 to 0.1 wt%, Tween 80 0.005 to 0.01 wt%, 0.01 to 0.05 wt% of magnesium sulfate, and 0.001 to 0.01 wt% of manganese sulfate.

In the method for producing the dead cell powder of the present invention, the culturing in the first step is a first to obtain a pre-culture by performing pre-culture for 6 to 10 hours in a first incubator containing a first medium. Step -1, and a step 1-2 of performing the first main culture for 10 to 13 hours by adding the pre-culture obtained in step 1-1 to a second incubator containing the first medium; characterized by including. If the incubation time in step 1-1 is less than 6 hours, the growth of the bacteria does not increase enough to inoculate for the culture in step 1-2, resulting in a problem in which the number of bacteria in the result in step 1-2 is reduced. When the culture time exceeds 10 hours, the stationary phase and death phase of the microorganisms rapidly reach due to lack of nutrients in the culture medium, and the activity of the cells decreases, making it difficult to exert effectiveness. In addition, if the incubation time in step 1-2 is less than 10 hours, there is a problem that the number of dead cells decreases during powder production, and when it exceeds 13 hours, the stationary phase and death phase of microorganisms rapidly reach due to lack of nutrients in the culture medium. It is difficult to exert effectiveness because the activity of the cells is reduced.

In the method for producing dead cell powder of the present invention, the culturing in the second step is characterized in that the second main culture is performed for 22 to 26 hours. When the second main culture time is less than 22 hours, a problem occurs in that the number of bacteria decreases, and when it exceeds 26 hours, the activity of the cells decreases, making it difficult to exert effectiveness.

In the method for producing the dead cell powder of the present invention, in the third step, the mixing ratio of the concentrate and the culture is 1:2 to 1:5. When the mixing ratio of the culture is the same as that of the concentrate, the viscosity of the mixture increases, making it difficult to transfer heat during the tyndyl process, making it difficult to proceed with the dead cell process. There may be a problem that the yield is lowered.

In the method for producing the dead cell powder of the present invention, the drying is -180°C to -195°C, more preferably -195°C to the tindylated mixed culture using a liquid nitrogen freezer (LNF). Step 4-1 of rapid freezing at ℃, and the step of freeze-drying the quick-frozen Tyndylation mixed culture solution at -35 ℃ to -45 ℃, more preferably -40 ℃ in the step 4-1. characterized in that In the case of rapid freezing using a liquid nitrogen freezer primarily in the drying step of the present invention, the freeze-drying operation time can be shortened, the browning phenomenon of the raw material can be prevented, and the active ingredient can be maintained as well as the strain of the strain. damage can be minimized.

According to another aspect of the present invention, the present invention is a first step of culturing L. Fermentum VIMPP04 KCTC14499BP in a first medium and then concentrating to obtain a concentrate, L. Plantarum VIOAP03 KCTC14498BP in a second medium by culturing the culture. The second step of obtaining, a third step of mixing the concentrate obtained in the first step with the culture obtained in the second step to obtain a mixed culture solution, and a third step of obtaining a mixed culture solution obtained in the third step after the Tyndil process It provides a dead cell powder prepared through the drying step.

According to another aspect of the present invention, the present invention is a first step of culturing L. Fermentum VIMPP04 KCTC14499BP in a first medium and then concentrating to obtain a concentrate, L. Plantarum VIOAP03 KCTC14498BP in a second medium by culturing the culture. The second step of obtaining, a third step of mixing the concentrate obtained in the first step with the culture obtained in the second step to obtain a mixed culture solution, and a third step of obtaining a mixed culture solution obtained in the third step after the Tyndil process It provides a food composition for the prevention or improvement of hypersensitivity immune disease comprising the dead cell powder prepared through the drying step as an active ingredient.

According to another aspect of the present invention, the present invention is a first step of culturing L. Fermentum VIMPP04 KCTC14499BP in a first medium and then concentrating to obtain a concentrate, L. Plantarum VIOAP03 KCTC14498BP in a second medium by culturing the culture. The second step of obtaining, a third step of mixing the concentrate obtained in the first step with the culture obtained in the second step to obtain a mixed culture solution, and a third step of obtaining a mixed culture solution obtained in the third step after the Tyndil process It provides a health functional food composition for the prevention or improvement of hypersensitive immune diseases comprising the dead cell powder prepared through the drying step as an active ingredient.

According to an experimental example of the present invention, the dead cell powder (Example) prepared by separating and culturing each of the deposited strains isolated from the Sangha mushroom in an optimal suitable medium and mixing them (Example) is mixed after separating and culturing commercial strains for each deposited strain. Compared to the dead cell powder (Comparative Example 1) and deposited strains prepared by integrating and culturing in a general medium (Comparative Example 2), the number of bacteria is greater, and the antioxidant effect is excellent as well as an active ingredient related to immune activity. It was confirmed that the content of β-glucan was significantly increased (see Experimental Examples 2 to 4). These results suggest that the selection and combination of the deposited strain isolated from the Sangha mushroom according to the present invention and the medium for culturing the deposited strain is an essential component for preparing dead cell powder for preventing or improving hypersensitive immune disease.

In the food composition or health functional food composition for preventing or improving hypersensitive immune disease of the present invention, the hypersensitive immune disease is allergic urticaria, allergic rhinitis, allergic conjunctivitis, allergic asthma, allergic dermatitis, autoimmune hepatitis, It is characterized in that at least one selected from the group consisting of allergic bronchopulmonary aspergillosis and allergic stomatitis.

As described above, the method for producing the dead cell powder according to the present invention improves the growth of each deposited strain isolated from the Sangha mushroom, thereby increasing the number of bacteria, and exhibiting an antioxidant effect as well as an active ingredient β- A dead cell powder having an increased glucan content can be prepared.

In addition, in the case of the dead cell powder prepared according to the preparation method of the present invention, since it has an excellent antioxidant effect and a high content of β-glucan, it is possible to prevent or improve hypersensitive immune diseases.

1 is a result of confirming the Gram staining and calcium carbonate decomposition ability of two strains isolated from Sanghwang mushroom.
2 is a result of confirming the morphological characteristics of the L. plantarum VIOAP03 strain.
3 is a result confirming the morphological characteristics of the L. fermentum VIMPP04 strain.
4 is a result confirming the antioxidant activity of L. plantarum VIOAP03 strain and L. fermentum VIMPP04 strain.
5 is a result confirming the IL-6-induced STAT-3 dependent luciferase inhibitory activity of L. plantarum VIOAP03 strain and L. fermentum VIMPP04 strain.
6 is a process diagram of a dead cell powder manufacturing process according to the present invention.
7 and 8 show the results of confirming the antioxidant activity of the dead cell powder prepared according to the present invention (FIG. 7: DPPH, FIG. 8: ABTS, AA: Ascorbic acid 100ug/ml, A: L. plantarum VIOAP03 5×10 ⁷ CFU/mL, B: L. fermentum VIMPP04 5×10 ⁷ CFU/mL).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention by these examples.

Preparation Example 1: Isolation of microorganisms

1) Separation source and sample preparation

In order to isolate lactic acid bacteria from Sanghwang mushroom, the most representative Phellinus linteus, Phellinus linteus , derived from mulberry, was used. Sanghwang mushrooms grown at domestic farms were purchased online, and the equipment used in all pre-treatment processes was sterilized and disinfected. The Sangha mushrooms were chopped and steeped in 0.85% NaCl solution for 12 to 24 hours, and only the supernatant was taken and used for the experiment.

2) Lactobacillus selection and identification

Samples obtained by pretreatment were diluted by concentration using 0.85% NaCl solution, inoculated on BCP plate count agar (EIKEN, Japan), and incubated at 37°C for 48 hours. Afterwards, the medium was yellow and the colonies with different phenotypes were purified using MRS agar (Difco, USA) plate medium. For the purely isolated strain, the characteristics of the colony were confirmed, the cell morphology was confirmed through microscopic observation (×1000), and only the strains showing Gram-positive were first selected through Gram staining. After that, it was inoculated on MRS agar medium containing 1% calcium carbonate (DAEJUNG, Korea) and cultured at 37° C. for 48 hours to decompose calcium carbonate with organic acid to finally select two strains with large clear zones. (Fig. 1).

The isolated strain was prepared as a stock using 30% glycerol and stored at -80°C. To check the biochemical properties such as glycolysis and arginin and esculin degradation of the two strains, use the API 50 CHL kit (Biomerieux, France) to change the color of the medium using the apiweb program (http://apiweb.biomerieux.com) was sympathized. 16s rRNA analysis was commissioned by Biofact Co., Ltd. and PCR was performed using 27F and 1492R primers and sequencing analysis was performed. The nucleotide sequence of each identified strain was compared for homology with the 16S ribosomal RNA gene sequence registered in GenBank using the NCBI blast program, and aligned using the MEGA-X program using the neighbor-joining method. For creation and confirmation of phylogenetic trees, MEGA-X's bootstrap N-J tree method was used.

3) Decoding the genome sequence (base sequence and phylogenetic tree appendix 1)

As a result of confirming the nucleotide sequence of 1,417bp through 16S rRNA gene analysis of strain VIOAP03 strain, it showed 99.79% homology with Lactobacillus plantarum JCM 1149 strain. Therefore, the VIOAP03 strain was named Lactobacillus plantarum VIOAP03 (SEQ ID NO: 1).

As a result of confirming the base sequence of 1429bp size through 16S rRNA gene analysis of strain VIMPP04 strain, it showed 99.72% homology with Lactobacillus fermentum CIP 102980 strain. Therefore, the VIMPP04 strain was named Lactobacillus fermentum VIMPP04 (SEQ ID NO: 2).

4) Identification through sugar usability survey

As a result of identification of strain VIOAP03 through sugar availability investigation using API 50 CHL kit of the isolated strain, 99.9% similarity with Lactobacillus plantarum was confirmed. Strain VIMPP04 was confirmed to have 99.7% similarity with Lactobacillus fermentum .

Preparation Example 2: Biochemical and Morphological Characteristics of the Strain

1) Biochemical properties

The biochemical properties of the isolated strain were measured with API 50 CHL kit. The colony cultured in MRS agar medium was diluted to an appropriate concentration in API 50 CHL medium and inoculated with API 50 CHL kit, and then cultured at 37°C for 24-48 hours to observe the color change of the inoculated medium. are shown in Tables 1 and 2.

No. sugar type availability No. sugar type availability One Glycerol - 26 Salicine + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 Maltose + 4 L-Arabions - 29 Lactose + 5 Ribose + 30 Melibiose + 6 D-Xylose - 31 Sanccharose + 7 L-Xylose - 32 Trehalose + 8 Adonitol - 33 Inuline - 9 β-Methl-xyloside - 34 Melezitose + 10 Galactose + 35 D-Raffinose - 11 D-Glucose + 36 Amidon - 12 D-Fructose + 37 Glycogen - 13 D-Mannose + 38 Xylitol - 14 L-Sorbose - 39 β Gentiobiose + 15 Rhamnose - 40 D-Turanose + 16 Dulicitol - 41 D-Lyxose - 17 Inositol - 42 D-Tagatose - 18 Mannitol + 43 D-Fucose - 19 Sorbitol + 44 L-Fucose - 20 α Methyl-D-mannoside + 45 D-Arabitol - 21 α Methyl-D-glucoside - 46 L-Arabitol - 22 N acetyl glucosamine + 47 Gluconate + 23 Amygdaline + 48 2 ceto-gluconate - 24 Arbutine + 49 5 ceto-gluconate - 25 Esculin +

No. sugar type availability No. sugar type availability One Glycerol - 26 Salicine + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 Maltose + 4 L-Arabions - 29 Lactose + 5 Ribose + 30 Melibiose + 6 D-Xylose - 31 Sanccharose + 7 L-Xylose - 32 Trehalose + 8 Adonitol - 33 Inuline - 9 β-Methl-xyloside - 34 Melezitose + 10 Galactose + 35 D-Raffinose - 11 D-Glucose + 36 Amidon - 12 D-Fructose + 37 Glycogen - 13 D-Mannose + 38 Xylitol - 14 L-Sorbose - 39 β Gentiobiose + 15 Rhamnose - 40 D-Turanose + 16 Dulicitol - 41 D-Lyxose - 17 Inositol - 42 D-Tagatose - 18 Mannitol + 43 D-Fucose - 19 Sorbitol + 44 L-Fucose - 20 α Methyl-D-mannoside + 45 D-Arabitol - 21 α Methyl-D-glucoside - 46 L-Arabitol - 22 N acetyl glucosamine + 47 Gluconate + 23 Amygdaline + 48 2 ceto-gluconate - 24 Arbutine + 49 5 ceto-gluconate - 25 Esculin +

As can be seen in Table 1, the L. plantarum VIOAP03 strain used 23 sugars, including D-galactose, D-glucose, D-fructose, D-mannose, mannitol, and sorbitol, among 49 sugars, starch, Xylitol was not used.

As can be seen in Table 2, the L. fermentum VIMPP04 strain contains 14 sugars, including L-arabionose, D-xylose, D-glucose, D-fructose, D-manose, maltose, lactose, sucrose, etc. among 49 sugars. was used, and starch, xylitol, mannitol, and sorbitol were not used.

2) Morphological Characteristics

For morphological characteristics, the colony shape and color of the strain cultured in MRS agar medium were confirmed, and the strain shape was observed under a microscope. The colony of L. plantarum VIOAP03 strain is 1.0-2.5 mm round and convex, white or yellowish, and glossy. As a result of observing the morphology of the strain under a microscope, single, pairs, and short chain morphologies were shown as bacilli (Fig. 2). The colony form of L. fermentum VIMPP04 strain is 1.5-2.5 mm round and flat, and has a milky white luster. As a result of microscopic observation, the bacilli showed a single or pairs chain form, but also formed a long chain (FIG. 3).

Preparation Example 3: Confirmation of strain characteristics

1) Check antioxidant activity

There is a report that lactic acid bacteria have antioxidant activity to protect themselves from free radicals and act as a food preservative, and it can increase the possibility of use as a probiotic preparation and thus exhibit an antioxidant effect in the body. Accordingly, the scavenging activity of DPPH free radicals and ABTS free radicals was confirmed in order to confirm the inhibition and removal potential of free radicals of lactic acid bacteria.

The test method for measuring DPPH free radical scavenging activity is a test method for measuring the degree of removal of active oxygen by using a DPPH reagent containing active oxygen that changes from purple to yellow when active oxygen is removed by an antioxidant. The ABTS free radical scavenging activity measurement test method is a test method to measure the change in color concentration caused by the reaction of ABTS, which has a bluish-green color, with an antioxidant, and turns into a transparent color due to active oxygen. As a control of the DPPH free radical and ABTS free radical scavenging activity test, 100 μg/mL of ascorbic acid, a representative antioxidant, was used, and a sample with a concentration of 5×10 ⁷ CFU/mL of the strain was used. The results are shown in FIG. 4 .

As a result, as can be seen in FIG. 4, the DPPH free radical scavenging ability of the L. plantarum VIOAP03 strain was 72.4% and the ABTS free radical scavenging ability was 57.1%, and the DPPH free radical scavenging ability of the L. fermentum VIMPP04 strain was 75.2%, and the ABTS free radical scavenging ability was 75.2%. The scavenging ability was confirmed to be 75.4%.

2) Evaluation of IL-6-induced STAT-3 dependent luciferase inhibitory activity

IL-6-induced STAT-3 dependent luciferase inhibitory activity was measured for L. plantarum VIOAP03 strain and L. fermentum VIMPP04 strain. For the measurement method, a Hep3B cell line expressing the luciferase gene dependent on STAT-3 activity was used. Hep3B cells were pretreated with samples of the pretreated strains at each concentration (10, 30, 60 ug/ml) for 1 hour, and then treated with IL-6 (10 ng/ml) for 18 hours to activate STAT-3, followed by luciferase Activity was measured, and the results are shown in FIG. 5 .

As a result, as can be seen in FIG. 5 , both L. plantarum VIOAP03 and L. fermentum VIMPP04 strains exhibited concentration-dependent inhibitory activity.

Preparation Example 4: Strain deposit

After confirming the 16S RNA sequence analysis and morphological characteristics, and finally confirming the strain, the deposit procedure was performed at the Institute of Biotechnology and Biotechnology Life Resource Center (KCTC) and an accession number was given (Table 3).

strain name deposit number L. plantarum VIOAP03 KCTC 14498BP L.fermentum VIMPP04 KCTC 14499BP

Experimental Example 1: Establishment of culture conditions in the method for producing dead cells

A strain test was performed to establish culture conditions for each isolated strain. The test was conducted by developing a medium composition in which a normal medium without lactic acid bacteria-derived Sanghang mushroom extract and an extract of Sanghyang mushroom derived from lactic acid bacteria were developed, and the composition of the medium is shown in Table 4 below.

plain badge Glucose, Yeast Extract, Soy Peptone, Sodium Acetate, Dipotassium phosphate, Sodium Citrate, Tween 80, Sulfuric Acid Magnesium sulfate, Manganese sulfate Extract from Sanghwang mushroom
containing medium Sanghwang mushroom extract, Glucose, Yeast Extract, Soy peptone, Sodium Acetate, Dipotassium phosphate, Sodium Citrate, Tween 80), magnesium sulfate, manganese sulfate

Each of the strains L. plantarum VIOAP03 KCTC14498BP and L. fermentum VIMPP04 KCTC14499BP isolated in the present invention was cultured in the medium of Table 4, and the number of strains according to the change of the culture medium was confirmed, and the results are shown in Table 5 below.

VIOAP03 KCTC14498BP VIMPP04 KCTC14499BP Extract from Sanghwang mushroom
containing medium 1.35*10 ¹¹ cfu/ml 5.34*10 ⁹ cfu/ml general badge 7.47*10 ⁹ cfu/ml 1.25*10 ¹⁰ cfu/ml

As a result, as can be seen in Table 5 above, the growth of each strain was found to be affected by the extract derived from Seonghuang mushroom. In the case of VIOAP03 KCTC14498BP, it was found that the growth of the number of bacteria was significantly increased in the medium containing the extract derived from Shanghai mushroom (origin) compared to the normal medium, whereas it grew up to 7.47*10 ⁹ cfu/ml in the normal medium, and the VIMPP04 KCTC14499BP strain In the case of , there was no significant difference between the general medium and the mixed medium with Sanghwang mushroom extract, but it was confirmed that the normal medium showed a higher number of bacteria than the mixed medium with the Sanghwang mushroom extract. Through this, culture conditions were established and dead cells were prepared by identifying the most suitable medium for each strain.

Example: Method for producing dead cell powder using novel strain derived from Sanghwang mushroom

After flask seed culture of L. Plantarum VIOAP03 KCTC14498BP and L. Fermentum VIMPP04 KCTC14499BP, respectively, novel strains derived from Sanghwang mushroom, each strain was cultured in the culture medium established in Experimental Example 1. That is, L. Plantarum VIOAP03 KCTC14498BP was main cultured in a medium containing the extract derived from Sanghwang mushroom, and L. Fermentum VIMPP04 KCTC14499BP was main cultured in a normal medium.

Specifically, as a first step, flask seed culture of L. Fermentum VIMPP04 KCTC14499BP was performed for 18 hours, pre-cultured in a 50L incubator containing normal medium for 8 hours, and then pre-cultured in a 5,000L incubator containing normal medium. The first main culture was performed for 12 hours by adding water. After the first main culture was completed, it was centrifuged for 3 hours to prepare a concentrate 130L (solid content about 10%).

As a second step, after flask seed culture of L. Plantarum VIOAP03 KCTC14498BP separately from L. Fermentum VIMPP04 KCTC14499BP for 18 hours, the second seed culture was carried out in a 700L incubator containing a medium containing an extract derived from Shanghai mushroom (origin) for 24 hours. The culture was carried out to prepare 350L of culture.

As a third step, 130L of the concentrate prepared in the first step was mixed with 350L of the culture prepared in the second step to prepare a mixed culture solution.

Finally, as a fourth step, the mixed culture solution prepared in the third step is subjected to a tindil process, and after the tindyll process is completed, a rapid freezing process is performed using LNF (Liquid Nitrogen Freezer), followed by freeze-drying to produce dead cell powder did.

The Tyndil process was carried out twice. In the first, it was carried out at 110° C. for 15 minutes, and in the second, it was carried out at 90° C. for 30 minutes.

Comparative Examples 1 and 2: Preparation method of dead cell powder

Table 6 below compares the composition and manufacturing method of the dead cell powder of Comparative Examples 1 and 2 with the composition and manufacturing method of the dead cell powder of Examples.

strain and medium Culture method Example L. Plantarum VIOAP03 + Medium containing extract derived from Sanghwang mushroom Separate culture on each medium and mix L. Fermentum VIMPP04 + Plain Medium Comparative Example 1 Commercial L. Plantarum + Medium containing extract derived from Sanghwang mushroom Separate culture on each medium and mix commercial L. Fermentum + normal badge Comparative Example 2 L. Plantarum VIOAP03 + L. Fermentum VIMPP04 + Normal medium Incorporate culture of all strains in one medium

The production of dead cell powder according to Comparative Example 1 was compared with a commercially available strain, not a strain isolated from Sanghwang mushroom, and was prepared in the same manner as in the manufacturing process of Example. Comparative Example 2 was cultured by adding two types of deposited strains according to the present invention to a general medium, respectively.

Experimental Example 2: Test for confirming the number of dead cells

The number of dead cells of the dead cells powder prepared in Examples and Comparative Examples 1 and 2 The number of dead cells was measured using a hemtocytometer. Dead cells were measured by staining the dead cells with a coomassie blue solution, followed by microscopic observation, and the number of dead cells in the hematocyte was directly counted using a microscope. was measured, and the results are shown in Table 7 below.

number of dead cells cells/g Example 7.14 ¹¹ Comparative Example 1 1.47 ¹⁰ Comparative Example 2 8.74 ⁹

As a result, as can be seen in Table 7, the number of dead cells was found to be large in the order of Example, Comparative Example 1, and Comparative Example 2. In the case of Comparative Example 2 (integrated culture) in which the culture method was different from the example (separation culture mixed), the number of bacteria cultured during culturing was a mixed culture of two types, but the dominant spawn was generated among the two strains, so the increase in the number of bacteria was significantly It did not appear, so the number of dead cells was small. In addition, the Example and the culture method were the same, but Comparative Example 1 using a commercial strain, not the deposited strain of the present invention, also showed a smaller number of dead cells than the Example, so that the growth of the deposited strain according to the present invention was more was found to be excellent. Moreover, by culturing the VIOAP03 strain with low activity of the strain using a medium containing an extract derived from Shanghai mushroom (Origin), the growth of the strain was facilitated and the number of bacteria was increased.

Experimental Example 3: Antioxidative activity confirmation test of dead cell powder

The antioxidant activity of the dead cell powder prepared according to Examples and Comparative Examples 1 and 2 was confirmed. Antioxidant activity was tested by DPPH and ABTS test methods. DPPH (2,2-dipheyl-1-picrylhydrazyl) has free radicals in its oxidized form and is a test method using the principle that when it meets an antioxidant, which is an electron donor, it gains electrons and is reduced. The absorbance was measured using a spectrophotometer to confirm the result.

ABTS+ shows strong absorption in the range of 600-750 nm, which makes it easy to measure by spectroscopic analysis. In the absence of phenolic compounds, ABTS+ is relatively stable, but reacts intensely with hydrogen donors such as phenolic compounds to convert to colorless ABTS. Thus, antioxidant activity measures the amount of ABTS+ consumed by reacting with samples containing phenolic compounds. After mixing the ABTS+ solution and the sample and reacting in the dark for 7 minutes, absorbance was measured using a spectrophotometer to confirm the result. The results are shown in FIGS. 7 (DPPH) and 8 (ABTS).

As a result, as can be seen in FIGS. 7 and 8 , it was found that the antioxidant effect of the dead cell powder of Examples prepared according to the preparation method of the present invention was the best.

Experimental Example 4: Active ingredient confirmation test

The content of β-gulcan, an active ingredient related to the immune activity of the dead cell powder prepared according to Examples and Comparative Examples 1 and 2, was confirmed according to the beta-glucan test method described in the Health Functional Foods Codex, and the results are shown below. Table 8 shows.

note β-gulcan content mg/g Example 2.75 Comparative Example 1 0.54 Comparative Example 2 0.12

As a result, as can be seen in Table 8, it was found that the content of β-gulcan, an active ingredient for immune activity, was significantly increased in the dead cell powder prepared in Examples.

<110> VITECH Co., Ltd. <120> MANUFACTURING METHODS OF HEAT-KILLED STRAIN POWDER ORIENTED FROM PHELLINUS LINTEUS, HEAT-KILLED STRAIN POWDER AND USES THEREOF <130> P21-0115 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1417 <212> DNA <213> Unknown <220> <223> Lactobacillus plantarum VIOAP03 KCTC14498BP <400> 1 tgcagtcgaa cgaactctgg tattgattgg tgcttgcatc atgatttaca tttgagtgag 60 tggcgaactg gtgagtaaca cgtgggaaac ctgcccagaa gcgggggata acacctggaa 120 acagatgcta ataccgcata acaacttgga ccgcatggtc cgagcttgaa agatggcttt 180 ggctatcact tttggatggt cccgcggcgt attagctaga tggtggggta acggctcacc 240 atggcaatga tacgtagccg acctgagagg gtaatcggcc acatgggac tgagacacgg 300 cccaaactcc tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg 360 agcaacgccg cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt taaagaagaa 420 catatctgag agtaactgtt caggtattga cggtatttaa ccagaaagcc acggctaact 480 acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggattt attgggcgta 540 aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc ttcggctcaa ccgaagaagt 600 gcatcggaaa ctgggaaact tgagtgcaga agaggacagt ggaactccat gtgtagcggt 660 gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgtaactg 720 acgctgaggc tcgaaagtat gggtagcaaa caggattaga taccctggta gtccataccg 780 taaacgatga atgctaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt 840 aagcattccg cctggggagt acggccgcaa ggctgaaact caaaggaatt gacgggggcc 900 cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct 960 tgacatacta tgcaaatcta agagattaga cgttcccttc ggggacatgg atacaggtgg 1020 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080 cccttattat cagttgccag cattaagttg ggcactctgg tgagactgcc ggtgacaaac 1140 cggaggaagg tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1200 gctacaatgg atggtacaac gagttgcgaa ctcgcgagag taagctaatc tcttaaagcc 1260 attctcagtt cggattgtag gctgcaactc gcctacatga agtcggaatc gctagtaatc 1320 gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc 1380 atgagagttt gtaacaccca aagtcggtgg ggtaacc 1417 <210> 2 <211> 1429 <212> DNA <213> Unknown <220> <223> L. Fermentum VIMPP04 KCTC14499BP <400> 2 tgcagtcgaa cgcgttggcc cagaattgat tgatggtgct tgcacctgat tagattttgg 60 tcgccaacga gtggcggacg ggtgagtaac acgtaggtaa cctgcccaga agcgggggac 120 aacatttgga aacagatgct aataccgcat aacaacgttg ttcgcatgaa caacgcttaa 180 aagatggctt ctcgctatca cttctggatg gacctgcggt gcattagctt gttggtgggg 240 taacggccta ccaaggcgat gatgcatagc cgagttgaga gactgatcgg ccacaatggg 300 actgagacac ggcccatact cctacgggag gcagcagtag ggaatcttcc acaatgggcg 360 caagcctgat ggagcaacac cgcgtgagtg aagaagggtt tcggctcgta aagctctgtt 420 gttaaagaag aacacgtatg agagtaactg ttcatacgtt gacggtattt aaccagaaag 480 tcacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat 540 ttattgggcg taaagagagt gcaggcggtt ttctaagtct gatgtgaaag ccttcggctt 600 aaccggagaa gtgcatcgga aactggataa cttgagtgca gaagagggta gtggaactcc 660 atgtgtagcg gtggaatgcg tagatatatg gaagaacacc agtggcgaag gcggctacct 720 ggtctgcaac tgacgctgag actcgaaagc atgggtagcg aacaggatta gataccctgg 780 tagtccatgc cgtaaacgat gagtgctagg tgttggaggg tttccgccct tcagtgccgg 840 agctaacgca ttaagcactc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac 960 cttaccaggt cttgacatct tgcgccaacc ctagagatag ggcgtttcct tcgggaacgc 1020 aatgacaggt ggtgcatggt cgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgtt actagttgcc agcattaagt tgggcactct agtgagactg 1140 ccggtgacaa accggaggaa ggtggggacg acgtcagatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacggtaca acgagtcgcg aactcgcgag ggcaagcaaa 1260 tctcttaaaa ccgttctcag ttcggactgc aggctgcaac tcgcctgcac gaagtcggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccatgagagt ttgtaacacc caaagtcggt ggggtaacc 1429

Claims (11)

A first step of culturing L. Fermentum VIMPP04 KCTC14499BP in a first medium and then concentrating to obtain a concentrate;
a second step of culturing L. Plantarum VIOAP03 KCTC14498BP in a second medium to obtain a culture;
a third step of mixing the concentrate obtained in the first step with the culture obtained in the second step to obtain a mixed culture solution; and
A method for producing dead cell powder comprising a; a fourth step of drying the mixed culture solution obtained in the third step after the Tyndil process to obtain a dead cell powder.
According to claim 1,
The second medium is a method for producing a dead cell powder, characterized in that it comprises an extract derived from Sanghwang mushroom.
According to claim 1,
The first medium and the second medium are glucose (Glucose), yeast extract (Yeast Extract), soy peptone (Soy peptone), sodium acetate (Sodium Acetate), dipotassium phosphate (Dipotassium phosphate), sodium citrate (Sodium Citrate) , Tween 80, a method for producing a dead cell powder, characterized in that it comprises one or more components selected from the group consisting of magnesium sulfate and manganese sulfate.
According to claim 1,
Culturing in the first step,
Step 1-1 to obtain a pre-culture by performing pre-culture for 6 to 10 hours in a first incubator containing a first medium;
A second step of adding the pre-culture obtained in step 1-1 to a second incubator containing the first medium and performing the first main culture for 10 to 13 hours; characterized in that it comprises; A method for producing a dead cell powder.
According to claim 1,
The culture in the second step is a method for producing dead cell powder, characterized in that the second main culture proceeds for 22 hours to 26 hours.
According to claim 1,
The method for producing dead cell powder, characterized in that the mixing ratio of the concentrate and the culture in the third step is 1:2 to 1:5.
According to claim 1,
The drying is
a 4-1 step of rapidly freezing the tindylated mixed culture solution at -180°C to -195°C using a liquid nitrogen freezer (LNF); and
Method for producing dead cell powder, characterized in that it is carried out through; lyophilizing the quick-frozen Tyndylation mixed culture solution in step 4-1 at -35°C to -45°C.
A dead cell powder prepared through the method of claim 1.
A food composition for preventing or improving hypersensitivity immune disease comprising the dead cell powder of claim 8 as an active ingredient.
10. The method of claim 9,
The hypersensitive immune diseases include allergic urticaria, allergic rhinitis, allergic conjunctivitis, allergic asthma, allergic dermatitis, autoimmune hepatitis, allergic bronchopulmonary aspergillosis and allergic stomatitis. ) Food composition for the prevention or improvement of hypersensitive immune disease, characterized in that at least one selected from the group consisting of.
A health functional food composition for the prevention or improvement of hypersensitive immune diseases comprising the dead cell powder of claim 8 as an active ingredient.

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