JP5535559B2 - Peroxidase - Google Patents

Description

  The present invention relates to a peroxide-degrading enzyme produced by lactic acid bacteria.

  In order to remove peroxides inside and outside the living body, organisms have various peroxide-degrading enzymes, and examples of typical peroxide-degrading enzymes include glutathione peroxidase.

  Glutathione peroxidase is widely present in all living organisms including animals, and converts peroxide into alcohols and water as shown in the following general formula (1) by a multi-step reaction via glutathione (non- (See Patent Documents 1 to 4).

  Thus, peroxide-degrading enzymes are widely present in organisms including animals and plants, and have an antioxidant ability as long as there is no fear of denaturation and deactivation due to strong acids, strong alkalis, heating, or the like.

Toshikazu Yoshikawa and three others, “All about active oxygen and free radicals”, Maruzen Co., Ltd., p.85-92 Naoyuki Taniguchi, “Reactive Oxygen Experiment Protocol”, Shujunsha, p.84-95 Akio Futaki, two others, "Antioxidants", Society Press, p79-86, p321-323 International Biochemical Union Enzyme Committee Report "List of Enzyme Names and Enzyme Reaction Symbols" Kyoritsu Publishing Co., Ltd., p70-88

  By the way, some lactic acid bacteria widely used in fermented foods such as yogurt are said to have antioxidant enzymes, but there are many unclear points about their properties, so the properties of the enzymes are There is a need to clarify.

  Therefore, the present inventors have isolated lactic acid bacteria from various fermented foods and conducted extensive studies on the properties of the cultured lactic acid bacteria. As a result, the enzyme produced by lactic acid bacteria belonging to the genus Lactobacillus is peroxide. The present invention has been completed.

That is, the gist of the present invention is as follows.
[1] A peroxide-degrading enzyme derived from Lactobacillus plantarum TY-1572 (NITE P-90) and having the following physicochemical properties:
(1) Substrate specificity In the presence of NAD (P) H, it exhibits high reactivity with peroxides and also reacts with hydrogen peroxide. (2) Molecular weight 43 kDa (measured by SDS polyacrylamide gel electrophoresis)
[2] A peroxide-degrading enzyme derived from Lactobacillus plantarum TY-1572 (NITE P-90) and having the following physicochemical properties:
(1) Substrate specificity In the presence of NAD (P) H, it exhibits high reactivity with peroxides and also reacts with hydrogen peroxide. (2) Molecular weight 48 kDa (measured by SDS polyacrylamide gel electrophoresis)
[3] A food or drink containing the peroxide-degrading enzyme according to [1] and / or [2], or a cosmetic.

According to the present invention, a novel peroxide-degrading enzyme derived from Lactobacillus plantarum TY-1572 strain (NITE P-90) among lactic acid bacteria can be provided. In addition, by incorporating the enzyme into a food or drink or cosmetics, it can be expected to utilize the peroxide removing ability of the enzyme.

2 is an electrophoretic diagram of enzyme A. FIG. FIG. 6 is an electrophoretic diagram of enzyme B.

  The lactic acid bacterium used for the production of the peroxide-degrading enzyme of the present invention is Lactobacillus plantarum TY-1572 strain (NITE P-90). When the present inventors examined, this strain produces two types of peroxide-degrading enzymes called “enzyme A” and “enzyme B” in the present invention. In the presence of NAD (P) H, enzyme A is highly reactive with peroxides and also reacts with hydrogen peroxide. (2) As a molecular weight, enzyme A is an SDS polyacrylamide gel. 43 kDa is shown by measurement by electrophoresis (SDS-PAGE). Enzyme B has (1) substrate specificity, high reactivity to peroxide in the presence of NAD (P) H, and also to hydrogen peroxide, and (2) molecular weight, SDS polyacrylamide gel 48 kDa is shown by measurement by electrophoresis (SDS-PAGE). That is, enzymes A and B both show the same substrate specificity, but have different molecular weights.

  In the present invention, hydrogen peroxide is a peroxide having a peracid group on a hydrogen atom, and the peroxide is a hydrocarbon other than normal lipid peroxide (eg, linoleic peroxide). This is a concept including a peroxide having a peracid group (for example, butyl peroxide, cumene peroxide, etc.) and does not include hydrogen peroxide. In the above enzyme reaction, it is presumed that the peroxide is decomposed (reduced) into alcohols and water, and that hydrogen peroxide is decomposed (reduced) into water.

  Lactobacillus plantarum TY-1572 strain (NITE P-90), which exhibits decomposition characteristics for peroxide, can be screened as follows. That is, first, various fermented foods as a separation source, the separation source is cultured in a solid medium to isolate lactic acid bacteria, and the cells are collected from a culture obtained by culturing the isolated lactic acid bacteria in a liquid medium, Subsequently, screening can be carried out by examining whether or not the test liquid has a decomposition property against peroxide, using the cell suspension containing the bacterial cells as the test liquid.

  In the present invention, the fermented food generally refers to animal and plant fermented foods containing lactic acid bacteria. Examples of typical fermented foods include, for example, miso, soybean cake, koji, koji. Pickled, miso pickled, pickled cucumber, pickled in Nozawa, pickled with sunflower, bettara, miso, soy sauce, sake lees, natto, rice vinegar, balsamic vinegar, apple vinegar, kimchi, milk, nanpura, tofuyo, wine lees, sake lees, yogurt , Vegemite, Kvass, Kumis, Givyac, Pickles, Kumis, Sawarout. In the culture, the fermented food is preferably crushed and, for example, a food suspension diluted with physiological saline.

  The solid medium may be any medium that is usually used for culturing lactic acid bacteria, and representative examples include, for example, yeast extract peptone medium, glucose medium, phenethyl alcohol medium, acetate medium, GYP medium, MRS medium, TITG medium, SL medium, cysteine milk medium, LBS medium, TATAC medium, MG medium, sodium chloride 18% -potassium nitrate medium, broth medium, BCP medium, thioglycolate medium, diluted grape juice medium, ESY medium, Yoshitake Mr. medium, salt-resistant lactic acid bacteria medium, soy sauce medium for salt-resistant lactic acid bacteria, Ueno medium, Iizuka-Yamazato medium, TYG medium, lactic medium, M17 medium, Lactic streptococci fractionation medium, Lactic streptococci fractionation medium for fermenting citric acid, PPYL medium, YPG medium, acidic tomato medium, Mayeux & Colmer Leuconostoc detection medium, Hamamoto et al. Leuconostoc detection medium, Pearce & Halig An an Leuconostoc measuring medium, an APT medium, a Briggs tomato juice medium, a Rogosa medium, a NAP medium and the like are added with 0.5 to 2.0% agar as a solidifying agent. In addition, since lactic acid bacteria are selectively grown on the medium, substances that inhibit the growth of aerobic bacteria (for example, sodium azide), substances that inhibit the growth of gram-negative bacteria (for example, polymyxin B), and fungal growth A substance that inhibits (for example, cycloheximide) or the like may be appropriately combined and contained in the solid medium. The liquid medium may be any medium that is usually used for culturing lactic acid bacteria. Typical examples include, for example, yeast extract peptone medium, glucose medium, phenethyl alcohol medium, acetate medium, GYP medium, MRS. Medium, TITG medium, SL medium, cysteine milk medium, LBS medium, TATAC medium, MG medium, sodium chloride 18% -potassium nitrate medium, broth medium, BCP medium, thioglycolate medium, diluted grape juice medium, ESY medium, Yoshihiro's medium, salt-resistant lactic acid bacteria medium, soy sauce medium for salt-resistant lactic acid bacteria, Ueno medium, Iizuka / Yamazato medium, TYG medium, lactic medium, M17 medium, Lactic streptococci fractionation medium, Lactic streptococci fractionation that ferments citric acid Medium, PPYL medium, YPG medium, acidic tomato medium, Mayeux & Colmer Leuconostoc detection medium, Hamamoto et al. Leuconostoc detection medium, Pea Examples include rce & Haligan's Leuconostoc measurement medium, APT medium, Briggs' tomato juice medium, Rogosa medium, and NAP medium.

  The lactic acid bacteria may be cultured according to a conventional method, for example, aerobic culture, stationary culture or neutralization culture under conditions of 30 to 40 ° C. and 10 to 40 hours.

After completion of the culture, it is preferable to isolate colonies of lactic acid bacteria one by one and purify them according to a conventional method. Then, the isolated lactic acid strain is cultured according to a conventional method, and the obtained culture is centrifuged to recover the cells, and a buffer solution is added to the cells to adjust the concentration to OD _{660 nm} = 1.5 to 1.7. Lactic acid bacteria suitable for the purpose of the present invention are prepared by examining whether or not the bacterial cell suspension exhibits degradation characteristics against peroxides. And

  Decomposition characteristics for peroxide include, for example, peroxide (final concentration: 1.0 to 3.0 mM) and, if necessary, saccharide (for example, glucose) (final concentration: 30 to 100 mM) in the cell suspension. Each is added, reacted at 37 ° C. for 3 hours, the reaction solution after completion of the reaction is centrifuged, and the remaining amount of peroxide contained in the supernatant is measured. In addition, although the said saccharide | sugar is added in order to provide the energy by metabolism of a lactic acid strain, it does not necessarily need to add.

  By evaluating the peroxide decomposition characteristics of the lactic acid strain isolated as described above, the lactic acid bacteria TY-1572 strain can be selected as exhibiting peroxide decomposition characteristics. From the results of mycological properties and genetic characteristics, the lactic acid bacterium strain TY-1572 was identified as Lactobacillus plantarum. The above strain is deposited as Lactobacillus plantarum TY1572 (NITE P-90) in the National Institute of Technology and Evaluation Patent Microorganism Depositary.

  In the present invention, the lactic acid bacterium strain TY-1572 isolated as described above was cultured according to a conventional method, and the resulting culture was centrifuged to recover the cells, and a buffer solution was added to the cells. It can be used in a crude enzyme state such as a bacterial cell suspension or a cell-free extract obtained by crushing the bacterial cell suspension by a mechanical method, an enzymatic method or the like. Since the enzymes A and B according to the present invention coexist in the crude enzyme, the enzymes A and B are used together when used in the state of the crude enzyme. Further, the crude enzyme solution may be used as a capsule by being micellized with a coating protective material (eg, lecithin, chitin, etc.) or adsorbed with an excipient (eg, dextrin, etc.) and used as a powder. Further, the crude enzyme solution can be used as a dried product by lyophilization, spray drying and the like according to a conventional method.

  When the enzymes A and B are used individually, the crude enzyme solution may be purified and used. In the purification of the crude enzyme solution, for example, a cell-free extract obtained by crushing the cell suspension is subjected to a centrifugal treatment, and uncrushed cells are removed from the cell-free extract. Used as a liquid. Then, a purified enzyme is obtained by appropriately combining, for example, salting-out treatment of ammonium sulfate, sodium sulfate, etc. and chromatography such as a hydrophobic butyl column, a weak anion exchange column, a gel filtration column, an affinity column, etc. with this crude enzyme solution. Can do. The purified enzymes A and B are purified to such an extent that each of them shows a substantially single band by SDS-PAGE.

  In addition, about each fraction obtained when said various chromatographic methods are applied, the fraction containing enzyme A or B is analyzed by analyzing the peroxide decomposition activity of each fraction and the peak pattern of protein. Collected. Peroxide decomposition activity was determined by adding NADH or NADPH (hereinafter sometimes referred to as “NAD (P) H”) as an electron donor and peroxide as an electron acceptor to each fraction at 37 ° C. The reaction is evaluated for 3 minutes, and the NAD (P) H concentration and peroxide concentration after the reaction are measured. The NAD (P) H concentration is measured by absorbance at 340 nm, and the peroxide concentration is measured by a color development method using a known thiocyanate-iron complex.

Enzyme A obtained as described above exhibits the following physicochemical properties.
(1) Substrate specificity In the presence of NAD (P) H, it exhibits high reactivity with peroxides and also reacts with hydrogen peroxide.
(2) Molecular weight 43 kDa (measured by SDS polyacrylamide gel electrophoresis).
In addition, the hydrogen peroxide decomposition activity is lower in enzyme A than in enzyme B.

In addition, enzyme B exhibits the following physicochemical properties.
(1) Substrate specificity In the presence of NAD (P) H, it exhibits high reactivity with peroxides and also reacts with hydrogen peroxide.
(2) Molecular weight 48 kDa (measured by SDS polyacrylamide gel electrophoresis).

Enzymes A and B according to the present invention can be used alone or in the presence of both, for example, in a state where they are contained in foods and beverages and cosmetics. By containing the enzyme in food or drink, it can be expected to utilize the peroxide removing ability of the enzyme.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the examples.

1. Screening for lactic acid bacteria
1-1. Preparation of food suspension As sources for separating lactic acid bacteria, Koji, bean paste, koji, koji pickled, miso pickled, koji pickled, Nozawa nazuke, sunki zuke, bettara pickled, miso, soy sauce, sake lees, Natto, rice vinegar, balsamic vinegar, apple vinegar, kimchi, roasted milk, nanpura, tofuyo, wine lees, sake lees, yogurt, vegemite, quas, cumis, gibiyac, pickles, kumis, sauerkraut, fruit, vegetables, seaweed, fish After obtaining and crushing each of the foods, it was diluted with physiological saline to obtain a food suspension.

1-2. Screening GYP medium (1% glucose, 1% yeast extract, 0.5% peptone, 0.2% sodium acetate _{· 3H 2 0,20ppm MgSO 4 · 7H} 2 0,1ppm MnSO 4 · 4H 2 0,1ppm FeSO 4 · 7H ₂ 0,1ppm NaCl, 50ppm Tween80 sodium azide) (final concentration 30 ppm), polymyxin B (the on selective agar medium containing final concentration 30 ppm) and cycloheximide (final concentration 30 ppm) "1-1. food suspension Each of the food suspensions prepared in “Preparation” was added and statically cultured at 30 ° C. for 16 hours. After completion of the culture, it was confirmed that colonies had grown from the food suspension-added sample prepared from the separation source described in “1-1. Preparation of food suspension”, and each colony was isolated. The obtained colonies of lactic acid strains were inoculated on a GYP agar medium and purified according to a conventional method. And the lactic acid strain isolate | separated from the said food group was named TY-1572 strain.

2. Bacteriological properties of lactic acid bacteria The bacteriological properties of the lactic acid bacteria strain (TY-1572) isolated in the above “1-2. Screening” are shown in the lactic acid bacteria experiment manual (supervised by Michio Kosaki, Yasushi Uchimura, Sanae Okada, Asakura). (Bookstore). The results are shown in Table 1.

3. Genetic characteristics of lactic acid bacteria For TY-1572 strain isolated in “1-2. Screening”, the base sequence of 16S rDNA was determined according to the conventional method, and the existing lactic acid bacteria were analyzed using BLAST program and Clustal W program. We searched which bacterial species was closest to the sequence. As a result, the base sequence of 16S rDNA of lactic acid bacteria strain TY-1572 was 100% identical to the base sequence of 16S rDNA of Lactobacillus plantarum. From the above results, lactic acid bacteria strain TY-1572 was identified as Lactobacillus plantarum.

4). Peroxide degradation characteristics of lactic acid bacteria Peroxide is added to the cell suspension of TY-1572 strain identified in “3. Genetic characteristics of lactic acid bacteria” and remains in the reaction solution after a predetermined time. The remaining amount of peroxide to be measured was measured, and the decomposition characteristics against peroxide were examined.

4-1. Preparation of cell suspension
Each strain of TY-1572 strain was inoculated into GYP medium and cultured at 37 ° C. for about 24 hours. After completion of the culture, the culture was centrifuged at 8,000 rpm for 10 minutes, and the cells were collected by removing the supernatant. Next, the collected bacterial cells were suspended in a phosphate buffer solution, and the concentration adjusted to OD _{660 nm} = 1.6 was used as the bacterial cell suspension.

4-2. Decomposition characteristics for peroxides Peroxide (cumene peroxide or t-butyl peroxide) (final concentration) in the cell suspension prepared in “4-1. Preparation of cell suspension”. 3.0 mM) and glucose (final concentration 50 mM) were added, respectively, and reacted at 37 ° C. for 3 hours on a horizontal shaker. After completion of the reaction, the reaction solution was centrifuged using a tabletop centrifuge, and the unreacted peroxide concentration contained in the supernatant was measured.

  For the measurement of the peroxide concentration, the Yagi method using the equimolar color reaction of peroxide and methylene blue derivative in the presence of hemoglobin was used. The measurement method follows the instructions of Determiner LPO (Kyowa Medex), which is a Yagi method. When the decomposition amount of peroxide was calculated based on the obtained data, it was about 2800 μM for cumene peroxide and about 800 μM for t-butyl peroxide.

4-3. Decomposition characteristics for peroxides In place of the peroxides used in “4-2. Decomposition characteristics for peroxides”, peroxides derived from linoleic acid (linoleic peroxide) were used in the experiments. . The peroxide was obtained by adding lipoxygenase to a linoleic acid solution and stirring vigorously while blowing oxygen in a hot water bath (30 ° C.) for about 5 minutes. In addition, thin layer chromatography was used for the confirmation of peroxidic linoleic acid (Reference: Experimental method for lipid peroxide, Naoshi Kaneda, Nobuo Ueda, Ishigaku Publishing Co., Ltd., Agric. Biol. Chem. 45, P587. -593,1981, General hygiene test method and explanation, edited by Japan Pharmaceutical Association, Nanzando, P33, Introduction to Lipid Analysis, Yasuhiko Fujino, Academic Publishing Center, P100). In the experiment, an emulsified dispersion of the obtained linoleic peroxide was used. The peroxide concentration of the emulsified and dispersed linoleic acid peroxide solution was about 1000 μM.

  Then, the above-described aqueous linoleic acid aqueous solution (final concentration 1 mM) and glucose (final concentration 50 mM) were added to the cell suspension prepared in “4-2. For 3 hours at 37 ° C. After completion of the reaction, the reaction solution was centrifuged with a tabletop centrifuge, and the unreacted linoleic acid concentration in the supernatant was measured. The measuring method of the concentration of peroxylinoleic acid was performed according to the above-mentioned “4-2. Decomposition characteristics for peroxide”. When the decomposition amount of linoleic peroxide was calculated based on the obtained data, it was about 850 μM.

5. Purification of peroxide-degrading enzyme The peroxide-degrading enzyme was purified as follows. In each purification step, NADH concentration, cumene peroxide (as peroxide) concentration, protein concentration and SDS-PAGE were measured by the following methods.

(NADH concentration)
In measuring the concentration of NADH, the activity was measured by decreasing the absorption wavelength (λ = 340 nm) of the substance. Specifically, about 10 mM in a micro black cell, final concentration 150 μM NADH dissolved in 500 μl of pH unadjusted Tris buffer, final concentration 500 μM cumene peroxide, 1 / 100-fold test sample (cell-free extract or each purification) Each fraction obtained in the step) was preheated at 37 ° C. for 3 minutes in advance, added sequentially, and mixed by inversion each time. The activity of the enzyme was measured as a decrease in NADH over time at 37 ° C. using a double beam absorptiometer (ε ₃₄₀ = 6.220 M ⁻¹ cm ⁻¹ ). The NADH decreasing activity accompanying the decomposition of cumene peroxide was calculated by subtracting the initial speed of the test sample immediately after the addition from the initial speed when the cumene peroxide was added. Measurement was performed using NADPH instead of NADH as necessary.

(Cumene peroxide concentration)
The measurement of the concentration of cumene peroxide was performed by a color development method using a thiocyanate-iron complex. Using the same experimental system as the “(NADH concentration)”, the peroxide-degrading enzyme reacted with cumene peroxide was appropriately diluted and collected in a 500 μl, 2.0 ml Eppendorf tube, and 10% trichloroacetic acid aqueous solution was added in 500 μl, 0.01%. N 100 mL of ammonium iron (II) sulfate dissolved in sulfuric acid and 100 μL of 2.5 M potassium thiocyanate were sequentially added, and the sample that reacted in red was measured at λ = 480 nm with a double beam spectrophotometer.

(Protein concentration)
The protein concentration of each fraction having a cumene peroxide decomposition activity was calculated using a conventional method called the Bradford method. The measurement principle uses the color change from red purple to blue when a dye called Coomassie Brilliant Blue (CBB) binds to a protein. When quantifying the protein concentration, a calibration curve was prepared with chicken egg white albumin, which is a known protein, and the protein concentration was calculated.

(SDS-PAGE)
SDS-PAGE was used for confirmation of the degree of purification in the enzyme purification process and determination of fractions useful for collection. Enzyme analysis is performed with reference to “Bio-experiment from the beginning” (Toru Oyama, Toshihiro Watanabe, Sankyo Publishing), and uses the modified method of the previous book as necessary.

  Gel preparation stock solution A solution prepared with 29.2 g of acrylamide, 0.8 g of N, N'-methylenebisacrylamide, 100 ml distilled water, and gel preparation with 0.4% SDS dissolved in 1.5 M Tris buffer adjusted to pH 8.8 Stock solution B, gel preparation stock solution C prepared by dissolving 0.4% SDS in 0.5M Tris buffer adjusted to pH 6.8, and gel preparation stock solution D comprising 10% ammonium persulfate solution were prepared and prepared. Dispense into plates. After the separation SDS polyacrylamide gel was cured, the concentration SDS polyacrylamide gel was overlaid.

  The prepared gel plate was set in an electrophoresis tank filled with a buffer for electrophoresis, and electrophoresis was performed by applying a current of 7.5 to 10 mA in the concentrated gel portion and 15 to 20 mA after passing through the concentrated gel. Electrophoresis stopped at about 1 cm before the lower end of the gel plate and moved to the staining step.

  The polyacrylamide gel after the electrophoresis was stained with Coomassie Brilliant Blue (CBB) staining solution, and then decolorization was performed until a clear staining band was obtained.

5-1. Preparation of Crude Enzyme Solution The method for culturing lactic acid bacteria was performed according to a conventional method used by those skilled in the art. Lactobacillus plantarum (LITEobacillus plantarum) TY-1572 strain (NITE P-90) was inoculated into 20 l of a GYP liquid medium normally used for lactic acid bacteria culture and cultured at 37 ° C. for 13 to 14 hours in a jar fermenter. . In the late logarithmic growth phase, the cultured cells were collected by centrifugation. The collected cultured cells were washed with pH 7.0, 50 mM phosphate buffer.

  A crude enzyme solution was obtained by lysozyme treatment, disruption by ultrasonic treatment, and disruption by an ultrahigh pressure cell disrupter.

5-2. Salting-out treatment, denucleic acid treatment and chromatography The crude enzyme solution obtained by the above operation was subjected to salting-out treatment with ammonium sulfate and denucleic acid treatment with streptomycin sulfate. The supernatant obtained in this step was applied to a column for purification.

The supernatant of the crude enzyme solution obtained by the above operation was subjected to a hydrophobic butyl column, a weak anion exchange column, and a gel filtration column to obtain enzyme A. Fraction collection was judged using the protein recovery amount by Abs _280nm and the degradation activity against cumene peroxide as indicators. The fraction was dialyzed for each collection. In the recovered fraction, the ability to decompose cumene peroxide was particularly high, and the purified band was subjected to SDS-PAGE. As a result, the presence of a single band at a position of about 43 kDa was confirmed, and this fraction was used as a purified enzyme. . The result of electrophoresis is shown (FIG. 1).

The supernatant of the crude enzyme solution obtained by the above operation was applied to a hydrophobic butyl column and an affinity column to obtain enzyme B. Fraction collection was judged using the protein recovery amount by Abs _{280 nm} and the decomposition activity of cumene peroxide as indicators. The fraction was dialyzed for each collection.

  In the recovered fraction, the ability to decompose cumene peroxide was particularly high, and the purified band was subjected to SDS-PAGE. As a result, the presence of a single band was confirmed at about 48 kDa, and this fraction was used as a purified enzyme. . The result of electrophoresis is shown (FIG. 2).

6). Substrate specificity In the experimental system of “5. Purification of peroxide-degrading enzyme (NADH concentration)”, purified enzymes A and B were used as test samples, and hydrogen peroxide was used instead of cumene peroxide. Except for the above, the enzymatic reaction was carried out in the same manner as described above to examine the decomposition activity of enzymes A and B against hydrogen peroxide. The concentration of hydrogen peroxide was measured according to “5. Purification of peroxide-degrading enzyme (cumene peroxide concentration)”. As a result of the measurement, both enzymes A and B reacted with hydrogen peroxide, and the hydrogen peroxide decomposition activity of enzyme A was lower than that of enzyme B.

In addition, using purified enzymes A and B, inhibitors were examined in the presence of cumene peroxide. Inhibitors were used KCN, NaOH, pCMB, HgCl _2, ethanol, quinine, and Quinacrine. The test conditions are the same as in the experimental system described in “5. Purification of peroxide-degrading enzyme (NADH concentration)”, but the inhibitor is a final concentration of 2 mM, the cumene peroxide concentration is 500 μM, and the NADH concentration is 150 μM. did. As a result of the measurement, when KCN, pCMB, HgCl ₂ , quinine, and quinacrine were added to both enzymes A and B, the peroxide decomposition reaction inhibitory effect could be confirmed.

7). Uses of peroxide-degrading enzymes
7-1. Production Example of Peroxide Degrading Crude Enzyme Solution Inoculate TY-1572 strain into a medium composed of 97.5 parts by weight of water, 1 part by weight of glucose, 0.5 part by weight of soybean peptide and 1 part by weight of yeast extract, The cells were statically cultured at 30 ° C. for 16 hours, and the cultured cells were collected by centrifugation. This microbial cell was suspended in a phosphate buffer solution having a volume 5 times the wet weight, and the microbial cell was crushed by a high-pressure treatment of 270 MPa or more to obtain a peroxide-decomposing crude enzyme solution.

7-2.Production example of yogurt 44.36 parts by weight of water, 0.1 part by weight of gelatin, 8.2 parts by weight of sugar, 0.05 part by weight of lactic acid bacteria starter, 5.81 parts by weight of skim milk powder, 41.4 parts by weight of milk, 0.03 part by weight of peroxide-degrading crude enzyme solution Then, 0.05 part by weight of a fragrance was mixed to obtain yogurt. In addition, the peroxide decomposition | disassembly crude enzyme liquid used what was manufactured above.

7-3. Production Example of Ice Cream 55.85 parts by weight of water, 8.0 parts by weight of sugar, 8.0 parts by weight of starch syrup, 4.0 parts by weight of liquid sugar, 13.0 parts by weight of vegetable oil and fat, 8.5 parts by weight of skim milk powder, sweetened egg yolk (20% egg yolk content) An ice cream was obtained by mixing 2.0 parts by weight, 0.05 parts by weight of a peroxide-degrading crude enzyme solution, 0.5 parts by weight of a stabilizer, and 0.1 parts by weight of a fragrance. In addition, the peroxide decomposition | disassembly crude enzyme liquid used what was manufactured above.

7-4. Example of lotion preparation 73.89 parts by weight of water, 5 parts by weight of glycerin, 5 parts by weight of solubilizer, 1 part by weight of surfactant, 15.0 parts by weight of ethanol, 0.1 part by weight of peroxide-degrading crude enzyme solution, antiseptic A lotion was prepared by mixing 0.005 parts by weight of the agent and 0.005 parts by weight of the fragrance. In addition, the peroxide decomposition | disassembly crude enzyme liquid used what was manufactured above.

Claims (3)

Lactobacillus plantarum (Lactobacillus plantarum) TY-1572 strain (NITE P-90) derived from a crude enzyme solution obtained by crushing the cell suspension 2 Among the types of peroxide-degrading enzymes, peroxide-degrading enzymes having the following physicochemical properties:
(1) Substrate specificity In the presence of NAD (P) H, it exhibits high reactivity with peroxides and also reacts with hydrogen peroxide.
(2) Molecular weight 43 kDa (measured by SDS polyacrylamide gel electrophoresis). Lactobacillus plantarum (Lactobacillus plantarum) TY-1572 strain (NITE P-90) derived from a crude enzyme solution obtained by crushing the cell suspension 2 Among the types of peroxide-degrading enzymes, peroxide-degrading enzymes having the following physicochemical properties:
(1) Substrate specificity In the presence of NAD (P) H, it exhibits high reactivity with peroxides and also reacts with hydrogen peroxide.
(2) Molecular weight 48 kDa (measured by SDS polyacrylamide gel electrophoresis). Food / beverage products or cosmetics containing the peroxide-degrading enzyme according to claim 1 and / or 2.