JP5296463B2 - Antibacterial agent - Google Patents

Description

  The present invention relates to an antibacterial agent containing fine silver particles and fermented radish juice.

  Conventionally, silver is known to have an antibacterial effect and a bactericidal effect, and many antibacterial agents containing silver ions and silver particles are known (for example, see Patent Document 1).

Among these antibacterial agents, a silver solution containing a polypeptide carrying fine silver particles (hereinafter also referred to as a silver-carrying peptide) has a low irritation to the human body because it uses a peptide as a carrier. Can be used.
JP 2000-198708 A

  However, it is difficult to say that the silver solution containing the above conventional silver-carrying peptide has a significant bactericidal and antibacterial effect on molds and yeasts.

  For example, even when a silver-carrying peptide is contained as an antibacterial agent for cosmetics, since many microorganisms and yeasts contaminate cosmetics, it effectively suppresses the growth of molds and yeasts. There was a case that could not be.

  In such a case, it may be possible to add a drug capable of remarkably suppressing mold and yeast together with the silver-carrying peptide. However, since many of these drugs are often highly irritating to the human body, It was a factor that hesitates to use.

  The present invention has been made in view of such circumstances, and can inhibit the antibacterial effect of silver-carrying peptides as much as possible, and can cause a growth inhibitory effect on fungi and yeast, To provide antibacterial agents that are less irritating to the human body.

  In order to solve the above conventional problems, the antibacterial agent according to the present invention is obtained by fermenting and squeezing a silver solution containing a polypeptide carrying fine silver particles and a radish in a predetermined immersion liquid. It was decided to mix with radish fermented liquor.

  The antibacterial agent according to the present invention is also characterized by the following points.

(1) The polypeptide carrying fine silver particles is reacted by adding a silver salt solution and a catalyst to an aqueous solution of collagen or a hydrolyzate of collagen, and irradiating the reaction solution with ultraviolet light having a wavelength of 400 nm or less. By carrying out reduction, fine silver particles are deposited in the vicinity of the α-amino group of the collagen or the hydrolyzate of collagen, and supported by electrostatic adsorption.
(2) The radish fermented liquid is obtained by fermentation with a microorganism belonging to the genus Leuconostoc.

In the cosmetic according to the present invention, the antibacterial agent according to claim 1 or 2 is added.

In the antibacterial agent according to claim 1, a silver solution containing a polypeptide carrying fine silver particles is mixed with a radish fermented liquid obtained by fermenting and squeezing radish in a predetermined immersion liquid. Therefore, it is possible to effectively perform antibacterial and sterilization on molds and yeasts that are unlikely to have an antibacterial effect and a bactericidal effect with a silver-carrying peptide alone or a radish fermentation broth alone.
Moreover, since the radish fermented liquid is characterized by being fermented by microorganisms belonging to the genus Leuconostoc, it can produce a remarkable antibacterial effect and bactericidal effect against mold and yeast. .

  Further, in the antibacterial agent according to claim 2, the polypeptide carrying the fine silver particles is reacted by adding a silver salt solution and a catalyst to an aqueous solution of collagen or a hydrolyzate of collagen, By carrying out reduction by irradiating with ultraviolet rays having a wavelength of 400 nm or less, fine silver particles are precipitated in the vicinity of the α-amino group of the collagen or collagen hydrolyzate and supported by electrostatic adsorption. Therefore, long-term dispersion stability can be imparted to the silver-carrying peptide.

Moreover, in the cosmetic of Claim 3 , since the antibacterial agent of Claim 1 or 2 was added, it can be set as the cosmetic which can prevent the contamination by mold | fungi or yeast over a long period of time.

  The present invention provides an antibacterial agent obtained by mixing a silver solution containing a polypeptide carrying fine silver particles and a radish fermented liquid obtained by fermenting and squeezing radish in a predetermined immersion liquid. Is.

  In addition, squeezing here includes not only obtaining the juice while squeezing with a filter press or the like, but also including obtaining the filtrate by filtering with a cloth, paper, a thread-wound filter or the like, for example. .

  According to the antibacterial agent according to the present invention, an antibacterial effect and a bactericidal effect can be produced against mold and yeast that contaminate cosmetics.

  Especially when cosmetics are manufactured or shipped at factories, etc., since a sanitary environment is maintained, there is almost no risk of microbial contamination, but users can take out the contents of cosmetics in their hands, When it is attached to the fingertip, mold or yeast adhering to the user's skin may contaminate the cosmetic outlet or directly contaminate the contents.

  In such a case, mold and yeast may grow in the contents as time passes, which is a major factor in deterioration of the cosmetic.

  Therefore, by using the antibacterial agent according to the present invention, an antibacterial effect and a bactericidal effect can be produced against mold and yeast, and contamination by mold and yeast can be prevented as much as possible.

  Here, the polypeptide (silver-carrying peptide) carrying fine silver particles is reacted with collagen or a hydrolyzate of collagen by adding a silver salt solution and a catalyst, and reacting the reaction solution with ultraviolet light having a wavelength of 400 nm or less. It is desirable that fine silver particles be deposited in the vicinity of the α-amino group of the collagen or the hydrolyzate of collagen and supported by electrostatic adsorption by performing reduction by irradiating with.

  The polypeptide for carrying silver is not particularly limited, but is required to be harmless even when the user ingests or applies it to the skin.

  Therefore, examples of such a polypeptide include collagen and collagen hydrolyzate, which have been used as cosmetics. Collagen or collagen hydrolyzate is harmless even when ingested or applied to the skin. It is said that it has the effect of improving the tension and gloss of the skin.

  Therefore, when the carrier of silver particles is collagen or a hydrolyzate of collagen, cosmetics can be given antibacterial and bactericidal effects against mold and yeast, and can also be expected to improve skin. it can.

  In addition, although details are still unclear, collagen and collagen hydrolyzate are more stable in water than other polypeptides, and can effectively suppress the precipitation of silver particles. Also have.

  This is considered to be due to the hydrophobicity derived from collagen and amino acid residues constituting the collagen hydrolyzate and the tertiary structure.

  Also from this viewpoint, there is an advantage that collagen or a hydrolyzate of collagen is selected as a carrier for silver particles.

  In other words, the silver-carrying peptide allows fine silver particles to be carried by electrostatic adsorption on the electronegative functional group (an α-amino group of the peptide group) of an organic polymer micelle that can take the state of a water-soluble or hydrophilic gel. It can also be said that it was added to the conductive polymer micelle.

  The silver salt solution used in preparing the silver solution may be at least one silver salt solution selected from the group consisting of an aqueous ammoniacal silver nitrate solution, an aqueous silver nitrate solution, and an aqueous silver sulfate solution.

  By using these silver salt solutions, fine silver particles can be efficiently precipitated.

  In addition, as described above, one kind of silver salt solution is selected from ammoniacal silver nitrate aqueous solution, silver nitrate aqueous solution and silver sulfate aqueous solution, and all of them are markedly different in the antibacterial effect of fine silver particles produced by reduction. It is not allowed.

  Any of them can exhibit the characteristic effects of the present invention, but it is preferable to select an appropriate silver salt by testing depending on the material and properties of the carrier. According to the knowledge of the inventors, the aqueous ammoniacal silver nitrate solution and the aqueous silver nitrate solution are suitable for loading on a relatively high molecular weight collagen or gelatin having a molecular weight of 100,000 or more, and are the most versatile silver salt solutions. The aqueous silver sulfate solution is suitable for supporting collagen or gelatin having a molecular weight of several thousand to less than 100,000.

The concentration (Ag ⁺ mol / l) of the aqueous silver salt solution used for loading is suitably in the range of 0.1 to 0.001. If the silver ion concentration is too high, the generated silver particles tend to aggregate, and the quantum size effect of the fine silver particles is reduced, so that the antibacterial effect tends to be reduced. When the silver ion concentration is set to a lower value, a result superior in antibacterial effect is obtained.

  Moreover, the catalyst used at the time of preparation of an antibacterial composition is good also as at least 1 type of aqueous solution selected from the group which consists of each aqueous solution of lithium hydroxide, calcium hydroxide, sodium hydroxide, and potassium hydroxide.

  By using these aqueous solutions, fine silver particles can be efficiently precipitated.

  As described above, as the catalyst, one type is selected from each of lithium hydroxide, calcium hydroxide, sodium hydroxide, and potassium hydroxide aqueous solutions. There is no significant difference in effect. The catalyst is used as a catalyst for temporarily substituting the silver ion of the aqueous silver salt solution previously added to the aqueous solution of the carrier with the hydrogen of the α carboxyl group generated by the cleavage of the peptide bond of the carrier. Is.

  The catalyst is preferably selected by testing an appropriate catalyst according to the type of carrier and molecular weight. Since calcium hydroxide has a relatively low solubility in water, it is suitable for loading on a relatively low molecular weight collagen having a molecular weight of less than 10,000.

  Lithium hydroxide is effective for application to high molecular weight (100,000 or more) collagen. Sodium hydroxide and potassium hydroxide are common catalysts that are effective when used for collagen in the entire molecular weight region. Therefore, when selecting collagen, a trial loading trial using sodium hydroxide or potassium hydroxide as the first supported catalyst is recommended.

  As the aqueous solution concentration of the catalyst to be added, an aqueous solution containing an amount of the catalyst component corresponding to 2 to 5% by weight with respect to the collagen used as the carrier is used. Since the solubility in water varies depending on the type of catalyst component, the concentration of the aqueous catalyst solution can be selected as appropriate, but it is preferably 10% by weight or less. In particular, when choosing strong alkaline lithium hydroxide, potassium hydroxide and sodium hydroxide, it is recommended to use each in an aqueous solution of less than 10% by weight.

  In addition, as a light source used for reduction by ultraviolet irradiation, one kind or two or more kinds selected from natural sunlight, a high-pressure or low-pressure xenon gas discharge tube, a high-pressure or low-pressure mercury lamp, or a light source group having equivalent ultraviolet radiation activity May be used.

  By using these light sources, fine silver particles can be efficiently precipitated.

  This irradiation with ultraviolet rays photoreduces silver ions bonded to the α-amino group and α-carboxyl group by coordination or substitution in situ to produce fine silver particles, and at the same time, the particles are surrounded by the α-amino group. This is a process for supporting fine silver particles to be electrostatically adsorbed in the negative electric field region.

  Any light source can be used as long as the light source includes a near ultraviolet region having a wavelength of 400 nm or less. Preferably, as described above, it is preferable to use a mercury lamp, a xenon gas discharge tube that radiates ultraviolet rays intensively, or a luminaire having an ultraviolet emission ability equal to or higher than those. As a result of conducting an experiment with sunlight, the present inventors selected a time zone from 11:00 am to 2:00 pm in fine weather, and were sufficiently satisfied as a result of the test on a sample irradiated with sunlight for 30 minutes. It was confirmed that a power reduction result was obtained.

  Confirmation of the photoreduction process by ultraviolet irradiation is a method of comparing the difference between absorption at a specific wavelength near 300 nm of a sample before ultraviolet irradiation and absorption at the same wavelength after ultraviolet irradiation reduction treatment using an ultraviolet absorption spectrum meter. Is in principle appropriate. However, since collagen derived from natural products has large variations in molecular weight, types of impurities, and contents thereof, measurement by an ultraviolet absorption spectrum is difficult. Therefore, a method using a colorimeter is recommended as a simple method for checking and managing the photoreduction process.

  Confirmation of the photoreduction process by a colorimeter is made by measuring and comparing the specific transmittance of light having a wavelength of 430 to 540 nm before and after the photoreduction process. This method is simple but has practical convenience and is industrially effective.

  On the other hand, the radish fermented liquid is obtained by fermenting and squeezing radish in a predetermined immersion liquid. Fermentation of this radish can be performed using Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, Leuconostoc, etc. Those fermented by lactic acid bacteria belonging to the above are preferred.

  Among them, a radish fermented liquid fermented and squeezed by Leuconostoc, which performs heterofermentation, is desirable, and can exert a remarkably antibacterial effect and bactericidal effect on mold and yeast.

  In addition, as described above, by adding the antibacterial agent according to the present invention to cosmetics, contamination of the cosmetics by molds and yeasts can be prevented safely and effectively on the human body.

  Next, embodiments and effects of the antibacterial agent according to the present invention will be specifically described.

[1. Preparation of antibacterial agent
Preparation of the antibacterial agent which concerns on this embodiment is demonstrated. Below, preparation of a silver solution is demonstrated first, Then, preparation of a radish fermented liquid is demonstrated, Furthermore, the process of preparing these and preparing an antibacterial agent is demonstrated.

  The silver solution was prepared according to the following procedures (1) to (6).

(1) 100 g of collagen powder was put into a 500 ml round bottom flask, 200 ml of water was added and completely dissolved while stirring at 50 ° C. with heating.

(2) Next, 70 ml of 0.5% aqueous silver nitrate solution was added to the collagen solution, and the reaction was stirred for 30 minutes while maintaining the temperature at 60 ± 2.0 ° C.

(3) To this, 100 ml of a 5.0% potassium hydroxide aqueous solution was added and stirred for 2.5 hours while maintaining the above temperature.

(4) Next, heating was stopped and the reaction solution was cooled to room temperature. During this time, stirring was continued. When this step was completed, about 5 ml of a part of the reaction solution was previously collected in a colorimetric tube (A) for colorimeter.

(5) Transfer the reaction liquid to a flat stainless steel vat (30 x 21 x 5 cm), hold it horizontally, and irradiate it with ultraviolet rays for 10 minutes from the center of the reaction liquid surface at a vertical distance of 30 cm directly below. went. About 5 ml of a part of the reaction solution was collected and stored in the colorimetric tube (B). The ultraviolet light source device used for the photoreduction is a stabilized xenon lamp 75W and a dedicated constant current power source C2576 manufactured by Hamamatsu Photonics.

(6) In order to confirm the photoreduction step, the colorimetric samples collected and stored in advance in the above step (4) and step (5) were compared for light transmittance at a wavelength of 430 nm to confirm the reduction reaction. At that time, the colorimetric tube (A) / colorimetric tube (B) transmission ratio was 25%, and it was confirmed that the reduction was sufficiently performed. The colorimeter used was Photo Uni5 manufactured by ATAGO Corporation.

  Next, preparation of a radish fermented liquid is demonstrated.

  A 10 L salt solution with a salt concentration of 10% was placed in a sealable 25 L container, 10 kg of daikon radish was added to the immersion solution, and inoculated with kimchi-derived microorganisms (Leuconostoc sp.). In this state, it was stored at 35 ° C. for 14 days for fermentation.

  Next, the obtained fermentation broth was squeezed while being filtered using a filter press to obtain a radish fermentation broth.

  Next, the process of preparing an antibacterial agent by blending the aforementioned silver solution and radish fermented liquid will be described.

  98 ml of water was placed in a 200 ml beaker, 1 ml of silver solution and 1 ml of radish fermentation broth were added, and the mixture was stirred for 5 minutes with a stirrer to obtain an antibacterial agent.

[2. Verification of the effect of the antibacterial agent according to this embodiment]
Next, the effect of the prepared antibacterial agent was verified. There are 6 types of bacteria: Eshrichia coli (E. coli), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Bacillus sbutilis (spore-forming bacteria), Candida albicans (yeast), and Aspergillus niger (mold). is there.

  The bacterial solution was prepared as follows. First, one platinum loop of each microorganism that has been lyophilized and stored in a tryptic soy agar medium (SCD agar medium) is cultured at 35 ± 1 ° C. for 16 to 24 hours (pre-culture). Aspergillus niger was transferred to a Sabouraud agar medium and cultured at 20 to 25 ° C. for 72 to 120 hours while observing the degree of growth.

  Next, one platinum loop of each pre-cultured bacterium is transferred to an SCD agar medium and cultured at 35 ± 1 ° C. for 16-24 hours (pre-culture). Aspergillus niger is not precultured.

Then, 1 strain of each strain was dissolved in 4 ml of sterile phosphate buffered saline to prepare a bacterial solution. The viable cell count in each bacterial solution at this time was 10 ⁷ to 10 ⁹ cells / ml and 10 ⁵ to 10 ⁷ cells / ml in bacteria.

  Moreover, as a test liquid, we decided to use three types, the silver solution, radish fermented liquid, and the antibacterial agent which concerns on this embodiment.

  These test solutions were dispensed into 7 sterilized tubes of 10 g each, 6 of which were inoculated with 100 μl of each bacterial solution, and 1 was used as a control not inoculated with the bacterial solution.

  And these tubes were preserve | saved at 20-25 degreeC for 2 weeks, and the antibacterial and bactericidal effect in the meantime was verified. The results are shown in Table 1.

  As shown also in Table 1, the antibacterial agent according to the present embodiment resulted in remarkable antibacterial and bactericidal effects in yeast and mold as compared to the case of a silver solution alone or a radish fermented liquid alone. In addition, in the above-mentioned control, since the growth of microorganisms was not confirmed, description in this Table 1 is abbreviate | omitted.

  Table 1 will be explained in detail. First, in Eshrichia coli (E. coli), after 1 hour of inoculation, in any of the silver solution, radish fermented liquid, and antibacterial agent, the number of viable bacteria calculated from the dilution factor was almost the same. Although viable bacteria were confirmed, the number of viable bacteria decreased after 3 hours and became below the detection limit after 1 day. Thereafter, viable bacteria were not confirmed until 2 weeks later.

  In Staphylococcus aureus (Staphylococcus aureus), after 1 hour from the inoculation, in the silver solution, the radish fermented liquid, and the antibacterial agent, viable bacteria that were almost the same as the viable cell count calculated from the dilution factor were confirmed. After 3 hours, only the silver solution tended to decrease. At the time after 1 day, the silver solution was below the detection limit, and the radish fermented broth and the antibacterial agent were able to detect viable bacteria slightly. After one week, no viable bacteria were confirmed in any of the test solutions.

  For Pseudomonas aeruginosa (Pseudomonas aeruginosa), both the silver solution and the fermented radish solution showed a significant decrease in the number of viable bacteria after 1 hour, but the antibacterial agent showed a significant decrease. I couldn't. However, after 3 hours, a decrease in the number of viable bacteria was observed even with the antibacterial agent, and after 1 day, it was below the detection limit in any test solution.

  In Bacillus subtilis (spore-forming bacteria), no significant decrease in the number of viable bacteria was observed in any of the test solutions until 3 hours after the inoculation, but after one day, there was a tendency to decrease in all the test solutions. It was recognized and no viable bacteria were detected after 1 week.

  For Candida albicans (yeast), no significant decrease in viable cell count was observed in the silver solution and radish fermented liquid until 3 hours later. However, as a remarkable point, in the antibacterial agent according to the present embodiment, a decreasing tendency has already been confirmed 1 hour after inoculation, and when 3 hours have passed, viable bacteria are below the detection limit.

  This indicates that the antibacterial agent according to the present embodiment has a remarkable antibacterial / bactericidal effect on yeast as compared with the case of a silver solution alone or a fermented radish solution alone.

  For Aspergillus niger, in the silver solution and the radish fermented liquid, no significant decrease in the viable cell count was observed until after 1 day. However, as a remarkable point, in the antibacterial agent according to the present embodiment, the number of viable bacteria is significantly reduced one day after inoculation.

  This indicates that the antibacterial agent according to the present embodiment has a remarkably antibacterial and bactericidal effect on fungi compared to the case of a silver solution alone or a fermentation radish solution alone.

  When these results are comprehensively examined, the antibacterial agent according to the present embodiment is more effective in bacteria (E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, spore-forming bacteria) than in the case of silver solution alone or fermentation radish solution alone. ) Causes an equivalent or slightly weaker antibacterial and bactericidal effect, but it has a significantly stronger antibacterial and bactericidal effect against yeast and mold than silver solution alone or fermented radish solution alone. confirmed.

  That is, it was shown that it is an antibacterial agent capable of producing a growth inhibitory effect against mold and yeast without inhibiting the antibacterial effect of the silver-carrying peptide as much as possible.

  Further, in the antibacterial agent according to the present embodiment, collagen is used as a carrier for silver particles, and since a fermented liquid of food (daikon radish) is used, compared to conventional fungicides for fungi or yeast, etc. It can be seen that there is little irritation to the human body and it is gentle on the skin.

[Cosmetic preservation test]
Next, a cosmetic containing the above-described antibacterial agent was prepared, and a storage test of the cosmetic was performed. Here, the cosmetic is lotion, and the preparation process of 100 kg will be described below. 93.28 kg of normal temperature natural water was put into a 150 L capacity mixing tank and the stirrer was operated.

  Next, 6 kg of 1,3-butylene glycol, 0.01 kg of sodium hyaluronate, and 0.05 kg of seaweed extract were added and dispersed uniformly.

  After becoming fully dispersed, steam was passed through the jacket of the blending tank, and the blended solution in the blending tank was heated to 80 ° C and allowed to stand for 30 minutes with stirring. The raw material was dissolved.

  After confirming that it was sufficiently dissolved, water was allowed to flow through the jacket portion of the preparation tank, and the liquid temperature of the preparation liquid was adjusted to 35 to 40 ° C. while stirring.

  Next, 1 kg of sodium pyrrolidonecarboxylate was added, and 2 L of the above-described antibacterial agent was further added.

  In a separate container, a chelate solution prepared by dissolving 0.08 kg of citric acid and 0.08 kg of sodium citrate in 5 kg of natural water in advance was put into the preparation tank and stirred.

  A small amount of citric acid was added to adjust the pH so that the pH was 7.

  Finally, the liquid temperature of the preparation liquid was adjusted to 35 ° C., and the preparation liquid was filtered through a 200-400 mesh filter to obtain 100 kg of lotion.

  Moreover, the lotion which added 2L of natural water instead of the antibacterial agent was also prepared as the lotion used as a comparison object.

  Each 300 ml of each lotion was dispensed into a sterilized medium bottle, and a storage test was conducted at 25 ° C. for 3 months with no lid attached (with falling bacteria mixed in). Since water evaporates, sterilized water was added every 2 weeks in a reduced amount.

  As a result, in the lotion to which the antibacterial agent was added, generation of mold that could be visually confirmed over 3 months was not observed, and no significant increase in turbidity was observed.

  On the other hand, in the lotion to which no antibacterial agent was added, a black mold-like floating substance was observed after one month, and the floating substance grew to a colony having a diameter of about 2 cm after three months. In addition, a precipitate was observed at the bottom of the bottle, and yeast was detected by a microscope of the precipitate.

  From these things, it was shown that the cosmetics added with the antibacterial agent according to the present embodiment can effectively suppress the growth of mold and yeast.

  The skin lotion added with the antibacterial agent was applied once a day for 3 months by 20 employees of the factory that produced the skin lotion, but no skin abnormality was reported.

  As described above, the antibacterial agent according to this embodiment includes a silver solution containing a polypeptide carrying fine silver particles, and a radish fermented liquid obtained by fermenting and squeezing radish in a predetermined immersion liquid. Can suppress the antibacterial effect of silver-carrying peptide as much as possible, and can cause growth suppression effect against mold and yeast, and it is less irritating to the human body. Antibacterial agents can be provided.

  Finally, the description of each embodiment described above is an example of the present invention, and the present invention is not limited to the above-described embodiment. For this reason, it is a matter of course that various modifications can be made in accordance with the design and the like as long as they do not depart from the technical idea according to the present invention other than the embodiments described above.

Claims (3)

An antibacterial agent obtained by mixing a silver solution containing a polypeptide carrying fine silver particles and a radish fermented liquid obtained by fermenting and squeezing radish in a predetermined immersion liquid ,
The antibacterial agent characterized in that the radish fermented liquor is obtained by fermentation with a microorganism belonging to the genus Leuconostoc.   The polypeptide carrying the fine silver particles is reduced by adding a silver salt solution and a catalyst to an aqueous solution of collagen or a hydrolyzate of collagen, and irradiating the reaction solution with ultraviolet light having a wavelength of 400 nm or less. The antibacterial agent according to claim 1, wherein fine silver particles are deposited in the vicinity of the α-amino group of the collagen or collagen hydrolyzate and supported by electrostatic adsorption. Cosmetics to which the antibacterial agent according to claim 1 or 2 is added.