JP2006325538A - Method for producing bacillus natto kinase and the resultant bacillus natto kinase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily producing Bacillus natto kinase as a thrombolytic enzyme at low cost, in a short time and in high yield without using any expensive and large-sized machine. <P>SOLUTION: The method for producing Bacillus natto kinase comprises binding the original Bacillus natto kinase to a phenolic compound to effect isolation of the aimed Bacillus natto kinase. Another version of this method comprises binding the original Bacillus natto kinase to a phenolic compound to effect extraction of crude Bacillus natto kinase followed by removing vitamin K and/or Bacillus natto-specific smell components from the extract in question using an alcohol. The bacillus natto kinase thus produced is provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing nattokinase, which is a thrombolytic enzyme produced by Bacillus natto. In particular, a method for producing nattokinase in a simple manner, in a short time, and at a low cost with a high yield, and a method for producing nattokinase by removing vitamin K which is a blood coagulation component and odor components peculiar to natto, It relates to nattokinase produced by these.

  Extracting and purifying nattokinase, a thrombolytic enzyme produced by Bacillus natto, from the culture solution of Bacillus natto and removing vitamin K, which is a blood coagulation component, using an ultrafiltration machine, removes vitamin K and odorous components from the culture solution. Thus, a method for concentrating nattokinase has been proposed (Japanese Patent Laid-Open No. 2001-352929 = Japanese Patent No. 3532503). In addition, a method has been proposed in which chitosan is added to a culture solution of Bacillus natto to remove vitamin K by coagulation and precipitation, and then nattokinase is concentrated using a reverse osmotic pressure concentrator (Japanese Patent Laid-Open No. 2001-299277).

However, since both methods use expensive and large-sized devices, the manufacturing cost increases. In addition, since extraction and purification takes time, nattokinase is degraded and the recovery rate is also reduced.
JP 2001-299277 A JP 2001-352929 A

  An object of the present invention is to propose a method for producing nattokinase that can produce nattokinase, which is a thrombolytic enzyme, at low cost, in a short time, and in high yield without using an expensive and large apparatus. It is said.

  In addition, nattokinase can be produced at a low cost, in a short time, and in a high yield without using an expensive and large apparatus, and from the produced nattokinase, vitamins that are blood coagulation components are produced. It aims at proposing the nattokinase manufacturing method from which K and the odor component peculiar to natto were removed.

  And it aims at providing the nattokinase from which the nattokinase manufactured by these manufacturing methods, vitamin K, and the odor component peculiar to natto were removed.

  The inventor of the present application paid attention to the fact that an impurity mainly composed of a protein called in the liquor brewing process is precipitated and removed by tannin, which is a kind of polyphenols, and completed the present invention. Is.

  That is, in order to solve the above-mentioned problems, the method for producing nattokinase proposed by the present invention is characterized in that nattokinase is combined with polyphenols and separated. Another nattokinase production method is a method of producing nattokinase by combining nattokinase with polyphenols and then separating the nattokinase from the extract using alcohols to remove odor components peculiar to vitamin K and / or natto. is there.

  And this invention removes the nattokinase manufactured by the manufacturing method of the said invention of this application, the nattokinase from which vitamin K was removed, the nattokinase from which the odor component peculiar to natto was removed, the vitamin K and the odor component peculiar to natto removed Nattokinase is provided.

  According to the present invention, nattokinase, which is a thrombolytic enzyme produced by Bacillus natto, can be produced in a simple, short time, high yield and low cost. For example, nattokinase can be produced by simply extracting nattokinase from a Bacillus natto culture solution in a short time, in a high yield and at a low cost.

  In addition, vitamin K and / or natto-specific odor components that are blood coagulation components can be easily removed from the nattokinase produced as described above, and nattokinase from which these components have been removed can be extracted and purified. Nattokinase can be produced with low yield and low cost.

  Thus, according to the present invention, for example, nattokinase can be produced by extracting and purifying nattokinase in high yield by concentrating from a culture solution of Bacillus natto.

  Thus, it is possible to produce nattokinase that can be taken without difficulty even by people who hate natto by removing vitamin K which inhibits the effect of warfarin, a therapeutic agent for heart diseases such as myocardial infarction, almost completely and removing odor components.

  In the production method of the present invention, catechin was used as a polyphenol, and nattokinase was produced by binding nattokinase and catechin from a culture solution of natto and precipitating to produce nattokinase. Catechin is bound to the nattokinase of the present invention. In recent years, catechins have been found to have physiological effects such as antiallergic action and antitumor action, and can exhibit effective effects on people with symptoms such as allergies.

  The method for producing nattokinase proposed by the present invention is characterized in that nattokinase is separated by binding with polyphenols.

  Here, as a form in which nattokinase is combined with polyphenols and separated, for example, polyphenols, for example, catechin which is a kind of tannin is added to a culture solution of natto, and catechin and nattokinase are combined and precipitated. The extraction method can be adopted. For example, catechin can be added to a culture solution of Bacillus natto, catechin and nattokinase can be combined, and nattokinase can be extracted as a precipitate by centrifugation.

  By lyophilizing the precipitate, a powder of nattokinase (which is a combination of nattokinase and catechin) of the present invention can be obtained.

  Next, according to another nattokinase production method proposed by the present invention, nattokinase is combined with polyphenols and separated, and thereafter, an alcohol is used to remove vitamin K and / or natto-specific odor components from the extract. Nattokinase is produced.

  That is, nattokinase produced as described above, for example, as described above, catechin is added to the culture solution of natto, catechin and nattokinase are combined, and the precipitate of nattokinase extracted by centrifugation is mixed with alcohols, for example, Ethyl alcohol is added and mixed well, and vitamin K and / or odor components peculiar to natto are removed to produce nattokinase.

  Alcohol is added to and mixed with the nattokinase precipitate (which is a combination of nattokinase and catechin), and vitamin K and / or natto-specific odor components are dissolved in the alcohol to remove them. Can recover nattokinase (which is a combination of nattokinase and catechin) from which vitamin K and / or natto-specific odor components have been removed by centrifugation, for example, as a precipitate.

  Examples of odor components unique to natto include dimethylpyrazine, trimethylpyrazine, tetramethylpyrazine, 2-methylbutyric acid, isovaleric acid, and ammonia. In the present invention, at least one of these is removed. Is.

  In consideration of the fact that the final product is distributed as food, the polyphenols can be separated from the combined product of nattokinase and polyphenols by binding with nattokinase, for example, in a culture solution of Bacillus natto Any substance can be used as long as it can be extracted by binding to nattokinase by precipitation and precipitation.

  The alcohols can be used as long as they do not dissolve the combination of nattokinase and polyphenols, but dissolve vitamin K and the odor components specific to natto exemplified above. Considering that it is a food, for example, ethyl alcohol can be used.

  As described above, nattokinase (which is a combination of nattokinase and catechin) from which vitamin K and / or natto-specific odor components have been removed is recovered as a precipitate, and then water is added to the precipitate and mixed. Then, after substituting the alcohol with water, the precipitate is recovered by centrifugation, and the precipitate is freeze-dried to remove vitamin K and natto-specific nattokinase of the present invention ( Powder of nattokinase and catechin).

  Hereinafter, by adding polyphenols to a culture solution of Bacillus natto, nattokinase is combined with polyphenols and extracted, so that a combined product of nattokinase and polyphenols is separated, and the nattokinase of the present invention is produced. A form is demonstrated.

(Manufacture of Bacillus natto culture)
The medium components are preferably foods and food additives since the final product is distributed as food, carbon sources such as starch and saccharides, nitrogen sources such as seasonings and meat extracts, inorganic salts such as calcium carbonate and magnesium sulfate Etc. are prepared in combination.

  The medium is put into a jar fermenter and sterilized, then inoculated with natto bacteria, and cultured with aeration and stirring at a temperature of 30 to 45 ° C., a stirring rate of 300 to 500 rpm, and an aeration rate of 0.3 to 2 vvm for 3 to 4 days. A Bacillus natto culture solution is obtained.

  In addition, the method for producing a Bacillus natto culture solution is not limited to this, and any conventional method can be used as long as it can produce a Bacillus natto culture solution containing a large amount of nattokinase in consideration of the fact that the final product is distributed as food. The natto bacteria culture solution manufactured by these various methods can be used.

  The subsequent steps are preferably carried out at as low a temperature as possible in order to avoid decomposition of nattokinase.

(Extraction of nattokinase)
From the Bacillus natto culture solution obtained as described above, natto bacillus and insoluble impurities are removed as a precipitate by centrifugation to obtain a culture supernatant.

  Add 0.5-2% by weight of catechin to the culture supernatant and mix well. Stir gently for about 1 hour. Next, this liquid is centrifuged to obtain a precipitate.

  This precipitate is freeze-dried to obtain a powder of nattokinase (combined nattokinase and catechin) of the present invention.

(Removal of vitamin K and odorous components)
Ethyl alcohol in an amount 10 times the weight of the precipitate is added to the precipitate and mixed well, and after gently stirring for about 30 minutes, the precipitate is obtained by centrifugation.

  Again, 10 times the amount of ethyl alcohol is added to the precipitate and mixed well. The mixture is gently stirred for about 30 minutes, and the precipitate is obtained by centrifugation.

  Since vitamin K and odor components peculiar to natto are dissolved in ethyl alcohol, 99% or more of vitamin K and odor components peculiar to natto are removed by this process.

(Purification)
Water of 10 times the weight of the precipitate obtained as described above is added to the precipitate and mixed well to replace ethyl alcohol with water. Thereafter, a precipitate is quickly obtained by centrifugation, and this is freeze-dried after preliminary freezing.

  Thereby, a powder of nattokinase (combined nattokinase and catechin) of the present invention from which vitamin K and odor components peculiar to natto are removed can be obtained.

  Based on the above-described embodiment in which nattokinase is combined with polyphenols and extracted by adding polyphenols to the Bacillus natto culture, and nattokinase of the present invention is manufactured, catechin is added to 750 g of the supernatant of Bacillus natto culture. Was added by 1% by weight, and 1.4 g of lyophilized powder after the purification step was obtained through the steps described above.

  The nattokinase activity (NKase) and vitamin K content (VK) of the supernatant of the Bacillus natto culture and the lyophilized powder were as follows.

NKase NKase recovery rate VK VK removal rate Culture supernatant 93000FU −3200μg −
Freeze-dried powder 82000FU 88% 0.21μg 99.99%
As described above, nattokinase was recovered at a high yield of 88%, and 99.99% of vitamin K was removed.

  Moreover, there was no odor unique to natto from the freeze-dried powder.

  In addition, the activity measurement of nattokinase and the measurement of vitamin K content were based on the following methods described in Experientia 43, 1110 (1987) (Sumi et al.).

(Measurement of nattokinase activity)
Nattokinase was allowed to act on fibrin, and the amount of acid-soluble low-molecular-weight degradation product that increased with fibrin degradation was measured by measuring the absorbance of ultraviolet rays (275 nm).

(Preparation of aqueous fibrinogen solution)
An equivalent amount of 72 mg of fibrinogen (manufactured by Sigma, bovine plasma-derived fibrinogen fraction I, TYPEI-S) was dissolved in 10 ml of 50 mM borax buffer solution (containing pH 8.5, 150 mM NaCl) to prepare a 0.72% fibrinogen aqueous solution. .

(Preparation of thrombin solution)
Thrombin (manufactured by Sigma, derived from bovine plasma) is dissolved in 50 mM borax buffer, adjusted to a concentration of 1000 U / ml, and diluted 50 times with the same borax buffer at the time of use (ie, 20 U / ml) To do.

(Activity measurement)
In a test tube, 1.4 ml of 50 mM borax buffer and 0.4 ml of fibrinogen aqueous solution are weighed and heated in a constant temperature bath at 37 ° C. ± 0.3 ° C. for 5 minutes, and then 0.1 ml of thrombin solution is added and stirred. After the mixed solution is left in this constant temperature water bath for 10 minutes, 0.1 ml of the sample solution is added to the mixed solution, stirred for 5 seconds and left in the constant temperature water bath. After 20 minutes and 40 minutes from the addition of the sample solution, the mixture is stirred for 5 seconds. After 60 minutes, 2 ml of 0.2 M trichloroacetic acid solution is added and stirred, and the mixture is further left for 20 minutes. The reaction solution is centrifuged at 15,000 × g for 5 minutes, and the absorbance (Ar) of the supernatant at 275 nm is measured.

  On the other hand, 1.4 ml of 50 mM borax buffer and 0.4 ml of fibrinogen aqueous solution are weighed and heated in a constant temperature bath at 37 ° C. ± 0.3 ° C. for 5 minutes, and then 0.1 ml of thrombin solution is added and stirred. The mixed solution is allowed to stand in this constant temperature bath for 10 minutes, and then 2 ml of 0.2 M trichloroacetic acid solution is added to the mixed solution and stirred. Next, 0.1 ml of the sample solution is added and stirred and left for 20 minutes. This reaction solution is centrifuged at 15,000 × g for 5 minutes, and the absorbance (Ac) at 275 nm of the supernatant is measured as a control.

The activity A (FU / ml) of nattokinase is obtained by the following formula: A (FU / ml) = {(Ar−Ac) / (0.01 × 60 × 0.1)} × D (D is the dilution factor) .

(Quantification of vitamin K)
Vitamin K was measured by HPLC by preparing a sample for HPLC by the following method.

(Preparation of sample for HPLC)
Mix 0.5 ml of measurement sample, 0.5 ml of water and 1.5 ml of isopropanol, and then add 5 ml of hexane and stir. Centrifugation (1700 × g, 10 minutes, 20 ° C.) is performed, 4 ml of the supernatant (organic layer) is concentrated to dryness, and dissolved in 100 μl of ethanol to obtain a sample solution.

(HPLC conditions)
HPLC conditions are as follows: Column: Sigma STR ODS-24.6 × 250 mm Eluent: 97% ethanol Flow rate: 0.7 ml / min Detection: UV254 nm The retention time of vitamin K under these conditions is the latter half of 16 minutes.

Claims (3)

  A method for producing nattokinase by combining nattokinase with polyphenols and separating them.   A method for producing nattokinase by combining nattokinase with polyphenols and then removing odor components peculiar to vitamin K and / or natto from the extract using alcohols.   A nattokinase produced by the production method according to claim 1 or 2.