KR102612095B1 - Anti-microbial composition comprising bioconversion product of Nostoc commune extract or extract thereof - Google Patents

Abstract

The present invention relates to an antibacterial composition comprising a biotransformation product of a Bacillus extract or an extract thereof. Specifically, the Bacillus sp. It relates to an antibacterial composition containing a bioconversion product bioconverted with JD3-7 or an extract of the bioconversion product. In addition, the present invention relates to a method for bioconversion of a bead mal extract and a fermentation strain used herein, Bacillus sp. It concerns strain JD3-7.
The antibacterial composition of the present invention utilizes bead, a biological resource that has been rarely utilized before, and can exhibit excellent antibacterial activity against Propionibacterium acnes, Escherichia coli, and Salmonella enterica. In particular, it can exhibit high antibacterial activity against Propionibacterium acnes, the causative agent of acne, and can be safely used on the skin, so it will be useful as a natural antibacterial material for functional cosmetics, especially for the prevention, treatment or improvement of acne.

Description

Antimicrobial composition comprising bioconversion product of Nostoc commune extract or extract thereof}

The present invention relates to an antibacterial composition comprising a biotransformation product of a Bacillus extract or an extract thereof. Specifically, the Bacillus sp. It relates to an antibacterial composition containing a bioconversion product bioconverted with JD3-7 or an extract of the bioconversion product. In addition, the present invention relates to a method for bioconversion of a bead mal extract and a fermentation strain used herein, Bacillus sp. It concerns strain JD3-7.

Antibacterial agents are drugs that inhibit or kill the growth of bacteria and are used in various fields. In particular, antibacterial agents are used to prevent the deterioration of foods, cosmetics and medicines and to maintain their purity during use or preservation. In this case, it is essential that they are not harmful to the human body and their addition must not damage their quality. .

Antibacterial agents can be divided into natural antibacterials using natural substances and synthetic antibacterials using synthetic compounds. Natural antibacterials have problems in various aspects such as color, odor, stability, antibacterial spectrum, ease of formulation, and economic feasibility, which are limitations of natural substances. Therefore, most of them are not commercialized. Synthetic antibacterial agents have a high affinity for lipids, so when ingested as food, they accumulate in the body's fatty tissue, which has the potential to cause long-term toxicity and have a negative impact on the environment. There are also problems due to changes in consumers’ perception of their preferences. Accordingly, there is a need to develop new natural antibacterial materials that can exhibit excellent antibacterial activity and can be easily manufactured.

Meanwhile, Nostoc commune is a cyanobacteria belonging to the genus Nostoc of the Nostocaceae family. Its bead-shaped cells are connected in a row, tangled, and wrapped in mucus to form a lump. It is easily found in soil, moist rocks, and the bottom of lakes and fountains, and its quantity is also a significant biological resource, but there is still almost no technology to utilize it.

The present inventor studied ways to utilize bead mal, a biological resource that is rarely utilized, particularly focusing on its potential as a natural antibacterial agent.

Shiels, K., N. Browne, F. Donovan, P. Murray and S.K. Saha. 2019. Molecular Characterization of Twenty-Five Marine Cyanobacteria Isolated from Coastal Regions of Ireland. Biology 8:59. Katoh H., J. Furukawa, K. Tomita-Yokotani, and Y. Nishi. 2012. Isolation and purification of an axenic diazotrophic drought-tolerant cyanobacterium, Nostoc commune, from natural cyanobacterial crusts and its utilization for field research on soils polluted with radioisotopes. Biochimica et Biophysica Acta 1817: 1499-1505. Hae-Ri Choi, Jin-Soo Park, Kyu-Min Kim, MinSun Kim, Kwang-Wook Ko, Chang-Gu Hyun, Joon Woo Ahn, Joo-Hyun Seo, and Seung-Young Kim. 2018. Enhancing the antimicrobial effect of genistein by biotransformation in microbial system. Journal of Industrial and Engineering Chemistry. 63: 255-261.

Therefore, the main purpose of the present invention is to provide a new antibacterial composition that can exhibit excellent antibacterial activity as a new natural antibacterial material using beads.

Another object of the present invention is to provide a method for producing the antibacterial composition.

Another object of the present invention is to provide a new strain necessary for producing the antibacterial composition.

According to one aspect of the present invention, the present invention provides an extract of Bacillus sp. An antibacterial composition comprising a bioconversion product bioconverted with JD3-7 or an extract of the bioconversion product is provided.

In the antibacterial composition of the present invention, it is preferable that the extract of the bead is extracted from the bead with water, ethanol, or a mixed solvent thereof.

In the antibacterial composition of the present invention, the bioconversion product contains 10 to 500 mg/l of the bead horse extract and the Bacillus sp. It is preferably obtained by incubating a composition containing cells of JD3-7, containing a pH buffer, and having a pH of 6.5 to 8 at 20 to 40° C. for 1 hour to 10 days.

In the antibacterial composition of the present invention, the extract of the bioconversion product is preferably an ethyl acetate extract of the bioconversion product.

According to another aspect of the present invention, the present invention provides a cosmetic composition containing the antibacterial composition.

According to another aspect of the present invention, the present invention provides an extract of Bacillus sp. Mixing the cells of JD3-7 to produce a composition for bioconversion reaction; and incubating the composition for bioconversion reaction at a temperature.

In the bioconversion method of the agar extract of the present invention, it is preferable that the amulet extract is extracted from the amulet with water, ethanol, or a mixed solvent thereof.

In the bioconversion method of the bead extract of the present invention, the step of producing the composition for the bioconversion reaction is performed using a biological agent containing 10 to 500 mg/l of the bead extract, a pH buffer, and a pH of 6.5 to 8. It is preferable that this step is to produce a composition for conversion reaction.

In the method for bioconversion of the snail extract of the present invention, the incubation step is preferably a step of incubating the composition for bioconversion reaction at 20 to 40° C. for 1 hour to 10 days.

According to another aspect of the present invention, the present invention relates to Bacillus sp. deposited under accession number KACC 92346P. Strain JD3-7 is provided.

The antibacterial composition of the present invention utilizes bead, a biological resource that has been rarely utilized before, and can exhibit excellent antibacterial activity against Propionibacterium acnes, Escherichia coli, and Salmonella enterica. In particular, it can exhibit high antibacterial activity against Propionibacterium acnes, the causative agent of acne, and can be safely used on the skin, so it will be useful as a natural antibacterial material for functional cosmetics, especially for the prevention, treatment or improvement of acne.

Figure 1 shows a photograph and a micrograph of Nostoc commune according to an embodiment of the present invention.
Figure 2 shows Bacillus sp. Shows the results of HPLC analysis of the bioconversion material of genistein by JD3-7.
Figure 3 shows Bacillus sp. It shows the neighbor-joining phylogenetic tree created based on the 16S rRNA gene base sequence of JD3-7.
Figure 4 shows the antibacterial activity of the bioconversion product according to an example of the present invention against Propionibacterium acnes CCARM 0081.
Figure 5 shows the antibacterial activity of the bioconversion product according to an example of the present invention against Propionibacterium acnes CCARM 9089.
Figure 6 shows the antibacterial activity of the bioconversion product according to an example of the present invention against E. coli ATCC 25922.
Figure 7 shows the antibacterial activity against Salmonella enterica ATCC 14028 according to an example of the present invention.
Figure 8 shows Bacillus sp. This is the deposit certificate of JD3-7.

The antibacterial composition of the present invention is a Bacillus sp. It is characterized in that it contains a bioconversion product bioconverted with JD3-7 or an extract of the bioconversion product.

In the present invention, Nostoc commune , which is the extraction raw material for the above-mentioned extract, is a cyanobacteria belonging to the genus Nostoc of the Nostocaceae family, and is preferably collected from the graveyard of the Daejeon National Cemetery, and is olive green to green. It is dark green and has the 16S rRNA gene sequence of SEQ ID NO: 1.

In the present invention, the bead horse extract is extracted using the bead horse as an extraction raw material, and can be prepared by conventional extraction methods in the art, such as boiling water extraction, ultrasonic extraction, and reflux extraction. Preferably, the extraction is performed using a solvent selected from the group consisting of C1 to C4 lower alcohols, water, and mixtures thereof, more preferably, the extraction is performed using water, ethanol, or a mixed solvent thereof, and even more preferably, water or water. It is extracted with a mixed solvent of water and ethanol, and more preferably, is extracted with a mixed solvent of water and ethanol. The mixed solvent of water and ethanol is preferably a 10 to 99% (v/v) ethanol solution, more preferably a 30 to 90% (v/v) ethanol solution, and more preferably a 40 to 90% (v/v) ethanol solution. % (v/v) ethanol solution, more preferably 50 to 90% (v/v) ethanol solution, more preferably 60 to 80% (v/v) ethanol solution, and even more preferably is a 65 to 75% (v/v) ethanol solution.

In the present invention, when preparing the extract, the collected beads are preferably dried and used, and more preferably, the beads are dried and then powdered before use. In addition, the amount of solvent when preparing the extract is preferably 20 ml to 10 liters, more preferably 100 ml to 5 liters, and more preferably 500 ml to 2 liters based on 10 g of the extraction raw material, that is, the weight (e.g. dry weight) of the beads. Do this. The extraction temperature is preferably 0 to 100°C, more preferably 5 to 90°C, and even more preferably 10 to 80°C. In particular, when using a mixed solvent of water and ethanol as a solvent, the extraction temperature is preferably 5 to 35°C, more preferably 10 to 30°C, and even more preferably 15 to 25°C. Additionally, when using water as a solvent, the extraction temperature is preferably 40 to 80°C, more preferably 50 to 70°C, and even more preferably 55 to 65°C. The extraction time is preferably 1 hour to 10 days, more preferably 12 hours to 5 days, and more preferably 1 day to 2 days.

The extract of the present invention may have undergone additional processes such as filtration, concentration, reduced pressure drying, and freeze-drying after the above extraction process.

Bacillus sp, the strain used for bioconversion of the present invention. JD3-7 was isolated from traditional soybean paste from Jeju Island and has been deposited in the Korean Agricultural Culture Collection (KACC) of the National Academy of Agricultural Sciences under the accession number KACC 92346P. The strain of the present invention is classified into strains such as Bacillus amyloliquefacience , B. siamensis , and B. velezensis , and is particularly similar to Bacillus siamensis. Although classified, there are clear differences in biochemical properties (Table 2). The strain of the present invention bioconverts the extract of the snail extract to exhibit antibacterial activity that was not present (for example, in the extract of the snail extract before bioconversion) (for example, which was not confirmed through the antibacterial activity test described in the Examples), or It can increase the antibacterial activity that was previously relatively low, and in this respect, it is differentiated from other similar existing strains.

Bacillus sp of the present invention. JD3-7 can be cultured according to normal culture conditions for cultivating microorganisms of the genus Bacillus. For example, it can be cultured in LB (Luria Bertani) medium or NB (nutrient broth) medium at a temperature of 25 to 35°C.

In the present invention, the bioconversion product is Bacillus sp. It has been bioconverted with JD3-7 and can be obtained using a common bioconversion method used to bioconvert the target material using microorganisms. For example, bead extract and Bacillus sp. It can be obtained by preparing a composition mixed with JD3-7, that is, a composition for bioconversion reaction to achieve bioconversion, and incubating this composition. At this time, the bead mal extract is added to the composition for bioconversion reaction preferably at 10 to 500 mg/l, more preferably at 20 to 400 mg/l, more preferably at 30 to 300 mg/l, more preferably at 50 to 50 mg/l. It is included at 200㎎/ℓ. Preferably, a pH buffer is included to adjust the initial pH of the composition for bioconversion reaction and control pH changes in the composition during subsequent reactions. Preferably, a pH buffer is included so that the initial pH of the composition for bioconversion reaction is 6.5 to 8, more preferably pH 7 to 7.5, and a pH buffer is included to maintain the pH during the bioconversion reaction. The incubation of the composition for bioconversion reaction to obtain a bioconversion product is preferably for 1 hour to 10 days, more preferably for 12 hours to 7 days, more preferably for 1 to 5 days, more preferably is carried out with an incubation time of 2 to 4 days, preferably at a constant temperature of 20 to 40 ℃, more preferably 25 to 35 ℃, more preferably 27 to 33 ℃, more preferably 29 to 31 ℃ Performed at processing temperature.

In the present invention, the extract of the bioconversion product can be obtained using a conventional extraction method that can extract a specific substance or a combination of specific substances that can exhibit antibacterial properties from the bioconversion product. For example, a method of preparing an extract from the bioconversion product using a common extraction solvent and confirming the antibacterial activity, for example, a method of measuring the antibacterial activity disclosed in the examples of the present specification to confirm the antibacterial activity of the extract. It can be obtained by this method. Preferably, extraction is performed using ethyl acetate as a solvent.

The antibacterial composition of the present invention may be composed solely of the bioconversion products or extracts of the bioconversion products as described above, and may further contain other ingredients. The antibacterial composition of the present invention contains the bioconversion product or extract of the bioconversion product in an amount of preferably 0.1% by weight or more, more preferably 1% by weight or more, more preferably 10% by weight or more, and more preferably 20% by weight. % or more, more preferably 50% by weight or more.

The antibacterial composition of the present invention exhibits excellent antibacterial activity against Propionibacterium acnes , a bacteria causing acne, Escherichia coli , a strain related to food hygiene, and Salmonella enterica , a bacteria causing food poisoning. You can. Therefore, it can be used as an antibacterial material in fields that require antibacterial activity against microorganisms as described above, especially in the food and cosmetic fields. In particular, the antibacterial composition of the present invention has been shown to be safe when applied to the human body, such as the skin, and will therefore be useful as an antibacterial material for cosmetics.

Accordingly, the cosmetic composition of the present invention is characterized by comprising the antibacterial composition of the present invention. The cosmetic composition of the present invention may be composed only of the antibacterial composition of the present invention, and may further contain cosmetically acceptable ingredients. When containing cosmetically acceptable ingredients, the cosmetic composition of the present invention may contain, for example, 0.01 to 50% by weight of the antibacterial composition of the present invention. The cosmetic composition of the present invention may be a functional cosmetic composition, and is preferably a cosmetic composition for preventing, treating or improving acne.

The present invention also provides a method for bioconversion of a snail extract that can impart antibacterial activity to the snail extract or increase its antibacterial activity.

The bioconversion method of the present invention includes the extract of Beadmal and Bacillus sp. Mixing the cells of JD3-7 to produce a composition for bioconversion reaction; and incubating the composition for bioconversion reaction at a temperature. At this time, the bead horse extract, Bacillus sp. For specific details on JD3-7, composition for bioconversion reaction, constant temperature treatment, etc., please refer to the specific details on the above antibacterial composition.

Matters suggested as preferred for each component of the composition and method of the present invention are the expected effects of the present invention, such as antibacterial activity, for example, antibacterial activity against Propionibacterium acnes, Escherichia coli and Salmonella enterica, use This is a desirable matter for expected effects such as safety, for example, safety in human application, and is not necessarily presented to limit the present invention.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.

[Example]

1. Method

1-1. Collecting and identifying beads

Bead mals were collected from some cemeteries at the Daejeon National Cemetery. This trichome is olive green to dark green and grows near the lawn. Under an optical microscope, it forms a colony of numerous trichomes made of round cells connected to each other surrounded by mucus. Among the filaments, heterocysts larger than normal cells were observed.

For molecular biological confirmation, 16S rRNA gene sequence analysis was performed. A portion of the sample was taken, washed with sterilized water, and then crushed with a homogenizer. It was mixed with 100㎕ of 10% Chelex 100 solution and heated at 99°C for 30 minutes. After cooling on ice, centrifugation was performed, and the supernatant was used as a DNA template (Walsh et al. 1991). The 16S rRNA gene region was amplified using cyanobacteria-specific primers CYA106F (CGGACGGGTGAGTAACGCGTGA, Nubel et al. 1997) and FDSKS_CyaR1 (CACCTTCCGGTACGGCTACCTTG, Shiels et al. 2019). The amplified DNA fragment of approximately 1.3 kb was requested from Genotech Co., Ltd. and the final 1,288 bp base sequence information was obtained. As a result of a homology search in the NCBI database based on the finally secured 1,288bp sequence information, it was 99.6% identical to Nostoc commune HK-02 (Katoh et al. 2012). Combining these results, the blue-green algae obtained from the Daejeon National Cemetery cemetery was identified as Nostoc commune .

16S rRNA gene base sequence of Beommalmae (1,288bp) (SEQ ID NO: 1):

5'-CCTGGAACGGTGGCTAATACCGGATGTGCCGAAAGGTGAAAGATTAATTGCCTGGAGATGAGCTCGCGTCTGATTAGCTAGTAGGTGTGGTAAAGGCGCACCTAGGCGACGATCAGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGAGCAATACCGCGTGAGGGAGG AAGGCTCTTGGGTCGTAAACCTCTTTTCTCAGGGAAGAAAAAAATGACGGTACCTGAGGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCAAGCGTTATCCGGAATGATTGGGCGTAAAGCGTCCGCAGGTGGCTGTGTAAGTCTGCTGTTAAAGAGTCTAGCTCAACTAGTAAGAGCAGTGGAAAACTACACGGCTAGAGTACGTTCGGGGCAGAGGGAATTCCTGGTGTAGCGG TGAAATGCGTAGAGATCAGGAAGAACACCGGTGGCGAAGGCGCTCTGCTAGGCCGTAACTGACACTGAGGGACGAAAGCTAGGGGAGCGAATGGGATTANATACCCCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGTGGCTTGTATCGACCCGAGCCGTGCCGTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGT ATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGTCGCGAATCTTCTCGAAAGGGAAGAGTGCCTTCGGGAGCGCGAACACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTTTTAGTTGCCAGCATTAAGTTGGGCACTCTAGAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCAGCAT GCCCCTTACGCCTTGGGCTACACACGTACTACAATGCTCCGGACAGAGGGCAGCAAGCGGGCGACCGCAAGCAAATCCCGGAAACCGGAGCTCAGTTCAGATCGCAGGCTGCAACTCGCCTGCGTGAAGGAGGAATCGCTAGTAATTGCAGGTCAGCATACTGCAGTGAATTCGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGCTGGTAGTGCCCGAAGTCATTACTCCAACTGTCAAAAGAGG AGGATGCCTAAGGCAGGACTG-3'

1-2. Manufacture of bead horse extract

1-2-1. Manufacture of bead mal alcohol extract

10 g of the dried powder of 1-1 was added to the extraction tank, 1 liter of 70% (v/v) ethanol was added, and extraction was performed at room temperature for 24 hours to recover the primary extract. Afterwards, 1 liter of 70% (v/v) ethanol was added to the extraction tank and extracted at room temperature for 24 hours to recover the secondary extract. The recovered primary and secondary extracts were mixed and filtered through a filter with a pore size of 5.0㎛ (TOYO, Japan) to remove residual solids. The filtered extract was concentrated under vacuum and used for the next bioconversion. The extract prepared in this way is indicated as 'NE'.

1-2-2. Manufacture of Geummal hydrothermal extract

10 g of the dried powder of 1-1 was added to the extraction tank, 1 liter of primary distilled water was added, and extraction was performed at 60°C for 48 hours to recover the primary extract. Afterwards, an additional 1 liter of primary distilled water was added to the extraction tank and extraction was performed at 60°C for 48 hours to recover the secondary extract. The recovered primary and secondary extracts were mixed and filtered through a filter with a pore size of 5.0㎛ (TOYO, Japan) to remove residual solids. The filtered extract was concentrated under vacuum and used for the next bioconversion. The extract prepared in this way is denoted as ‘NW’.

1-3. bioconversion

1-3-1. Fermenter preparation

Jeju Island soybean traditional soy sauce was diluted from 10 ^-1 to 10 ^-5 using a serial dilution method, and then 100 ㎕ of each dilution was spread on LB (Luria Bertani) solid medium and cultured at 30°C for 48 hours. Eleven strains were pure cultured, and the 16S rRNA gene sequence of each colony was analyzed using bacterial universal primers 27F, 518F, 800R, and 1492R, and all were classified as strains of the genus Bacillus .

Using 11 isolated strains, bioconversion was performed based on the bioconversion method using genistein as a substrate described by Choi et al. (2018), and then each supernatant was concentrated and analyzed by HPLC. did. As a result of HPLC analysis, strain JD3-7 with the best bioconversion ability was selected (Figure 2).

The final selection of Bacillus sp. The JD3-7 strain is Bacillus amyloliquefacience and Bacillus siamensis ( B siamensis ) and B. velezensis (Table 1 and Figure 3).

Strain name Similarity (%) Bacillus siamensis KCTC 13613 (AJVF01000043) 99.93 Bacillus velesensis CR-502 (AY603658) 99.93 Bacillus subtilis NCIB 3610 (ABQL01000001) 99.79 Bacillus amyloliquefaciens DSM 7 (FN597644) 99.72 Bacillus nematocida B-16 (AY820954) 99.72 Bacillus nakamurai NRRL B-41091 (LSAZ01000028) 99.65

The biochemical characteristics of the JD3-7 strain were also investigated using the 20NE API kit, and the results were compared with those of Bacillus siamensis and are shown in Table 2 below.

enzyme JD3-7 B. siamensis PD-A10 Alkaline phosphatase + + Esterase (C4) + + Esterase Lipase (C8) + + Lipase (C14) - - Leucine arylamidase - - Valine arylamidase - - Cystine arylamidase - - Trypsin + - α-chymotrypsin - - Acid phosphatase + - Naphtol-AS-BI-phosphohydrolase + + α-galactosidase - - β-galactosidase - - β-glucuronidase - - α-glucosidase +- - β-glucosidase +- - N-asetyl-β-glucosaminidase - - α-mannosidase - - α-fucosidase - - Nitrate reduction + + Urease + + Gelatinase + +

Strain JD3-7 was cultured for 20 hours in 1 liter of nutrient medium (beef extract 3.0 g/l, peptone 5.0 g/l), and then centrifuged at 5,000 rpm for 15 minutes to obtain bacterial cells. This fungus was used for bioconversion of the bead mal extract.

1-3-2. Bioconversion of bead mal extract

Bacillus sp. obtained in 1-3-2 above. JD3-7 cells were washed twice with PG buffer (50mM phosphate, pH 7.2, 2% glyerol) and then suspended in 50 ml of PG buffer to prepare a cell suspension. Mix 10 mg of the malt alcohol extract (NE) obtained in 1-2-1 or the hot water extract (NW) obtained in 1-2-2 and 100 ml of the bacterial cell suspension and keep at 30°C for 72 hours. Processed. After incubation was completed, the supernatant from which the bacteria were removed was centrifuged, and 50 ml of the supernatant was extracted twice with 100 ml of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure and dissolved in DMSO to a concentration of 100 mg/ml to prepare samples for subsequent antibacterial activity tests.

The bioconversion sample in NE was named NE-BC, and the bioconversion sample in NW was named NW-BC. Additionally, in order to be used as a negative control, a sample that underwent the above bioconversion process without adding the extract was prepared and was named PG-BC.

1-4. Antibacterial activity evaluation

1-4-1. Strains and media used

Propionibacterium acnes CCARM 0081, Propionibacterium acnes CCARM 9089, Escherichia coli ATCC 25922, and Salmonella enterica ATCC 14028 were used as indicator bacteria in the antibacterial activity test.

CCARM 0081 and 9089 were cultured under anaerobic conditions at 37°C for 18 hours using LB medium (tryptone 10.0g, yeast extract 5.0g, sodium chloride 10.0g), and ATCC 25922 and ATCC 14028 were cultured at 37°C using LB medium. Cultured under aerobic conditions for 18 hours.

1-4-1. Antibacterial activity measurement

Minimum inhibitory concentration (MIC), which refers to the minimum concentration that inhibits bacterial growth, was measured. Each indicator bacteria was cultured and used under optimal growth conditions, and the absorbance was measured at 600 nm, and bacteria with an OD value of 0.08 to 0.13 were adjusted to 1x10 ⁴ cfu/ml before use. The antibacterial activity test was conducted using a 96-well continuous dilution method, and the concentration range of the sample was set from 10 mg/ml to the lowest concentration of 0.04 mg/ml and diluted two-fold. As a negative control group, PG-BC, a sample that had undergone a bioconversion process without the addition of bead horse extract, was used, and ampicillin was used as a positive control group. In addition, the bioconverted samples NE-BC and NW-BC obtained in 1-3-2, as well as the extract samples (non-bioconverted) NE and NW obtained in 1-2-1 and 1-2-2. Antibacterial activity was also measured. After culturing for 18 hours, the absorbance was measured at 600 nm to determine the minimum inhibitory concentration.

1-5. Skin safety evaluation

The skin stability of the NE-BC sample of 1-3-2, that is, a bioconversion sample of bead mal alcohol extract, was evaluated. The evaluation test was commissioned to the Cosmetics Clinical Research Support Center of Semyung University.

1-5-1. Selection of test subjects

Adults who met the selection criteria and did not meet the exclusion criteria were selected. In order to evaluate the effectiveness of the test product, documented information was provided to the test subject, the test director explained it in detail orally, and the test subject was allowed to participate in the test if he or she freely agreed in writing to become a test subject.

30 test subjects were selected and tested as those who fit the selection criteria and were not subject to the exclusion criteria (Basis: Article 5, 1 of the Ministry of Food and Drug Safety Notice No. 2019-47, ‘Regulations on the Evaluation of Functional Cosmetics’ (See Paragraph B, Section 2 A, ‘Toxicity Test Method, Section 7 Human Use Test’ in ‘Appendix 1’).

1-5-2. Test Methods

The test site was the back. A skin patch test was conducted on test subjects using IQ Ultra. 6㎕ of the test product diluted to 10% with distilled water was added dropwise to IQ Ultra. The patch was attached for 24 hours, and 30 minutes after patch removal, 24 hours after patch removal, and 48 hours after patch removal, the degree of irritation was assessed by two experts according to the criteria of the International Contact Dermatitis Research Group (ICDRG). was observed.

2. Results

2-1. Antibacterial activity against Propionibacterium acnes CCARM 0081

As a result of MIC measurement for CCARM 0081, PG-BC and NW did not show an antibacterial effect, and NE showed an effect at a high concentration of 10 mg/ml, while NW-BC showed an effect at 0.625 mg/ml and NE-BC had an antibacterial effect. The effect was shown at a concentration of 0.156 mg/ml. This means that during the bioconversion process, components in natural products are converted by microbial metabolism, resulting in new or improved antibacterial activity. In particular, NE-BC showed the highest antibacterial activity among the samples (Figure 4).

2-2. Antibacterial activity against Propionibacterium acnes CCARM 9089

As a result of MIC measurement for CCARM 9089, PG-BC, NW and NE did not show antibacterial activity. On the other hand, NW-BC showed antibacterial activity at a concentration of 0.625 mg/ml and NE-BC at a concentration of 0.313 mg/ml. This means that during the bioconversion process, components in natural products are converted by microbial metabolism, resulting in new or improved antibacterial activity. Also, as in CCARM 0081, NE-BC showed greater antibacterial activity than NW-BC (Figure 5).

2-3. Antibacterial activity against E. coli ATCC 25922

As a result of MIC measurement for ATCC 25922, PG-BC, NW and NE did not show antibacterial activity. On the other hand, NW-BC and NE-BC showed effectiveness at concentrations of 2.5 mg/ml and 1.25 mg/ml, respectively. This means that during the bioconversion process, components in natural products are converted by microbial metabolism, resulting in new or improved antibacterial activity. Additionally, NE-BC showed greater antibacterial activity than NW-BC (Figure 6).

2-4. Antibacterial activity against Salmonella enterica ATCC 14028

As a result of MIC measurement for ATCC 14028, PG-BC, NW, and NE did not show antibacterial activity. On the other hand, NW-BC and NE-BC showed effectiveness at concentrations of 5 mg/ml and 2.5 mg/ml, respectively. This means that during the bioconversion process, components in natural products are converted by microbial metabolism, resulting in new or improved antibacterial activity. Additionally, NE-BC showed greater antibacterial activity than NW-BC (Figure 7).

2-5. skin safety

The test subjects who participated in the human application test were 30 healthy adult women with an average age of 42.4 ± 4.6 years, and 30 participating test subjects completed the final test without dropping out. The test subject's subjective skin condition (Table 3), test subject's lifestyle (smoking and drinking) (Table 4), and skin sensitivity (Table 5) are shown in Tables 3 to 5 below.

After evaluation according to the evaluation criteria, the average irritation index was calculated according to the formula, and the intensity of skin irritation was evaluated according to the judgment criteria. Whether or not the final skin irritation is caused is determined based on three criteria: safety zone depending on the product, products that cause irritation at a frequency exceeding 20% of test subjects, and test products in which irritation reactions of +2 or more are observed in more than 10% of total test subjects at each reading. The final prediction of product suitability was determined and shown in Table 6.

As a result of the human application test, no irritation was observed at 30 minutes, 24 hours, and 48 hours after patch removal (30 minutes after patch removal, ± was 0 people, + was 0 people, ++ was 0 people, +++ was 0 people) 24 hours after removal of the patch, 0 patients with ±, 0 patients with +, 0 patients with ++, 0 patients with +++ were observed, and 48 hours after removal of the patch, 0 patients with ±, 0 patients with +, ++ was observed in 0 people, +++ was observed in 0 people). The average skin reactivity was 0.00, which was judged to be non-irritating according to the skin patch test criteria.

In addition, when the closed patch test used in the primary skin irritation test was conducted, it was found to be suitable for the three final judgment criteria, so it could be classified as a safe product with a low possibility of causing irritation.

Agricultural Biotechnology Research Institute KACC92346P 20210401

<110> National Institute of Biological Resources <120> Anti-microbial composition comprising bioconversion product of Nostoc commune extract or extract thereof <130> ALP21010 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1288 <212> DNA <213> Nostoc commune <400> 1 cctggaacgg tggctaatac cggatgtgcc gaaaggtgaa agattaattg cctggagatg 60 agctcgcgtc tgattagcta gtaggtgtgg taaaggcgca cctaggcgac gatcagtagc 120 tggtctgaga ggacgatcag ccacactggg actgagacac ggcccagact cctacgggag 180 gcagcagtgg ggaattttcc gcaatgggcg aaagcctgac ggagcaatac cgcgtgaggg 240 aggaaggctc ttgggtcgta aacctctttt ctcagggaag aaaaaaatga cggtacctga 300 ggaataagca tcggctaact ccgtgccagc agccgcggta atacggagga tgcaagcgtt 360 atccggaatg attgggcgta aagcgtccgc aggtggctgt gtaagtctgc tgttaaagag 420 tctagctcaa ctagtaagag cagtggaaac tacacggcta gagtacgttc ggggcagagg 480 gaattcctgg tgtagcggtg aaatgcgtag agatcaggaa gaacaccggt ggcgaaggcg 540 ctctgctagg ccgtaactga cactgaggga cgaaagctag gggagcgaat gggattanat 600 accccagtag tcctagccgt aaacgatgga tactaggcgt ggcttgtatc gacccgagcc 660 gtgccgtagc taacgcgtta agtatcccgc ctggggagta cgccggcaac ggtgaaactc 720 aaaggaattg acgggggccc gcacaagcgg tggagtatgt ggtttaattc gatgcaacgc 780 gaagaacctt accaaggctt gacatgtcgc gaatcttctc gaaagggaag agtgccttcg 840 ggagcgcgaa cacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 900 agtcccgcaa cgagcgcaac cctcgttttt agttgccagc attaagttgg gcactctaga 960 gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaagtcagc atgcccctta 1020 cgccttgggc tacacacgta ctacaatgct ccggacagag ggcagcaagc gggcgaccgc 1080 aagcaaatcc cggaaaccgg agctcagttc agatcgcagg ctgcaactcg cctgcgtgaa 1140 ggaggaatcg ctagtaattg caggtcagca tactgcagtg aattcgttcc cgggccttgt 1200 acacaccgcc cgtcacacca tggaagctgg tagtgcccga agtcattact ccaactgtca 1260 aaagaggagg atgcctaagg caggactg 1288

Claims (10)

The bead mal extract was mixed with Bacillus sp. An antibacterial composition comprising a bioconversion product bioconverted to JD3-7 or an extract of the bioconversion product,
The above bead mal extract is extracted from bead mal with a 30 to 90% (v/v) ethanol aqueous solution,
Antibacterial composition. delete According to paragraph 1,
The bioconversion product contains 10 to 500 mg/l of the bead mal extract and the Bacillus sp. An antibacterial composition obtained by incubating a composition containing cells of JD3-7, a pH buffer, and having a pH of 6.5 to 8 at 20 to 40° C. for 1 hour to 10 days. According to paragraph 1,
An antibacterial composition wherein the extract of the bioconversion product is an ethyl acetate extract of the bioconversion product. A cosmetic composition comprising the antibacterial composition of any one of claims 1, 3, and 4. Bead mal extract and Bacillus sp. Mixing the cells of JD3-7 to produce a composition for bioconversion reaction; and
A bioconversion method of a bead extract comprising the step of incubating the composition for bioconversion reaction,
The above bead mal extract is extracted from bead mal with a 30 to 90% (v/v) ethanol aqueous solution,
Bioconversion method of bead horse extract. delete According to clause 6,
The step of producing a composition for a bioconversion reaction is a step of producing a composition for a bioconversion reaction containing 10 to 500 mg/l of the extract of the bead extract, containing a pH buffer, and having a pH of 6.5 to 8. Bioconversion method. According to clause 6,
The incubation step is a step of incubating the composition for bioconversion reaction at 20 to 40° C. for 1 hour to 10 days. Bacillus sp. deposited with accession number KACC 92346P. Strain JD3-7.