CN1407093A - Selenium Nadou enzyme and preparing process thereof - Google Patents

Abstract

A process for preparing selem' Nattokinas (SNK) includes such steps as immersing soybean in Se source solution, steaming, inocualting Bacillus subtilis natto strain, and fermenting. Its advantages are high Se content, high absorption rate and utilization rate of Se, low toxic, long half-life period, and high medical effect.

Description

Selem and preparation method thereof

Technical field

The present invention relates to Selem and preparation method thereof.More particularly, relate to, obtain Selem by fermentation with Bacillus subtilis Natto inoculation selenium soybean.

Background technology

Current, in the medication of rescuing the thrombus patient, select streptokinase (SK), urokinase (UK) and tissue plasminogen activator (t-PA) etc. more widely for use.But these medicines easily produce perfusion property more in actual applications, can occur untoward reactions such as thrombus or hemorrhage, allergy once more, the carrying out of influence treatment.And these medicines can only be injected, and are oral invalid, and the transformation period of its medicine is also short, even dozens of minutes only, in treatment, cause very big inconvenience and difficult.

Natto is the traditional food of Japan, and it is by obtaining soybean boiling secondary fermentation.Owing to natto is to use the strain fermentation to the human body beneficial to obtain, therefore, it is rich in various compositions to the human body beneficial, is that a kind of doctor eats homologous food, for a long time, in Japan always as the senior nutrition of folk tradition.(Nattokinase NK) is the fibrinoclase that the fabulous height of a kind of security is tired to the Nattokinase that extracts from natto, uses as oral, the same effectively thrombolytics of injection at present.Nadou enzyme mainly by the scleroproein in the thrombus, increases the t-PA activity indirectly, and activates uPA (pro-UK) activity, makes it to be converted into approach such as urokinase, carries out the treatment of thrombotic diseases.

Summary of the invention

The objective of the invention is, provide a kind of can reduce inorganic selenium toxicity and enlarge the Nadou enzyme range of application selenium and the complex body of Nadou enzyme.Promptly, use the bio-transformation synthesis method, infiltration by biology, adduction, absorption etc. transform synthesis step, have selenium (Selenium) element (Luo Haiji etc. anticancer, anti-oxidant activity with known, the biological action of selenium and meaning thereof, trace element and health research, the 70th page of the 17th the 2nd phase of volume, 2000) insert the Nadou enzyme body, make it become rich selenium enzyme body.

That is, the invention provides a kind of method for preparing Selem, this method may further comprise the steps:

(1) soybean is flooded in selenium source liquid, obtained the selenium soybean after the boiling;

(2) Bacillus Subtilis Natto inoculation is fermented in above-mentioned selenium soybean.

In the method for the invention, can directly use the Bacillus Subtilis Natto bacterial strain without domestication, but the present inventor finds that Bacillus Subtilis Natto is tamed with selenium cultivate the back use, the form of lawn is better, the bacterium number is more.

In the method for the invention, promptly can adopt solid fermentation method, also can adopt solution fermentation to ferment the selenium fermented soybean.But the employing solution fermentation can make the mycelia thing separate out in liquid, thereby can obtain the higher Selem of purity.

In addition, in the better embodiment of method of the present invention, used selenium source is a Sodium Selenite.

The present invention also provides the Selem that obtains with above-mentioned preparation method.

Because Nadou enzyme has inserted the selenium element, becomes the selenium enzyme complex of enrichment selenium element, thereby can bring into play the synergy of selenium enzyme.And, the selenium element is inserted the enzyme body, make inorganic selenium become biological selenium.Known organism selenium is compared with inorganic selenium, and its specific absorption and bioavailability can significantly improve, and can reduce its original toxicity (Xu Chengshui, trace element and disease, trace element and health research, the 64th page of the 16th the 4th phase of volume, 1999 years greatly; Chen Xiaoxia etc., algae is inquired into the biomagnification and the mechanism thereof of trace element, food and fermentation industries, the 56th page of the 25th the 4th phase of volume, 1999 years).Simultaneously the selenium element is inserted the range of application that has also enlarged the preventing and treating diseases of Nadou enzyme in the Nadou enzyme, have the advantages that a medicine is used more.The same with Nadou enzyme, Selem (Selem ' Nattokinase, SNK) have injection, oral equal effectively, the advantage of long half time (8 days), and have no side effect.

Embodiment

Elaborate with regard to the concrete operations in the above steps of the present invention below.

At first, bacterial classification is tamed.Concrete operations are as follows:

(1) cultivation of lawn: will dissolve with the sterilization physiological water by the Bacillus SubtilisNatto (ASI.1086) that Beijing institute of microbiology of the Chinese Academy of Sciences buys, be inoculated in the following Bpy substratum, at 37 ℃ of slant culture 24-48 hours.The Bpy substratum:

Extractum carnis 5 gram glucose 5 grams

Yeast extract paste 5 gram agar 10-20 grams

Peptone 10 gram deionized waters are added into 1000mL

Sodium-chlor 5 gram pH 7.0-7.6

(2) culture transferring: get the inclined-plane lawn, be inoculated in the selenium source substratum, hatched 12-18 hour, carry out strain domestication, obtain first-generation bacterial classification thus at 37 ℃.Above-mentioned selenium source substratum is that 1000ml makes by add above-mentioned Bpy substratum to cumulative volume in standard selenium liquid 1.0ml.

(3) inoculation of mother starter: get above-mentioned first-generation tube lawn, plant in following mother starter, mixing was hatched 24-48 hour at 37 ℃.Mother starter:

Extractum carnis 5 gram wood sugars 5 grams

Yeast extract paste 5 gram standard selenium liquid 1.0ml

Soy peptone 10 gram deionized waters are added into 1000mL

Sodium-chlor 5 gram pH 7.0-7.6

(4) inoculation of sub-starter: before the growth of Bacillus Subtilis Natto reaches the logarithmic phase stage casing (at this moment, the form of Bacillus Subtilis Natto lawn is relatively neat, rule), get the culture of mother starter, amount culture transferring by 5% is formed the sub-starter identical with above-mentioned mother starter in it, mixing was hatched 18-30 hour at 37 ℃.Obtain can be used for the starter that contains Bacillus Subtilis Natto of selenium fermented soybean thus.Be placed in the refrigerator below 0 ℃ stand-by.

Choose the granule beans that particle diameter is 6.0-6.5mm with crossing sieve method, remove foreign matters such as sandstone, iron filings, and rejecting there are breakage or mildew and rot, spotted beans.

Then, with the soybean of selected mistake dipping in 18-26 ℃ of 1/2 standard selenium liquid (preparation of standard selenium liquid: Sodium Selenite is dissolved in the water, and making selenium concentration is 100mg/L) that is being equivalent to soybean weight, till steeping fluid is blotted.And then the standard selenium liquid of interpolation and soybean identical weight, continuation is at 18-26 ℃ of dipping, and when continuing dipping after about 2.5-3.5 hour, steeping fluid is almost blotted usually, the beans volumetric expansion, soft sense is arranged and the crack is arranged slightly, the plasma selenium in the plasma selenium of beans body and the aqueous solution aqueous solution reaches balance.Stop dipping this moment.

To put into autoclave by the selenium soybean that above-mentioned steps obtains, boiling in 110 ℃, the high compressed steam of 0.1-0.11MPa, sterilization 30 minutes.Take out beans then, after making it be cooled to below about 40-50 ℃, with spray method the starter of the above-mentioned Bacillus of containing Subtilis Natto is sprayed at selenium soybean surface, mixing immediately, placing thermostat container, is that 30-34 ℃, pH are that 5-8, relative humidity are solid fermentation under 60-85%, oxygen capacity＞70%, peak＜50 ℃ the condition in temperature.Usually reaction is after about 18-25 hour, and the growth of BacillusSubtilis Natto reaches the logarithmic phase stage casing, has tangible haircuts thing to form.Interrupt reaction this moment, and cooling immediately.

Then, by steps such as high pressure pulverizing, freeze-drying, conditioning, powder process, obtain being rich in the pulvis of Selem (plasmin).

In liquid fermenting, screen out beans after the fermentation, with liquid concentrating under reduced pressure, freeze-drying, can obtain the higher Selem powder of purity.Qualitative evaluation to the fibrinolytic of Selem pulvis

With reference to Arch.Biochem.Biophys., 1957,40:346 and Biochem.Biophys.Acta., 1957,24:279-82 carries out qualitative evaluation with the fibrin plate method to the fibrinolytic of Selem pulvis.That is, above-mentioned Selem pulvis is done suitably dilution with the Tris damping fluid, be added to blood clot that 1 human blood forms in vitro.The blood clot that has added above-mentioned Selem sample is molten entirely in 60 minutes, and it is insoluble to have added the blood clot of physiological saline in contrast.Quantitative evaluation to the fibrinolytic of Selem pulvis

With reference to Arch.Biochem.Biophys., 1957,40:346 and Biochem.Biophys.Acta., 1957,24:279-82 quantitatively identifies the fibrinolytic of Selem pulvis with the fibrin plate method.That is, survey the flat board of living and contain Fibrinogen, zymoplasm and agarose.With its short mix in glass dish, be put in level attitude, it is solidified, form the thick scleroproein plate of about 1.5mm.After the dilution of above-mentioned Selem pulvis usefulness Tris damping fluid, point sample 10 μ L in kind use urokinase (UK) to compare in demarcating circular hole in addition.In 37 ℃ of incubators, placed 18 hours.Take out and measure the fibrinolysis area, obtain the fibrinolytic unit (μ) of Selem with respect to urokinase.

In qualitative, the quantitative test of above-mentioned Selem cellulolytic activity, three batches of Selem pulvis that used different batches are as sample.Concrete outcome sees Table 1.

Table 1

	Qualitative	Quantitatively
			Sample 1	Clot is molten entirely	????800u/g
Sample 2	Clot is molten entirely	????800u/g
			Sample 3	Clot is molten entirely	????600u/g
Physiological saline	Insoluble	????-

The Selem that obtains with above-mentioned preparation method of the present invention is a white powder, free from extraneous odour.It is a kind of fibrinoclase of enrichment selenium element, has higher fibrinolytic and activities of antioxidant enzymes.It both can be used as protective foods, was used to prevent heart and brain thrombus, tumour, aging, coronary heart disease and atherosclerosis, heavy metal (cadmium, lead, silver) to poison and flavacin B ₁Toxicity, Keshan disease, Kaschin-Beck disease and the other diseases that causes by lacking selenium in the body after purification process, also can be used as medicine and are used for above-mentioned treatment of diseases.

The preparation method of Selem of the present invention has also that cost is low, income is high, easy and simple to handle, the advantage that is easy to grasp.

Be to specify above, but these explanations should not thought limitation of the invention to of the present invention.Those skilled in the art will appreciate that according to top explanation of the present invention, can make various modifications and changes to the specific embodiment of the present invention and do not depart from essence of the present invention and by the determined protection domain of the claims that attach.

Claims (4)

1. the preparation method of Selem, it may further comprise the steps:

(1) soybean is flooded in selenium source liquid, obtained the selenium soybean after the boiling;

(2) Bacillus Subtilis Natto inoculation is fermented in above-mentioned selenium soybean.

2. preparation method as claimed in claim 1, wherein, described Bacillus Subtilis Natto bacterial strain is the bacterial strain of cultivating with the selenium domestication.

3. the method for claim 1, wherein used selenium source is a Sodium Selenite.

4. Selem, it obtains with each described preparation method among the claim 1-3.