JPH05320068A - Antibacterial agent, tyrosinase activity-inhibiting agent containing lactoferrin decomposition product as active ingredient, and treatment of article with lactoferrin decomposition product - Google Patents

Abstract

PURPOSE:To provide the lactoferrin hydrolysate more stable against heat and the change in pH than the natural lactoferrin, having a remarkably excellent antibacterial property and furthermore having a tyrosinase activity-inhibiting effect. CONSTITUTION:This antibacterial agent or tyrosinase activity-inhibiting agent contains as an active ingredient the lactoferrin of a mammalian, an apolactoferrin produced by removing iron from the lactoferrin with hydrochloric acid, citric acid, etc., a metal- saturated lactoferrin produced by chelating the apolactoferrin with a metal such as iron, copper, zinc or manganese, and a lactoferrin decomposition product obtained by hydrolyzing a lactoferrin selected from a group consisting of their arbitrary mixtures. The hydrolysis of the lactoferrin is performed by an acidic hydrolysis at a pH of 1-2 at 90-120 deg.C or at a pH of 2-4 at 100-120 deg.C or by an enzymatic hydrolysis using an protease in an amount of 0.5-3.0% based on the lactogerrin at 30-50 deg.C. The hydrolysis rate determined by the formal titration is preferably 7-15% in the case of the antibacterial agent or 6-40% in the case of the tyrosinase activity-inhibiting agent.

Description

Detailed Description of the Invention

The present application is filed under Japanese Patent Application No. Hei 2-169636 by the same applicant as the present applicant (filing date: June 2, 1990).
This is an application claiming priority on the 6th, based on an antibacterial agent and a tyrosinase activity inhibitor whose names are lactoferrin degradation products as active ingredients.

[0002]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial agent and a tyrosinase activity inhibitor containing a decomposed product of lactoferrin as an active ingredient.

[0003]

2. Description of the Related Art Lactoferrin is an iron-binding protein contained in tears, saliva, peripheral blood, milk and the like in the living body, and is 0.1% to E. coli, Staphylococcus and Enterococcus.
It is known to have an antibacterial effect at a concentration of 5 to 30 mg / ml [Nonecke and Smith; Journal of
Mary Science (Nonnecke, BJ & Smith, K.
L .; Journal of Dairy Science) 67 volumes; 606 pages: 1
984].

[0004] It is generally considered that the antibacterial action of lactoferrin is due to the fact that iron, which is necessary for the growth of bacteria, binds to lactoferrin so that the bacteria cannot utilize iron.
However, the antibacterial action of lactoferrin cannot be said to be sufficiently strong. Therefore, when lactoferrin is used, especially lactoferrin is compounded with other articles, impregnated,
Attached or coated with lactoferrin,
Since a large amount of lactoferrin is required to exert an antibacterial effect, there is a limit to the use of lactoferrin in the conventional techniques.

[0005] In order to improve this point, various studies have conventionally been conducted to enhance the antibacterial action of lactoferrin.
For example, a method of synergistically enhancing the antibacterial action by coexistence of lactoferrin and lysozyme [JP-A-62-2499]
31], a method of synergistically enhancing the antibacterial action by coexistence of lactoferrin and secretory IgA [Stephens, S., et al; Immunolog
y); 41; 597; 1980] and the like are reported.

However, as far as the inventors of the present invention know, there has been no attempt to chemically treat lactoferrin itself to improve its activity in order to obtain high antibacterial activity.

Furthermore, lactoferrin is heat-labile, and is almost inactivated by heating at 62.5 ° C. for 30 minutes.
It is known that heating at 0 ° C. for 15 minutes causes complete deactivation. [Ford, JE, et al; Journal of Pediatric
s); 90; 29; 1977] Therefore, when a lactoferrin-containing solution is conventionally treated and heat treatment is included in the process, lactoferrin may be deactivated, The reality is that a simple heat treatment cannot be adopted.

[0008] The effect of tyrosinase activity inhibition of lactoferrin and its hydrolyzate is not known.

Tyrosinase is an enzyme that catalyzes the oxidation of tyrosine, other monohydric phenols and the corresponding ortho-dihydric phenols with molecular oxygen.
Widely present in many plants such as potatoes and apples, and also widely in animal tissues. It is known that in plants, it is involved in the blackening phenomenon of damaged parts of tissues, and in animals, it is involved in the formation of melanin pigments of tissues, in particular skin epidermal cells (edited by the Chemistry Dictionary Editorial Committee, Chemistry Dictionary,
Volume 5, page 976, Kyoritsu Shuppan, 1960). It is also known that melanin deposition on the skin or mucous membrane in Addison's disease is caused by a decrease in secretion of adrenocortical hormone that antagonizes melanotropin that promotes tyrosinase activity (edited by the Chemistry Dictionary Editorial Committee, Chemistry Dictionary, Volume 1, page 65, Kyoritsu Shuppan, 1960). Furthermore, tyrosinase is also said to be involved in reducing the freshness of foods.

As described above, there has been a strong demand for the development of tyrosinase activity inhibitors for preventing, preventing or treating the undesirable effects of tyrosinase in the fields of food, cosmetics and pharmaceuticals. Particularly in the cosmetics industry, active research is being conducted on cosmetics or external preparations for skin that effectively suppress the production of melanin pigments and impart a whitening effect, and products containing tyrosinase activity inhibitors have been developed one after another. Examples of tyrosinase activity inhibitors include cysteine, vitamin C (Yutaka Mishima et al., Basic Dermatology, page 258, Asakura Shoten, 1973), kojic acid (Nikkei Sangyo Shimbun, May 24, 1988), arbutin (Kenichi Tomita, 20th FJ Seminar Proceedings, page 21, Fragrance Journal,
March 14, 1990), a product of a microorganism belonging to the genus Trichoderma (JP-A-2-145189) is known.

[0011]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As a result of intensive studies on the enhancement of antibacterial action and improvement of heat resistance of lactoferrin, the present inventors have found that lactoferrin derived from mammals and aposomes derived from mammals chemically treated with them. A lactoferrin degradation product obtained by hydrolyzing lactoferrin selected from the group consisting of lactoferrin, metal-saturated lactoferrin derived from mammals, or any mixture thereof is superior to natural lactoferrin in antibacterial action, and is resistant to heating and pH change. On the other hand, they found that they were stable, and completed the present invention.

Further, conventional tyrosinase activity inhibitors are
It is unstable in the product, has too strong an effect on melanocyte that synthesizes melanin pigment, and is expensive because it is difficult to obtain the raw material. It was not satisfactory as a cosmetic or an external preparation for skin.

The present inventors have found that lactoferrin, particularly its hydrolyzate, has a strong tyrosinase activity inhibitory effect, and completed the present invention.

OBJECT OF THE INVENTION An object of the present invention is to provide an antibacterial agent containing a lactoferrin degradation product as an active ingredient.

Another object of the present invention is to provide an antibacterial composition containing a lactoferrin degradation product as an antibacterial active ingredient.

[0016] Yet another object of the present invention is to provide a method for treating an article with a lactoferrin degradation product.

Another object of the present invention is to provide a tyrosinase activity inhibitor containing a lactoferrin degradation product as an active ingredient.

Still another object of the present invention is to provide an antibacterial agent / tyrosinase activity inhibitor containing a lactoferrin degradation product as an active ingredient, which is safe for humans and animals.

[0019]

In the present invention, lactoferrin used as a starting material is commercially available lactoferrin, colostrum of mammals (eg, human, cow, sheep, goat, horse, etc.), transitional milk, normal milk, end stage. Lactoferrin separated from milk or the like, or skim milk that is a processed product of these milks, whey or the like (hereinafter, collectively referred to as milk and the like) by a conventional method (for example, ion exchange chromatography), and hydrochloric acid, It may be apolactoferrin deironed with citric acid or the like, metal saturated lactoferrin chelated with metals such as iron, copper, zinc, manganese, or a mixture thereof (hereinafter collectively referred to as LF). ..

The lactoferrin hydrolyzate according to the present invention can be obtained by hydrolyzing the above LF with an acid or an enzyme.

Hydrolysis with an acid gives LF of 0.1 to 20.
% (Weight; hereinafter the same unless otherwise specified), preferably dissolved in water or purified water at a concentration of 5 to 15%, and the resulting solution is an inorganic acid such as hydrochloric acid, phosphoric acid, or citric acid. Organic acid is added to adjust the pH of the solution to 1 to 4, preferably 2 to 3
Adjust to. The solution thus obtained is hydrolyzed by heating at a suitable temperature for a predetermined time according to the adjusted pH.
For example, if the pH is adjusted to 1-2, then 80-13
0 ° C., preferably 90-120 ° C., 100-130 ° C., preferably 100-130 ° C. when adjusted to pH 2-4
120 ° C., 1 to 120 minutes, preferably 5 to 60 minutes
Heat for minutes.

When hydrolyzing with an enzyme, LF is dissolved in water, purified water or the like at a concentration of 0.5 to 20%, preferably 5 to 15%, and the resulting solution is optimized for the enzyme used. p
It hydrolyzes as H.

The enzyme used is not particularly limited, and a commercially available enzyme such as Morcin F (trademark, manufactured by Seishin Pharmaceutical Co., Ltd., optimum p
H2.5-3.0), pork pepsin (manufactured by Wako Pure Chemical Industries, Ltd., optimum pH 2-3) Sumiteam AP (trademark, manufactured by Shin Nippon Kagaku Co., Ltd.
Optimum pH 3.0, Amano M (trademark, manufactured by Amano Pharmaceutical Co., Ltd.).
Optimum pH 3.0, Amano A (trademark, manufactured by Amano Pharmaceutical Co., Ltd.
Optimum pH 7.0, trypsin (Novo. Optimal pH
8.0) etc. are used alone or in combination. In the production of the antibacterial agent, particularly good results were obtained by using porcine pepsin and Sumiteam AP. In addition to the above-mentioned enzymes, a soy sauce enzyme (manufactured by Tanabe Seiyaku Co., Ltd.) containing a commercially available exopeptidase may be used in combination.

The amount of enzyme used is 0.
1-5.0% range, especially. 0.5-3.0% is desirable.

After adjusting the pH of the LF solution and adding the desired amount of the desired protease, the temperature of the resulting solution was adjusted to 15
The LF is hydrolyzed by holding at ˜55 ° C., preferably 30 to 50 ° C. for 30 to 600 minutes, preferably 60 to 300 minutes.

Next, the solution as it is or after being neutralized, the enzyme is inactivated by heating by a conventional method.

The reaction solution obtained by the above method is cooled by a conventional method and, if necessary, neutralized, desalted and decolorized, and the obtained solution is directly or concentrated to give a liquid product, or after concentration. It can be dried to a powder product.

The above-mentioned hydrolysis conditions are not strict, and appropriate conditions may be taken into consideration in consideration of production costs such as temperature, time, type and amount of acid or enzyme, reaction apparatus (whether or not pressurization). Can be set.

The LF decomposed product obtained by the above method is a mixture of decomposed products having various molecular weights.

From the viewpoint of the loss of antigenicity of proteins, the rate of hydrolysis is 6 to 20%, particularly 7 to 15 in terms of the degree of decomposition by formol titration when used as an antibacterial agent.
% Is preferable, and when used as a tyrosinase activity inhibitor, the decomposition rate by formol titration is preferably 4 to 50%, particularly preferably 6 to 40%.

The LF hydrolyzate thus obtained is extremely stable against changes in heat and pH, and is superior in antibacterial properties to lactoferrin which is not hydrolyzed.
It also has a tyrosinase activity inhibitory effect.

Therefore, the lactoferrin hydrolyzate according to the present invention can be used as an antibacterial agent as it is or by mixing it with an excipient or another drug.

This antibacterial agent is not only used as a medicine,
Antibacterial property when incorporated into products such as foods, feeds, cosmetics, etc. that are taken into the body of humans and animals or applied to the surface of the body and other products that are generally desired to prevent or suppress the growth of bacteria. It can be used as a composition, or can be used as an article having an antibacterial treatment by treating those products with the antibacterial agent or the antibacterial composition.

The lactoferrin hydrolyzate according to the present invention can be used as a tyrosinase activity inhibitor as it is or after being mixed with an excipient or another drug.

This tyrosinase activity inhibitor is not only used as a drug, but also as a food, feed, cosmetic, quasi drug or a material thereof (for example, an emulsifier, a perfume, or other food, feed, cosmetic, drug, quasi drug). (Ingredients that are allowed as a material for tyrosinase) and other products that are taken into the body of humans and animals or applied to the surface of the body, and other products that are generally desired to inhibit tyrosinase activity. It can be used as an inhibitory composition, or can be used as a product that has been subjected to tyrosinase activity inhibition treatment by treating the product with the inhibitor or the inhibitory composition.

The antibacterial agent and the antibacterial composition containing the lactoferrin hydrolyzate of the present invention as an active ingredient are effective in treating infectious diseases when used as an internal medicine, when ingested by adding them to food or feed. It is effective for treatment or prevention and suppression of growth of harmful bacteria in the intestine.

The antibacterial agent, the antibacterial composition, the tyrosinase activity inhibitor and the tyrosinase activity inhibitory composition containing the lactoferrin hydrolyzate of the present invention as an active ingredient are applied to human or animal body surfaces as cosmetics or external preparations. In this case, it is effective not only for treating and preventing infectious diseases but also for preventing melanin pigmentation.

Since the lactoferrin hydrolyzate of the present invention has its antibacterial properties and tyrosinase activity inhibitory properties,
By treating articles that humans or animals ingest, such as food and feed, it is effective for the deterioration of the quality of those articles (prevention, suppression of discoloration, prevention of discoloration, freshness, etc.) and further treatment. It is effective in treating or preventing infectious diseases and suppressing the growth of harmful bacteria in the intestine by ingesting used articles, and treating articles that come into contact with the human or animal body surface (for example, mixing or impregnating those articles). , Adhere, coat) to maintain the hygiene of these articles, and when such articles are applied to the surface of human or animal body, they are effective in treating and preventing infectious diseases and preventing melanin pigmentation. is there.

Next, the usefulness of the antibacterial agent containing the LF degradation product of the present invention as an active ingredient will be illustrated by showing test examples.

[Test Example 1] This test was carried out in order to investigate the relationship between the degree of decomposition of the LF decomposed product decomposed by an acid and the antibacterial property.

1) Test method Preparation of sample Commercially available LF (manufactured by Olefina, Belgium) as separated from milk or the like was dissolved in purified water at a concentration of 5%, and 1M hydrochloric acid was added to adjust pH to 1. Adjusted to 2, 3 and 4. Here, the phrase "as separated from milk or the like" means that lactoferrin is only separated, and chemical treatments such as iron removal and metal saturation are not performed (the same applies hereinafter). Next, the solutions of each pH were heated at temperatures of 60 ° C. to 130 ° C. for 5 minutes to 60 minutes, respectively, to prepare LF decomposed products with various degrees of decomposition hydrolyzed under various decomposition conditions.

Degradation degree measurement Degradation degree (%) of each obtained LF degradation product was calculated by the following formula from the measured values of formol nitrogen in each sample by the formol titration method. Degradation rate = 100 x (formol nitrogen amount / total nitrogen amount) Preparation of medium and preculture liquid [Preparation of preculture liquid] One platinum loop was taken from a preserved slant of Escherichia coli, and standard agar medium (day Sui Pharmaceutical Co., Ltd.) and aerobically cultivated at 35 ° C. for 16 hours. The colonies grown on the surface of standard agar medium are scraped off with a platinum loop, suspended in sterile physiological saline, and then spectrophotometer (Hitachi Ltd.). The turbidity (OD; 660 nm) measured by the company) was 1.
The preculture liquid was adjusted to 0.

[Preparation of basal medium] Bactocaditon (manufactured by Difco) was dissolved in purified water at a concentration of 1%, the pH was adjusted to 7.0 with 1M sodium hydroxide, and the temperature was adjusted to 115 ° C at 15 ° C.
The basal medium (liquid medium) was prepared by sterilizing for a minute.

[Preparation of test medium and control medium] The LF decomposed product solution and the undecomposed LF solution having various degrees of decomposition prepared by the same method as described above were sterilized with a sterilizing filter (manufactured by Advantech). After 25ppm, 50ppm,
100ppm, 250ppm, 500ppm and 100
Test medium and control medium were prepared by adding each to the basal medium at a concentration of 0 ppm.

[Antibacterial Effect Test] Each of the above test medium and each control medium was inoculated with the above preculture liquid at a concentration of 1% and aerobically cultured at 35 ° C. for 16 hours. The growth inhibition rate of E. coli was calculated from the following formula. Growth inhibition rate (%) = 100− (A / B × 100) [where A is the difference in turbidity of the test culture solution (turbidity of the test culture solution after 16 hours of culture and turbidity of the test culture solution before culture). Difference from degree)
B shows the difference in turbidity of the control culture solution (the difference between the turbidity of the control culture solution after 16 hours of culture and the turbidity of the control culture solution before culture). 2) Test Results The results of this test are shown in Table 1.

[0046]

[Table 1] The undegraded LF (control) began to show antibacterial action from the addition amount of 250 ppm, but even with the addition amount of 1000 ppm, it was not possible to completely inhibit the growth of E. coli (weak antibacterial property). On the other hand, LF with a resolution of 10%
The decomposed product shows a strong antibacterial action from the addition amount of 25 ppm,
The addition amount of 100 ppm or more completely inhibited the growth of E. coli. From the results of Table 1, among LF decomposed products obtained by heating LF at an acidic pH and hydrolyzing it, the degree of decomposition is 6 to
It was found that 20%, especially 7-15%, had a significantly stronger antibacterial activity against E. coli than undegraded LF.

[Test Example 2] This test was carried out using an enzyme-based LF.
Was carried out to investigate the decomposition conditions.

1) Test method Commercially available LF (manufactured by Oleofina, Belgium) as separated from milk or the like was dissolved in purified water at a concentration of 5%, and commercially available Morcin F, Porcine pepsin, Sumiteam AP, Amano M Or trypsin is added to the substrate (LF) in an amount of 0.1 to 6%.
Add and adjust the pH to the optimum pH of those enzymes, 37
Hold at 0 ° C. After 30 to 360 minutes, adjust the pH to 7,
L was obtained by inactivating the enzyme by heating at 80 ° C for 10 minutes.
The solution of the F degradation product was freeze-dried to obtain various LF degradation products (1
2 samples were obtained for each enzyme). The degree of decomposition (%) of the obtained LF decomposed product was measured by the same method as in Test Example 1,
Calculated.

2) Test results The results of this test are shown in Table 2.

[0050]

[Table 2] Degradation degree of degradation products using acidic proteases such as Morcin F, porcine pepsin, Sumiteam AP and Amano M is 5.
It was 9 to 14.7%. In addition, the decomposition degree of LF degradation product using trypsin which is a neutral protease is 10.3 to
It was 12.5%.

[Test Example 3] This test was carried out in order to investigate the antibacterial property of the LF decomposed product decomposed by an organic acid.

Using the same method as in Example 2 (decomposed with citric acid), an antibacterial LF decomposed product with a decomposition degree of 12% and undecomposed LF, were obtained by the same method as in Test Example 1.
The antibacterial activity against E. coli was tested.

The results are shown in Table 3.

[0054]

[Table 3] From the above test examples, it was proved that the decomposition of LF in the production of the antibacterial LF decomposition product of the present invention may be performed with any of inorganic acid, organic acid and enzyme.

[Test Example 4] This test was conducted to confirm the antibacterial effect of the method for treating an article using the antibacterial LF decomposition product according to the present invention.

1 commercially available primary processed vegetable (cut vegetable)
The antibacterial LF decomposition product (decomposition with inorganic acid, decomposition degree 9
%) Was immersed in a 1% solution for 30 seconds, thoroughly drained, stored at 5 ° C., and changes in the viable cell count were measured over time by a conventional method.

As a control, the same test as above was carried out on a solution in which undecomposed LF was dissolved at the same concentration and water to which nothing was added.

The test results are shown in Table 4.

[0059]

[Table 4] This test demonstrated that the antibacterial LF decomposed product of the present invention has a remarkably high antibacterial action as compared with undecomposed LF when the antibacterial LF decomposed product of the present invention is used [Test Example 5] This test Was carried out in order to demonstrate the antibacterial effect of the composition when the antibacterial LF degradation product according to the present invention was incorporated into a food as an active ingredient.

10 ml of raw milk heated at 65 ° C. for 30 minutes
Each was dispensed into a test tube. An antibacterial LF decomposed product having a decomposition degree of 9% (manufactured by the same method as in Example 1) or undecomposed LF was added to each of these at a concentration of 0.1%, uniformly mixed and sealed. Samples (Sample 1 and Sample 2) and similar samples (control) were prepared except nothing was added. Each sample was stored at 25 ° C., and the number of days until solidification was measured.

As a result of this test, sample 1 coagulated in 9 days, while sample 2 and control coagulated in 4 days and 2 days, respectively, and the antibacterial LF degradation product of the present invention was not degraded. LF
It was found that the coagulation of milk was remarkably prolonged as compared with.
In addition, when a sensory test was performed on milk immediately after the addition of the antibacterial LF hydrolyzate of the present invention and milk without the addition, there was no difference in flavor, appearance or the like between them. confirmed.

[Test Example 6] This test was carried out in order to investigate the antibacterial property of the LF degradation product decomposed by the enzyme. 1) Test method Various LF decomposition product solutions (decomposed by various enzymes) and undecomposed LF solutions (control) prepared by the same method as in Test Example 2
Sterilized with a sterile filter (Advantech),
Different concentrations (5%) in basal medium (same as in Test Example 1)
Test medium and control medium added at 0, 100, 250, 500 and 1000 ppm) were prepared. The antibacterial activity was tested in the same manner as in Test Example 1 except for the above.

2) Test Results The results of this test are shown in Table 5.

[0064]

[Table 5] Undegraded LF (control) showed a weak antibacterial action from 250 ppm, but could not completely inhibit the growth of E. coli even with the addition amount of 1000 ppm. Decomposition products decomposed with Morcin F, Porcine pepsin, Sumiteam AP and Amano M (all of which are acidic proteases) to a degree of decomposition of 10% or more show a strong antibacterial action even at an addition amount of 100 ppm, and completely at an addition amount of 250 ppm or more. The growth of E. coli was blocked. On the other hand, the decomposition product of trypsin (neutral protease) did not show any antibacterial action even at the addition amount of 1000 ppm.

From the results shown in Table 5, among the LF degradation products obtained by degrading LF with acidic protease, the degree of degradation was 1
It was confirmed that 0% or more had stronger antibacterial activity against Escherichia coli than undegraded LF.

[Test Example 7] The purpose of this test is to investigate the antibacterial activity of a composition containing a decomposition product of metal-saturated LF.

The same as in Test Example 5 except that an antibacterial LF decomposition product (iron-saturated LF decomposition product) produced by the same method as in Example 5 was used as the active ingredient, and that commercially available milk was used. The test was conducted.

That is, 10 ml of commercially available milk was dispensed into a test tube, and 1 mg / ml of the above-mentioned antibacterial LF decomposition product (iron-saturated LF decomposition product) or undecomposed LF (control) was dispensed.
Test samples were added at the same concentration, mixed uniformly, and sealed, and a control sample to which nothing was added was prepared in triplicate. These samples were stored at 25 ° C. and the change in appearance was visually observed.

The results of this test are shown in Table 6.

[0070]

[Table 6] With regard to the milk added with the antibacterial LF degradation product of the present invention, no change was observed in any of the three samples until 14 days after the start of the test.

On the other hand, the milk to which undegraded LF was added did not change until the 10th day from the start of the test, but on the 14th day, all 3 samples were coagulated. In the control group, 2 samples coagulated on the 10th day and all samples coagulated on the 14th day.

From these results, it was proved that the antibacterial LF decomposition product of the present invention has an antibacterial property superior to that of undecomposed LF.

TEST EXAMPLE 8 The purpose of this test is to investigate the antibacterial activity of LF degradation products in the presence of iron.

Some samples used in Test Example 6, that is, sample No. 1, No. 2, No. 5, No. For 6 and undegraded LF (control), 0.01m ferrous sulfate was added to the medium.
The antibacterial activity was tested in the same manner as in Test Example 6 except that M was added.

The test results are shown in Table 7.

[0076]

[Table 7] Undegraded LF, in the presence of 0.01 mM ferrous sulfate,
Although the antiproliferative effect was lost, each of the degradation products of the present invention by LF acidic protease maintained the antibacterial activity even in the presence of 0.01 mM ferrous sulfate.

[Test Example 9] This test was carried out to examine the antibacterial effect of a hydrolyzate of LF with an enzyme against various microorganisms.

(1) Preparation of Sample Sample 4 showing the highest growth inhibition rate in Test Example 6 was prepared by the same method as in Test Example 6. Before use, it was filtered with a filter (0.45 μm Mylex filter) to remove bacteria.

(2) Test method Various microorganisms shown in Tables 8 and 9 were prepared by adding 2 ml of a peptone medium containing 1% Bactopeptone (manufactured by Difco Laboratory) and 1% Bactopeptone (manufactured by Difco Laboratory). The cells were cultured in 2 ml of PYG medium consisting of 1% glucose and 0.05% yeast extract for 16 to 20 hours. LF enzyme hydrolyzate was added to each medium at various ratios of 0 to 1600 μg / ml. Standard strains of various microorganisms in the logarithmic phase were inoculated into each medium at a bacterial concentration of 10 ⁶ / ml, and cultured at 37 ° C except for the strains described in the table. The growth of various microorganisms was monitored by measuring the absorbance at 660 nm. The minimum concentration of HF enzyme hydrolyzate that completely inhibits the growth of various microorganisms is the minimum growth inhibitory concentration (MIC.
μg / ml).

(3) Test Results The results of this test are shown in Tables 8 and 9.

[0081]

[Table 8]

[Table 9] As is clear from Table 8 and Table 9, the enzymatic hydrolyzate of LF was 940 against various types of Gram-positive bacteria, including aerobic and anaerobic bacteria and yeast, Gram-negative bacteria, and yeast.
The antibacterial action was exhibited at a low concentration of μg / ml or less. The concentration of LF enzymatic hydrolyzate that completely inhibits the growth of microorganisms is
It varied depending on the medium. Among the microorganisms tested, Pse
udomonas fluorescens IFO-
141602 and Enterococcus faec
alis ATCC-E19433 was resistant to the enzymatic hydrolyzate of LF under these test conditions.

Next, the antibacterial agent according to the present invention will be described in more detail with reference to examples.

[0083]

[Example 1] 50 g of commercially available LF (manufactured by Olefina, Belgium) as separated from milk or the like was added to purified water 9
Dissolved in 50 g, adjusted the pH to 2 by adding 1 M hydrochloric acid to the obtained solution, heated at 120 ° C. for 15 minutes, cooled, and contained a LF decomposition product at a concentration of 5% to obtain a liquid antibacterial agent. 1000 g of a sex decomposition product was obtained.

The degree of decomposition of this LF decomposed product was 9% (measured by the same method as in Test Example 1).

[Example 2] 150 g of commercially available LF (manufactured by Olefina, Belgium) as separated from lactose was dissolved in 850 g of purified water, and 1 M citric acid was added to the resulting solution to adjust the pH to 3. The temperature was adjusted and heated at 130 ° C. for 60 minutes. After heating, the solution was cooled, 1 M sodium hydroxide was added to adjust the pH to 7, and then filtered, desalted and freeze-dried to obtain about 45 g of a powdery antibacterial LF decomposition product. ..

The decomposition degree of this antibacterial LF decomposition product was 12% (measured by the same method as in Test Example 1).

Example 3 20 g of commercially available LF (manufactured by Olefina, Belgium) as separated from milk or the like was dissolved in 180 g of purified water, and 200 m of ferrous sulfate heptahydrate was dissolved in the solution.
g, and after reacting at 25 ° C. for 12 hours, unreacted iron was removed by an ultrafiltration module SEP-1013 (trademark, manufactured by Asahi Kasei Corp.), freeze-dried, and iron-saturated LF (about 19 g)
Got The obtained iron-saturated LF (15 g) was dissolved in purified water (285 g), the pH was adjusted to 1.0 with 2M hydrochloric acid, the mixture was heated at 90 ° C. for 15 minutes, then cooled, and then cooled to a liquid concentration of 5% antibacterial LF. About 300 g of a decomposed product was obtained.

The degree of decomposition of the obtained antibacterial LF decomposition product is 7%.
(Measured by the same method as in Test Example 1).

[Example 4] 500 ml of Sepabeads FP-CM13 (trademark, manufactured by Mitsubishi Kasei) having an ion-exchange group of carboxymethyl group was filled in a column having an inner diameter of 10 cm, and 2 l of 10% saline was passed through the column. Then, it was washed with water to prepare a Na-type ion exchanger. Next, 60 l of cheese whey (pH 6.5) obtained from goat was passed through the column at a temperature of 4 ° C. and a flow rate of 4 l / hour. After that, wash the column with water,
The non-adsorbed components of the cheese whey were removed, and 5 l of 10% saline was passed at a flow rate of 5 l / hour to desorb the components of the cheese whey adsorbed on the ion exchanger to obtain 5 l of an eluate.
This eluate was concentrated using an ultrafiltration module SEP-1013 (trademark, manufactured by Asahi Kasei Corporation), water was added to carry out diafiltration to remove salt, and a solution containing 1% goat LF. About 200 ml was obtained. 1M hydrochloric acid was added to this solution to adjust the pH to 2.0, heated at a temperature of 120 ° C. for 20 minutes, and then cooled to 1% liquid antibacterial LF.
About 200 g of a decomposed product was obtained. The degree of decomposition of the obtained antibacterial LF degradation product was 10% (measured by the same method as in Test Example 1).

[Example 5] 1 kg of a commercially available LF (manufactured by Olefina, Belgium) as separated from milk etc. was dissolved in 9 kg of purified water, and 10 g of ferrous sulfate heptahydrate was added,
Allow to react for 12 hours at 25 ℃, then ultrafiltration module S
Unreacted iron was removed by EP-1013 (trademark, manufactured by Asahi Kasei), pH was adjusted to 3.5 by adding 0.5 N hydrochloric acid, and commercially available Morcine F (trademark, manufactured by Seishin Pharmaceutical Co., Ltd.) ) 10g
(42,000 units / 1 g protein) was added and mixed uniformly, and the mixture was kept at 37 ° C for 180 minutes to neutralize, and the mixture was mixed at 85 ° C for 1
The enzyme was inactivated by heating for 0 minutes and then cooled to obtain about 10 kg of a liquid antibacterial LF decomposition product.

The degree of decomposition of the obtained antibacterial LF decomposition product is 1
It was 3.5% (measured by the same method as in Test Example 1).

Example 6 1 kg of commercially available LF (manufactured by Olefina, Belgium) as it was separated from milk etc. was dissolved in 9 kg of purified water, and 2M citric acid was added to adjust the pH to 2.
Adjust to 5, and add 30 g of commercially available pig pepsin (1: 10,000, manufactured by Wako Pure Chemical Industries, Ltd.) and mix evenly.
The mixture was kept at 180 ° C. for 180 minutes, heated at 85 ° C. for 10 minutes to inactivate the enzyme, then cooled and concentrated by a conventional method to obtain about 10 kg of liquid antibacterial LF decomposition.

The degree of decomposition of the obtained antibacterial LF decomposition product was 1
It was 1.3% (measured by the same method as in Test Example 1).

[Embodiment 7] In 50 l of defatted goat milk, 0.0
5 l of 0.1 M sodium citrate solution containing 03 M ferric chloride was added and mixed uniformly. Then CM-
Sephadex C-50 (H ⁺ type, manufactured by Pharmacia)
About 5 l was added and stirred for about 1 hour. After stirring, the resin is thoroughly washed to remove unadsorbed goat milk components, and then 0.0
It was suspended in 5M Tris-hydrochloric acid buffer (pH 8.0), packed in a column (20 × 50 cm), and the resin was thoroughly washed with the same buffer. Then, elution was performed with a gradient containing 0 to 2 M sodium chloride in 0.05 M Tris-hydrochloric acid buffer solution to obtain about 800 ml of an LF fraction. This LF fraction was concentrated to about 150 ml using an ultrafiltration membrane PM-10 (trademark, manufactured by Amicon), and dialyzed against 0.05 M Tris-acetate buffer (pH 8.2) containing 0.5 M sodium chloride. .. Copper dialyzing Sepharose 6B (trademark, manufactured by Pharmacia) in which this dialysate is equilibrated with the same buffer.
The solution was passed through a column (10 × 30 cm) to adsorb LF. The column was washed with the same buffer solution, and the acetate buffer solution containing 0.5 M sodium chloride and having a pH of 4.0 was passed through to pass the LF.
Was eluted, then dialyzed against pure water and freeze-dried to obtain about 2 g of powdered LF.

2 g of this powdery LF was dissolved in 17 g of purified water, pH was adjusted to 3.5 by adding 1 M lactic acid, and 60 mg of commercially available Sumiteam AP (trademark, manufactured by Shin Nippon Chemical Industry Co., Ltd.)
(50,000 units / 1 g protein) was added and mixed uniformly, decomposed at 50 ° C for 180 minutes, neutralized, and heated at 85 ° C for 1 minute.
The mixture was heated for 0 minutes to inactivate the enzyme, cooled, then concentrated by a conventional method and freeze-dried to obtain about 2 g of a powdery antibacterial LF decomposition product.

The decomposition degree of the obtained antibacterial LF decomposition product was 1
It was 3.8% (measured by the same method as in Test Example 1).

Next, the usefulness of the tyrosinase activity inhibitor containing the LF degradation product of the present invention as an active ingredient will be illustrated by showing test examples.

[Test Example 10] This test was carried out in order to examine the tyrosinase activity inhibitory effect of the hydrolyzate by the acid of LF.

(1) Preparation of sample Commercially available LF (manufactured by Olefina) was dissolved in purified water at a concentration of 5% and 1M hydrochloric acid was added to adjust pH to 1, 2, 3 and 4.
4 types of solutions adjusted to were prepared. Each solution is heated at a temperature of 60 ° C to 130 ° C for 5 minutes to 60 minutes to perform hydrolysis, and then pH is adjusted with a 1M caustic soda solution.
Was adjusted to 7 and freeze-dried to prepare various LF degradation product samples with a degradation rate of 4 to 30%.

(2) Test method 1) Measurement of decomposition rate The decomposition rate (%) of the LF decomposed product was measured by measuring the amount of formol nitrogen in each sample by the formol titration method and expressed as a percentage with respect to the total amount of nitrogen in each sample. Was calculated from the following formula.

Decomposition rate (%) = 100 × (A / B) Here, A represents the amount of formol nitrogen and B represents the amount of total nitrogen.

2) Measurement of tyrosinase activity inhibitory effect a) Preparation of various solutions Preparation of substrate solution L-tyrosine of special grade reagent (manufactured by Wako Pure Chemical Industries, Ltd.) was used.
It was dissolved in M phosphate buffer at a concentration of 0.045% (W / V).

Preparation of Enzyme Solution Mushroom-derived tyrosinase (manufactured by Sigma. 3,
000 units / mg) in 0.1M phosphate buffer (pH
It was dissolved in 7.0 at a concentration of 0.1% (W / V).

Preparation of Copper Ion Solution 1% of reagent grade copper sulfate (Wako Pure Chemical Industries, Ltd.) was added to purified water.
It was dissolved at a concentration of (W / V).

Preparation of Sample Solution Each sample prepared in the above (1) was added with 0.1 M phosphate buffer (pH 7.0) at a concentration twice that of each concentration shown in Table 10.
Dissolved in.

B) Enzyme reaction 0.9 ml of substrate solution preheated to 37 ° C., sample solution 1.
Collect 0 ml and 0.02 ml of copper ion solution into a test tube,
0.08 ml of the enzyme solution heated to the same temperature was added (total amount 2.0 ml), and the mixture was reacted at 37 ° C. for 3 minutes. Then 30
% Ml of acetic acid solution was added to stop the reaction, and the absorbance at a wavelength of 640 nm was measured with a spectrophotometer (this absorbance value is referred to as B). 0.1M instead of the sample solution as a control
An enzyme reaction was carried out in the same manner as above except that 1.0 ml of phosphate buffer was added, and the absorbance was measured in the same manner as above (this absorbance value is designated as A). When the sample solution is cloudy, 0.08 ml of 0.1M phosphate buffer solution is added instead of the enzyme solution and the absorbance is measured in the same manner (this absorbance value is C), The absorbance derived from the turbid portion was removed. The tyrosinase activity inhibition rate (%) was calculated from each measured absorbance value by the following formula.

Inhibition rate (%) = 100 [1- (BC) / A] (3) Test results The results of this test are shown in Table 10. Sample 1 having a decomposition rate of 4% exhibited a tyrosinase activity inhibitory effect of about 10% at the addition of 0.3%. The inhibitory effect was improved as the amount of addition was increased, and the inhibitory rate was about 30% at 1% addition and about 50% at 2% addition.

[0108]

[Table 10] On the other hand, in Samples 2 to 8 having a decomposition rate of 6 to 30%, all of them are 0.
Addition of 05% already showed a tyrosinase activity inhibitory effect of about 30%. The inhibitory effect improves as the amount of addition increases, and it shows about 60% inhibition rate at 0.1% addition, about 80% at 0.3% addition, and 100% at 0.5% addition. It was

A similar tyrosinase activity-inhibiting effect was obtained with an acid hydrolyzate of metal lactoferrin obtained by chelating a metal such as apolactoferrin, zinc, copper or iron.

[Test Example 11] This test was carried out in order to examine the tyrosinase activity inhibitory effect of the hydrolyzate of LF with an enzyme.

(1) Preparation of sample Commercially available LF (manufactured by Oleofina) was dissolved in purified water at a concentration of 5%, and the pH was adjusted to about the optimum pH of the enzyme with 1M hydrochloric acid or 1M caustic soda. A commercially available soy sauce enzyme (manufactured by Tanabe Seiyaku Co., Ltd.) containing pig pepsin, amano A, or peptidase was combined, and the amount was 0.1 to 6 with respect to the LF solution.
%, And maintained at 37 ° C. After reacting for different times from 10 minutes to 24 hours, the pH was adjusted to 7 and 8
Heat at 0 ° C for 10 minutes to inactivate the enzyme, freeze-dry,
An LF hydrolyzate having a decomposition rate of 6 to 50% was prepared.

(2) Test Method The decomposition rate and the tyrosinase activity inhibitory effect were measured by the same method as in Test Example 10.

(3) Test Results Table 11 shows the tyrosinase activity inhibitory effect of the LF hydrolyzate by the enzyme.

[0114]

[Table 11] Samples 9 to 14 having a decomposition rate of 6 to 40% are all 0.0
A tyrosinase activity inhibitory effect of about 30% was already shown at the addition amount of 5%. The inhibitory effect improves as the amount of addition increases, and it shows about 60% inhibition rate at 0.1% addition, about 80% at 0.3% addition, and 100% at 0.5% addition. It was

On the other hand, Sample 15, whose decomposition rate is 45 to 50%,
No. 16 had a tyrosinase activity inhibitory effect of about 10% when 0.3% was added. The inhibitory effect improves as the amount of addition increases, about 30% at 1% addition and about 50% at 2% addition.
The inhibition rate was shown.

Incidentally, apolactoferrin, zinc, copper,
A similar tyrosinase activity-inhibiting effect was obtained with a hydrolyzate by the enzyme of metal lactoferrin chelated with a metal such as iron.

Next, examples of the tyrosinase activity inhibitor according to the present invention will be shown.

Example 8 80 g of a commercially available LF (manufactured by Oleofina) was dissolved in 1000 mg of purified water, and 1M hydrochloric acid was added to adjust the pH to 2. Then add the solution to 1
After heating at a temperature of 15 ° C. for 10 minutes, the pH was adjusted to 7 with a 1 M sodium hydroxide solution, freeze-dried, and the LF hydrolyzate 7 having a decomposition rate of 15% measured by the same method as in Test Example 7 was used.
7 g was obtained. 50 g of this LF hydrolyzate, 900 g of glycine (manufactured by Wako Pure Chemical Industries, Ltd.) and 50 g of lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) were uniformly mixed to obtain about 1000 g of a tyrosinase activity inhibitor for keeping the food fresh.

The tyrosinase activity inhibitor for maintaining the freshness of the obtained food was dissolved in water at a concentration of 20%, and the tyrosinase activity inhibitor was measured by the same method as in Test Example 10.
It was 100%.

[Example 9] 270 g of commercially available LF (manufactured by Olefina) was dissolved in 6300 ml of purified water, and then 10
% Citric acid aqueous solution was added to adjust the pH to 2.5, and the mixture was reacted at room temperature for 1 hour. The reaction product was subjected to ultrafiltration, and the cake was freeze-dried to obtain about 260 g of apolactoferrin. 60g of this apolactoferrin
Was dissolved in 1000 ml, and the pH was adjusted to 3 by adding a 2M phosphoric acid solution. Then, the solution was heated at a temperature of 121 ° C. for 25 minutes, the pH was adjusted to 7 with 1 M sodium hydroxide, and the solution was freeze-dried. 55 g of an LF hydrolyzate having a decomposition rate of 23% measured by the same method as in Test Example 10 Got 20 g of this LF hydrolyzate, 400 g of propylene glycol (manufactured by Wako Pure Chemical Industries), 4 g of oleyl alcohol (manufactured by Wako Pure Chemical Industries), ethanol (manufactured by Wako Pure Chemical Industries) 2
00 g and 3376 g of purified water were uniformly mixed to obtain about 4000 g of a tyrosinase hydrolysis inhibitor for cosmetics.

The tyrosinase activity inhibition rate of the obtained cosmetic tyrosinase activity inhibitor was measured by the same method as in Test Example 10. As a result, the apparent inhibition rate was 75%. The rate was 100%.

[Example 10] 100 g of commercially available LF (manufactured by Olefina) was dissolved in 1000 ml of purified water to give 1M.
After adjusting the pH to 2 by adding the hydrochloric acid of 1., the mixture was maintained at 37 ° C., and 5 g of commercially available pig pepsin (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted for 60 minutes. Then, it was heated at 80 ° C. for 10 minutes to inactivate the enzyme and freeze-dried to obtain about 95 g of an LF hydrolyzate having a decomposition rate of 12% measured by the same method as in Test Example 10. 30 g of this LF hydrolyzate, 200 g of propylene glycol (manufactured by Wako Pure Chemical Industries), 2 g of oleyl alcohol (manufactured by Wako Pure Chemical Industries), 100 g of ethanol (manufactured by Wako Pure Chemical Industries) and 1668 g of purified water are uniformly mixed. About 2,000 tyrosinase activity inhibitors for cosmetics
g was obtained.

The tyrosinase activity inhibitory rate of the obtained cosmetic tyrosinase activity inhibitor was measured by the same method as in Test Example 10, and as a result, it was 100%.

Example 11 Commercially available LF (manufactured by Oleofina) (150 g) was dissolved in purified water (1000 ml), 1 M caustic soda was added to adjust the pH to 6, and the mixture was heat-sterilized at 60 ° C. for 10 minutes. This solution was cooled and kept at 50 ° C., and a commercially available soy sauce enzyme (manufactured by Tanabe Pharmaceutical Co., Ltd.) 30 containing 15 g of commercially available trypsin (manufactured by Novo Co.) and peptidase 30.
g was added and reacted for 5 hours. Then, the enzyme was inactivated by heating at 80 ° C. for 10 minutes and freeze-dried to obtain about 145 g of an LF hydrolyzate having a decomposition rate of 38% measured by the same method as in Test Example 10. 27 g of this LF hydrolyzate, 1 g of sodium hyaluronate (manufactured by Wako Pure Chemical Industries, Ltd.), 10 g of glycerin (manufactured by Wako Pure Chemical Industries, Ltd.) and 962 g of purified water were uniformly mixed, and about 100 tyrosinase activity inhibitors for cosmetics were used.
0 g was obtained.

The tyrosinase activity inhibition rate of the obtained cosmetic tyrosinase activity inhibitor was measured by the same method as in Test Example 10, and as a result, it was 100%.

[Example 12] 90 g of commercially available LF (manufactured by Olefina) was dissolved in 2100 ml of purified water, and this was mixed with 755 ml of 2.6 mM iron sulfate aqueous solution at room temperature for 24 hours.
Reacted for hours. The reaction product was ultrafiltered and the cake was lyophilized to give about 78 g of lactoferrin iron.
40 g of this lactoferrin iron was dissolved in 500 ml of purified water, and the pH was adjusted to 3 by adding 1 M hydrochloric acid.
Hold at ℃, commercially available Sumiteam AP (manufactured by Shin Nippon Chemical Co., Ltd.)
Was added and reacted for 3 hours. Then 1 at 80 ° C
Heat for 0 minutes to inactivate the enzyme, freeze-dry and test Example 1
About 35 g of an LF hydrolyzate having a decomposition rate of 18% measured by the same method as that of 0 was obtained. 30% of this LF hydrolyzate
g, 450 g of glycine (manufactured by Wako Pure Chemical Industries, Ltd.) and 20 g of lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) were uniformly mixed to obtain about 500 g of a tyrosinase activity inhibitor for keeping the food fresh.

The tyrosinase activity inhibitor for maintaining the freshness of the obtained food was dissolved in water at a concentration of 20%, and the tyrosinase activity inhibitory rate was measured by the same method as in Test Example 10.
It was 100%.

[0128]

The effects of the present invention are as follows.

1) An antibacterial agent, an antibacterial composition, a tyrosinase activity inhibitor, and a tyrosinase activity inhibitory composition containing the LF degradation product of the present invention as an active ingredient are degradation products of natural antibacterial substances derived from milk and the like. Therefore, it is safer than chemically synthesized antibacterial substances.

2) The antibacterial agent and the antibacterial composition containing the LF degradation product of the present invention as an active ingredient have remarkably superior antibacterial properties to natural LF (undegraded).

3) The antibacterial agent, the antibacterial composition, the tyrosinase activity inhibitor, and the tyrosinase activity inhibitory composition containing the LF degradation product of the present invention as an active ingredient can be used in any form of liquid or powder, and they can be used. Extensive.

4) An antibacterial agent containing the LF hydrolyzate of the present invention as an active ingredient, an antibacterial composition, a tyrosinase activity inhibitor,
The tyrosinase activity inhibitory composition is stable even when added to various products and stable against heating, and thus has a wide range of applications.

5) An antibacterial agent containing the LF hydrolyzate of the present invention as an active ingredient, an antibacterial composition, a tyrosinase activity inhibitor,
By applying the tyrosinase activity-inhibiting composition to human and animal foods, feeds, etc. (for example, washing of foods, feeds, addition to foods, feeds, blending, impregnation), maintenance of their quality (discoloration, deterioration In addition to the prevention, when they are inoculated, they have the effects described for the above-mentioned antibacterial agents, and they are applied (mixed) to articles that come into contact with human skin (for example, absorbent cotton, wet tissues, etc.). , Impregnation)
The hygiene of these articles is maintained, and when they are used, a whitening effect is obtained.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical indication location A61K 37/14 AED 8314-4C 37/64 8314-4C (72) Inventor Kozo Kawase Urawa, Saitama Prefecture 761-1 Shirae-shi, Japan (72) Yoshitaka Tamura 1-4-5, Fujizuka, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture (72) Mitsunori Takase 138-10 Minamichumaru, Omiya-shi, Saitama Prefecture (72) Tsuneharu Yamauchi Kamakura, Kanagawa Prefecture 4-2-2 Ichitamanawa Garden Heights Kamakura 405 Tamagawa, Kamakura (72) Inventor Hitoshi Saito 2-7-4 Yurigaoka, Aso-ku, Kawasaki, Kanagawa Prefecture 301 (72) House of Yurigaoka Morinaga, Ina, Hiroaki Abe 3-Urago-cho, Yokosuka City, Kanagawa Prefecture 43

Claims (6)

[Claims] 1. A mammalian lactoferrin, a mammalian apolactoferrin, a mammalian metal-saturated lactoferrin,
And an antibacterial agent containing a lactoferrin degradation product obtained by hydrolyzing lactoferrin selected from the group consisting of any mixture thereof as an active ingredient. 2. Mammalian lactoferrin, mammalian apolactoferrin, mammalian metal-saturated lactoferrin,
And an antibacterial composition containing a lactoferrin degradation product obtained by hydrolyzing lactoferrin selected from the group consisting of any mixture thereof as an active ingredient. 3. Mammalian lactoferrin, mammalian apolactoferrin, mammalian metal-saturated lactoferrin,
And a tyrosinase activity inhibitor containing, as an active ingredient, a lactoferrin degradation product obtained by hydrolyzing lactoferrin selected from the group consisting of any mixture thereof. 4. The tyrosinase activity inhibitor according to claim 4, wherein the lactoferrin degradation product is contained at a concentration of at least 0.05% (weight). 5. The tyrosinase activity inhibitor according to claim 4 or 5, wherein the decomposition rate of the lactoferrin degradation product is at least 6% (by weight). 6. Mammalian lactoferrin, mammalian apolactoferrin, mammalian metal-saturated lactoferrin,
And a method for treating an article with a lactoferrin degradation product obtained by hydrolyzing a lactoferrin selected from the group consisting of any mixture thereof.