JP7320826B2 - Bacteriostatic agent, sericin-containing aqueous composition, and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a bacteriostatic agent, a sericin-containing aqueous composition, and a method for producing the same.

Cocoons produced by silkworms are mainly composed of two types of proteins, fibroin inside and sericin covering the outside (Non-Patent Documents 1 to 3).

The inner fibroin fiber is a protein that makes up about 75% of the cocoon and is made in the posterior silk gland of the silkworm. On the other hand, sericin, which is an outer glue-like protein, is produced in the middle silk gland (Non-Patent Documents 2, 4, 5). Sericin is composed of several kinds of proteins including sericins 1 to 4 as main components.

For a long time, sericin has been decomposed into low-molecular-weight substances, removed, and discarded during the process of refining raw silk. However, in recent years, it has become clear that sericin has biocompatibility, antioxidant action, UV protection action, anticancer action, etc., and has been attracting attention in the fields of medicine and cosmetics (Non-Patent Documents 3 and 6). ).

The present inventors have established a silkworm strain in which the function of the posterior silk gland is lost by expressing Piericin 1A, a toxic protein derived from cabbage butterfly, in the posterior silk gland (Patent Document 1, Non-Patent Document 7). In this silkworm strain, the posterior silk gland shows an abnormal morphology and the expression of the fibroin gene is suppressed.

Japanese Patent Application Laid-Open No. 2018-007569

Inoue S. et al., J. Biol Chem, (2000), 275 (51), 40517-28 Tashiro T. et al., J. Cell Biol, (1970), 46 (1), 1-16 Kunz R.I. et al., Bio Med Research International, (2016), 8175701, Equb 2016 Nov 14 Takasu Y. et al., Biosci. Biotechnol. Biochem. (2002), 66 (12), 2715-2718. Takasu Y., et al., Insect Biochem. Melec. Biol. (2010) 40, 339-344. Joseph B. et al., Frontiers in Life Science, (2012), 6, Nos. 3-4, 55-60. Otsuki R. et al., Proc Natl Acad Sci U.S.A. (2017), 114 (26): 6740-6745.

A main object of the present invention is to provide a bacteriostatic agent containing a new active ingredient.

Item 1. A bacteriostatic agent containing at least one sericin as an active ingredient.
Item 2. A sericin-containing aqueous composition containing full-length sericin contained in cocoons.
Item 3. The sericin-containing aqueous composition according to Item 2, further comprising cocoon-derived fibroin.
Item 4. A method for producing an aqueous composition containing sericin, comprising the steps of treating cocoons containing at least one type of sericin with an aqueous solution of a chaotropic agent, followed by dialysis to obtain an aqueous composition containing at least one type of full-length sericin. .
Item 5. The production method according to Item 4, wherein the aqueous solution of chaotropic agent is an aqueous solution of lithium bromide of 5M or more.

In the past, sericin was removed in the process of scouring silk composed of fibroin, but at that time, sericin was hydrolyzed, and an aqueous composition of non-hydrolyzed full-length sericin was not obtained. .

According to the present invention, cocoons are treated with an aqueous solution of a chaotropic agent such as lithium bromide and dialyzed to yield an aqueous composition containing unhydrolyzed full-length sericin, which exhibits potent bacteriostatic activity. revealed to have

A graph showing the effect of a sericin-containing aqueous composition on the colony numbers of Escherichia coli and Candida albicans. *P<0.05, ***P<0.001 Photographs showing colony sizes of Escherichia coli, Salmonella enterica, Staphylococcus aureus, Bacillus subtilis, and Candida albicans. Graph showing the effect of a sericin-containing aqueous composition on colony size of Escherichia coli, Salmonella enterica, Staphylococcus aureus, Bacillus subtilis, and Candida albicans. *P<0.05, ***P<0.001 Fig. 2 is a photograph showing the results of electrophoresis for confirming the presence or absence of sericins 1 to 4 in the sericin-containing aqueous composition (sericin) of the present invention and commercially available pure sericin. It was clarified that commercially available pure sericin does not contain undegraded sericins 1 to 4, and that the sericin-containing aqueous composition (sericin) of the present invention contains all of sericins 1 to 4. . Graph showing the effect of aqueous compositions containing sericin on colony size of Escherichia coli, Salmonella enterica and Candida albicans. *P<0.05, ***P<0.001

As used herein, chaotropic agents include, for example, calcium chloride, calcium bromide, calcium iodide, potassium thiocyanate, sodium thiocyanate, calcium thiocyanate, magnesium thiocyanate, lithium chloride, lithium bromide, lithium iodide, hydrochloric acid. Guanidine, guanidine hydrobromide, guanidine hydroiodide, guanidine thiocyanate and the like can be mentioned, and among these, lithium bromide is particularly preferred.

Cocoons obtained from silkworms are fibroin-free as described in Patent Document 1 and Non-Patent Document 7, and cocoons containing sericin as a main component are preferably used. An aqueous sericin composition having a bacteriostatic effect can also be obtained using cocoons containing about 20-30% by weight of fibroin and about 70-80% by weight of fibroin.

As used herein, the term "aqueous composition containing sericin" includes forms such as an aqueous solution containing sericin, a sol, and a gel. Sericin-containing aqueous compositions are useful as bacteriostats.

When a cocoon containing fibroin as a main component and having a sericin content lower than the fibroin content is treated with an aqueous solution of a chaotropic agent, the protein content in the resulting sericin-containing aqueous composition reflects the protein content contained in the cocoon. Although the sericin content is lower than the fibroin content, even such an aqueous composition has a bacteriostatic effect and is included in the sericin-containing aqueous composition of the present invention.

The sericin contained in the aqueous composition containing sericin is composed mainly of sericins 1 to 4, and may contain at least one sericin other than sericins 1 to 4.

As used herein, "at least one type of sericin" means containing at least one type selected from the group consisting of sericin 1, sericin 2, sericin 3, and sericin 4. Since cocoon sericin contains 1 to 4 sericins, the bacteriostatic agent of the present invention preferably contains 1 to 4 sericins. Since cocoon sericin is almost or completely dissolved by a chaotropic agent, when cocoons are used as a raw material for sericin, the sericin composition in the sericin-containing aqueous composition substantially reflects the sericin composition of cocoons. Become. The bacteriostatic agent of the present invention may contain recombinant sericin produced in a host such as Escherichia coli.

The concentration of sericin in the sericin-containing aqueous composition of the present invention is preferably about 0.001 to 4% by mass, more preferably about 0.01 to 3% by mass, still more preferably about 0.1 to 2% by mass, particularly preferably 0.5 to 1% by mass. %. If the sericin concentration is too low, the bacteriostatic action will not be exhibited.

In the present invention, sericin with bacteriostatic action is full-length sericin that has not been degraded. The above concentrations are those of full-length sericin and do not consider hydrolyzed sericin.

Microorganisms targeted for bacteriostatic action include Gram-negative bacteria, fungi and some Gram-positive bacteria. It has no significant bacteriostatic action against staphylococci. Gram-negative bacteria include Neisseria, Neisseria gonorrhoeae, Meningococcus, Bordetella pertussis, Bordetella bronchiseptica, Escherichia coli, Citrobacter, Salmonella, Salmonella typhi, Paratyphi A, Paratyphi B, Enteritidis, Shigella, Enterobacter, Plague Vibrio, Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas, Pseudomonas aeruginosa, and the like. Fungi include Aspergillus, Candida, dermatophytes, and the like.

The bacteriostatic agent of the present invention can inhibit the growth of Gram-negative bacteria and fungi, among others.

A sericin-containing aqueous composition having a bacteriostatic action is prepared by the following steps of treatment with a chaotropic agent and dialysis.
(1) Treatment with chaotropic agent The cocoons are immersed in an aqueous solution of chaotropic agent and treated at 20-40°C for 1-24 hours to solubilize the cocoons and obtain an aqueous solution containing sericin and a chaotropic agent. The concentration of the chaotropic agent is preferably about 5-7M, more preferably about 5.7-6.3M. Sericin dissolves best at around 6M, and fibroin dissolves more rapidly at around 8-9M.

A treatment step with a chaotropic agent can accelerate dissolution of the cocoons by vortexing, stirring or applying ultrasound. The resulting solution may be filtered through a filter. For example, when a cocoon containing a large amount of fibroin is treated with an aqueous solution of a chaotropic agent for a short period of time, sericin may be dissolved but some of the fibroin may not be dissolved. In such cases, the sericin content may be increased by filtering. can be done.

(2) Dialysis step The solution obtained by treating the cocoons with a chaotropic agent can then be subjected to a dialysis step to remove the chaotropic agent or other low-molecular-weight impurities. Dialysis may be performed in a buffer. Examples of buffers include phosphate buffer, Tris-HCl buffer, citrate buffer, acetate buffer, glycine buffer and the like. Water exchange may be performed in the dialysis step.

The sericin concentration in the sericin-containing aqueous composition obtained by dialysis is, for example, about 0.5 to 1.2% by mass. The sericin-containing aqueous composition after dialysis may be centrifuged to remove precipitates and further purified. Moreover, the sericin concentration in the sericin-containing aqueous composition may be concentrated or diluted.

EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the examples.
Example 1
(1) Materials As cocoons, the cocoons of Piericin strain silkworms that express Piericin 1A in their posterior silk glands bred at Kyoto Institute of Technology were used. The cocoons of the Piericin strain silkworms used did not contain fibroin and consisted mostly of sericin.

Bacteriostatic activity against Escherichia coli, Salmonella enterica, Staphylococcus aureus, Bacillus subtilis, Candida albicans was measured.

(2) Method
2-1 Extraction of sericin
To 24 ml of 6 M lithium bromide solution, 650 mg of cocoons of Piericin strain silkworm were added, allowed to stand at room temperature for 16 hours, and dissolved by vortexing. After adding 6 ml of 1 M Tris-HCL buffer to this solution and mixing by inversion, the mixture was centrifuged at 7000 rpm for 30 minutes. The obtained supernatant was dialyzed against pure water for 30 hours. On the way, water exchange of 2 L x 4 times was performed. The dialyzed solution was transferred to a 50 ml tube, centrifuged at 7000 rpm for 30 minutes, and the supernatant was used as a sericin-containing aqueous composition.
2-2 Investigation of effects on microorganisms
Sterilized water or a sericin-containing aqueous composition was dropped onto an LB agar medium, spread over the entire surface with a spreader, and air-dried for 10 minutes. Next, the bacterial solution was dropped, spread over the entire surface with a spreader, and air-dried for 10 minutes.

After drying, E.coli was cultured at 37°C for 48 hours, S.enterica, S. aureus and B.subtilis at 37°C for 16 hours, and C.albicans at 25°C for 4 days. A photograph of the cultured colony was taken, and the area of the colony was measured using image software (ImageJ). In addition, the number of colonies was counted for Escherichia coli and Candida.

(3) Results When cultured in a medium coated with a sericin-containing aqueous composition (denoted as "sericin" in Fig. 1) obtained from cocoons consisting only of sericin produced by transgenic silkworms of the Piericin strain, E.coli and C. No change was observed in the number of .albicans colonies (Fig. 1). In FIG. 1, "control" is obtained by applying sterilized water instead of the sericin-containing aqueous composition.

The colony areas of E.coli, S.enterica, B.subtilis, and C.albicans decreased when cultured on a medium coated with sericin compared to when cultured on a medium coated with sterilized water. In particular, growth inhibition against E.coli and S.enterica was remarkable (Figs. 2, 3, Table 1). On the other hand, the colony area of S. aureus did not change (FIGS. 2 and 3).

From the results of FIGS. 1 to 3 and Table 1, it was revealed that the sericin-containing aqueous composition had no bactericidal action (FIG. 1) and had a significant bacteriostatic action (FIGS. 2, 3, Table 1). .

On the other hand, as shown in FIG. 4, electrophoresis of cocoons composed only of sericin produced by transgenic silkworms of the Piericin line revealed sericin 1 (S1), sericin 2 (S2), sericin 3 (S3), sericin 4 (S4 ) was observed, whereas in the case of commercially available pure sericin, none of the bands S1 to S4 could be confirmed. This is because pure sericin was recovered from the wastewater from the process of removing sericin contained in cocoon filaments and raw silk (this process is called scouring), and sericin was decomposed during this scouring process. showing. The sericin subjected to this decomposition did not exhibit a bacteriostatic effect (Fig. 5).

These results indicate that sericin with its unbroken structure (full-length sericin) does not kill these microorganisms, but has the ability to inhibit their growth.

Human epidermis is inhabited by various microorganisms, some of which have positive and some of which have negative effects.

Claims (4)

A bacteriostatic agent comprising at least one full-length sericin as an active ingredient. 2. The bacteriostatic agent of claim 1, which inhibits the growth of Gram-negative bacteria and fungi. 3. The bacteriostatic agent according to claim 2, which suppresses the growth of E. coli and Salmonella. Fibroin-free bacteriostatic agent according to any one of claims 1 to 3.