WO2011142300A1 - バチルス属微生物由来の還元剤及びその用途 - Google Patents
バチルス属微生物由来の還元剤及びその用途 Download PDFInfo
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- WO2011142300A1 WO2011142300A1 PCT/JP2011/060622 JP2011060622W WO2011142300A1 WO 2011142300 A1 WO2011142300 A1 WO 2011142300A1 JP 2011060622 W JP2011060622 W JP 2011060622W WO 2011142300 A1 WO2011142300 A1 WO 2011142300A1
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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- A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
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- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
- A23L13/48—Addition of, or treatment with, enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/40—Colouring or decolouring of foods
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/08—Vasodilators for multiple indications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
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- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01284—S-(hydroxymethyl)glutathione dehydrogenase (1.1.1.284), i.e. nitroreductase
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- C12Y—ENZYMES
- C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
- C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
- C12Y108/01004—Dihydrolipoyl dehydrogenase (1.8.1.4), i.e. lipoamide-dehydrogenase
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
Definitions
- the present invention relates to a reducing agent derived from a Bacillus microorganism and its use.
- the reducing agent of the present invention is particularly useful for the purpose of improving the color tone of meat or processed meat products.
- Meat color is an important factor when consumers evaluate meat quality. Meat color has a great influence on consumers' willingness to purchase and evaluation of meat. For example, if it is bright red, it is judged as good quality meat, and if it is brown it is considered old.
- the color of the meat reflects the proportion of myoglobin derivatives present in the meat.
- this myoglobin When this myoglobin is oxidized, it becomes metmyoglobin and changes its color to brown, which is a major cause of significantly reducing the commercial value of meat products.
- nitrate and nitrite color formers are generally used in processed meat products such as ham and sausage.
- nitrates and nitrites have acute toxicity that causes methemoglobinemia in humans, the amount used is limited to 70 ppm or less as residual nitrite.
- nitrous acid may react with secondary amines to form nitrosamines, which are carcinogenic substances. For this reason, from the viewpoint of safety, a search has been made for a substance having a coloring effect and a coloring method instead of nitrate and nitrite coloring agents.
- Patent Document 1 For example, a method for preventing browning by adding raffinose (see Patent Document 1), a method for preventing browning by adding an enokitake extract (see Patent Document 2), and using ingredients contained in vegetables And a method of developing the color (see Patent Document 3) has been found.
- Patent Literature 1 and the method of Patent Literature 2 do not have sufficient coloring effects, and the method of Patent Literature 3 uses a nitrate contained in vegetables and has a safety problem.
- Patent Document 4 a method for maintaining the color of meat by replacing iron in myoglobin with zinc to form a myoglobin zinc protoporphine IX complex (Patent Document 4), or generation of myoglobin zinc protoporphine IX complex using ferrokeratase or yeast Has also been proposed (Patent Documents 5 and 6) for maintaining the bright red color of meat.
- Patent Documents 5 and 6 it is not possible to act on metmyoglobin once generated, and the color development or tone maintenance effect is limited.
- An object of the present invention is to provide a reducing agent effective for improving the color tone of meat or processed meat products and its use (color tone improving method without using a color former such as nitrite).
- the present inventors conducted screening mainly for microorganisms belonging to the genus Bacillus in order to find substances that improve the color tone of meat.
- a microbial strain that produces a substance having a high meat coloring effect was identified.
- the substance expected to be highly useful exhibits a reducing activity against metmyoglobin. That is, it has been found that microorganisms belonging to the genus Bacillus produce a substance that promotes color development of meat through metmyoglobin reducing activity.
- the substance exhibits a reducing action on heme, and is not limited to the color development of meat, but can be used in other applications where reduction of heme or heme protein is effective or necessary.
- the substance can be used for the purpose of reducing methemoglobin having a structure similar to metmyoglobin.
- a reducing agent comprising a heme reductase derived from a Bacillus microorganism.
- microorganism according to any one of [1] to [4], wherein the microorganism belonging to the genus Bacillus is a microorganism selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus natto, Bacillus thuringiensis, and Bacillus mycoides.
- the reducing agent according to 1.
- the reducing agent according to [1] wherein the heme reductase is dehydrolipoyl dehydrogenase or nitroreductase.
- the reducing agent according to [1] including dehydrolipoyl dehydrogenase and nitroreductase as the heme reductase.
- a color tone improving agent comprising a combination of the reducing agent according to any one of [1] to [9] and a substance exhibiting an action of substituting iron in the heme group of myoglobin with zinc.
- the color tone improving agent according to [11] wherein the substance is ferrochelatase.
- a color tone improving agent for meat or processed meat products comprising dehydrolipoyl dehydrogenase and / or nitroreductase.
- a medicament comprising the reducing agent according to any one of [1] to [9].
- the medicament according to [16] which is a preparation for oral administration.
- a method for producing a reducing agent comprising the following steps (1) and (2): (1) culturing a Bacillus microorganism that produces heme reductase under conditions under which the enzyme is produced; (2) A step of recovering the enzyme from the culture product.
- step (2) includes the following steps: (2-1) collecting microbial cells from the culture product; (2-2) A step of preparing a crushed bacterial cell. [21] The process according to [19] or [20], wherein the heme is metmyoglobin heme. [22] The method according to [19] or [20], wherein the heme is methemoglobin heme.
- a crushed cell of Bacillus selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus natto, Bacillus thuringiensis and Bacillus mycoides, which is characterized in that it acts on meat or processed meat products. To improve color tone.
- a blood circulation disorder a hypoxia or a hypoxia state, a disease accompanied by one or more of these conditions or symptoms, Or a method for preventing or treating a disease caused by one or more of these disease states or symptoms.
- DLD dehydrolipoyl dehydrogenase
- the R, G, and B values were 178, 104, and 102 for the negative control (left) and 210, 104, and 114 for the purified recombinant DLD (right). It is a figure which shows the result of the meat color development test using recombinant DLD.
- the R, G and B values are 201, 117 and 123 for sample 1, 185, 121 and 105 for sample 2, 217, 97 and 93 for sample 3, 160, 108 and 83 for sample 4, and 156 for sample 5. 84 and 65. It is a figure which shows the result of the meat color development test using recombinant DLD.
- the R, G and B values are 168, 122 and 106 for sample 1, 185, 152 and 145 for sample 2, 172, 123 and 119 for sample 3, 187, 153 and 144 for sample 4, and 173 for sample 5.
- 148 and 123 It is a figure which shows the elution pattern and metmyoglobin reductase activity which were obtained by the phenyl chromatography in the refinement
- Marker MDH / pET20b / BL21 (DE3pLysS) (Bacillus subtilis), yodC / pET20b / BL21 (DE3pLysS) (Bacillus subtilis), pET20b / BL21 (DE3pLysS), MDH / pET20b / BL21 (DE3pLysS) , YodC / pET20b / BL21 (DE3pLysS) (natto), marker. It is a figure which shows the result of the meat color development test using refined recombinant yodC and recombinant MDH.
- heme refers to a complex (iron porphyrin complex) composed of an iron atom and a porphyrin.
- Heme protein is a general term for proteins containing heme.
- heme reductase refers to a protein that exhibits a reducing activity on iron atoms in heme. The strength (degree) of the activity is not particularly limited. Typically, heme reductase exhibits an activity of reducing a meth compound of a heme protein. When attention is paid to this activity, heme reductase can also be referred to as heme protein reductase.
- metalmyoglobin reductase refers to a protein that exhibits an activity of reducing metmyoglobin, which is a myoglobin derivative.
- the strength (degree) of the activity is not particularly limited. Therefore, even if the other enzyme activity is more dominant, it corresponds to “metmyoglobin reductase” in the present specification as long as it shows a reducing activity against metmyoglobin.
- the “color tone improving agent” refers to a substance or composition used for improving the “color tone” in which the metal porphyrin complex is involved in the formation thereof.
- the metalloporphyrin complex there are a copper porphyrin complex, a cobalt porphyrin complex, an iron porphyrin complex, and the like, but there is no particular limitation as long as the metal in the porphyrin complex can be reduced.
- a preferred metal porphyrin complex is an iron porphyrin complex, and a composition containing the iron porphyrin complex is a heme protein.
- meat or processed meat products can be mentioned.
- the color tone improving agent of the present invention improves the target color tone by color development, color development promotion and / or fading prevention.
- the color tone improving agent of the present invention can improve the color tone by reducing the metal in the metal porphyrin complex.
- dye can be maintained by preventing the oxidation of the pigment
- a preferred object to which the color tone improving agent of the present invention is applied is meat or processed meat products. That is, in a preferred embodiment, the color tone improving agent of the present invention is used for coloring, maintaining color tone, or preventing fading of meat or processed meat products. “Meat coloring” means that a red color characteristic of meat or processed meat is developed.
- the first aspect of the present invention relates to a reducing agent.
- the reducing agent of the present invention contains heme reductase produced by Bacillus as an active ingredient.
- Bacillus subtilis As a result of large-scale screening by the present inventors, Bacillus subtilis, Bacillus amiloliquefaciens, Bacillus natto, which are microorganisms belonging to the genus Bacillus, It has been clarified that Bacillus thuringiensis and Bacillus mycoides produce polypeptides having excellent metmyoglobin reducing activity.
- metmyoglobin reductase produced by any of these microorganisms is used.
- These microorganisms must be obtained from, for example, public preservation institutions (NBRC (National Institute for Product Evaluation and Technology), JCM (RIKEN BioResource Center), ATCC (American Type Culture Collection), etc.) Can do.
- Bacillus natto is commercially available and can be easily obtained. It can also be obtained from the Miyagino Natto fungus factory.
- the reducing agent of the present invention includes active ingredients (polypeptides), excipients, buffers, suspending agents, stabilizers, pH adjusting agents, preservatives, preservatives, fragrances, thickeners, fats and oils, brighteners. , Binders, binder reinforcing agents, emulsion stabilizers, physiological saline, and the like.
- active ingredients polypeptides
- excipients starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used.
- Phosphate, citrate, acetate, etc. can be used as the buffer.
- pH adjusters include itaconic acid, succinic acid, tartaric acid, fumaric acid, citric acid, malic acid, adipic acid, gluconic acid, pyrophosphoric acid, acetic acid, lactic acid, ⁇ -ketoglutaric acid, phytic acid and other organic acids or organic acid salts
- Inorganic acids such as carbonic acid or inorganic acid salts; acidic amino acids such as aspartic acid and glutamic acid; basic amino acids such as arginine, lysine and histidine can be used.
- preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
- preservatives ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
- Perfumes include animal perfumes such as musk, civet, castorium, ambergris; anise essential oil, angelica essential oil, ylang ylang essential oil, iris essential oil, fennel essential oil, orange essential oil, cananga essential oil, caraway essential oil, cardamom essential oil, guayakwood essential oil, cumin essential oil , Black letter essential oil, cinnamon essential oil, cinnamon essential oil, geranium essential oil, copaiba balsam essential oil, coriandel essential oil, perilla essential oil, cedarwood essential oil, citronella essential oil, jasmine essential oil, gingergrass essential oil, cedar essential oil, spearmint essential oil, western peppermint essential oil Essential Oil, Tuberose Essential Oil, Clove Essential Oil, Orange Flower Essential Oil, Winter Green Essential Oil, Truval Sam Essential Oil, Batule Essential Oil, Rose Essential Oil, Palmarosa Essential Oil, Agate Essential Oil, Hiba Essential Oil, Sandalwood Essential Oil, Petit Glen Essential Oil, Bay Essential Oil, Vetiva Essential Oil
- natural polymers such as fucoidan and carrageenan
- seed exudates such as guar gum
- resin-like adhesives such as gum arabic
- microorganism-generated adhesive substances such as xanthan gum.
- starch-based or cellulose-based natural polymer derivative examples include starch-based starch-based starch-based or cellulose-based natural polymer derivatives such as methylcellulose.
- oils and fats include avocado oil, linseed oil, almond oil, fennel oil, sesame oil, olive oil, orange oil, orange rafa oil, cacao oil, chamomile oil, carrot oil, cucumber oil, coconut oil, sesame oil, rice oil, Safflower oil, shea fat, liquid shea fat, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, sunflower oil, camellia seed oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, Palm oil, palm kernel oil, owl, coconut oil, beef tallow, lard, etc. can be used.
- oils and fats modified by hydrogenation, fractionation, transesterification and the like can be used.
- waxes whether plant or animal
- waxes such as beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, squalene, squalane, pristane, etc.
- Mineral oils such as liquid paraffin, petrolatum, paraffin, ozokelide, ceresin, microcrystalline wax and the like can be used.
- soybean protein, egg protein, milk protein, blood protein, casein, starch, transglutaminase and the like can be used.
- a polymerized phosphate or the like can be used as the binder reinforcing agent.
- emulsion stabilizer sodium caseinate or the like can be used.
- Other additives include lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 1 2-hydroxystearic acid, undecylenic acid, tall oil, lanolin Natural fatty acids such as fatty acids; fatty acids such as synthetic fatty acids such as isononanoic acid, caproic acid, 2 ⁇ ⁇ -ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid and isopentanoic acid.
- the reducing agent of the present invention is composed of a crushed cell of a microorganism that produces the polypeptide of the present invention. That is, the reducing agent of this aspect includes a microbial cell disruption product of a predetermined microorganism.
- the microbial cell disruption liquid (usually obtained by a series of steps consisting of microbial culture, collection and microbial cell disruption) can be used as it is as a microbial cell disruption product.
- the cell disruption solution can be used as a cell disruption product after being subjected to further processing (purification, freezing, drying, addition of other components, etc.).
- the active ingredients derived from Bacillus microorganisms are dehydrolipoyl dehydrogenase (DLD) and nitroreductase (yodC).
- DLD dehydrolipoyl dehydrogenase
- yodC nitroreductase
- a reducing agent containing dehydrolipoyl dehydrogenase or nitroreductase derived from a Bacillus microorganism as a heme reductase In a preferred embodiment, both dehydrolipoyl dehydrogenase and nitroreductase are included.
- the amino acid sequence of dehydrolipoyl dehydrogenase is shown in SEQ ID NO: 3.
- the amino acid sequence of nitroreductase is shown in SEQ ID NO: 12.
- an enzyme prepared by genetic engineering that is, a heme reductase comprising a recombinant protein is used.
- a reducing agent comprising recombinant dehydrolipoyl dehydrogenase and / or recombinant nitroreductase as an active ingredient is provided.
- Recombinant protein refers to a protein that is artificially produced by a genetic recombination technique.
- the second aspect of the present invention relates to the use of the reducing agent of the present invention.
- Applications provided by the present invention are broadly classified into color improvement and other applications.
- the former use that is, use as a color tone improving agent is important.
- Those in which the metal porphyrin complex is involved in the formation of the color tone are the targets of the color tone improvement according to the present invention.
- Preferred objects include meat and processed meat products.
- the color of the meat reflects the proportion of myoglobin derivatives present in the meat.
- the reducing agent of the present invention contains heme reductase (preferably metmyoglobin reductase) as an active ingredient.
- the reducing agent of the present invention when allowed to act on meat, metmyoglobin in the meat is reduced and reduced myoglobin is produced. Reduced myoglobin is converted to oxymyoglobin which exhibits a bright red color tone by oxygenation.
- the reducing agent of the present invention is allowed to act, the amount of metmyoglobin in the meat or processed meat product is reduced, and as a result, oxymyoglobin is generated, resulting in an improvement in color tone.
- the oxidation of reduced myoglobin or oxymyoglobin is prevented, and as a result, the effect of preventing fading of meat can be expected.
- the reducing agent of the present invention not only color development but also color tone maintenance, that is, an effect of preventing fading can be exhibited.
- the target of action when the anti-fading effect is exhibited is a metal porphyrin complex, and is not particularly limited as long as the metal in the complex can be oxidized.
- a preferred metalloporphyrin complex is an iron porphyrin complex. Therefore, a preferable target when antifading effect is expected is a heme protein containing an iron porphyrin complex.
- the most preferred subject is a reduced myoglobin or oxymyoglobin, and meat or processed meat containing either or both of these two myoglobins (reduced myoglobin, oxymyoglobin).
- the reducing agent of the present invention is used for improving the color tone of meat or processed meat products (that is, the color tone improving method using the reducing agent of the present invention as a color tone improving agent)
- the reducing agent of the present invention is used for processing meat or meat.
- the goods will be processed.
- the treatment conditions may be such that the metmyoglobin reductase constituting the reducing agent works well (preferably the optimum conditions).
- Preferred treatment conditions can be easily specified or set by a preliminary experiment using processed meat or processed meat.
- specific examples of the treatment method when the microbial cell disruption solution is used for improving the color tone of meat are shown.
- a cell disruption solution of a predetermined microorganism (Bacillus genus microorganism that produces metmyoglobin reductase) is prepared, and the pH is adjusted to around 5.5. This is to reproduce the pH in the meat. Subsequently, contact with meat at 4 ° C.
- the contact method includes a method of injecting and tumbling a suspension for block meat, and a method of mixing the suspension for minced meat. By performing a suitable contact method, the suspension penetrates the whole meat.
- the processing temperature color development is possible even at around 40 ° C, but considering the quality of meat, it is preferable to carry out at around 4 ° C or around 4 ° C.
- the type of meat to be processed is not limited. As described above, the color of the meat reflects the proportion of the myoglobin derivative present in the meat.
- the reducing agent of the present invention affects the proportion of myoglobin derivative in meat and promotes color development. Therefore, the present invention can be applied to all meat or processed meat products containing myoglobin. Specifically, livestock meat such as pork, beef and chicken or processed products thereof, or fish meat such as tuna, bonito and salmon, or processed products thereof can be processed. However, it can be said that meat having a red color is a preferable processing target.
- the processed meat food which is one of the treatment targets, is not particularly limited as long as it is a food manufactured using meat as a raw material. Examples of processed meat products include raw ham, sausage, and roast ham. There is no particular limitation on the shape of the processed meat or processed meat food. Block meat, minced meat, etc. can be selected as appropriate according to the application.
- the reducing agent of the present invention is used in combination with a substance that exhibits an action of substituting iron in the heme group of myoglobin with zinc (hereinafter referred to as “iron / zinc substitute substance”).
- iron / zinc substitute substance a substance that exhibits an action of substituting iron in the heme group of myoglobin with zinc
- ferrochelatase can be used as the iron / zinc substitute substance (see JP-A-2006-61016 for details).
- Ferrochelatase is present in animal tissues (particularly built-in), plant tissues (mushrooms, bean sprouts, peas, etc.), yeasts (bakers yeast, beer yeast, sake yeast, wine yeast, shochu yeast, etc.), bacteria and the like. Ferrochelatase extracted from these natural products can be used. In addition, since the mitochondrial fraction contains a large amount of ferrochelatase, it is particularly preferable to use the mitochondrial fraction. Saccharomyces genus yeast (beer yeast, baker's yeast, sake yeast, shochu yeast, etc.) can also be used as the iron / zinc substitute substance (see JP-A-2005-87058 for details).
- the reducing agent of the present invention is used in combination with an iron / zinc substitute.
- the color tone improving agent of the present invention will be provided as a compounding agent obtained by mixing the reducing agent of the present invention and an iron / zinc substitute substance.
- the color tone improving agent of the present invention can be provided in the form of a kit comprising the reducing agent (first component) of the present invention and an agent (second component) containing an iron / zinc substitute substance.
- the processing target is processed simultaneously or separately by the first component and the second component.
- “Simultaneous” here does not require strict simultaneity. Therefore, it is necessary to use both elements, such as using both elements after mixing them, such as when using them after mixing them. The case of being carried out under a condition without substantial time difference is also included in the concept of “simultaneous” here.
- the active ingredient (polypeptide) and the above additives excipient, buffer, suspending agent, stabilizer, pH adjusting agent, preservative, preservative, flavoring agent
- seasonings spices, masking agents, softeners and the like
- soy sauce, miso, vinegar, sake, miso, salt, bonito, kombu, etc. meat extract, vegetable extract and the like can be used.
- Spices include pepper, laurel, thyme, clove, oregano, octagon, yam, sage, parsley, nutmeg, mustard, ginger, cinnamon, basil, paprika, rosemary, spearmint, lemongrass, tarragon, chervil, cardamom, cumin, Coriander, dill, fennel, marjoram, allspice, etc. can be used.
- the masking agent saccharides such as sucrose and cyclodextrin; herbs such as clove, allspice, bay leaves, cinnamon, and nutmeg can be used.
- proteases such as protease, trypsin, chymotrypsin, papain, bromelain and ficin can be used.
- the reducing agent of the present invention can be used in fields other than meat.
- the reducing agent of the present invention may be used for the purpose of reducing or preventing (including preventing discoloration) a composition containing a hemoprotein other than myoglobin (for example, hemoglobin).
- the reducing agent of the present invention can be expected to be applied to the measurement of hemoglobin concentration and the treatment of hemoglobinemia. That is, the reducing agent of the present invention is also useful as an active ingredient for reagents and pharmaceuticals.
- the cyanmethemoglobin method is frequently used as a method for measuring blood hemoglobin concentration. In this method, a mixture of potassium ferricyanide and potassium cyanide is allowed to act on methemoglobin to obtain cyan methemoglobin, which is then measured by a colorimetric method. If the reducing agent of the present invention is used, the amount of methemoglobin or the total amount of hemoglobin in blood or the like can be measured as an alternative method.
- Methemoglobinemia develops because the methemoglobin is excessively accumulated in the body for some reason, resulting in an oxygen-deficient state in the body.
- intravenous injection of methylene blue is most effective.
- methylene blue cannot be used when cyan poisoning is accompanied because it promotes cyan poisoning.
- Other treatment methods include oral administration of ascorbic acid and intravenous injection (can also be administered in combination with riboflavin), but neither method is very effective.
- the reducing agents of the present invention can provide new therapeutic strategies that replace these conventional therapies.
- the treatment method using the reducing agent of the present invention can also be applied to patients who cannot use methylene blue (such as those who develop cyan poisoning).
- G6PD glucose-6-phosphate dehydrogenase
- G6PD deficiency is one of the most common disorders worldwide. About 10% of black men in the United States are affected. There are also many affected individuals among Africans and Mediterranean residents. Therefore, a significant number of subjects are at risk for (oxidative) drug-induced methemoglobinemia. Administration of methylene blue itself to such patients is ineffective (since their G6PD deficiency causes NADPH deficiency) and even the potential for adverse effects exists.
- the reducing agent of the present invention can be expected to have an effect on the same patient.
- the reducing agent of the present invention can exert pharmacological and physiological effects such as reduction of the burden on the heart due to abnormal heart rate, blood pressure, and cardiac output, and increased metabolism of each tissue.
- the medicament containing the reducing agent of the present invention can be used as a durability enhancer that enhances the durability of a living body, for example, in physiological conditions with high oxygen demand of each tissue, for example, in an environment such as intense labor or exercise. I can expect.
- heart failure cardiomyopathy, myocarditis, myocardial infarction, pericarditis, perimyocarditis, transient ischemic attack, coronary heart disease, congenital anomalies with left-right vascular shunt ( vitia), Fallot tetralogy / pentadism, Eisenmengel syndrome, shock, peripheral ischemia, arterial occlusive disease (AOD), peripheral AOD (pAOD), carotid artery stenosis, renal artery stenosis, microcirculatory disorder in the brain (Arteriole sclerosis), intracerebral hemorrhage, cerebral venous and intracranial sinus thrombosis, vascular dysplasia, subarachnoid hemorrhage, vascular dementia, Biswanger's disease, subcortical atherosclerotic encephalopathy, embolism Multiple cortical infarction, vasculitis, diabetic retinopathy, prognosis of anemia
- the medicament of the present invention before, during and / or after the occurrence of an ischemic event in order to supply oxygen to the ischemic tissue to prevent ischemic cell damage (and protect the tissue from reperfusion injury) can also be administered.
- a vasoactive oxygen carrier eg, an oxygen carrier based on hemoglobin
- a vasoactive carrier heme protein-based oxygen carrier, etc.
- gaseous nitric oxide heme protein-based oxygen carrier, etc.
- a vasoactive carrier may be administered to the mammal to exert the desired therapeutic effect.
- Mammals to be treated by the methods described herein may have ischemic heart disease prior to treatment and have an acute ischemic condition (e.g., myocardial infarction, stroke, or renal ischemia).
- an acute ischemic condition e.g., myocardial infarction, stroke, or renal ischemia.
- vasospasm may be shown in organs (brain, heart, kidney, liver, gastrointestinal tract, etc.).
- the medicaments of the present invention can also be used to treat hypoxic tissue in vertebrates resulting from a variety of causes, including reduced red blood cell flow through part or all of the circulatory system, anemia and stroke.
- the medicament of the present invention may be used prophylactically.
- the medicament of the present invention can be applied for the purpose of treating or preventing hypoxia resulting from partial arterial occlusion or partial blockage in the microcirculation.
- hemoglobin for the administration of hemoglobin, reference is made to US patent application Ser. No. 08 / 409,337.
- the dosage and administration period of the medicament of the present invention are not particularly limited. It can be appropriately selected depending on the dosage form, age, weight, symptoms and the like.
- the administration target of the medicament of the present invention is not limited.
- administration subjects include humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, monkeys, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, horses. , Chickens, sheep, whales, dolphins, dogs, cats, etc.).
- the subject to be treated may be normal blood volume, excessive blood volume, or excessive blood volume before, during and / or after administration of the medicament of the present invention.
- the dosage form of the medicament of the present invention is not particularly limited. It can be administered by any oral or parenteral administration method.
- Parenteral administration as used herein is not particularly limited, but includes, for example, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, percutaneous Mention may be made of intraductal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intrathecal, and intrasternal injection and infusion. Intravenous injection is used as a preferred method of administration.
- formulations suitable for oral administration include, for example, tablets, capsules, powders, fine granules, granules, liquids, and syrups.
- preparations suitable for parenteral administration include injections, suppositories, inhalants, patches and the like.
- the medicament of the present invention may be produced by adding pharmacologically and pharmaceutically acceptable additives as necessary.
- pharmacologically and pharmaceutically acceptable additives include, for example, excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, and dissolution. Examples include agents or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, and pressure-sensitive adhesives.
- Preparations suitable for oral or parenteral administration include excipients such as glucose, lactose, D-sorbitol, D-mannitol, starch, kaolin, xylitol, dextrin, corn starch, potato starch, hydroxypropyl cellulose, or crystalline cellulose Agents; disintegrating agents or disintegrating aids such as carboxymethylcellulose, starch, or carboxymethylcellulose calcium; binders such as hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, or gelatin; light anhydrous silicic acid, synthetic aluminum silicate, stearic acid , Lubricants such as calcium stearate, magnesium stearate or talc; hydroxypropyl methylcellulose, sucrose, polyethylene glycol or titanium oxide Coating agent; Vaseline, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water, hard fat, etc.
- excipients such as glucose, lactos
- base such as Freon, diethyl ether, propellant such as compressed gas, etc .
- propellant such as compressed gas, etc .
- Adhesives such as polyisobutylene and polybutene
- additives for preparation such as a base cloth such as cotton cloth or plastic sheet can be added.
- Preparations suitable for injection include aqueous solutions such as distilled water for injection, physiological saline, propylene glycol, or solubilizers or solubilizers that can constitute injectable solutions for use; glucose, sodium chloride, D-mannitol, glycerin Tonicity agents such as: organic acids (itaconic acid, succinic acid, tartaric acid, fumaric acid, citric acid, malic acid, adipic acid, gluconic acid, pyrophosphoric acid, lactic acid, ⁇ -ketoglutaric acid, phytic acid, etc.) or organic PH adjusters such as acid salts, inorganic acids (carbonic acid, etc.) or salts of these inorganic acids, acidic amino acids (aspartic acid, glutamic acid, etc.), basic amino acids (arginine, lysine, histidine, etc.); soothing agents such as lidocaine Additives such as may be added.
- aqueous solutions such as distilled water for
- hemoglobin One of the typical objects of action of the medicament of the present invention is hemoglobin, but the hemoglobin here is not particularly limited. Natural (unmodified) hemoglobin, genetically modified hemoglobin, intramolecular or intermolecular crosslinking, polymerization, or addition of chemical groups (e.g. polyalkylene oxide, polyethylene glycol, superoxide dismutase or other adducts) Hemoglobin modified by a chemical reaction such as
- the medicament of the present invention can also be applied to hemoproteins other than the above hemoglobin. It can also be applied to metal porphyrin complexes whose structure is similar to that of the heme protein.
- a further aspect of the present invention provides a method for producing the reducing agent of the present invention.
- a step (step (1)) of culturing a Bacillus microorganism that produces heme reductase, preferably metmyoglobin reductase, under the conditions for producing the enzyme, and recovering the enzyme from the culture product Step (step (2)) is performed.
- any microorganism selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus natto, Bacillus thuringiensis, Bacillus mycoides may be used as the microorganism belonging to the genus Bacillus in step (1).
- the culture method and culture conditions are not particularly limited as long as the target enzyme is produced. That is, on the condition that a polypeptide exhibiting heme reduction activity is produced, a method and culture conditions suitable for the culture of the microorganism to be used can be appropriately set.
- examples of the culture conditions include a medium, a culture temperature, and a culture time.
- a medium capable of growing the microorganisms to be used is employed.
- carbon sources such as arabinose, xylose, glucose, fructose, galactose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or corn gluten
- Nitrogen sources such as meal, soy flour, casamino acid, coffee lees, cottonseed lees, peptone, yeast extract, corn steep liquor, casein hydrolyzate, bran, meat extract, and potassium, magnesium, sodium, phosphate , Manganese salts, iron salts, zinc salts and other inorganic salts can be used.
- vitamins, amino acids and the like may be added to the medium.
- the pH of the medium is adjusted to, for example, about 3 to 8, preferably about 5 to 7, and the culture temperature is usually about 10 to 50 ° C., preferably about 25 to 35 ° C. for 1 to 15 days, preferably 3 to Culture under aerobic conditions for about 7 days.
- the culture method for example, stationary culture, shaking culture, and aerobic deep culture using jar fermenter can be used.
- the target enzyme is recovered from the culture product (step (2)).
- a crushed microbial cell is prepared (step (2-2)). Centrifugation, filter filtration, and the like can be used to collect bacterial cells.
- a solid component other than cells such as when a solid medium is used, the solid component may be removed in advance.
- mechanical crushing processing using a French press, dynomill, etc., ultrasonic processing, freeze crushing processing, etc. can be used.
- the prepared microbial cell crushed material is used as the reducing agent of the present invention as it is (that is, without special treatment) or after additional treatment.
- additional treatment here are concentration (concentration with an ultrafiltration membrane, etc.), purification (salting out, various chromatographies, etc.), addition of other components, dilution and drying. Two or more processes may be performed as the additional process.
- the final form may be liquid or solid (including powder).
- Meat coloring test 1 0.1 g of the lyophilized powder was dissolved in 50 ⁇ L of 0.5 M phosphate buffer (pH 5.5). Subsequently, 2 g of pork thigh meat minced was mixed with the above-mentioned powdered solution and 50 ⁇ L of 4% (w / v) myoglobin, degassed and sealed, and left at 4 ° C. for 17 hours. The red color change (degree of color development) of the meat was visually observed. As a control test, the same test was performed for the sample without adding the powder solution and the sample obtained by boiling the powder solution for 10 minutes. The results are shown in Table 2.
- Meat coloring test 2 10 mg of the frozen powder (Bacilli subtilis crushed material) was dissolved in 100 ⁇ L of 0.5 M phosphate buffer (pH 5.5). To the lysate was added 10 mg of pork thigh minced freeze-dried powder, 30 ⁇ L of 4% (w / v) myoglobin, 30 ⁇ L of 0.2M NADH, 400 ⁇ L of sterilized water, and left at room temperature for 30 minutes. Subsequently, the reaction solution was centrifuged at 15,000 rpm for 15 minutes, and the supernatant was collected. The absorption spectrum of the obtained supernatant at a wavelength of 700 nm to 400 nm was measured using a spectrophotometer.
- Meat coloring test 3 3. According to the method shown in 1. A meat color development test was performed using each of the freeze-dried powders prepared in (1). As a control, the same test was carried out on a sample to which no powder solution was added. The absorbance at a wavelength of 580 nm of each sample is shown in FIG. It can be seen that all samples tested developed more intensely than the control after 5 days.
- the metmyoglobin reductase activity was measured as follows. First, 1. Each freeze-dried powder prepared in 1 was dissolved in water to 10 mg / mL to prepare an enzyme solution. Subsequently, 100 ⁇ L of 0.1% (w / v) myoglobin and 150 ⁇ L of enzyme solution were added to 200 ⁇ L of 0.1 M phosphate buffer (pH 5.5), and preincubated at 30 ° C. for 5 minutes. Then, 50 ⁇ L of 1 mM NADH was added, and the change in absorbance at a wavelength of 406 nm was measured for 5 minutes. Activity was expressed in units (U).
- the amount of enzyme that reduces metmyoglobin equivalent to 1 ⁇ M per minute under these conditions was 1 U.
- the enzyme activity of lyophilized powder of Pichia farinose IAM12223 was also examined.
- the obtained ammonium sulfate salting-out sample (5 mL) was subjected to DEAE chromatography (DEAE column (HiTrap TM DEAE FF (5 mL); GE Healthcare)) under the following conditions. As a result, the protein yield was 45.4%.
- FIG. 4 shows the elution pattern and metmyoglobin reductase activity obtained by the DEAE chromatography. The activity was confirmed to decrease A406, which is the maximum absorption of metmyoglobin.
- DEAE Fr. No. 61-63 which had particularly high activity, was subjected to hydroxyapatite chromatography (hydroxyapatite column (1 ⁇ 5 cm) (Type I 20 ⁇ m; Bio-Rad)) under the following conditions. Was purified.
- the protein yield of DEAE Fr. No. 61-63 by hydroxyapatite chromatography was 98.5%.
- FIG. 5 shows the elution pattern and metmyoglobin reductase activity obtained by subjecting DEAE Fr. No. 61-63 to hydroxyapatite chromatography.
- Hyapa Fr. No. 19 having a particularly high specific activity in FIG. 5 was dialyzed with 20 mM KPB (pH 6) and then subjected to gel filtration chromatography (Superfiltration TM 75; GE Healthcare) under the following conditions. (Gel filtration chromatography conditions) Carrier: Super dex 200 (120mL) Charge: Hiapa Fr.19 Buf A: 20mM KPB (pH6) Flow rate: 1mL / min Fraction: 5mL
- Fig. 6 shows the elution pattern and metmyoglobin reductase activity obtained by subjecting Hyapa Fr. No. 19 to gel filtration chromatography. Furthermore, the gel filtration Fr.No.12-16 including the gel filtration Fr.No.13-15 having a particularly high specific activity in FIG. 6 was subjected to SDS-PAGE, and a band was confirmed (FIG. 7: gel filtration Fr. .No.12-16). A single band was obtained for gel filtration Fr. No. 13-15 in FIG.
- Meat coloring test by DLD A meat coloring test was performed on purified DLD. Using purified DLD frozen powder samples, meat samples were prepared as follows (Table 3) and stored overnight at 4 ° C. for comparison (FIGS. 9 and 10).
- FIG. 9 shows meat samples (meat samples 1 to 4) with different amounts of enzyme side by side.
- DLD contributes to the improvement of meat color.
- FIG. 10 compares the color of meat with and without NADH (from left to right, no frozen powder (meat sample 1), NADH (meat sample 2), no NADH (meat sample 5)). It was confirmed that the color was sufficiently developed even without NADH. This is probably because NADH is sufficiently contained in the meat.
- Table 4 shows the Km values of other origins of Diapholase having high affinity with potassium ferricyanide.
- Table 4 shows that DLD obtained from Bacillus subtilis has higher affinity for potassium ferricyanide than DLD derived from C. kluyveri.
- the optimum temperature of DLD was measured as follows. Mix 0.5M citrate buffer (pH 6) 50 ⁇ L, 2mM potassium ferricyanide 50 ⁇ L, MilliQ water 100 ⁇ L and 1mM NADH 100 ⁇ L, add 200 ⁇ L of enzyme sample to each sample pre-incubated for 5 minutes, change the absorbance of A420 for 5 minutes The reaction was confirmed by measuring. The results are shown in FIG. From this, it was confirmed that the optimum temperature was 40 ° C. and it was deactivated at 50 ° C.
- the thermal stability was measured as follows.
- the enzyme sample was treated in advance at each temperature (30 ° C., 40 ° C., 60 ° C.) for 30 minutes and then ice-cooled to prepare a treated sample.
- To this treated sample 30 ⁇ L, add 0.5 M citrate buffer (pH 6.0) 50 ⁇ L, 2 mM potassium ferricyanide 50 ⁇ L and MilliQ water 270 ⁇ L and incubate at 30 ° C. for 5 minutes, then add 100 ⁇ L of 1 mM NADH solution, and adjust the absorbance of A420. Changes were measured at 1 second intervals for 30 seconds. From the results in FIG. 16, it was confirmed that there was a high residual activity even at 60 ° C.
- DLD Large-scale expression system for DLD
- the DLD gene was obtained from Bacillus subtilis, and studies were made to construct a large-scale expression system in E. coli.
- Genomic extraction from Bacillus subtilis strain 7417 Genome extraction from Bacillus subtilis strain 7417 was performed as follows. Bacillus subtilis 7417 strain was inoculated into a liquid medium (pH 6.5) containing 0.5% peptone, 1.0% yeast extract and 1.0% glucose, and cultured overnight at 30 ° C. and 300 rpm. Genomic DNA was extracted from the obtained culture using QIAquick TM Gel Extraction Test Kit (manufactured by QIAGEN).
- Amplification of DLD gene by PCR DLD gene amplification by PCR was performed as follows. Mix 10 ⁇ buffer 5 ⁇ L, dTNP 4 ⁇ L, Bacillus subtilis genome 1 ⁇ L, the following 10 ⁇ M primers (2 types) 5 ⁇ L each, EX. Taq (DNA polymerase, manufactured by Takara Bio Inc.) 0.1 ⁇ L, and add purified water to this 50 ⁇ L. In addition, 2 patterns (pattern 1, pattern 2) were prepared as primer combinations. The PCR reaction was performed in two steps. First, as step 1, heat denaturation was performed at 98 ° C. for 30 seconds.
- step 2 the following cycle (thermal denaturation: 98 ° C., 10 seconds, annealing: 46 ° C., 30 seconds, extension reaction: 72 ° C., 90 seconds) was performed 25 cycles to obtain a PCR product.
- TA cloning of DLD gene was performed as follows. To 3 ⁇ L of the PCR product obtained by PCR, add 2 ⁇ Liation buffer 5 ⁇ L, pGEM-T easy vector 1 ⁇ L, T4 ligase 1 ⁇ L, and react overnight at 4 ° C. Then add this to competent cell DH5 ⁇ , Heat-shocked at 42 ° C. for 30 seconds and ice-cooled for 2 minutes. To this, 150 ⁇ L of SOC medium was added and incubated at 37 ° C. for 20 minutes. The entire amount was cultured on an LB / Amp medium plate to obtain colonies.
- DLD was cloned into a vector as follows.
- the culture obtained by TA cloning was subjected to plasmid extraction using GenElute TM plasmid Miniprep Kit (manufactured by SIGMA).
- GenElute TM plasmid Miniprep Kit manufactured by SIGMA
- Nde I To the obtained plasmid extract, 10 ⁇ buffer and Nde I were added and treated at 37 ° C. for 2 hours.
- 1 ⁇ L of BamH I was added to this and treated at 37 ° C. for 1 hour as an inserted gene.
- the pET20b vector was prepared by adding 1 ⁇ L of BamH I and treating at 37 ° C. for 1 hour.
- samples were prepared as follows (unit: ⁇ L) and incubated at 16 ° C. for 30 minutes. Then, add the entire amount to competent cell DH5 ⁇ and dissolve on ice for 1 hour. Heat shock (42 ° C, 30 seconds), add SOC medium, incubate at 37 ° C for 20 minutes, and plate on LB / amp medium. .
- a beads shocker MULTI-BEADS SHOCKER, manufactured by Yasui Instruments Co., Ltd.
- FIG. 19 shows the metmyoglobin reducing activity of the transformant without His-tag and FIG. 20 with the His-tag.
- pET20b empty vector and IPTG vector data are also shown. All showed 10 times or more metmyoglobin reducing activity compared to the empty vector. Further, as can be seen by comparing FIG. 19 and FIG. 20, it seems that the control by IPTG is not applied.
- Meat coloring activity of recombinant DLD The recombinant DLD obtained above was crushed by beads, purified on a Ni-Sepharose column under the following conditions, and dialyzed against 20 mM KPB (pH 6). Meat coloring test 1 and meat coloring test 2 were conducted using the lyophilized product as a sample.
- Carrier Ni Sepharose (25mL) Sample: About 20mL of crushing supernatant Bind Buf: 20 mM KPB, 0.3 M NaCl (pH 6) Elute Buf: 20 mM KPB, 0.3 M NaCl, 0.4 M imidazole (pH 6) Flow rate: Charge: 5mL / min, others: 10mL / min Fraction: 10mL Programs: (1) Bind Buf wash 6cv, (2) Elute Buf 10% wash 10cv, (3) Elute Buf 100% / 20cv gradient, (4) Elute Buf wash 10cv
- a product without a frozen powder negative control
- FIG. Left is negative control
- right is meat with frozen powder. The color change was verified not only by visual observation but also by the RGB value of the image, but it can be seen that the color tone of the meat was more reddish by the recombinant DLD.
- the color tone of the meat without heat treatment in FIG. 21 will be described below. Good red color tone was observed in the samples with DLD added (sample (1) and sample (3)). In particular, the color tone of sample (3) to which DLD and sodium nitrite were added was good. In sample (4) to which sodium nitrite and zinc gluconate were added, the color turned brown. Sample (2) to which only sodium nitrite was added showed the same color tone as sample (5) with no additive.
- Phenyl Fr. No. 26-31 obtained by the above phenyl chromatography was subjected to hydroxyapatite chromatography under the following conditions.
- FIG. 25 shows the elution pattern and metmyoglobin reductase activity obtained by the hydroxyapatite chromatography.
- the highly active HiApa Fr.No.16 was dialyzed against 20 mM KPB, 0.3 M NaCl (pH 6) and applied to a Cu affinity column under the following conditions. Chromatography was performed. The elution pattern and metmyoglobin reductase activity obtained by Cu affinity chromatography are shown in FIG. Among these fractions obtained by Cu affinity chromatography, SDS-PAGE was performed on Cu.Fr.No. The results are shown in FIG. (Cu affinity chromatography conditions) Carrier: Cu2 + HP (1mL) Charge: Hi-Apa-Fr.
- Buf A 20 mM KPB, 0.3 M NaCl (pH 6)
- Buf B 20 mM KPB, 0.3 M NaCl, 0.4 M imidazole (pH 6)
- Flow rate 1mL / min
- FIG. 27 shows the results of SDS-PAGE of Hiapa Fr. No. 13-17 obtained by the above hydroxyapatite chromatography and Cu.Fr. No. 8-15 obtained by Cu affinity chromatography. .
- N-terminal amino acid sequence analysis was performed by the method described above.
- the protein with the larger molecular weight was found to be MGNTRKKVSVI. (SEQ ID NO: 8)
- the smaller molecular weight protein was MTNTLDVLKA (SEQ ID NO: 9).
- a BLAST search of the protein with the larger molecular weight based on the N-terminal amino acid sequence showed 100% homology with mdh (SEQ ID NO: 10) encoding malate dehydrogenase (MDH).
- the protein with the smaller molecular weight showed 100% homology with yodC (SEQ ID NO: 11) encoding putative NAD (P) H nitroreductase (Putative NAD (P) H nitroreductase: yodC).
- the amino acid sequence of yodC is shown in SEQ ID NO: 12. Cu.Fr.No.13 was presumed to be the same as Dehydrolipoyl dehydrogenase from the molecular weight.
- Mass Expression System for Meat Chromogenic Enzymes (MDH, yodC) Genes were obtained from Bacillus subtilis and Bacillus natto, and a mass expression system for MDH and yodC was constructed. Primers were created from the gene information of Bacillus subtilis and Bacillus natto, and the corresponding genes were excised by PCR. Using pET20b as the vector and BL21 (DE3 pLysS) as the host, 6 ⁇ His-tag was added to the C-terminus of each enzyme for expression. Expression confirmation culture was performed as follows.
- Carrier Ni Sepharose (25mL) Sample: About 20mL of crushing supernatant Bind Buf: 20 mM KPB, 0.3 M NaCl (pH 6) Elute Buf: 20 mM KPB, 0.3 M NaCl, 0.4 M imidazole (pH 6) Flow rate: Charge: 5mL / min, others: 10mL / min Fraction: 10mL Programs: (1) Bind Buf wash 6cv, (2) Elute Buf 10% wash 10cv, (3) Elute Buf 100% / 20cv gradient, (4) Elute Buf wash 10cv
- a meat coloring test was conducted as follows.
- Samples (3) and (5) were prepared for confirming deactivation by heat treatment (100 ° C., 30 minutes). The results are shown in FIG. Although yodC had a meat coloring effect, MDH was not observed at all.
- the reducing agent of the present invention is particularly useful as a color improving agent for meat or processed meat products. According to the reducing agent of the present invention, it is possible to color meat without using a color former such as nitrite, and therefore it becomes possible to produce a processed meat product with high commercial value.
- Sequence number 4 Description of artificial sequence: Primer DLD-Nde1-FW SEQ ID NO: 5: Description of artificial sequence: Primer DLD-BamH1-RV SEQ ID NO: 6: Description of artificial sequence: primer DLD-Nde1-FW Sequence number 7: Artificial sequence description: Primer DLD-BamH1-Histag-RV
Abstract
Description
[1]バチルス属微生物由来のヘム還元酵素を含む還元剤。
[2]前記ヘムがメトミオグロビンのヘムであることを特徴とする、[1]に記載の還元剤。
[3]前記ヘムがメトヘモグロビンのヘムであることを特徴とする、[1]に記載の還元剤。
[4]バチルス属微生物の菌体破砕物からなることを特徴とする、[1]~[3]のいずれか一項に記載の還元剤。
[5]前記バチルス属微生物がバチルス ズブチリス、バチルス アミロリケファシエンス、納豆菌、バチルス スリンギエンシス及びバチルス ミコイデスからなる群より選択される微生物である、[1]~[4]のいずれか一項に記載の還元剤。
[6]前記ヘム還元酵素がデヒドロリポイルデヒドロゲナーゼ又はニトロレダクターゼである、[1]に記載の還元剤。
[7]前記ヘム還元酵素として、デヒドロリポイルデヒドロゲナーゼ及びニトロレダクターゼを含む、[1]に記載の還元剤。
[8]前記デヒドロリポイルデヒドロゲナーゼのアミノ酸配列が配列番号3のアミノ酸配列を含み、前記ニトロレダクターゼのアミノ酸配列が配列番号12のアミノ酸配列を含む、[6]又は[7]に記載の還元剤。
[9]前記デヒドロリポイルデヒドロゲナーゼ及び前記ニトロレダクターゼがリコンビナントタンパク質である、[6]~[8]のいずれか一項に記載の還元剤。
[10][1]~[9]のいずれか一項に記載の還元剤からなる色調改善剤。
[11][1]~[9]のいずれか一項に記載の還元剤と、ミオグロビンのヘム基中の鉄を亜鉛に置換する作用を示す物質を組み合わせてなる色調改善剤。
[12]前記物質がフェロケラターゼである、[11]に記載の色調改善剤。
[13]食肉又は食肉加工品の色調の改善用である、[10]~[12]のいずれか一項に記載の色調改善剤。
[14]デヒドロリポイルデヒドロゲナーゼ及び/又はニトロレダクターゼを含む、食肉又は食肉加工品用の色調改善剤。
[15]発色作用、発色促進作用及び/又は退色防止作用により色調を改善する、[10]~[14]のいずれか一項に記載の色調改善剤。
[16][1]~[9]のいずれか一項に記載の還元剤を含むことを特徴とする医薬。
[17]経口投与製剤である、[16]に記載の医薬。
[18]非経口投与製剤である、[16]に記載の医薬。
[19]以下のステップ(1)及び(2)を含む還元剤の製造法:
(1)ヘム還元酵素を産生するバチルス属微生物を、該酵素が産生される条件下で培養するステップ;
(2)培養産物から前記酵素を回収するステップ。
[20]前記ステップ(2)が以下のステップからなる、[19]に記載の製造法:
(2-1)培養産物から菌体を収集するステップ;
(2-2)菌体破砕物を調製するステップ。
[21]前記ヘムがメトミオグロビンのヘムであることを特徴とする、[19]又は[20]に記載の製造法。
[22]前記ヘムがメトヘモグロビンのヘムであることを特徴とする、[19]又は[20]に記載の製造法。
[23]前記バチルス属微生物がバチルス ズブチリス、バチルス アミロリケファシエンス、納豆菌、バチルス スリンギエンシス及びバチルス ミコイデスからなる群より選択される微生物である、[19]~[22]に記載の製造法。
[24][10]~[15]のいずれか一項に記載の色調改善剤を食肉又は食肉加工品に作用させることを特徴とする色調改善方法。
[25]バチルス ズブチリス、バチルス アミロリケファシエンス、納豆菌、バチルス スリンギエンシス及びバチルス ミコイデスからなる群より選択されるバチルス属微生物の菌体破砕物を食肉又は食肉加工品に作用させることを特徴とする色調改善方法。
[26][1]~[9]のいずれか一項に記載の還元剤を用いた、血行障害、低酸素症若しくは血中酸素減少状態、これらの一つ以上の病態ないし症状を伴う疾患、又はこれらの一つ以上の病態ないし症状に起因する疾病の予防または治療方法。
本明細書において「ヘム」とは、鉄原子とポルフィリンから構成される錯体(鉄ポルフィリン錯体)をいう。「ヘム蛋白質」とはヘムを含む蛋白質の総称である。また、「ヘム還元酵素」とは、ヘム中の鉄原子に対して還元活性を示す蛋白質のことをいう。当該活性の強さ(程度)は特に限定されない。典型的には、ヘム還元酵素は、ヘム蛋白質のメト化合物を還元する活性を示す。この活性に注目した場合、ヘム還元酵素をヘム蛋白質還元酵素と呼ぶこともできる。
本発明の第1の局面は還元剤に関する。本発明の還元剤はバチルス属が産生するヘム還元酵素を有効成分とする。後述の実施例に示す通り、本発明者らによる大規模なスクリーニングの結果、バチルス属微生物である、バチルス ズブチリス(Bacillus subtilis)、バチルス アミロリケファシエンス(Bacillus amiloliquefaciens)、納豆菌(Bacillus natto)、バチルス スリンギエンシス(Bacillus thuringiensis)及びバチルス ミコイデス(Bacillus mycoides)が、メトミオグロビン還元活性に優れたポリペプチドを産生することが明らかとなった。この知見に基づき本発明の好ましい一態様では、これらの微生物のいずれかが産生するメトミオグロビン還元酵素が用いられる。尚、これらの微生物は例えば公共の保存機関(NBRC(独立行政法人製品評価技術基盤機構 生物遺伝資源部門)、JCM(理化学研究所バイオリソースセンター)、ATCC(American Type Culture Collection)等)から入手することができる。納豆菌については市販されており、容易に入手可能である。また宮城野納豆菌製造所から入手することも可能である。
本発明の第2の局面は本発明の還元剤の用途に関する。本発明が提供する用途は色調の改善及びその他の用途に大別される。特に前者の用途、即ち色調改善剤としての利用が重要である。その色調の形成に金属ポルフィリン錯体が関与しているものが、本発明による色調改善の対象となる。好ましい対象として食肉及び食肉加工品が挙げられる。食肉の色調は肉中に存在するミオグロビン誘導体の割合を反映する。上記の通り、本発明の還元剤はヘム還元酵素(好ましくはメトミオグロビン還元酵素)を有効成分とする。従って、本発明の還元剤を食肉に作用させると、食肉中のメトミオグロビンが還元され、還元型ミオグロビンが生成する。還元型ミオグロビンは酸素化によって鮮赤色の色調を示すオキシミオグロビンに変換される。本発明の還元剤を作用させると、食肉又は食肉加工品中のメトミオグロビン量が低減し、併せてオキシミオグロビンが生成する結果、色調が改善する。また、還元型ミオグロビンまたはオキシミオグロビンの酸化が防止され、その結果として食肉の退色を防止できるという効果も期待できる。このように、本発明の還元剤によれば、発色のみならず色調維持、つまり退色防止の効果も発揮され得る。退色防止効果が奏される際の作用対象は金属ポルフィリン錯体であり、当該錯体中の金属が酸化されうるものであれば特に制限されない。好ましい金属ポルフィリン錯体は鉄ポルフィリン錯体である。従って、退色防止効果を期待する場合の好ましい対象は、鉄ポルフィリン錯体を含有するヘム蛋白質である。最も好ましい対象は、還元型ミオグロビンまたはオキシミオグロビン、並びにこれらミオグロビン2種(還元型ミオグロビン、オキシミオグロビン)の何れか/もしくは双方を含む食肉又は食肉加工品である。
本発明の更なる局面は本発明の還元剤の製造法を提供する。本発明の製造法ではヘム還元酵素、好ましくはメトミオグロビン還元酵素を産生するバチルス属微生物を、該酵素が産生される条件下で培養するステップ(ステップ(1))及び培養産物から前記酵素を回収するステップ(ステップ(2))が行われる。
表1に示す液体培地10 mLを試験管に分注し、120℃、20分間滅菌した。前培養として前記試験管にバチルス ズブチリス(Bacillus subtilis JCM1465株(=ATCC6051株、IAM12118株、IFO13719株))、バチルス アミロリケファシエンス(Bacillus amyloliquefaciens NBRC15535株(=ATCC23350株))、納豆菌(Bacillus natto)、バチルス スリンゲンシス(Bacillus thuringiensis NBRC13865株(=ATCC13366株))、バチルス ミコイデス(Bacillus mycoides IAM1190株(=IFO3039株))をそれぞれ1エーゼ接種し、30℃、300rpmで一晩振とう培養した。
前記凍結乾燥粉末0.1gを0.5 Mリン酸バッファー(pH 5.5)50μLで溶解した。続いて豚もも肉ミンチ2gに、前述の粉末溶解液、4%(w/v)ミオグロビン50μLを混ぜ合わせ、脱気密封し、4℃で17時間放置した。肉の赤色の変化(発色度)を視覚的に観察した。対照試験として粉末溶解液を加えないものと、粉末溶解液を10分間煮沸処理したものについても同様の試験を行った。その結果を表2に示す。尚、ピキア ファリノサ(Pichia farinose IAM12223株(=IFO0465株、JCM1634株))の凍結乾燥粉末溶解液で処理した場合の結果(未処理のデータのみ)も併せて示す。
前記凍結粉末(バチルス ズブチリスの菌体破砕物)10mgを0.5Mリン酸バッファー(pH 5.5)100μLで溶解した。その溶解液に豚もも肉ミンチ凍乾粉末10mg、4%(w/v)ミオグロビン 30μL、0.2M NADH 30μL、滅菌水400μLを添加して、室温で30分放置した。続いて反応液を15,000rpm、15分、遠心分離し、上清を回収した。得られた上清の波長700nmから400nmの吸収スペクトルを、分光光度計を用いて測定した。対照試験として粉末溶解液を10分間煮沸処理したものについても同様の試験を行った。その結果を図1に示す。結果から明らかなように、未処理サンプルは545nmと580nmに吸収極大を有し、赤色を呈していることが分かる。
3.に示した方法に従って、1.で調製した各凍結乾燥粉末用いて食肉発色試験を行った。コントロールとして粉末溶解液を加えないものについても同様の試験を行った。各サンプルの波長580nmにおける吸光度を図2に示す。試験した全てのサンプルで5日後もコントロールよりも強く発色していることが分かる。
メトミオグロビン還元酵素活性は以下の通り測定した。まず、1.で調製した各凍結乾燥粉末を水で10mg/mLに溶解し、酵素溶液とした。続いて0.1Mリン酸バッファー(pH 5.5) 200μLに0.1%(w/v)ミオグロビン100μLと酵素溶液150μLを添加し、30℃、5分間プレインキュベートした。そして1mM NADH 50μLを添加して、波長406nmにおける吸光度の変化を5分間測定した。活性はユニット(U)で示した。本条件下で1分間に1μM相当のメトミオグロビンを還元させる酵素量を1Uとした。尚、比較のため、ピキア ファリノサ(Pichia farinose IAM12223株(=IFO0465株、JCM1634株))の凍結乾燥粉末についても酵素活性を調べた。
メトミオグロビン還元酵素は以下の通り精製した。1.で培養して得られたバチルス ズブチリス菌体をフレンチプレスにて破砕し遠心後、上清を硫安で塩析した。30%飽和で処理して上清をとり70%飽和で処理して遠心した後、沈殿物を回収した。これを20mM KPB(pH=6.0)溶液に溶解させ透析したものを硫安塩析サンプルとした。
(DEAEクロマトグラフィー条件)
担体:DEAE HP(5mL)
チャージ:硫安塩析サンプル(5mL)
Buf A:20mM KPB(pH6)
Buf B:20mM KPB(pH6),1M NaCl
流速:5mL/分
分画:5mL
プログラム:(1)Buf A洗浄 8cv、(2)Buf B 10% 洗浄 8cv、(3)Buf B勾配 30%/25cv、(4)Buf B 100% 8cv
(ハイドロキシアパタイトクロマトグラフィー条件)
担体:ハイドロキシアパタイト(5mL)
チャージ:DEAE精製Fr.No.61-63
Buf A:5mM KPB,0.3M NaCl(pH6)
Buf B:400mM KPB,0.3M NaCl(pH6)
流速:1mL/分
分画:4mL
プログラム:(1)Buf A洗浄 7cv、(2)Buf B勾配 100%/20cv、(3)Buf B 100% 10cv
(ゲルろ過クロマトグラフィー条件)
担体:Super dex 200(120mL)
チャージ:ハイアパFr.19
Buf A:20mM KPB(pH6)
流速:1mL/min
分画:5mL
図7のSDS-PAGEで得られたシングルバンドについてPDVF膜に転写しポンソー試薬で染色後、切り出しを行い、N末端アミノ酸配列解析をプロテインシークエンサーにて行った。その結果、N末端アミノ酸配列がVVGDFPIETDTLVIG(配列番号1)であることが見出された。さらに、当該アミノ酸配列をもとにBLAST内のバチルス・ズブチリスデータベースから該当遺伝子を検索した。その結果、デヒドロリポイルデヒドロゲナーゼ(Dehydrolipoyl dehydrogenase、以下DLDとする。)をコードするpdhD(配列番号2)と100%の相同性を有することが見出された。尚、DLDのアミノ酸配列を配列番号3に示す。
精製したDLDについて食肉発色試験を行った。精製したDLDの凍結粉末サンプルを使用し、食肉サンプルを以下の通り調製し(表3)、4℃で一晩保存した後、比較した(図9、図10)。
本酵素と高い親和性をもつフェリシアン化カリウムを基質として用い、諸性質を検討した。まずはDLDの基質反応性を確認した。酵素添加量を15μLとし基質濃度(終濃度)を0.025,0.05,0.075,0.1,0.15,0.2,0.3,0.4,0.5,0.6,0.7,0.8,1.0,1.25,1.5,1.75,2.0,2.5(mM)となるように、測定時間30秒間で、レイトアッセイ(Rate assay)を行った(pH=6.0)。フェリシアン化カリウムのモル吸光係数は1.02×103(M-1・cm-1)とし、速度(v)の単位はμM/分を用いた。結果を図11、図12に示す。図11の基質飽和曲線の立ち上がりから[s]/v~[s]プロットを作成し(図12)、速度パラメーター(Kinetic parameter)を算出した(Km=0.19(mM)、Vmax=26.2(μM/分))。比較として、フェリシアン化カリウムと親和性の高い他起源のDiapholaseのKm値を表4に示す。表4より、バチルス ズブチリスから得られたDLDは、C.kluyveri由来のDLDよりもフェリシアン化カリウムに対して親和性が高いことがわかる。
バチルス ズブチリスよりDLD遺伝子を取得し、大腸菌にて大量発現系を構築すべく検討を行った。
バチルス ズブチリス7417株からのゲノム抽出は次の通り行った。バチルス ズブチリス7417株を0.5%ペプトン、1.0%酵母エキス、1.0%グルコースを含む液体培地(pH6.5)に接種し、30℃、300rpmにて一晩振とう培養した。得られた培養物からQIAquickTM Gel Extraction Test Kit(QIAGEN社製)を用いてゲノムDNAの抽出を行った。
PCRによるDLD遺伝子増幅は次の通り行った。10×バッファー 5μL、dTNP 4μL、バチルス ズブチリスのゲノム 1μL、以下の10μMプライマー(2種)各5μL、EX. Taq(DNA polymerase, タカラバイオ社製) 0.1μLを混合し、これに精製水を加えて50μLとした。なお、プライマーの組み合わせとしては2パターン(パターン1、パターン2)用意した。PCR反応は2ステップで行った。まずステップ1として98℃で30秒間、熱変性させた。続いてステップ2として下記サイクル(熱変性:98℃,10秒間、アニーリング:46℃,30秒間、伸長反応:72℃,90秒間)を25サイクル行い、PCR産物を得た。
(プライマーの配列)
パターン1
DLD-Nde1-FW:GGCGTAATCATATGGTAGTAGGAG(配列番号4)
DLD-BamH1-RV:GATAGGATCCTTATTTTACGATG(配列番号5)
パターン2
DLD-Nde1-FW:GGCGTAATCATATGGTAGTAGGAG(配列番号6)
DLD-BamH1-Histag-RV:GATAGGATCCTTAGTGGTGGTGGTGGTGGTGTTTTACGATG(配列番号7)
続いてDLD遺伝子のTAクローニングを次の通り行った。PCRで得られたPCR産物3μLに、2×Liationバッファー 5μL、pGEM-T easyベクター 1μL、T4 リガーゼ 1μLを添加し4℃で一晩反応させた後、これをコンピテントセルDH5αに全量添加し、42℃で30秒間ヒートショックをかけて2分間氷冷した。これにSOC培地150μLを添加し、37℃で20分間インキュベートした。そして全量をLB/Amp培地プレートで培養し、コロニーを得た。
続いてDLDを次の通りベクターにクローニングした。TAクローニングによって得られた培養物をGenEluteTM plasmid Miniprep Kit(SIGMA社製)を用いてプラスミド抽出を行った。得られたプラスミド抽出物に10×バッファー、Nde Iを添加し37℃で2時間処理した。さらにこれにBamH I 1μLを添加し、37℃で1時間処理したものを挿入遺伝子とした。一方、pET20bベクターについてもBamH I 1μLを添加し、37℃で1時間処理したものを準備した。His-tagの有無によるDLDの酵素活性を確認するために、それぞれ下記の通り試料を調製し(単位はμL)、16℃で30分間インキュベートした。その後、全量をコンピテントセルDH5αに添加し氷上で1時間溶解したものをヒートショックにかけ(42℃、30秒間)、SOC培地を添加し37℃で20分間インキュベートし、LB/amp培地にプレートした。
上記の通り得られた形質転換体の活性測定を次の通り行った。DLD(+)Histag/pET20b/BL21の5コロニーとDLD(-)Histag/pET20b/BL21の4コロニーをとりLB/Amp培地にて30℃で一晩振とう培養した(前培養)。この前培養液60μLを3mLのLB/Amp培地に植菌し37℃で一晩振とう培養した(本培養)。OD600=0.4~0.5になったら、IPTGを終濃度0.1mMになるよう加え、30℃で4時間振とう培養した。こうして得られた菌体を集菌し、50mM Tris-HCl(pH=7.0)に懸濁させた。これをビーズショッカー(MULTI-BEADS SHOCKER、安井器械社製)にて破砕し遠心した後、上清をサンプルとした。
上記により得られたリコンビナントDLDをビーズ破砕し、以下の条件でNi-Sepharoseカラムにて精製して、20mM KPB(pH6)で透析した。これを凍結乾燥したものをサンプルとして用い食肉発色試験1、食肉発色試験2を行った。
(クロマトグラフィー条件)
担体:Ni Sepharose(25mL)
サンプル:破砕上清 約20mL
Bind Buf:20mM KPB,0.3M NaCl(pH6)
Elute Buf:20mM KPB,0.3M NaCl,0.4Mイミダゾール(pH6)
流速:チャージ:5mL/分,その他:10mL/分
分画:10mL
プログラム:(1)Bind Buf洗浄 6cv、(2)Elute Buf 10%洗浄 10cv、(3)Elute Buf 100%/20cv勾配、(4)Elute Buf洗浄 10cv
バチルス ズブチリス菌体由来の食肉発色酵素のうち、DLD以外の食肉発色酵素について精製を行った。精製工程はDEAEクロマト前半ピーク(図4のDEAE.Fr.No.27-35)を出発材料にして、フェニルクロマトグラフィー、ハイドロキシアパタイトクロマトグラフィー、Cuアフィニティークロマトグラフィーを行った。
(フェニルクロマトグラフィー条件)
担体:Phenyl HP(5mL)
サンプル:DEAE前半Fr.( 100210) UF 10mL
Buf A:20mM KPB,30%飽和硫安(pH6)
Buf B:20mM KPB(pH6)
流速:5mL/分
分画:5mL
プログラム:(1)Buf A洗浄 5cv、(2)Buf B勾配 100%/20cv、(3)Buf B 100% 洗浄 5cv
(ハイドロキシアパタイトクロマトグラフィー条件)
担体:ハイドロキシアパタイト(5mL)
チャージ:フェニル精製Fr.No.26-31
Buf A:5mM KPB,0.3M NaCl(pH6)
Buf B:400mM KPB,0.3M NaCl(pH6)
流速:2mL/分
分画:5mL
プログラム:(1)Buf A洗浄 5cv、(2)Buf B勾配 100%/25cv、(3)Buf B 100% 6cv
(Cuアフィニティークロマトグラフィー条件)
担体:Cu2+ HP(1mL)
チャージ:ハイアパ-Fr. 16
Buf A:20mM KPB,0.3M NaCl(pH6)
Buf B:20mM KPB,0.3M NaCl,0.4M イミダゾール(pH6)
流速:1mL/分
分画:2mL
プログラム:(1)Buf A洗浄 6cv、(2)Buf B勾配 10%/20cv、(3)Buf B洗浄 10cv
バチルス ズブチリスと納豆菌から遺伝子を取得し、MDHとyodCの大量発現系を構築した。バチルス ズブチリスと納豆菌の遺伝子情報からプライマーを作成しPCRにかけて該当遺伝子の切り出しを行った。ベクターにpET20b、宿主にBL21(DE3 pLysS)を用い、各酵素のC末端に6×His-tagを付加して発現させた。発現確認培養は次の通り行った。各酵素(MDH又はyodC)(+)Histag/pET20b/BL21のコロニーをとり、30℃、300rpmで一晩振とう培養した(前培養)。この前培養液の2%当量を10mLのLB/Amp培地に植菌し、ODが約0.5~0.7になったらIPTGを終濃度0.5mMになるよう加え、37℃,300rpmで4時間振とう培養した(本培養)。こうして得られた菌体を集菌し、50mM Tris-HCl(pH=7.0)に懸濁させた。これをビーズ破砕し遠心した後、上清をサンプルとした。
上述の方法で得られたリコンビナントyodC(バチルス ズブチリス)とMDH(バチルス ズブチリス)を下記条件でNi-Sepharoseカラムにて精製し、20mM KPB(pH6)で透析した。透析したサンプルを凍結乾燥し、リコンビナントyodCとMDHの酵素粉末を得た。
(クロマトグラフィー条件)
担体:Ni Sepharose(25mL)
サンプル:破砕上清 約20mL
Bind Buf:20mM KPB,0.3M NaCl(pH6)
Elute Buf:20mM KPB,0.3M NaCl,0.4M イミダゾール(pH6)
流速:チャージ:5mL/分,その他:10mL/分
分画:10mL
プログラム:(1)Bind Buf洗浄 6cv、(2)Elute Buf 10% 洗浄 10cv、(3)Elute Buf 100%/20cv勾配、(4)Elute Buf洗浄 10cv
yodCの酵素学的性質を検討した。DLDと同様に至適pH(図31)、pH安定性(図32)、至適温度(図33)、熱安定性(図34)、NADPHへの反応性(図35)、金属塩(金属カチオン)が活性に与える影響(図36)を調べた。至適pHについてはDLDと同様にpH6.0付近であるため食肉アプリケーション上支障はないものと思われる。また幅広いpH条件で安定であった。至適温度を考えても、20℃付近という低温条件下でよく作用することから食肉アプリケーション上好ましいといえる。熱安定性試験においても40℃まで活性を維持することを確認できた。補酵素特異性試験においてはNADHを用いた場合よりもNADPHを用いた場合で良好な活性が得られた。金属カチオンについてはMg、Na又はKを加えた場合に酵素活性の向上が見られた。さらに、基質反応性についても検証した。DLDと同条件で、フェリシアン化カリウムへの反応性、さらにはミオグロビンへの反応性について調べた。DLDの結果を併せて各々図37、図38に示す。DLDと比べ、フェリシアン化カリウムに対しては約2.6倍、ミオグロビンに対しては約22倍の反応性を示した。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
配列番号5:人工配列の説明:プライマーDLD-BamH1-RV
配列番号6:人工配列の説明:プライマーDLD-Nde1-FW
配列番号7:人工配列の説明:プライマーDLD-BamH1-Histag-RV
Claims (26)
- バチルス属微生物由来のヘム還元酵素を含む還元剤。
- 前記ヘムがメトミオグロビンのヘムであることを特徴とする、請求項1に記載の還元剤。
- 前記ヘムがメトヘモグロビンのヘムであることを特徴とする、請求項1に記載の還元剤。
- バチルス属微生物の菌体破砕物からなることを特徴とする、請求項1~3のいずれか一項に記載の還元剤。
- 前記バチルス属微生物がバチルス ズブチリス、バチルス アミロリケファシエンス、納豆菌、バチルス スリンギエンシス及びバチルス ミコイデスからなる群より選択される微生物である、請求項1~4のいずれか一項に記載の還元剤。
- 前記ヘム還元酵素がデヒドロリポイルデヒドロゲナーゼ又はニトロレダクターゼである、請求項1に記載の還元剤。
- 前記ヘム還元酵素として、デヒドロリポイルデヒドロゲナーゼ及びニトロレダクターゼを含む、請求項1に記載の還元剤。
- 前記デヒドロリポイルデヒドロゲナーゼのアミノ酸配列が配列番号3のアミノ酸配列を含み、前記ニトロレダクターゼのアミノ酸配列が配列番号12のアミノ酸配列を含む、請求項6又は7に記載の還元剤。
- 前記デヒドロリポイルデヒドロゲナーゼ及び前記ニトロレダクターゼがリコンビナントタンパク質である、請求項6~8のいずれか一項に記載の還元剤。
- 請求項1~9のいずれか一項に記載の還元剤からなる色調改善剤。
- 請求項1~9のいずれか一項に記載の還元剤と、ミオグロビンのヘム基中の鉄を亜鉛に置換する作用を示す物質を組み合わせてなる色調改善剤。
- 前記物質がフェロケラターゼである、請求項11に記載の色調改善剤。
- 食肉又は食肉加工品の色調の改善用である、請求項10~12のいずれか一項に記載の色調改善剤。
- デヒドロリポイルデヒドロゲナーゼ及び/又はニトロレダクターゼを含む、食肉又は食肉加工品用の色調改善剤。
- 発色作用、発色促進作用及び/又は退色防止作用により色調を改善する、請求項10~14のいずれか一項に記載の色調改善剤。
- 請求項1~9のいずれか一項に記載の還元剤を含むことを特徴とする医薬。
- 経口投与製剤である、請求項16に記載の医薬。
- 非経口投与製剤である、請求項16に記載の医薬。
- 以下のステップ(1)及び(2)を含む還元剤の製造法:
(1)ヘム還元酵素を産生するバチルス属微生物を、該酵素が産生される条件下で培養するステップ;
(2)培養産物から前記酵素を回収するステップ。 - 前記ステップ(2)が以下のステップからなる、請求項19に記載の製造法:
(2-1)培養産物から菌体を収集するステップ;
(2-2)菌体破砕物を調製するステップ。 - 前記ヘムがメトミオグロビンのヘムであることを特徴とする、請求項19又は20に記載の製造法。
- 前記ヘムがメトヘモグロビンのヘムであることを特徴とする、請求項19又は20に記載の製造法。
- 前記バチルス属微生物がバチルス ズブチリス、バチルス アミロリケファシエンス、納豆菌、バチルス スリンギエンシス及びバチルス ミコイデスからなる群より選択される微生物である、請求項19~22のいずれか一項に記載の製造法。
- 請求項10~15のいずれか一項に記載の色調改善剤を食肉又は食肉加工品に作用させることを特徴とする色調改善方法。
- バチルス ズブチリス、バチルス アミロリケファシエンス、納豆菌、バチルス スリンギエンシス及びバチルス ミコイデスからなる群より選択されるバチルス属微生物の菌体破砕物を食肉又は食肉加工品に作用させることを特徴とする色調改善方法。
- 請求項1~9のいずれか一項に記載の還元剤を用いた、血行障害、低酸素症若しくは血中酸素減少状態、これらの一つ以上の病態ないし症状を伴う疾患、又はこれらの一つ以上の病態ないし症状に起因する疾病の予防または治療方法。
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JP2012514778A JP5814914B2 (ja) | 2010-05-12 | 2011-05-09 | バチルス属微生物由来の還元剤及びその用途 |
EP11780557.2A EP2570478B1 (en) | 2010-05-12 | 2011-05-09 | Reducing agent from microorganism belonging to genus bacillus and application for same |
DK11780557.2T DK2570478T3 (en) | 2010-05-12 | 2011-05-09 | REDUCTION AGENT OF MICROORGANISM RELATED TO BACILLUS GENES AND USE OF SAME |
US13/696,446 US20130058911A1 (en) | 2010-05-12 | 2011-05-09 | Reducing agent from microorganism belonging to genus bacillus and application for same |
CN2011800231689A CN102892882A (zh) | 2010-05-12 | 2011-05-09 | 源自杆菌属微生物的还原剂及其用途 |
US14/048,538 US9241507B2 (en) | 2010-05-12 | 2013-10-08 | Reducing agent from microorganism belonging to genus Bacillus and application for same |
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US201213696446A Continuation | 2010-05-12 | 2012-11-06 | |
US14/048,538 Division US9241507B2 (en) | 2010-05-12 | 2013-10-08 | Reducing agent from microorganism belonging to genus Bacillus and application for same |
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Cited By (3)
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WO2014013795A1 (ja) * | 2012-07-18 | 2014-01-23 | 天野エンザイム株式会社 | 食肉の色調改善方法 |
WO2015045664A1 (ja) * | 2013-09-24 | 2015-04-02 | マルハニチロ株式会社 | ミオグロビン含有生食用赤身魚肉の加工食品及びその製造方法 |
CN106854629A (zh) * | 2015-12-08 | 2017-06-16 | 上海泓宝绿色水产股份有限公司 | 一种高浓度复合芽孢杆菌及其制备和使用方法 |
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KR101780348B1 (ko) * | 2016-07-22 | 2017-09-22 | 매그나칩반도체 유한회사 | Cmos 트랜지스터 형성 방법 |
CN111117920B (zh) * | 2020-01-07 | 2021-04-02 | 山东农业大学 | 一种产蛋白酶、产铁载体的蕈状芽孢杆菌及其应用 |
CN112608871B (zh) * | 2021-01-12 | 2022-11-01 | 江南大学 | 一种苏云金芽孢杆菌高密度发酵生产益生活性物质的方法 |
WO2023096255A1 (ko) * | 2021-11-25 | 2023-06-01 | 주식회사 헤모랩 | 미생물 헴단백 추출물을 포함하는, 정장 및 비만 개선용 조성물 |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2014013795A1 (ja) * | 2012-07-18 | 2014-01-23 | 天野エンザイム株式会社 | 食肉の色調改善方法 |
WO2015045664A1 (ja) * | 2013-09-24 | 2015-04-02 | マルハニチロ株式会社 | ミオグロビン含有生食用赤身魚肉の加工食品及びその製造方法 |
JP2015062345A (ja) * | 2013-09-24 | 2015-04-09 | マルハニチロ株式会社 | ミオグロビン含有生食用赤身魚肉の加工食品及びその製造方法 |
CN106854629A (zh) * | 2015-12-08 | 2017-06-16 | 上海泓宝绿色水产股份有限公司 | 一种高浓度复合芽孢杆菌及其制备和使用方法 |
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DK2570478T3 (en) | 2018-08-06 |
EP2570478A1 (en) | 2013-03-20 |
EP2570478A4 (en) | 2015-12-16 |
US20130058911A1 (en) | 2013-03-07 |
JP5814914B2 (ja) | 2015-11-17 |
JPWO2011142300A1 (ja) | 2013-07-22 |
CN107259310A (zh) | 2017-10-20 |
EP2570478B1 (en) | 2018-06-27 |
US9241507B2 (en) | 2016-01-26 |
CN102892882A (zh) | 2013-01-23 |
US20140037791A1 (en) | 2014-02-06 |
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