Antibacterial cellulose fiber containing spore-forming bacteria

Abstract

The objective is to develop a cellulose-based fiber that allows spore-forming bacteria to express their function more readily than the antibacterial rayon fiber containing spore-forming bacteria described in Japanese Patent Publication No. 7323742. Furthermore, the objective is to develop a new manufacturing method that does not expose spore-forming bacteria to harsh environments during the manufacturing process. [Solution] An antimicrobial cellulose fiber containing spore-forming bacteria, having a colony occupancy rate of 50% or more in a predetermined culture test. Furthermore, a method for producing an antimicrobial cellulose fiber containing spore-forming bacteria, comprising the step of mixing a cellulose fiber surface-treated with a dilute strong alkaline aqueous solution with spore-forming bacteria.

Description

Technical Field

[0001] The present invention relates to an antibacterial cellulose-based fiber containing spore-forming bacteria.

Background Art

[0002] The applicants of the present application developed an antibacterial rayon fiber containing spore-forming bacteria on which microorganisms having antibacterial activity were colonized in Patent Document 1.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The antibacterial rayon fiber containing spore-forming bacteria described in Patent Document 1 includes a process of mixing spore-forming bacteria in a spore state with viscose and then spinning. This production method is, so to speak, a method of kneading spore-forming bacteria into the fiber, and the spore-forming bacteria are scattered and colonized from the surface to the center of the fiber. Therefore, compared with the spore-forming bacteria colonized on the surface of the fiber, the spore-forming bacteria colonized inside the fiber may have a delayed or insufficient functional expression.

[0005] In addition, the alkalinity of viscose is a harsh environment for vegetative cells that have broken out of the dormant state (germinated). When germinated from the spore state due to an unexpected situation during the production process and contacted with viscose, there was also a risk of reducing the number of bacteria that could colonize on the rayon fiber.

[0006] An object of the present invention is to develop an antibacterial cellulose-based fiber containing spore-forming bacteria in which the functions of spore-forming bacteria are more easily expressed than the antibacterial rayon fiber containing spore-forming bacteria described in Patent Document 1. Another object is to develop a new production method that does not expose spore-forming bacteria to a harsh environment during the production process. [Means for solving the problem]

[0007] As a result of diligent research into the above-mentioned problems, the inventors discovered that the above-mentioned problems can be solved by surface-treating cellulose fibers with a dilute, strong alkaline aqueous solution and then mixing them with spore-forming bacteria in a spore state. Based on this finding, the inventors completed the present invention.

[0008] [1] Antimicrobial cellulose fiber containing spore-forming bacteria, having a colony occupancy rate of 50% or more in the following culture test. [Culture test] (1) In a petri dish with an inner diameter of 80 mm equipped with a standard agar medium, a cylinder with an inner diameter of 35 mm is placed on the standard agar medium in the center when the petri dish is viewed from above, and 0.05 g of antimicrobial cellulose fiber containing spore-forming bacteria is distributed approximately evenly within the cylinder, and then the cylinder is removed. (2) Add sterile water to the antimicrobial cellulose fiber containing spore-forming bacteria and mix it with the standard agar medium, then incubate at 35°C for 24 hours. (3) After culturing, the area of ​​colonies formed by spore-forming bacteria on the standard agar medium (hereinafter referred to as "colony area") is measured, and the colony occupancy rate is calculated using the following formula. Colony occupancy rate (%) = Colony area ÷ Inner diameter area of ​​petri dish × 100 [2] A nonwoven fabric composed of antimicrobial cellulose fibers containing spore-forming bacteria as described in [1] above. [3] Agricultural materials made using the nonwoven fabric described in [2] above. [4] A method for producing antimicrobial cellulose fibers containing spore-forming bacteria, comprising the step of mixing cellulose fibers surface-treated with a dilute strong alkaline aqueous solution with spore-forming bacteria. [Brief explanation of the drawing]

[0009] [Figure 1] This is a photograph of the antibacterial rayon fiber containing spore-forming bacteria from Example 1 after the culture test. [Figure 2]This is a photograph of the antibacterial rayon fiber containing spore-forming bacteria from Comparative Example 1 after the culture test. [Figure 3] This is a photograph taken after the completion of the antibacterial test using black mold in the example. [Figure 4] This is a schematic diagram of Figure 3(d). [Modes for carrying out the invention]

[0010] The present invention will be described in detail below based on preferred embodiments, but the present invention is not limited to the following embodiments, and various modifications are possible within the scope of the claims. In this invention, the notation "value 1 to value 2" in relation to a numerical range means a numerical range that includes both values ​​1 and 2, with value 1 as the lower limit and value 2 as the upper limit, and is synonymous with "value 1 or greater and value 2 or less".

[0011] The spore-forming bacteria-containing antibacterial cellulose fiber of the present invention (hereinafter referred to as "the fiber of the present invention") exhibits a colony occupancy rate of 50% or more in the following culture test. [Culture test] (1) In a petri dish with an inner diameter of 80 mm equipped with a standard agar medium, a cylinder with an inner diameter of 35 mm is placed on the standard agar medium in the center when the petri dish is viewed from above, and 0.05 g of antimicrobial cellulose fiber containing spore-forming bacteria is distributed approximately evenly within the cylinder, and then the cylinder is removed. (2) Add sterile water to the antimicrobial cellulose fiber containing spore-forming bacteria and mix it with the standard agar medium, then incubate at 35°C for 24 hours. (3) After culturing, the area of ​​colonies formed by spore-forming bacteria on the standard agar medium (hereinafter referred to as "colony area") is measured, and the colony occupancy rate is calculated using the following formula. Colony occupancy rate (%) = Colony area ÷ Inner diameter area of ​​petri dish × 100

[0012] The fibers of the present invention are those on which spore-forming bacteria with antibacterial activity have colonized. In the following description, even when simply referred to as "spore-forming bacteria", these "spore-forming bacteria" have antibacterial activity.

[0013] Cellulosic fibers are fibers composed mainly of cellulose and / or derivatives of cellulose. Generally, they can be classified into natural cellulosic fibers and regenerated cellulosic fibers. Examples of natural cellulosic fibers include hemp, cotton, etc. Examples of regenerated cellulosic fibers include viscose rayon, cuprammonium rayon, solvent-spun cellulose fibers, and high-strength cellulose fibers.

[0014] Preferred examples of spore-forming bacteria include spore-forming bacteria belonging to the genus Bacillus and spore-forming bacteria belonging to the genus Sporolactobacillus. Preferably, they are non-pathogenic, and a preferred example is of biosafety level 1.

[0015] Examples of spore-forming bacteria belonging to the genus Bacillus include Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus azotoformans, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus flexus, Bacillus Bacillus fusiformis, Bacillus globisporus, Bacillus glucanolyticus, Bacillus infermus, Bacillus licheniformis, Bacillus marinus, Bacillus megaterium, Bacillus mojavensis, Bacillus mycoides, Bacillus pallidus, Bacillus parabrevis, Bacillus pasteurii, Bacillus polymyxa, Bacillus popiliae, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis, Bacillus thermoamylovorans, Bacillus thuringiensis, etc. can be mentioned.

[0016] Examples of spore-forming bacteria belonging to the genus Sporolactobacillus include Sporolactobacillus inulinus, Sporolactobacillus kofuensis, Sporolactobacillus laevolacticus, Sporolactobacillus laevus, Sporolactobacillus nakayamae, Sporolactobacillus racemicus, Sporolactobacillus shoreicorticis, and Sporolactobacillus terrae.

[0017] A good example of a spore-forming bacterium belonging to the genus Bacillus is the BS-T2 strain (hereinafter referred to as "BS-T2 strain") deposited under accession number NITE P-02700. The BS-T2 strain is a spore-forming bacterium belonging to Bacillus subtilis and has a biosafety level of 1. The antibacterial activity of the BS-T2 strain includes direct antibacterial activity and indirect antibacterial activity. Direct antibacterial activity, also called contact antibacterial activity, refers to the antibacterial activity that the BS-T2 strain exhibits against bacteria in direct contact. Indirect antibacterial activity, also called remote antibacterial activity, spatial antibacterial activity, or non-contact antibacterial activity, refers to the antibacterial activity that the BS-T2 strain exhibits against bacteria through the space, that is, in situations where it does not directly come into contact with bacteria. In the fibers of the present invention containing the BS-T2 strain, the direct antibacterial activity is considered to contribute particularly to the exertion of its antibacterial properties.

[0018] In addition to antibacterial activity, the BS-T2 strain also possesses deodorizing properties. Therefore, the fibers of the present invention containing the BS-T2 strain can also exhibit a deodorizing effect.

[0019] (Culture test) The culture test examines the growth of spore-forming bacteria in antimicrobial cellulose fibers containing spore-forming bacteria. As is common practice in bacterial testing, it goes without saying that sterilized equipment, culture media, and chemicals should be used, and a clean bench or similar environment should be used to prevent contamination by other bacteria.

[0020] In the culture test described in (1) above, the method for preparing a petri dish with an inner diameter of 80 mm equipped with standard agar medium is as follows: 23.5 g of Nissui standard agar medium (granules for total viable cell count measurement, Code 05618) manufactured by Nissui Pharmaceutical Co., Ltd. is mixed with 1 L of purified water, sterilized by autoclaving, dispensed into a petri dish with an inner diameter of 80 mm, and cooled to solidify.

[0021] When placing the cylinder with an inner diameter of 35 mm on the standard agar medium at the center of the petri dish when viewed from above, it is preferable that the center of the cylinder coincides with the center of the petri dish. However, in practice, such precision is not necessary, so it is sufficient to place it so that the center of the petri dish is inside the cylinder. The cylinder should be made of a material that does not easily generate static electricity, or a material that removes static electricity. It is preferable to use the one that has been removed.

[0022] Regarding the arrangement of 0.05 g of antimicrobial cellulose fibers containing spore-forming bacteria on a standard agar plate, distributing them approximately evenly within the cylinder, "distributing them approximately evenly" means distributing them as evenly and without bias as possible.

[0023] In the culture test described in (2) above, the amount of sterile water added should be such that the antimicrobial cellulose fibers containing spore-forming bacteria are moistened and come into overall contact with the standard agar medium.

[0024] In the culture test described in (3) above, the method for measuring the colony area may be a conventional method, such as the graph paper method, parallel line method, or gravimetric method, or software capable of measuring area may be used.

[0025] A higher colony occupancy rate indicates a higher level of functional expression of spore-forming bacteria. As shown in Comparative Example 1 below, the colony occupancy rate of the antibacterial rayon fiber containing spore-forming bacteria manufactured according to the description in Manufacturing Example 1 of Patent Document 1 was a low 23.0%. One possible reason for this is that the spore-forming bacteria were kneaded into the fiber, and therefore the spore-forming bacteria that settled inside the fiber could not contribute to colony formation. In contrast, the colony occupancy rate of the fiber of the present invention is 50% or higher. As is clear from the manufacturing method described below, it is presumed that in the fiber of the present invention, the majority of spore-forming bacteria settle not inside the fiber but in the surface region (the surface and the part slightly inward from the surface), and this is thought to contribute to the colony occupancy rate of 50% or higher. The colony occupancy rate of the fiber of the present invention is preferably 55% or higher, and more preferably 60% or higher.

[0026] In the fibers of the present invention, spore-forming bacteria can survive as vegetative cells and / or spores depending on the environment in which the fibers are placed. Under normal activity, when spore-forming bacteria repeatedly divide and multiply, they exist as vegetative cells and can move within the fibers or multiply by coming into contact with suitable nutrients. On the other hand, under unfavorable conditions for growth, such as low moisture, poor nutrition, low oxygen, and high temperature, they form spores and remain in a spore state. For example, during washing or high-temperature drying in a dryer, the conditions are unfavorable for spore-forming bacteria, so it is thought that the spore-forming bacteria survive as spores.

[0027] Furthermore, spore-forming bacteria can colonize the fibers of the present invention for a long period of time, thereby enabling the long-term maintenance of antibacterial properties and the suppression of musty odors even after repeated washing.

[0028] The number of spore-forming bacteria present in the fibers of this invention is not particularly limited, as it increases or decreases depending on environmental conditions, and any number can be taken. From the viewpoint of exhibiting antibacterial properties, for example, 1.0 × 10⁶ bacteria per gram of fiber. 3 ~1.0×10 10 A range of CFU is preferred, and more preferably 1.0 × 10⁻⁶. 5 ~1.0×10 10This falls under the scope of CFU.

[0029] A preferred embodiment of the fibers of the present invention is a nonwoven fabric. Methods for manufacturing a nonwoven fabric composed of the fibers of the present invention include dry methods (chemical bonding method, thermal bonding method, needle punching method, water flow entanglement method), wet methods, and airlaid methods. A good example is a method in which fleece is formed by the dry method and then the fibers are entangled by the needle punching method.

[0030] In recent years, nonwoven fabrics have been used in various fields, and are often used as agricultural materials. A typical example is their use in direct covering. Direct covering is a method of covering the ridges of a field with nonwoven fabric after sowing, and this is expected to have effects such as heat retention, water retention, frost prevention, and temperature rise suppression. The nonwoven fabric that is covered will eventually decompose, and it is thought that soil microorganisms play a major role in this process. The nonwoven fabric composed of the fibers of the present invention (1) spore formation The protective effect against various bacteria is enhanced by two mechanisms: (1) physical elimination due to the bacteria settling as a preferred species on the fibers of the present invention, and (2) chemical elimination due to the antibacterial activity of spore-forming bacteria, thereby slowing down the decomposition process.

[0031] The present invention provides a method for producing fibers, which includes a step of mixing cellulosic fibers, surface-treated with a dilute strong alkaline aqueous solution, with spore-forming bacteria.

[0032] A dilute strong alkaline aqueous solution is a dilute aqueous solution of a strong alkali. Examples of strong alkalis include sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium carbonate. The concentration of the strong alkali in the dilute strong alkaline aqueous solution is preferably in the range of 0.01 to 0.5 N.

[0033] A preferred method for surface-treating cellulose fibers with a dilute strong alkaline aqueous solution is to immerse the cellulose fibers in the dilute strong alkaline aqueous solution for a predetermined time. The immersion time can be appropriately set according to the concentration of the dilute strong alkaline aqueous solution and the desired degree of surface treatment, but it is preferably in the range of 10 minutes to 1 hour. Examples of immersion times within the above range include 15 minutes, 20 minutes, 30 minutes, 45 minutes, etc.

[0034] While vegetative cells may be used as spore-forming bacteria, it is preferable to use spores from the viewpoint of increasing the survival rate.

[0035] The method for mixing cellulose fibers, which have been surface-treated with a dilute strong alkaline aqueous solution, with spore-forming bacteria is not particularly limited, but one preferred method is to keep the cellulose fibers immersed in the dilute strong alkaline aqueous solution even after surface treatment, add the spore-forming bacteria thereto, and then stir.

[0036] The reason why surface treatment of cellulose fibers with a dilute, strong alkaline aqueous solution makes it easier for spore-forming bacteria to colonize them is not entirely clear. However, it is thought that the surface of the cellulose fibers becomes negatively charged after surface treatment with the dilute, strong alkaline aqueous solution, and this is presumed to contribute to the colonization of spore-forming bacteria. In particular, since spores are said to be positively charged according to one theory, the negative charge on the fiber surface is thought to be advantageous for colonization.

[0037] It is preferable to include a neutralization step with an acid after the above steps. A dilute aqueous acid solution is preferred as the acid; for example, a 0.1 mL / L aqueous acetic acid solution is preferred. Furthermore, a dehydration step and a drying step may be included as needed. [Examples]

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0039] [Example 1] Viscose (cellulose content: 9% by mass, alkali content: 5% by mass, pH 14) was used as the spinning stock solution. A spinneret (pore size: 0.06 mm, number of nozzles: 13,000) was impregnated in a 47°C coagulation bath (sulfuric acid aqueous solution containing 95 g / L sulfuric acid, 350 g / L sodium sulfate, and 12.5 g / L zinc sulfate). The spinning stock solution was then extruded from the nozzle of the spinneret at a spinning speed of 60 m / min and coagulated to spin the fibers. After being stretched, the fibers were cut. This yielded rayon fibers (hereinafter referred to as "ordinary rayon") with a fineness of 1.4 decitex and a fiber length of 38 mm. Ordinary rayon is immersed in a 20g / L sodium carbonate aqueous solution for 15-20 minutes, and then heated to 50°C. The temperature was then raised. Next, while maintaining the immersion state, a suspension of BS-T2 strain was added. The BS-T2 strain suspension was prepared by adding the suspension of BS-T2 strain (bacterial count: 4.2 × 10) to 100 parts by mass of ordinary rayon. 8 The CFU / mL was added in a proportion of 10 parts by mass. After addition, the mixture was thoroughly stirred for 30 minutes. Next, it was neutralized with a 0.1 mL / L aqueous acetic acid solution, dehydrated, and dried at approximately 100°C. This yielded the spore-forming bacteria-containing antibacterial rayon fiber of Example 1.

[0040] [Comparative Example 1] In accordance with the description in Manufacturing Example 1 of Patent Document 1, the spore-forming bacteria-containing antibacterial rayon fiber of Comparative Example 1 was obtained by the following manufacturing method. A suspension of BS-T2 strain in spore form (bacterial count: 4.2 × 10⁴) is prepared in 100 parts by mass of viscose (cellulose content: 9% by mass, alkali content: 5% by mass, pH 14). 8A spinning stock solution was prepared by mixing a suspension of viscose and BS-T2 strain so that the amount of CFU / mL was 10 parts by mass. Next, a spinneret (pore size: 0.06 mm, number of nozzles: 13,000) was impregnated in a 47°C coagulation bath (sulfuric acid aqueous solution containing 95 g / L sulfuric acid, 350 g / L sodium sulfate, and 12.5 g / L zinc sulfate). The spinning stock solution was then extruded from the nozzle of the spinneret at a spinning speed of 60 m / min and coagulated to spin the fiber, which was then stretched and cut. This yielded the spore-forming bacteria-containing antibacterial rayon fiber of Comparative Example 1, with a fineness of 1.4 decitex and a fiber length of 38 mm.

[0041] The antimicrobial rayon fibers containing spore-forming bacteria from Example 1 and the antimicrobial rayon fibers containing spore-forming bacteria from Comparative Example 1 were subjected to the following culture tests.

[0042] [Culture test] (1) 23.5 g of Nissui standard agar medium (granules, for measuring total viable cell count, Code 05618) manufactured by Nissui Pharmaceutical Co., Ltd. was mixed with 1 L of purified water and sterilized by autoclaving. The mixture was then dispensed into a sterilized petri dish (φ86 x 13.5 mm) manufactured by Atect Co., Ltd. (product number: 3101011-70101; inner diameter of petri dish body for measurement: 80 mm) up to the line provided on the inner wall of the petri dish body, and cooled to solidify. A cylinder with an inner diameter of 35 mm was placed on the standard agar medium in the center of the petri dish when viewed from above. 0.05 g of antimicrobial rayon fibers containing spore-forming bacteria from Example 1 or Comparative Example 1 was distributed approximately evenly within the cylinder, and then the cylinder was removed. (2) An appropriate amount of sterile water was added to the antimicrobial rayon fiber containing spore-forming bacteria, and after mixing it with the standard agar medium, it was incubated at 35°C for 24 hours. (3) After culturing, the colony area was measured. The image analysis software "A-zo-kun" sold by Asahi Kasei Engineering Corporation was used to measure the colony area. The colony occupancy rate was calculated using the following formula. Colony occupancy rate (%) = Colony area ÷ Inner diameter area of ​​the petri dish × 100

[0043] Photographs used to measure the colony area are shown in Figures 1 and 2. Figure 1 is Example 1, and Figure 2 is Comparative Example 1.

[0044] The colony occupancy rate was 66.9% for Example 1 and 23.0% for Comparative Example 1. This indicates that the antimicrobial rayon fiber containing spore-forming bacteria in Example 1 exhibits greater functional expression than the antimicrobial rayon fiber containing spore-forming bacteria in Comparative Example 1.

[0045] [Antibacterial testing] The spore-forming bacteria-containing antibacterial rayon fiber from Example 1 (hereinafter also referred to as "antibacterial rayon") and ordinary rayon were blended in the proportions shown in the "Mixing Ratio (mass ratio)" column of Table 1 to prepare a nonwoven fabric. Test specimens were prepared by punching out this nonwoven fabric with a 17 mm inner diameter hole punch.

[0046] The test organism used was black mold (Cladosporium cladosporioides). The black mold spore solution (concentration: 1.0 × 10⁻⁶) 4 ~ 6 100 μL of CFU / mL was spread onto PDA (potato dextrol agar) medium in a petri dish with an inner diameter of 80 mm. Next, the test specimen was placed on the medium in the center of the petri dish and incubated at 25°C for 72 hours.

[0047] Photographs after culturing are shown in Figure 3. In test specimen No. 1, which contained only ordinary rayon, black mold was scattered on the specimen (see Figure 3(a)). On the other hand, in test specimens No. 2 to 4, a halo (zone of inhibition) was formed due to the inclusion of antibacterial rayon, and the clear zone of the inhibition became clearer as the proportion of antibacterial rayon increased (see Figures 3(b) to (d), Figure 4).

[0048] Furthermore, since the halo width increased as the proportion of antibacterial rayon increased (see the "Halo Width" column in Table 1), it was concluded that the magnitude of the antibacterial effect by spore-forming bacteria depends on the number of bacteria.

[0049] The halo widths in Table 1 were calculated using the following formula. Harrow width (mm) = (Harrow diameter - Specimen length) / 2 Here, the halo diameter is the longest line segment among those whose ends intersect with the clearly defined outer edge of the halo (growth inhibition zone) in a straight line passing through the center of the specimen.

[0050] The criteria for evaluating antimicrobial activity in Table 1 are as follows: -: No antimicrobial activity, +: Halo width greater than 0 mm and 3.0 mm or less, ++: Halo width greater than 3.0 mm and 6.0 mm or less, +++: Halo width greater than 6.0 mm.

[0051] [Table 1]

Claims

1. Antimicrobial cellulose fibers containing spore-forming bacteria, exhibiting a colony occupancy rate of 50% or more in the following culture tests. [Culture test] (1) In a petri dish with an inner diameter of 80 mm equipped with a standard agar medium, a cylinder with an inner diameter of 35 mm is placed on the standard agar medium in the center when the petri dish is viewed from above, and 0.05 g of antimicrobial cellulose fiber containing spore-forming bacteria is distributed approximately evenly within the cylinder, and then the cylinder is removed. (2) Add sterile water to the antimicrobial cellulose fiber containing spore-forming bacteria and mix it with the standard agar medium, then incubate at 35°C for 24 hours. (3) After culturing, the area of ​​colonies formed by spore-forming bacteria on the standard agar medium (hereinafter referred to as "colony area") is measured, and the colony occupancy rate is calculated using the following formula. Colony occupancy rate (%) = Colony area ÷ Inner diameter area of ​​petri dish × 100

2. A nonwoven fabric composed of antimicrobial cellulose fibers containing spore-forming bacteria as described in claim 1.

3. Agricultural material comprising the nonwoven fabric described in claim 2.

4. A method for producing antimicrobial cellulose fibers containing spore-forming bacteria, comprising the step of mixing cellulose fibers surface-treated with a dilute strong alkaline aqueous solution with spore-forming bacteria.

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