JPS6283877A - Inhibitor of growth and proliferation of bacteria - Google Patents

Abstract

PURPOSE:The titled inhibitor that contains, as an active ingredient, chitosan or slightly decomposed chitosan, thus being effective in prevention and inhibition of bacterial growth and proliferation safely without injury to human. CONSTITUTION:The titled inhibitor contains chitosan or slightly decomposed chitosan as an active ingredient. The chitosan may have any deacetylation degree, but 50-100% is preferred. The slightly decomposed chitosan preferably contains less than 120mg D-glucose/1g of chitosan as reducible saccharides. The concentration of chitosan or slightly decomposed chitosan is 0.2g/l, preferably ranging from 0.2-2g/l in the place where inhibition and proliferation of bacteria are not desired.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a bacterial growth and proliferation inhibitor, and more particularly to a bacterial growth and proliferation inhibitor comprising chitosan or a mildly degraded product thereof. The bacterial growth and proliferation inhibitor of the present invention can inhibit the growth and proliferation of not only bacteria but also mold. It can be used for.

[Technical Background and Summary of the Invention]

Benzoic acid, benzoate, sorbic acid, sorbate, dehydroacetic acid, dehydroacetate, hydroxybenzoic acid ester, propionic acid, propionate, salicylic acid, salicylate, phenol as a preservative and disinfectant for food or cosmetics, etc. There are many synthetic products such as parachlormetacresol, hexachlorophene, trichlorocarbanilide and benzalkonium chloride, but because these preservatives are synthetic, they cannot be used as food or cosmetic additives. Recently, there has been a strong tendency to restrict the use of synthetic preservatives and sterilizers, as they may pose safety problems for the human body when used as antiseptics.

On the other hand, chitosan is a saccharide obtained by deacetylating the chitin contained in shrimp, crabs, etc., and D-glucosamine is bonded to a straight chain via a β-1,4 chain. Chitosan, which is a multi-U class compound and has a low degree of polymerization obtained by decomposing chitosan, is also known. Methods for decomposing chitosan include hydrolysis with hydrochloric acid, oxidative decomposition with nitrous acid, and oxidative decomposition with salt accumulation. chemical methods such as
and enzyme (chitosanase) methods. Bacillus (llaci) is a microorganism that produces chitosanase.
llus Sp, ) R-4 (h Minaga et al.: Biohimi Power, Engineering, Biofiji Power, Acta (y, Tomin
agaet al: Biochimica et al.
Blophysica Acta) M1110 No. 145-155 (1957)], Penicillium
Penicilliumislan
(Dicum) (D.M. Fenton et al.: Journal of General Microbiology (Skin M-Fenton et at Journal of
enera1Microbiology) 12th
6 volumes m 151- +6500 (1981)], Bacillus ([1aeillus 5p, ) 99-5 (
Kutsunai: Japanese Society of Agricultural Chemistry, Abstracts of the 1981 Conference No. 550), Streptomyces
es) No, 6 C J. Nis. Price et al.:
Journal of Bacteriology (J, S, Pr
1ce et al: Journal of Bact
criologY) Volume 124, No. 1574-1584
N (1975)], Streptomyces griseus
(Ohtakara et a.
l: Chltin+ Chitosanand R
e1ated Enzymes) No. 147-16
0 page (1985) Academic Press] and Bacillus (Bacillus sp, ) Na 7-M
' (Patent Application No. 120673) and that chitosan affects the growth of plant-pathogenic molds [B. Niss-Stoessel et al.
:Phytopathologysche Zett
sehrift) Volume 1II, pages 82-89 (19
1984), C.R.A11an et at.
: Experimental Mycology
) 3rd y 285-287 F! (1979)
] and affecting the suppression of mold growth in the decomposition product of chitosan [D.F.Kendra++! 1
: Experimental Mycology (Hada F, Ken
Dra et at ExperiIIIental
Mycology) m8Q 1276-281 ffl
41984)] However, nothing is yet known about the inhibition of bacterial growth by chitosan and chitosan decomposition products.The present inventors have been conducting research on chitosan for many years. In that research, chitosan was found to be effective not only in inhibiting the growth and proliferation of mold, but also in inhibiting the growth and proliferation of IH, and that Bacillus no.
7- H and produced '': Chitokun decomposed by tosanase jσ'T) 'tJ n: Yotoj + E for suppressing the growth and proliferation of Yoribi and 1 so 7 + T ul i 1 = and feather 1 blade; Summary of the Invention The object of the present invention is to provide an inhibitor of bacterial growth and proliferation that is not only effective in inhibiting and inhibiting the growth of bacteria, but also effective in inhibiting the growth and inhibition of mold. Specifically, the object is to provide an inhibitor of bacterial growth and proliferation that is harmless to the human body and can be used safely.

The present invention is a bacterial growth and proliferation inhibitor containing chitosan or a decomposed product of chitosan as an active ingredient. In the present invention, the amount of chitosan produced is 12
0■・D-glucosamine/19・It is preferable that the amount is not more than that of chitosan, and the mildly degraded chitosan can be used to convert chitosan into Bacillus No., 7-@ (R1 Lab. It is preferable that the enzyme be one that has been degraded by chitosanase produced by

[11 Specific details of the invention] The chitosan with the efficacy of the III agent for bacterial growth and proliferation of the present invention has the following properties: Although this can be used, it is preferred to use a degree of deacetylation of 50 to 100%. The mild decomposition product of chitosan is its reduced MHk
It is preferable to use one in which the amount is not more than 120 .D-glucosamine/I9.chitosan.

The reduction MWk shown by m9・D-glucosamine/Ig・chitosan is a value obtained by converting the reducing power of the mildly degraded chitosan of 11 to the reducing power of D-glucosamine, and is 1112711g・D-glucosamine/I9・chitosan. is a numerical value indicating that chitosan has been decomposed 100% into its constituent D-glucosamine, and has been decomposed into D-glucosamine.

Chitosan or a mildly degraded chitosan product can be used at least o
, zy/l (preferably in the range of 0.2 to 2 g/l)
It is used in an amount of Chitosan or a mildly decomposed product of chitosan can be used alone, but also in the form of a composition with a solid or liquid inert carrier.

The solid or liquid inert carrier in the composition for inhibiting bacterial growth and proliferation can be any of the carriers in common food additives, but depending on the use of the composition. However, it is not necessarily limited to carriers used for food additives, and clay, kagirin, vermiculite, perlite, etc. can also be used.

To produce a mildly decomposed product of chitosan using chitosanase, first, chitosan is dissolved in an acid aqueous solution to prepare a chitosan aqueous solution, which is preincubated at a reaction temperature. In addition, the temperature is 30 to 80°C (preferably 4
Chitosan is decomposed by chitosanase at a reaction temperature of around 0"C). The pH of the reaction solution varies depending on the action pH of chitosanase, but is usually 3 to 9 (preferably around 6). Colloidal chitosan is used as the raw material chitosan. If so, it may be suspended in water.Any acid used to prepare the chitosan solution may be used as long as it can dissolve chitosan. However, it is preferred to use dilute solutions of hydrochloric or nitric acid, formic acid, acetic acid, glutamic acid or ascorbic acid.

Since the degree of decomposition of chitosan varies depending on the reaction conditions such as temperature, pII, and reaction time, it is preferable to determine the reaction conditions necessary to obtain the desired degree of decomposition in a preliminary experiment and use these. It is easy to determine the degree of decomposition of chitosan by the production-reduction electrification of the reaction product (rv・D-glucosamine/19・chitosan), and usually, the production-reduction Wffi
It is preferable that the amount is not more than 120 .D-glucosamine/Ig.chitosan.

The decomposition of chitosan is carried out by Bacillus sp.
, )lh7-M (Feikoken Bacterial Serial No. 8139).

Bacillus No. 7-M is a bacillus (Aeillus sp- ) No, 7F
Using J as the parent strain, this strain was treated with N-methyluda-nitroso-N-nitrosoguanidine (NTC) to induce mutations, and highly active chitosanase was produced from among the resulting streptomycin-resistant mutants. It is a mutant strain that has been axiomatically considered to be capable of
No. (FERM P-8139) and has been deposited with the Microbial Technology Research Institute of the Ministry of International Trade and Industry.

The mycological properties of Bacillus No. 7-H are shown below.

A. Cell morphology (1) Cell shape and size: Brachybacillus, (broth and broth agar slant culture, culture at 37°C for 124-72 hours) (2) Presence or absence of cell pleomorphism: None, (3 ) With or without motility, (broth agar semi-fluid high-layer puncture culture) (4) With or without spores: present, endospores and naked spores, spherical, [Dorner's a! One-color method and modified Wttz method] (5) Durham Q color: positive, [broth agar slant culture, 37°6118 hours, Hüyükka-
(Stained by a modified method of (1lucker)) B. Growth status in each medium ()
Time): Forms a round, raised, milky-white colony with a filamentous periphery. The surface of the colony is uneven, slightly shiny, and translucent. It will increase as time passes. No pigment is produced. (2) Juicy agar slant culture (37°C, 124 to 168 hours). Two M1 milky white colonies are formed in the rice field.

Colonies have convex circular ridges and are shiny. It grows well and will spread over time. Does not produce pigment.

(3) Broth liquid culture (37°C, 24-168 hours): No film is formed on the surface. The whole thing becomes cloudy over time. Granular sediments are formed at the bottom and gradually increase in number.

(4) Meat juice gelatin puncture culture (25°C124-168
Time): Grows along the perforated lateral line and liquefies. The surface and inside of the dome grow in a Q$ shape and liquefy. The liquefied part becomes cloudy.

(5) Lidmus milk (378C 124-168 hours)
= After 2 days, the upper part gradually liquefied, and on the 4th day, the color completely changed and became acidic. It does not coagulate.

As time passed, liquefaction progressed and it became translucent.

C0 Physiological Properties (1) Reduction of nitrate (nitrate broth medium, 37°0124-120 hours) (2
) Denitrification reaction knee (■ method of Kata et al., using fermentation tube, 37℃, 24-120℃)
(3) MR test: + (37℃, 24-168 hours) (4) VP test (acetyl methyl carbinol formation test 20 (37℃, 124-168 hours) (5) Indole formation knee (37℃) , 24-168 hours) ([5) Generation of hydrogen sulfide (TSr agar method, 37°C, 24-168 hours) (7) Hydrolysis of desi powder: + (37°C, 24-168 hours) (8) Utilization of citric acid (Koser's medium, 37°C, 24-168 hours): - (Christensen's medium, 37°C, 124-168 hours) Utilization of Nitrogen source (37°C, 124-168 hours) ) Nitrate: undetermined, ammonium salt: undetermined, (10) Pigment production (mannitol/yeast extract agar slant 11!1): [King A agar slant medium Coni (11)
Presence or absence of fluorescence: None 12) Urease: + (Christensen-urea agar medium, 37°0124~
(168 hours) (13) Oxidase: + (Meat juice agar medium, 37°C 124-48 hours) (14)
Catalase: + (broth agar, 37°0124-48 hours) (15)
Growth range = (gravy agar medium) Temperature: undetermined, pH: 5-10, Saline concentration: undetermined, (16) Attitude towards oxygen: aerobic (1% glucose gravy high-rise agar medium, 37°C 124~
72 hours) (17) 0-F test [Hugh Lifeson (Ilug)
h-Leifson) method, 37°C1D-glucose]: Acid is produced fermentatively.

(fermentative) (1s) Presence or absence of acid and gas generation from M group (378
C124-168 hours): Sugar Acid Gas D-glucose + - D-mannose - D-galactose - D-fructose + - L-arabinose - D-xylose - D-sorbitol - D-mannitol - − Wild boar − − Maltose 10 − Saducalose ′+− Lactose − − Starch + − Cellulose − Glycerin − − Regarding the above mycological properties, please refer to the Virgies Manual of Determinative Bacteriology.
rgey's! 4annual of Determinition
When I searched for the 8th edition (1974) of Inative Oral Actiology, No. 7-M
It was found that B corresponds to Bacillus K.

The enzymatic chemical properties of chitosanase produced by Bacillus No. 7-H are as shown below.

(1) Action: Acts on chitosan and optionally removes β-1,4 from the internal chain of the molecule.
By decomposing the bonds, mainly chitosan oligosaccharide (C; 1c
N) (n==2 to 8) (lfi bodies to 8 sacrifice bodies) are generated. Chitosan oligosaccharides can be separated from the chitosan decomposition solution using high performance liquid chromatography.

The decomposition degree of chitosan in this decomposition liquid is about 45%. It also acts on carboxymethyl cellulose (CMC),
It decomposes this to some extent, but it has no effect on chitin.

(2) Working temperature range and optimal working temperature: When soluble chitosan is used as a substrate, it works up to 80' (:), and the optimal working temperature is 506C.

FIG. 1 shows the relationship between temperature and specific activity when reacting for 10 minutes at pl+ 6.0.

(3) Working pH range and optimum pH: It acts in the pH range of 3 to 9, and the optimum pH is p116.
It is.

Add 9 liters of 1% soluble chitosan to 2 liters of buffer at each pH.
FIG. 2 shows the relationship between pFI and specific activity of the enzyme when a reaction solution to which 1 rnll of enzyme solution was added was reacted at 376C for 10 minutes.

(4) Thermal stability: Stable for up to 15 minutes at 50°C;
Heating for 15 minutes at 0.06C reduced the enzyme to about 409
6 was deactivated by heating at 70 °C for 15 min.
Completely deactivated.

Figure 3 shows the relationship between temperature and specific activity.

(5) pH stability: Residual enzyme activity was measured after 2 hours at 30°C in 0.1 M buffer, and was stable in the pH range of 5 to 11. One of the major characteristics of chitosanase produced by Bacillus No. 7-H is that it is stable at pH 10-11. The relationship between pl+ and specific activity is shown in FIG.

(6) Inhibitors: Chitosanase produced by Bacillus Na7-M is inhibited by lIgcI, PbCl at a final concentration of IxlOH.
, AgN0, and PCMB resulted in 100% inhibition.

(7) Substrate specificity: When different substrates are used and the final concentration of the substrate is 0.25%, the total reductive shearing rate is determined after 1 hour by 4 nLi of the enzyme reaction solution and 1 liter of enzyme-rich white matter. and hexosamine@C
m9/rrLIB white matter/hour) was measured.

The results are shown in the IT table.

(Leaving space below) Table 1 1/4 size *: Chitosanase produced by Bacillus No. 7-H by the Reissig method is highly effective at dissolving colloidal chitosan, soluble chitosan, and glycol chitosan. It decomposed, and carboxymethylcellulose (CMC) was also slightly decomposed, but it did not affect powdered chitosan. In addition, co-idal chitin, glycol chitin, powdered chitin, and methylcellulose were not decomposed at all.

(8) Molecular weight: Figure 5 shows the results of measuring one molecule by 5DS-polyacrylamide electrophoresis. In Figure 5 (○)
is the molecular weight of chitosanase produced by Bacillus No. 7-H, which is approximately 41,000.

The results of molecular weight measurements by gel filtration using Sephadex G-100 are shown in Figure 6. In Figure 6, (O) is the molecular weight of chitosanase produced by Bacillus No. 7-H, approximately 30,000.

(9) Enzyme titer measurement method: 1g of powdered chitosan (28 mesh) at 50rrLI!
After dissolving the solution in 0.1M acetic acid aqueous solution and adjusting the pH to 6.0 with O.1M sodium acetate aqueous solution, add O.1M acetic acid solution i? 2 (IIH: 6.0) to make the total 10
At 0n'LA, a. Prepare a 1% soluble chitosan solution. Incubate at 37°C for 5 minutes. Add 1 mJ of 1% soluble chitosan diagnostic solution to
Add 1 ml of the enzyme solution that was incubated in the same manner as in Step 14, and allow the enzyme reaction to occur at 37°C for exactly 10 minutes. After that, the reaction solution was boiled for 3 minutes to stop the enzyme reaction.
The reducing sugar produced in the reaction solution is stabilized.

Under these conditions, the enzyme that transilluminates reducing sugars corresponding to 1 B+ moles of glucosamine is defined as 1 unit of chitosanase activity.

〔Effect of the invention〕

Chitosan and mildly degraded chitosan products of the present invention can not only prevent or suppress the growth and proliferation of bacteria, but also the growth and proliferation of mold.

Since chitosan and chitosan l1iI decomposition products are derived from materials that have been used for food since ancient times, they are not harmful even when used as food additives.

The present invention will be explained in more detail below using reference examples and test examples that can be substituted for the examples.

Reference Example 1 (Preparation of seed culture) 250 ml; In a Erlenmeyer flask, yeast extract 0.8%
, a liquid medium containing 0.4% peptone, 0.2% meat extract, and 0.5% colloidal chitosan (pH: 7.2)
After adding 50 TrLil and sterilizing it by a conventional method, Bacillus sp.
) No. 7-M (FERMP-8139) was inoculated and cultured with shaking at 30°C for 1 day.

(Preparation of culture solution for enzyme production) 51! Fill two Erlenmeyer flasks with 1 liter of a liquid medium having the same composition as above, and sterilize them using the usual method.
This was inoculated with 40rni of the seed culture obtained above, and
The cells were incubated at 0°C for 4 days with shaking. 5. Culture solution.
The microbial cells were removed by centrifugation at 00 Or, pom, and the chitosanase activity of the obtained supernatant was determined by the enzyme titer ffl! It was measured by the standard method. Supernatant liquid 1 rr
L7! The hit was about 0.991Ji.

(Purification of enzyme solution) Mix the supernatant obtained above and mix the obtained mixture i1.
81/L: Solid ammonium sulfate 1,015 fi (ammonium sulfate 8
(corresponding to 0% saturation) was added, filtered, and the resulting precipitate was dissolved in distilled water to make 177 ml. This enzyme solution was mixed with steamed water and then 0.02 M phosphate buffer (pH
: After dialysis against 6.0), the obtained enzyme solution was
Chitosanase was adsorbed by flowing it through a column (2.6 cm (diameter) x 45 m' (length)) packed with CM-Sephadex C-50 equilibrated with 0.02 M phosphate buffer in advance.Haton Any impure protein was collected in the flow-through section. After washing the column with 350 ml of 0.02M phosphate buffer, the enzyme protein was eluted with 0-0.5M sodium chloride using a linear concentration gradient.

Next, the 218th to 240th waracin 3 that showed chitosanase activity were combined and concentrated 17 times using an ultrafiltration device using a diaflow membrane filter PM-10 (product of Amicon). Sephadex G in liquid
Gel filtration using -100 was performed.

Nos. 50 to 63, which showed chitosanase activity of this gel filtration.
Combine the fractions and add again CM-Sephadex C-
Column chromatography using 50 was performed. The enzyme was adsorbed under the same conditions as the previous time, and the enzyme protein was eluted using a linear FA gradient using 0 to 0.5 M sodium chloride.

Reference Example 2 (Preparation of chitosan decomposition product) (Preparation of sample of chitosan decomposition product A) Add 50% of distilled water to 1g of chitosan, and add 1M acetic acid 9% to this.
- was added and sufficiently stirred to dissolve it, 1M sodium acetate aqueous solution was added thereto, the pH was adjusted to 6.0, distilled water was added to bring the total volume to 100-, and a 1% chitosan solution was added. Prepared.

Take 1% chitosan solution 5- into a test tube and add Reference Example 1 to it.
Dilute the chitosanase solution obtained in 30 units with water.
Add 0.1- of chitosanase solution with an activity of /-,
After reacting for 3 hours in a 37°C incubator,
The test tube was immersed in a boiling bath and boiled for 5 minutes to stop the reaction, and chitosan decomposition product A was prepared.

The reducing sugar ff1 (*g・D-glucosamine/1g・chitosan) produced from chitosan decomposition product A was
s) K method [T. Imoto: Agricultural Natural
Biological Chemistry (T, imoto:
Agricultural Biological Ch.
emistry ) Volume 35 No. 1154-1156 m
(1971)], the result was 59 (L19
・D-Glucosamine/19・Chitosan.

(2) Preparation of sample of chitosan decomposition product B Using a chitosanase solution with an activity of 5 units/incubation, proceed in the same manner as described above.
Production of z6amg.O-glucosamine/Ig.chitosan A sample of decomposed product 8 of reduced candied chitosan was prepared.

(3) Preparation of sample of chitosan decomposition product C Using a chitosanase solution with an activity of 3 units/- as the chitosanase solution, in the same manner as in (1),
Production of 100 mg D-glucosamine/1.9 chitosan A sample of chitosan decomposition product C was prepared in an amount of reduction W. (4) Preparation of sample of chitosan decomposition product Using the solution, proceed as in (1) for 40
(2) Production of 0-glucosamine/Ll.Chitosan A sample of chitosan decomposition product with reduction activity was prepared.

(5) Preparation of sample of chitosan decomposition product E Using a chitosanase solution with an activity of 0.5 units/ml, prepare a sample of 8.D-glucosamine/1g.chitosan in the same manner as in (1). A sample of chitosan decomposition product E of produced reduced Wffi was prepared.

(6) Preparation of sample of chitosan decomposition product F 0.05 unit 7ml as chitosanase solution
Using the activated chitosanase solution, a sample of chitosan decomposition product F of the reduced Wffi was prepared in the same manner as in (1).

(7) Preparation of chitosan sample Instead of chitosanase solution, 0.05 M acetate buffer (
pH: 6.0) and 4% in the same manner as in (1).
Production of g-D-glucosamine/1g of chitosan A sample of chitosan was prepared in an amount of reduced M.

Test Example 1 A test was conducted on the effect of chitosan on bacterial growth.

(1) Preparation of sample (+-1) Preparation of sample (1-1) After dissolving 10 g of meat extract, 10 g of peptone, and 5 g of sodium chloride in distilled water and adjusting the pI+ to 6.0,
A bouillon medium was prepared by adding distilled water and bringing the total volume to 1 liter. Sample 1- of chitosan of Reference Example 2, which had been appropriately diluted, was added to this bouillon medium 8.9- to prepare a sample (1-1) having a final chitosan concentration of 0.015%.

(1-2) Preparation of sample (t-2) Except that the final concentration of chitosan was 0.02% (+
Sample (1-2) was prepared in the same manner as in -1).

(+-3) Preparation of Sample (G-1) (1-1) Bouillon medium 8.9 A solution of glucosamine hydrochloride in water! A sample (G-1) with a final concentration of glucosamine hydrochloride of 5% was prepared.

(1-4) Preparation of Sample H (G-2) Sample (C-2) was prepared in the same manner as in (1-3) except that the final concentration of glucosamine hydrochloride was 0.6%. .

(1-5) Preparation of sample ((, -3) Sample (G-3) was prepared in the same manner as (t-3) except that the final concentration of glucosamine hydrochloride was 0.7%.

(1-6) Preparation of control sample Add 1 ml of the solvent used in (1) trial preparation of Reference Example 2 to the bouillon medium 8.9- in (1-1),
This was used as a control sample.

(2) Test method 0.1- of the culture of the test bacteria in Table 2 was applied to each sample, cultured with shaking at 30°C for 24 hours, and the formation of turbidity was observed. (+) and no turbidity (-). The occurrence of turbidity indicates the growth of the test bacteria.

(3) Test results The results of the test were as shown in Table 2.

(Left below) According to Table 2, chitosan inhibits the growth of all test bacteria and exhibits antibacterial properties at a final concentration of 0.02%, while glucosamine hydrochloride exhibits antibacterial properties at a final concentration of 0.7%. %, it inhibited the growth of all the tested plants and only exhibited @bacterial properties.

This shows that chitosan exhibits antibacterial properties at a concentration approximately 1/40 that of glucosamine hydrochloride.

Test Example 2 A test was conducted on the effect of chitosan on the proliferation of Escherichia coli.

(1) Preparation of sample (1-1) Preparation of control sample Dissolve the meat extract log, peptone 1θy and 5 g of sodium chloride in distilled water, adjust the pI to 6.0 4 times a week, then add distilled water. Adjust the total volume to 500 ml to prepare a double concentration bouillon medium, take 2.5 ml of it into a test tube,
A control sample containing no chitosan was prepared by adding 0.04 M Mm acetate solution (pH: 6.0) to make the total amount 5-.

(1-2) Preparation of sample (2-1) Take the bouillon culture [115-] of (1-1) into a test tube, and add a 0.08% solution of the chitosan sample of Reference Example 2 0.62-(
(equivalent to a final concentration of chitosan of 0.01%), and further added 0.04 M acetate buffer (pH: 6.0) to make the total concentration 5- and prepare sample H (2-1) from M. did.

(l-3) Preparation of sample (2-2) Add 0.75% solution of chitosan sample of Reference Example 2
- the final concentration of chitosan used in the amount of 0.012
%), do the same as (1-2) and try! (2-
2) was prepared.

(1-4) Preparation of sample (2-3) Add 0.08% solution of the chitosan sample of Reference Example 2 to 1.25%
- final concentration of chitosan to be used in the amount o, oz9
(corresponding to 6) and (+-2), sample (2-
3) was prepared.

(1-5) Preparation of sample (z-4) A 0.08% solution of the chitosan sample of Reference Example 2 was added to 1.87%
The maximum Ma degree of chitosan to be used in the amount of 5-0.03
96) and (l-2), try B (2).
-4) was prepared.

(+-6) Preparation of sample (2-5) Add a 0.08% solution of the chitosan sample of Reference Example 2 to 2.5-
Sample (2-5) was prepared in the same manner as in (1-2).
) was prepared.

(2) Test method Sterilize each sample and add Escherichia coli B
(Eseheriehia eolio) was inoculated and cultured with shaking at 30°C.

The turbidity of the sample after incubation was determined by absorption at 660 nm.

(3) Test results The results of the test were as shown in Figure 7.

The horizontal axis in Figure 7 is the culture time (hours), and the vertical axis is the absorption at 660 nm, which indicates the turbidity of the sample. A sample with high turbidity indicates growth of Escherichia coli. show.

In Fig. 7, (-X-) in the solid line indicates the control sample;
(-Δ-) on the dotted line is sample (2-1), (-Δ-) on the dashed line is sample (2-23), and (-) on the solid line
0-) indicates the results for sample (2-3), (-0-) on the dotted line indicates sample B (2-4), and (-0-) on the dashed-dotted line indicates the results for sample (2-5).

According to the results shown in Figure 7, compared to the control sample to which no chitosan was added, sample (2-t) and sample (2-2)
The proliferation of Escherichia coli was suppressed for 1 day, but the trial @ (
2-3), 4 for sample (2-4) and sample (2-5)
Escherichia coli did not grow at all during the two-day culture. From this, the final concentration of chitosan was set at 0.02%.
The above results show that it exhibits excellent antibacterial properties.

Test Example 3 A test was conducted on the effect of chitosan on the proliferation of Bacillus subtilis.

(1) Sample preparation The same sample as in Test Example 2 was used.

(2) Test method A test was conducted in the same manner as in Test Example 2, using Bacillus subtilis instead of Escherichia coli in Test Example 2.

(3) Test results The results of the test were as shown in Figure 8.

The horizontal axis in Figure 8 is the culture time (hours), and the vertical axis is the absorption at 660 nm, which indicates the turbidity of the sample. A sample with high turbidity indicates the proliferation of Bacillus subtilis. show.

The sample in FIG. 8 is the same as in FIG.

The results in Figure 8 are also the same as in Figure 7, and when the final concentration of chitosan is 0.02% or more, chitosan
It can be seen that it exhibits excellent antibacterial properties against Iris subtilis.

Test Example 4 A test was conducted on the effect of chitosan on the growth of Fusarium oxysporum.

(1) Preparation of sample (1-1) Preparation of sample (4-1) Potato dextrose agar medium @ (manufactured by Eiken Chemical Co., Ltd.)
Add 5.46g to 70g of distilled water and dissolve by heating.
Take the sample A into a test tube and add 0.5-1% solution of chitosan to it.
(equivalent to a final concentration of 0.05%), and further icO
105M acetic acid f, L4? E (pH: 6.0) was added to make 10 baskets. After sterilizing this, it was transferred to a Petri dish and gelatinized to prepare a sample (5-1).

(1-2) Preparation of sample (4-2) The amount of 1% solution of chitosan used was ld (final concentration 0.1%).
Sample (5-2) was prepared in the same manner as (+-1) except that the sample was changed to (corresponding to).

(+-3) Preparation of sample (4-3) The amount of 1% chitosan melt used was 2 crowns (final concentration 0.2%).
Sample (5-3) was prepared in the same manner as (1-1) except that the sample (corresponding to ) was changed.

(1-4) Preparation of control sample A control sample was prepared in the same manner as in (1-1) except that 1% M tTl of chitosan was not used.

(2) Test method Each sample contains Fusarium oxysporum (Fusarium oxysporum).
arium oxysporum) to ff1i, 3
Static jj/l culture was performed at 7°C. 3, 40, and 6 days after inoculation, the diameter of the colony on the surface of the sample was calculated by tt t+'JJ, and the ratio (%) to the diameter of the colony on the side sample was calculated.

(3) Test results The test results are shown in Table 3.

Table 3 Regarding the growth of Fusarium oxysporum According to Table 3, when the concentration of chitosan was 0.2%, no slow growth was observed even after 6 days, and its antifungal properties were 11'
It can be seen that ff=, ff.

Test Example 5 Fusarium oxysporum eapae
) was tested for the effect of chitosan on the growth of 01° i.

(1) Sample preparation test example 1 and 1. (2) Test method A test was conducted in the same manner as in Test Example 4 except that Fusarium oxysporum capae was used in place of Fusarium oxysporum in Test Example 4.

(3) Test results The results of the test were as shown in Table 4.

(Leaving space below) Table 4 Effect of chitosan on the proliferation of Fusarium oxysporum cabbage According to Table 4, when the concentration of chitosan becomes 0.2%,
No bacterial growth was observed even after 6 days, indicating that its antifungal properties are remarkable.

Test Example 6 A test was conducted on the influence of chitosan and chitosan decomposition products on the growth of Escherichia coli.

(+) Preparation of sample (lt) Preparation of sample (6-A) Bouillon culture a of Test Example 1 (meat extract = 1%, peptone =
Sample 1 of chitosan decomposition product A of Reference Example 2, which had been appropriately diluted, was added to 8.9% of sodium chloride (1% and sodium chloride 0.5%), and sample 9 with a final concentration of chitosan decomposition product A of 0.004% was prepared.
(6-A) was prepared.

(1-2) Preparation of sample (6-8) Using the sample of chitosan decomposition product 8 of Reference Example 2 as the chitosan decomposition product, the final concentration of chitosan decomposition product B was determined in the same manner as in (1-1). 0.004% samples (6-8) were prepared.

(1-3) Preparation of sample (6-C) Using the sample of chitosan decomposition product C from Reference Example 2 as the chitosan decomposition product, the final concentration of chitosan decomposition product C was determined in the same manner as in (1-1). A 0.004% sample (6-C,) was prepared.

(1-4) Preparation of sample (a-O) The sample of chitosan decomposition product from Reference Example 2 was used as the chitosan decomposition product, and the final concentration of chitosan decomposition product was determined in the same manner as in (1-1). A 0.004% sample (6-D) was prepared.

(1-5) Trial @ Preparation of (6-E) As a chitosan decomposition product, chitosan decomposition laE of Reference Example 2
A sample (6-E) with a final concentration of chitosan decomposition product E of 0.004% was prepared in the same manner as H-B.

(+-6) Preparation of sample (6-F) Using the sample of chitosan decomposition product F from Reference Example 2 as the chitosan decomposition product, the final concentration of chitosan decomposition product F was determined in the same manner as in (1-t). A 0.004% sample (6-F) was prepared.

(1-7) Preparation of test a (6-C,) Using the chitosan sample of Reference Example 2 instead of the sample of chitosan decomposition product A, the final concentration of chitosan was adjusted to 0 in the same manner as in (11). .004% test! (6-G) was prepared.

(1-8) Preparation of control sample Add broth medium 8.9- of Reference Example 2 (1) to (1-1).
A control sample was prepared by adding the solvent 1- used in sample preparation.

(2) Test method Escherichia coli culture 0.1- was inoculated into each sample, cultured with shaking at 306C for 24 hours, the formation of turbidity was observed wIJ, and those with turbidity (+
), and those that did not produce turbidity were marked (-). A sample that produced turbidity indicates the proliferation of Escherichia coli, and a sample that did not produce turbidity indicates that Escherichia coli did not proliferate.

(3) Test results The results of the test were as shown in Table 5.

(Left space below) Table 5 Effects of chitosan and chitosan decomposition products on the growth of Escherichia coli According to Table 5, chitosan decomposition products C, D, and E.

It can be seen that the samples of Fl and chitosan inhibit the growth of Escherichia coli and have antibacterial properties.

Test Example 7 A test was conducted on the influence of chitosan and chitosan decomposition products on bacterial growth.

(1) Preparation of sample (+-1) Preparation of sample (7-E) Bouillon medium of Test Example 1 (meat extract: 1%, peptone:
1% and sodium chloride = 0.5%) Sample H 1ml of chitosan decomposition product E of Reference Example 2 appropriately diluted to 8.9ml
was added to prepare a sample (7-E) in which the final concentration of chitosan decomposition product E was 0.02%.

(1-2) Preparation of sample (7-F) Using the sample of chitosan decomposition product F from Reference Example 2 as the chitosan decomposition product, the final concentration of chitosan decomposition product F was determined in the same manner as in (1-1). A 0.02% sample (7-F) was prepared.

(+-3) Trial! Preparation of (7-G) Using the chitosan sample of Reference Example 2 as the chitosan decomposition product, prepare the sample (7-G) with a final chitosan concentration of 0.02% in the same manner as (+-1). Prepared.

(1-4) Preparation of sample (7-C) As the chitosan decomposition product, the chitosan component of Reference Example 2, decomposition product C
A sample (7-C) in which the final concentration of chitosan decomposition product C was 0.02% was prepared in the same manner as in (1-1).

(1-5) Preparation of sample (7-D) Using the sample of chitosan decomposition product A from Reference Example 2 as the chitosan decomposition product, the final concentration of chitosan decomposition product A was determined in the same manner as in (1-1). A 0.02% sample (7-D) was prepared.

(1-6) Preparation of control sample (1-t) broth @8.9- to (1) of Reference Example 2
A control sample was prepared by adding 1 ml of the solvent used for sample preparation.

(2) Test method 0.1 mjl of the culture of the test bacteria listed in Table 4 was inoculated into each sample, cultured with shaking at 30°C for 24 hours, and the formation of turbidity was observed. The results were evaluated as +), and those that did not generate turbidity were evaluated as (-). A case where turbidity occurs indicates the growth of the test bacteria, and a case where no turbidity occurs indicates that the test bacteria did not grow.

(3) Test results The results of the test were as shown in Table 6.

(Margins below) According to Table 6, the test @ (7-C) in which chitosan was added had the effect of inhibiting the growth of all bacteria, and when chitosan decomposition product was added, chitosan decomposition product C It can be seen that the sample (7-C) to which 0 was added had the effect of suppressing the growth of all bacteria except that it had no effect of suppressing the growth of Streptomyces aureus.

Test Example 8 A test was conducted on the effect of chitosan decomposition products on the growth of Escherichia coli.

(1) Preparation of sample (preparation of t-B test H (8-A) Add the sample of chitosan decomposition product A of Reference Example 2 to the bouillon medium of Test Example 1 so that the final concentration is 0.01%. , 5- was placed in a test tube and sterilized to prepare sample (8-A).

(1-2) Preparation of sample (8-B) Using the sample of chitosan decomposition product B of Reference Example 2 as the chitosan decomposition product, prepare sample (8-B) in the same manner as in (1-1).
) was prepared.

(+-3) Preparation of sample a (8-E) Using the sample of chitosan decomposition product E of Reference Example 2 as the chitosan decomposition product, the sample (8-E) was prepared in the same manner as in (1-1).
E) was prepared.

(1-4) Preparation of sample (8-F) Using the sample of chitosan decomposition product F of Reference Example 2 as the chitosan decomposition product, the sample (s-p
) was prepared.

(1-5) Preparation of sample a (8-(1;) Using the chitosan sample of Reference Example 2 instead of the chitosan decomposition product, prepare sample (8-C,) in the same manner as Nt) did.

(+-6) Preparation of control sample A control sample was prepared in the same manner as in (1-1) without adding chitosan and chitosan decomposition products to the bouillon medium of Test Example 1.

(2) Test method: Inoculate each sample with Escherichia coli,
After incubation for 91 hours at C, the turbidity of each sample was reduced to 6.
Measured by absorption at 60 nm.

High turbidity indicates that Escherichia coli has grown, and low turbidity indicates that Escherichia coli has not grown.

(3) Test results The results of the test were as shown in Figure 9.

The horizontal axis in FIG. 9 is the culture time (hours), and the vertical axis is the absorption at 660 nm, which indicates the turbidity of the sample. A sample with high turbidity indicates that Escherichia coli has grown, and a low sample turbidity indicates that Escherichia coli has not grown.

In Figure 9, (-) on the dotted line is the sample (8-).
A), (-△-) in the dashed line is sample (8-B),
(-0-) on the solid line is the sample (8-E), (-0-) on the dashed line is the sample (8-F), and (-
〇-) is the sample (8-G), and (-X-) in the solid line
) indicate the respective results for the control sample.

According to FIG. 9, sample (8-F) C chitosan decomposition product F]
, and sample (8-) (chitosan), the growth of Escherichia coli was observed in sample
-8), it was delayed by 24 hours, and in test B (8-E), the growth of Escherichia coli was delayed by 48 hours.

Test Example 9 A test was conducted on the bactericidal effect of chitosan and chitosan decomposition products against Escherichia coli.

(1) Sample preparation (1-1) Trial! Preparation of (9-A) Bouillon culture of Test Example 1 @5ml. was placed in a test tube, inoculated with sterilized Escherichia coli, cultured at 30°C for 18 hours, and 0.1 d of the culture solution was added to the bouillon medium of Test Example 1, and incubated at 30°C for 4 hours. A culture solution of Escherichia coli was obtained by culturing. Dilute this Escherichia coli culture solution 10 times, take 0.5-fold of it into a test tube, and add sample a of chitosan decomposition product A of Reference Example 2 to this.
(corresponds to 1% final concentration of chitosan decomposition product A) 0.5
- and 0.05 M acetate buffer (pH: 6.0)
4.0- was added and cultured with shaking at 37°C for 1 hour to prepare a sample (9-A).

(1-2) Preparation of sample (9-11) Using the sample of chitosan decomposition product B of Reference Example 2 as the chitosan decomposition product, in the same manner as in (1-11).

Sample (9-13) was prepared.

(1-3) Preparation of sample (9-E) Using the sample of chitosan decomposition product E of Reference Example 2 as the chitosan decomposition product, in the same manner as (+-1), sample (9-,
E) was prepared.

H-a) Preparation of Trial B (9-P) Using the sample of chitosan decomposition product F of Reference Example 2 as the chitosan decomposition product, prepare Trial B (9-F) in the same manner as in (1-1).
) was prepared.

(1-5) Preparation of sample (9-G) Instead of chitosan decomposition product, chitosan sample G of Reference Example 2 was used.
Sample (9-C) was prepared in the same manner as (1-1).

(1-6) Preparation of Control Sample Control Sample 6 was prepared in the same manner as in (1-+) without adding chitosan and chitosan decomposition products to the bouillon medium of Test Example 1.

(2) Test method: Dilute each sample 10 times, and the dilution wave is 0.1-
Melt agar T3 (meat extract = 1%, peptone = 1%,
Sodium chloride: 0.5%, pH: 6.0) was added to 10 crowns, stirred, and spread in a Petri dish. This vetri dish is 37
The cells were cultured at 8C for 24 hours, and the number of viable bacteria in the colonies formed on the Petri dish was counted.

(3) Test results The results of the test were as shown in Table 7.

Table 7: Chitosan against Escherichia coli According to Table 7, all chitosan and chitosan decomposition products had a bactericidal effect on Escherichia coli, but chitosan decomposition product E1, chitosan decomposition product F, and chitosan It can be seen that the bactericidal effect in the samples using .

Test Example 10 Fusarium oxysporum
The effects of chitosan and chitosan decomposition products on the growth of P. sporum were tested.

(+) Preparation of sample (+-1) Preparation of sample (to-A) Potato dextrose agar medium for collection (manufactured by Eiken Chemical Co., Ltd.)
Add 5.46 g to 70 d of distilled water, dissolve by heating, then take the 5- in a test tube and add 1 sample of chitosan decomposition product A from Reference Example 2 so that the final concentration is 0.1%. , and further added 0.05 M acetate buffer (pH: 6.0) to prepare sample (10-A).

(1-2) Preparation of sample (to-8) Using the sample of chitosan decomposition product B of Reference Example 2 as the chitosan decomposition product, in the same manner as in (1-1), tcHHo
-n) was prepared.

(1-3) Preparation of sample (10-E) Chitosan decomposition product, chitosan decomposition vlE of Reference Example 2
Using sample (10) in the same manner as (+-1)
-E) was prepared.

(1-4) Preparation of Trial 1 (10-F) Using the sample of chitosan decomposition product F of Reference Example 2 as the chitosan decomposition product, the sample (10-F) was prepared in the same manner as in (It).
) was prepared.

(1-5) Preparation of sample (10-G) Using the chitosan sample of Reference Example 2 instead of the chitosan decomposition product, the same procedure as in (t t) was carried out to prepare sample (10-C,
) was prepared.

(2) Test method Sterilize each sample, transfer them to their respective petri dishes, gel them, and then add Fusarium to these samples.
Oxysporum was inoculated and statically cultured at 37°C. The diameter of Fusarium oxysporum colonies 3, 4, and 6 days after inoculation was adjusted to the IT side, and the same test was conducted using a control sample prepared without adding chitosan or chitosan decomposition products. The ratio to the colony diameter in the sample was calculated.

(3) Test results The results of the test were as shown in Table 8.

Table 8 Effect of chitosan and chitosan decomposition products on the growth of Fusarium oxysporum According to Table 8, all samples containing chitosan and chitosan decomposition products have an inhibitory effect on the growth of Fusarium oxysporum. However, the growth inhibitory effect was observed in sample (10-E) and sample (10-F).
It can be seen that the resistance and force of chitosan and mildly decomposed products of chitosan are large.

[Brief explanation of drawings]

Figure 1 is a chart showing the relationship between salinity and specific activity in chitosanase produced by Bacillus No. 7-M, and Figure 2 is a chart showing the relationship between pH and specific activity in chitosanase produced by Bacillus No. 7-H. Figure 3 is a diagram showing the relationship between salinity and specific activity in chitosanase produced by Bacillus No. 7-H, and Figure 4 is a diagram showing the relationship between salinity and specific activity in chitosanase produced by Bacillus No. 7-M. Figure 5 is a diagram showing the relationship between pH and specific activity;
The figure is a chart showing the molecular weight of chitosanase produced by Bacillus No. 7-H by gel filtration method.
FIG. 7 is a chart showing the relationship between the turbidity of the sample and culture time showing the growth state of Escherichia coli in the test results of Test Example 2, and FIG. A chart showing the relationship between the turbidity of the sample showing the growth condition of E. subtilis and the culture time, and FIG. 9 shows the relationship between the turbidity of the sample and the culture time showing the growth failure of E. It would be a diagram showing the relationship.

Claims (3)

[Claims] (1) A bacterial growth and proliferation inhibitor characterized by containing chitosan or a mildly decomposed product of chitosan as an active ingredient. (2) The light decomposition product of chitosan is characterized by being a light decomposition product of chitosan in which chitosan is decomposed by chitosanase to an extent that the amount of reducing sugar produced (mg D-glucosamine/1g chitosan) is not more than 120 mg. An agent for inhibiting bacterial growth and proliferation according to claim 1. (3) Chitosanase is produced by Bacillus s
p. ) No. The inhibitor of bacterial growth and proliferation according to claim 2, characterized in that it is produced by A. 7-M (Keikoken Kyokuyori No. 8139).