CN1799627A - Natto kinase oral liquid and its manufacturing method - Google Patents
Natto kinase oral liquid and its manufacturing method Download PDFInfo
- Publication number
- CN1799627A CN1799627A CN 200510000158 CN200510000158A CN1799627A CN 1799627 A CN1799627 A CN 1799627A CN 200510000158 CN200510000158 CN 200510000158 CN 200510000158 A CN200510000158 A CN 200510000158A CN 1799627 A CN1799627 A CN 1799627A
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- oral liquid
- fermentation
- nattokinase
- liquid
- natto kinase
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Landscapes
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Abstract
The invention relates to a nattokinase oral liquid, which is prepared from Bacillus subtilis fermentation liquor, and contains 0.5-3% of cyclodextrin or cyclodextrin derivative, 1ml of the oral liquid contains 10-400IU of reactive fibrinolytic nattokinase. The invention also provides the process for preparing the nattokinase oral liquid.
Description
Technical field
The present invention relates to a kind of health food, specifically, relate to a kind of natto kinase oral liquid and manufacture method thereof with cardiovascular health-care function effect.
Background technology
Cardiovascular and cerebrovascular disease is one of principal disease of serious harm human health, and according to The World Health Organization's statistics, annual nearly 1,500 ten thousand people in the whole world die from cardiovascular and cerebrovascular disease.The number that China dies from cardiovascular and cerebrovascular disease every year also surpasses 50% of whole death tolls, and high especially at the big city sickness rate.Thrombotic disease (for example acute myocardial infarction etc.) is as the height morbidity of middle-aged and elderly people, it is one of disease of death threats maximum, present old man more than 60 years old has reached more than 100,000,000, account for 8.7% of population ratio, China will enter aging society in 20 years from now on, and the control of cardiovascular and cerebrovascular disease must cause enough attention.
The existing blood embolization patient in the whole world is about 1,500 ten thousand, about 2,000,000,000 dollars of the potential market of required thrombolytic medicine.And all there are several tangible deficiencies in the thrombolytic drug streptokinase (SK) that uses on the domestic market now, urokinase (UK), tissue-type plasminogen activator (tPA) etc.: anti reperfusion, produce thromboembolism and hemorrhagic side effect again, and cost an arm and a leg.
Nattokinase (Nattokinase, NK) be to see by Japanese professor physiology that the doctor of foreign firm waited a kind of molecular weight at first find and to name to be far smaller than UK from 200 numerous food in 1987, SK, the protein of tPA, it is a kind of serine protease that produces in the Bacillus natto sweat, isoelectric point, IP is 8.7, molecular weight is 25000, has very strong fibrinolysis ability, and has an activity of activation Elastase and uricase, correlational study after this shows, nattokinase can effectively be removed the thrombosis in the blood of human body, be the highest protease of present thrombolytic vigor (also have report to think have nattokinase only in the food can dissolve the thrombosis that has formed), and to hypertension, aging, apoplexy, muscle spasm and cardiovascular and cerebrovascular disease also have better curative effect, have been used as thrombolytic drug.And, because nattokinase derives from the food (natto) of japanese traditional, safety is good, simultaneously also can be by intestinal absorption, acting duration is long in vivo, the most important be it can also human activin in rt-PA (RtPA), make it gentle, improve the fibrinolytic of blood constantly, what be main component with the nattokinase as the R﹠D work progress of medicine, functional food, food additive or health product therefore in recent years, is very fast.The nattokinase product " NKCP " (45 dollars/box) that Japan is big and pharmaceutical Co. Ltd produces has soft friendship capsule, hard capsule and 3 kinds of forms of tablet as medicinal; " natto essence " that Wen Cuipu-Sheng Te company produces has soft capsule, two kinds of forms of hard capsule, as food additive and functional food in Japan with sell all over the world; Anaphylaxis research group company produces contains the nattokinase product, is compound preparation, and name of product is nattokinase (65 dollars/box), at home and abroad sells as food tonic and functional food; What the Korea S dimension longevity, enzyme company produced is that the dimension longevity enzyme of main component is medicinal enteric coated capsule with the nattokinase, and every day, dose was 2000IU/ day.
From above introduction as can be seen, present nattokinase product all is capsule or tablet in form, perhaps be processed to contain the health food of nattokinase, its reason mainly is because the abnormal smells from the patient that natto or nattokinase itself have a kind of uniqueness, it for a lot of people a kind of little foul smell flavor of discomfort, so mostly present production is to be processed into concentrated dosage form for swallowing.But, nattokinase is a kind of protease of thermal sensitivity, under the condition of heating (being higher than 60 ℃), be easy to the degeneration inactivation, cause its cellulolytic activity to reduce even disappearance, therefore, in the nattokinase production, desire keeps higher nattokinase fibrinolytic, realizes that again the height of product concentrates, for production technology very high requirement has been proposed, the method that G-sphendex Gel filtration purification that for example adopts mostly at present and lyophilization obtain dry product, the production cost height, the production cycle is long, production scale is little, and this also is the main cause that causes this series products high price.
Summary of the invention
Main purpose of the present invention is to provide a kind of natto kinase oral liquid, can be used as the health product or the auxiliary therapeutic agent of prevention cardiovascular and cerebrovascular vessel, by proper formula design, has both guaranteed the fibrinolytic of oral liquid, also has the mouthfeel that can be accepted by consumer.
The present invention also provides the manufacture method of this natto kinase oral liquid.
Natto kinase oral liquid provided by the invention, it comes the tunning of the bacillus subtilis of self-produced nattokinase, and contains cyclodextrin or the cyclodextrin derivative of 0.5-3%, and the nattokinase fibrinolytic in every milliliter of oral liquid is 100-400IU.
This case inventor has carried out macromethod research back to existing nattokinase health-product market and has found, by the end of at present, the interior not relevant report of natto kinase oral liquid of world wide does not have Related product to come out yet.The inventor gropes through testing repeatedly, think the fermentation liquid of nattokinase fibrinolytic in the 100-400IU/ml scope, the relatively good acceptance of its mouthfeel, and the product of this concentration range can satisfy daily health care intake substantially as oral formulations, especially by the regulating action of cyclodextrin, can directly needn't become highly purified dry product by refabrication as oral liquid.Owing to do not need to have improved security of products yet, and can save the nutritional labeling of tunning from damage by refinement treatment such as various chemical reagent precipitate.Based on such conclusion, the nattokinase health product of drink form provided by the invention have been filled up the market vacancy of this series products.
Reach more fully care treatment effect for making oral liquid have better mouthfeel, during forming, natto kinase oral liquid of the present invention can contain suitable flavoring agent and other functional additive, comprise sweeting agent, for example sucrose, stevioside, Mel, protein sugar, high fructose syrup etc.; Acidic flavoring agent, for example citric acid or sodium citrate etc.; Functional additive, for example isoflavone, lecithin, low polyxylose alcohol, VE, VC etc.; Antiseptic can be sodium benzoate or potassium sorbate etc.Described additive all should satisfy the related standards of food additive, and their content also should be in prescribed limit, makes oral liquid have more ideal mouthfeel and comprehensive nutritional health function through suitably allocating.Generally speaking, gross weight with the oral liquid product is a benchmark, wherein also can comprise the acidic flavoring agent of sucrose, the 0.15-0.25% of 6-8%, an amount of functional additive and the antiseptic of 0.005-0.01%, also can adopt other sweeting agents to substitute sucrose, to make things convenient for diabetic to take.
This case the inventor find in exploratory experiment, adds the Fructus Crataegi water extract and will improve the mouthfeel of oral liquid more significantly in above-mentioned concentrated solution, simultaneously by the adding of health-care components in the Fructus Crataegi, also can assist a ruler in governing a country the effect of nattokinase.So the present invention preferably also provides a kind of compound natto kinase oral liquid, in above-mentioned oral liquid, also added the Fructus Crataegi water extract of oral liquid gross weight 15-30%, this Fructus Crataegi water extract can be the supernatant that directly uses after the pulp that obtains after the haw berry grinding adds flooding, purpose is to utilize health-care components such as mountain terpene in the Fructus Crataegi and flavone, has more improved the effect of nattokinase.Certainly this nattokinase fibrinolytic that has added in the oral liquid of Fructus Crataegi water extract still should be 100-400IU/ml.
For the application of nattokinase product, the nutritionist advises that the preventive dose of taking every day is 1500-2000IU, so take natto kinase oral liquid 10-20 milliliter of the present invention every day, just can reach prophylactic treatment, salubrious purpose fully.As seen natto kinase oral liquid of the present invention all is very easily on taking and carrying, and because the change of dosage form has reduced production cost, the demand that more meets ordinary consumer, it is popular that consumer groups are turned to from particularization, has great economic benefit and social benefit.
On the other hand, under the prerequisite of the health care that does not reduce described natto kinase oral liquid product, the present invention also provides a kind of production method of natto kinase oral liquid, can effectively reduce cost, shorten the production cycle, increase output, thereby solve the high problem of current nattokinase health care product production process complicated production cost.
The manufacture method of natto kinase oral liquid of the present invention mainly may further comprise the steps:
(1) utilize the bacillus subtilis that produces nattokinase to prepare the nattokinase fermentation liquid;
(2) centrifugal with the rotating speed of 3000-5000rpm, remove thalline and impurity in the fermentation liquid;
(3) supernatant after centrifugal adopts sand rod filter to take off charcoal and handles;
(4) be concentrated into predetermined concentration being lower than under 60 ℃ the temperature, add cyclodextrin or cyclodextrin derivative then, its content is that the 0.5-3% of oral liquid is (in practical operation, being converted into the quality that concentrates liquid measure is that the benchmark interpolation is more convenient), stirred 1-2 hour being lower than under 60 ℃ the temperature, and allocate; With
(5) be lower than under 60 ℃ concentrated solution enforcement microfiltration sterilization or irradiation sterilization.
Main active nattokinase in the oral liquid of the present invention is from the tunning of the bacillus subtilis that can produce nattokinase.Bacillus subtilis is as a kind of probiotic bacteria, in disclosed patent and the document a large amount of reports are arranged at home and abroad, also can be commercially available on the market, Bacillus Subtilis AK (PN2003-210136 for example, FERM P-18291 JP), Bacillus Subtilis MR141 (PN 08-154616, FERM P-14692 JP), (PN 5486467 for Bacillus Subtilis IAM1026, FERM BP-6713US), (PN 6420145 for Yunnan SL-001, FERM BP-4844 US), (PN 99/28441 for Bacillus SubtilisAS2, FERM BP-6139 PCT), Bacillus natto KA145 (PN2001-238667, FERM P-17659 JP), Bacillus natto T0-9 (PN 06-261744 FERMP-13164 JP), (PN 6767729 for Bacillus Subtilis M2-4, FERM BP-7155 US) (PN 2003/013565 for Bacillus Subtilis TOYO-11 TO 13, FERM BP-8011 TO 8013PCT), Bacillus natto NLSSe (application number 02121352.6, CGMCC NO.0724 CN), Bacillus Subtilis natto HW4-1 (application number 02116667.6, CGMCC NO.0718 CN), Bacillus natto X-501 (application number 00107589.6, CGMCC NO.0449 CN), BacillusSubtilis natto (ASI.1086), Bacillus SP.CK 11-4 (Wonkeuk kim.etl.AmericanSociety for Microbiology, VOL 62.NO.7) etc., these strains are during the fermentation except that producing the nattokinase, their metabolites at different levels all contain the composition to the human body beneficial, therefore all can be used as raw material strain of the present invention, the present invention preferably adopts bacillus natto, and it can produce the nattokinase or the similar streptokinase of efficient fibrinolytic by solid or fluid matrix fermentation.
According to selected strain, select its suitable condition of culture to carry out fermentation culture, wherein, can select solid fermentation method for use, strain is made bacteria suspension, be inoculated in the solid medium, under suitable condition, cultivate.But because the present invention finally need make a kind of oral liquid, and when adopting solid fermentation, the nattokinase dissolving that fermentation need be produced extracts, and can make complex manufacturing on the contrary, increase production cost.Therefore, the present invention preferably adopts liquid fermentation method, and its concrete technology is as follows:
Test tube strains → flat board activation → inoculation → seed liquor fermentation → production liquid fermentation.
Select the nattokinase superior strain for use, directly on flat board, carry out separation screening, separating plate is preferably LB-agarose-fibrin plate: dissolve with 0.01M kaliumphosphate buffer (PB pH7.4) during the preparation of LB culture medium, when making flat board, Cryodesiccant Human Fibrinogen and thrombin are dissolved in wherein the same agarose-fibrin plate of its manufacture method.
Carry out the optimization of the liquid fermentation medium of nattokinase afterwards, among the present invention, optimal culture condition according to selected different strain, nitrogenous source in the used fermentation medium, carbon source and inorganic salt can be selected, for example, nitrogenous source can select one or more mixing in soy peptone, tryptone, yeast powder, caseinic acid hydrolysate and the dried powdered soybean etc. to use, and the mass concentration of use is preferably about 0.5-4.0%; Carbon source can be one or more in glucose, xylose, soluble starch, maltose, lactose and the sucrose, and the mass concentration of use is preferably about 1.0%-10.0%; Inorganic salt can be selected KH
2PO
4About 0.2%, K
2HPO
4About 0.4%, MgSO
4About 0.05% and CaCl
2About 0.02% etc.
The preferable methods according to the present invention uses bacillus natto to carry out the fermenting process of preparing seed liquor, ferments to OD=0.3-1.0, carries out liquid submerged fermentation with the inoculum concentration about 1.0-2.0%, and fermentation temperature 25-40 ℃, fermentation period 12-35 hour, pH was 6-8.
Liquid after the fermentation at first needs to carry out low-speed centrifugal, to remove most of thalline and the impurity in the fermentation liquid.Centrifugal rotation speed is preferably 3000-5000rpm; Then, to the supernatant after centrifugal, adopt sand rod filter to take off charcoal and handle, to remove bitter taste wherein; Afterwards, concentrate again, because some beneficial products that the bacterial strain of the production nattokinase among the present invention produces during the fermentation are thermal sensitivity, therefore, the present invention is not suitable for adopting heating to concentrate, be applicable to that method for concentration of the present invention is preferably the room temperature ultrafiltration and concentration, the molecular cut off of ultrafilter membrane is 50000-200000, ultrafiltration pressure 0.1-0.3MPa, rejection 〉=90%, cycles of concentration are looked the fibrinolytic of nattokinase in the fermentation liquid and are determined, generally about 10-30 times, suitable is about 20 times, is required the concentrated solution of concentration to obtain.
Directly oral this concentrated solution can be felt distinctive salty, the bitterness of natto, still feel bad acceptance for majority, so add the cyclodextrin or derivatives thereof of 1-3% again, suitably heating (is not higher than 60 ℃, preferred 30-60 ℃) stirred 1-2 hour, can reach the purpose of improving local flavor, employed cyclodextrin or derivatives thereof is meant in the food processing normally used, for example beta-schardinger dextrin-(β-CD), HP-(hp-β-CD) etc.Certainly also can add suitable flavoring agent simultaneously suitably allocates, for example, gross weight with the natto kinase oral liquid that finally obtains is a benchmark, adds the acidic flavoring agent of sucrose, the 0.15-0.25% of 6-8%, an amount of functional additive and the antiseptic of 0.005-0.01%, and stirs.
The preferred scheme according to the present invention, in adding the also deployed concentrated solution of cyclodextrin or derivatives thereof, add the Fructus Crataegi water extract, the preparation method of described Fructus Crataegi water extract comprises ground back adding distil water stirring and leaching 12-24 hour with haw berry, supernatant is collected in centrifugalize, and this Fructus Crataegi water extract accounts for the 15-30% of oral liquid weight in the oral liquid of making.
Next, for prolonging the shelf-life of final products, need sterilize and fill to prepared oral liquid.Same existence owing to nattokinase and some other heat-sensitive substances, the present invention can not adopt pressure sterilizing, must adopt a kind of " cold sterilization ", that is, require the temperature of sterilization preferably not to be issued to effective sterilizing in lower temperature above 60 ℃.The present invention preferably can adopt microfiltration degerming or irradiation sterilization.Micro-filtration membrane aperture 0.05-0.45 μ m, antibacterial and virus that can the effectively elimination overwhelming majority be carried out sterile filling afterwards.Perhaps, adopt irradiation sterilization (adopt this processing method should first fill sterilize together with packing again), radioactive source can be Co60, and irradiation dose is 1-12Kgy, is preferably 3-8Kgy.
The natto kinase oral liquid product that obtains by above-mentioned steps, outward appearance is to be close to colourlessly (to adopt the LB culture medium for yellow substantially, if it then is colourless adopting the culture medium of optimizing, if add the Fructus Crataegi water extract then blush) transparent, the abnormal smells from the patient of natto slightly after the allotment, the oral liquid that adds Fructus Crataegi extract then has the sour-sweet taste of Fructus Crataegi, does not have the bitter salty taste of natto substantially.This oral liquid product can be preserved 6 months at normal temperatures at least.
Simultaneously, have good fibrinolytic for verifying in the oral liquid product of the present invention, the present invention has also carried out qualitative and quantitative evaluation to the fibrinolytic of the natto kinase oral liquid product that obtains, and concrete grammar is as follows:
1, to the qualitative evaluation of the fibrinolytic of natto kinase oral liquid
Reference literature Acta Nutrimenta Sinica, 2003,01 (25), the method among the 1:46-51 is carried out qualitative evaluation with solidifying the fibrinolytic of clot dissolution method to natto kinase oral liquid.That is, from above-mentioned packaged natto kinase oral liquid finished product, draw 1ml and do suitably dilution, be added to blood clotting that 1 human blood forms in vitro with the PB buffer of pH=7.4.Add blood clotting all dissolvings in 1-2 hour of above-mentioned natto kinase oral liquid sample, and added the not dissolving of blood clotting of normal saline in contrast accordingly.
2, to the quantitative evaluation of the fibrinolytic of natto kinase oral liquid
Reference literature Arch Biochem Biophys, 1952, the method among the 40:346-351 is quantitatively identified the fibrinolytic of natto kinase oral liquid with the fibrin plate method.Manufacture method is as follows: the 0.1g agarose is dissolved in the 10ml PB buffer, after the heating for dissolving, places 45 ℃ of insulations 45 minutes.The pure product of 10mg Cryodesiccant Human Fibrinogen are dissolved in the 10ml PB buffer 45 ℃ of water-baths 10 minutes.The thrombin of getting 1IU adds mixing in the agarose solution, simultaneously fibrinogen solution is poured in the above-mentioned mixed liquor, and mixing is poured in the culture dish of 9cm * 9cm immediately, and room temperature was placed at least one hour, and it is fully solidified.With the plate punching, last sample 10 μ l placed 10 minutes under the room temperature, put into 37 ℃ of constant incubators again and cultivated 18 hours.With its solusphere diameter of phi of vernier caliper measurement, according to area formula of circle S=π * Φ
2/ 4 calculate the solusphere area, represent enzyme activity, or calculate the units (being defined as IU) of suitable urokinase vigor.
3, the mensuration of urokinase standard curve
Get the urokinase of different iu numbers (IU), its point sample on fibrin plate, was cultivated 18 hours for 37 ℃,, calculate the solusphere area with its solusphere diameter of vernier caliper measurement.With the solusphere area is abscissa (X-axis), with log
10IU+2 is vertical coordinate (Y-axis), makes the urokinase canonical plotting with Microsoft Excel, and draws computing formula y=0.0071x-0.2354.
After measured, the fibrinolytic unit of nattokinase if take every day about the 10-20 milliliter, can reach prevention and treatment cardiovascular and cerebrovascular disease, salubrious purpose at 100-400IU/ml fully in the oral liquid of the present invention.
By the natto kinase oral liquid of the invention described above and production method thereof as can be known, the present invention has the following advantages:
1. do not have the relevant report of natto kinase oral liquid in the present world wide, therefore, the present invention is the achievement of an initiative in nattokinase health product development field, has enlarged the application of nattokinase as health product.
2. in the oral liquid of the present invention, contain the highly active nattokinase that biofermentation produces, as the health product long-term drink, framework ingredient-fibrin of the human thrombus that can effectively degrade, thereby thrombus plays the health-care effect that prevents and treat cardiovascular and cerebrovascular disease.
3. because the simplification of processing method, and adopt rational sterilization means, the loss of product nutritional labeling is few, has kept biological activity and other thermal sensitivity bioactive substance of nattokinase in the oral liquid to greatest extent, that is the more common liquid fermentation production height of the nattokinase fibrinolytic in the product.For example, application of sample 1ml in the qualitative detection, clot all dissolves in 90min, and sample 50 μ l in the fibrin plate detection by quantitative, the fibrinolytic of nattokinase can reach a 150-400 urokinase unit/ml nattokinase fermentation and concentrate sample.
4. the present invention allocates by suitable means and interpolation, has effectively covered nattokinase and the special abnormal smells from the patient of other useful tunning, and mouthfeel is greatly improved, and is fit to crowd widely.Utilize health-care components such as mountain terpene in the Fructus Crataegi and flavone simultaneously, the oral liquid product of preparing that contains the Fructus Crataegi water extract has more improved the effect of nattokinase.
5. liquid fermentation medium raw material and the food additive wide material sources selected for use of the present invention, low price, safe and reliable.
6. in the process of screening superior strain, traditional screening technique is generally the dull and stereotyped grown cultures of carrying out bacterial strain of growth, then adopt separating plate to screen, and the present invention is through research experiment, adopted a step separation to carry out growth, separation and the screening of bacterial strain, good separating effect not only, and simplified the step of bacterial strain screening greatly, saved the production time.
7. in the process of producing, omitted the step that traditional polydextran gel (G-sphendex Gel) or column chromatography filtration purification and lyophilization obtain dry product, thereby production technology is simple, low production cost, with short production cycle, constant product quality, and be easy to realize suitability for industrialized production, have great economic benefit and social benefit.
The specific embodiment
Following embodiment will be further explained the present invention, but the present invention is not limited only to these examples, the scope that these examples do not limit the present invention in any way, some change that those skilled in the art is made within the scope of the claims and adjustment also should be considered as belonging to scope of the present invention.
Embodiment 1
1, fermentation
Test tube strains → flat board activation → inoculation → seed liquor fermentation → production liquid fermentation
Plating medium wherein and seed fermentation culture medium are LB (Luria-Bertani) culture medium, and its prescription is: antibacterial culturing tryptone 10g/L, antibacterial culturing yeast extract 5g/L, NaCl10g/L; Producing fermentation medium is soy peptone 1.5%, sucrose 2%, KH
2PO
40.2%, K
2HPO
40.4%, MgSO
40.05%, CaCl
20.02%.
The seed liquor fermentation temperature is 37 ℃, stops when fermenting to OD660=0.3-1.0;
Get 1.0% seed liquor and be inoculated into 5L and produce fermentation liquid and carry out enzymatic production, 35 ℃ of fermentation temperatures, fermentation period 12h, pH6, fermentation stops fermentation during to the viscosity filament that produces a large amount of whites.
2, fermentation liquor treatment
After fermentation finishes, carry out low-speed centrifugal earlier, remove thalline and impurity in the fermentation liquid, rotating speed is 3000rpm;
Supernatant after centrifugal adopts sand rod filter to take off charcoal and handles, and removes the bitter taste of fermentation liquid in advance.
3, concentrate
Fermentation liquid carries out ultrafiltration and concentration through taking off after charcoal is handled, and to remove too much solvent at normal temperatures, makes nattokinase in the fermentation liquid and other active component obtain effective enrichment.The ultrafilter membrane molecular cut off is 100000, ultrafiltration pressure 0.1MPa, and the ultrafiltration temperature is a room temperature, cycles of concentration is 15 times, approximately obtains the 0.3L concentrated broth.
4, allotment is filtered
Add the beta-schardinger dextrin-of about 3g in the concentrated solution, keep about 40 ℃ and stirred 1-2 hour, cyclodextrin is dissolved fully.
Add the 60ml hawthorn extract again, addition is 20% (is about final oral liquid gross weight 16%) of above-mentioned concentrated solution quality, simultaneously according to weight proportion take by weighing respectively sodium benzoate, the 0.6g of sucrose, the 0.03g of 24g sodium citrate, be sequentially added in the nattokinase concentrated solution of 0.3L, after in material-compound tank, mixing, carry out pre-filtering, to remove the impurity in the mixed liquor.
The preparation method of hawthorn extract wherein is: take by weighing a certain amount of haw berry (through screening and cleaning), grind the back and add 5 times of water, 50 ℃ of left and right sides stirring and leaching (150-240rpm) about 12-15 hour, centrifugalize behind the elimination marc is collected supernatant and is hawthorn extract.Because the existence of pectin, extracting solution cooling back is thick, so should keep 40-50 ℃ to add as early as possible in the above-mentioned concentrated solution, the oral liquid after the allotment has certain clarity, and the thickness sense is arranged after the cooling, and color is little red.
5, sterilization
The microfiltration degerming is adopted in sterilization, and micro-filtration membrane aperture 0.1 μ m with the microorganism (comprising antibacterial, mycete, yeast and virus) and the macromolecular substances (molecular weight surpasses 50000) that remove in the mixed liquor, prolongs the shelf-life of product.
6, fill
After the product sterilization,, be in the ampere bottle of 10ml to the went out capacity of bacterium of ultraviolet, promptly obtain final products liquid medicine filling according to the requirement of sterile working.
Final products outward appearance in the present embodiment is erythroid bright liquid, sour and sweet palatability, and fibrinolytic vigor height reaches 2300IU/ and props up, and can effectively prevent and treat cardiovascular and cerebrovascular disease.This oral liquid product room temperature can be preserved more than 6 months.
Embodiment 2
1, fermentation
Test tube strains → flat board activation → inoculation → seed liquor fermentation → production liquid fermentation
Plating medium wherein, seed fermentation culture medium are LB (Luria-Bertani) culture medium, and its prescription is: antibacterial culturing tryptone 10g/L, antibacterial culturing yeast extract 5g/L, NaCl10g/L; Producing fermentation medium is soy peptone 0.5%, sucrose 1.5%, KH
2PO
40.2%, K
2HPO
40.4%, MgSO
40.05%, CaCl
20.02%.
The seed liquor fermentation temperature is 37 ℃, stops when fermenting to OD660=0.3-1.0;
Get 2% seed liquor and be inoculated into 5L and produce fermentation liquid and carry out enzymatic production, 30 ℃ of fermentation temperatures, fermentation period 30h, pH8, fermentation stops fermentation during to the viscosity filament that produces a large amount of whites.
2, fermentation liquor treatment
After fermentation finishes, carry out low-speed centrifugal earlier, remove thalline and impurity in the fermentation liquid, rotating speed is 5000rpm;
Supernatant after centrifugal adopts sand rod filter to take off charcoal and handles, and removes the bitter taste of fermentation liquid.
3, concentrate
Fermentation liquid carries out ultrafiltration and concentration through taking off after charcoal is handled, and mainly is in order to remove too much solvent at normal temperatures, makes nattokinase in the fermentation liquid and other active component obtain effective enrichment.The ultrafilter membrane molecular cut off is 80000, ultrafiltration pressure 0.3MPa, and the ultrafiltration temperature is a room temperature, cycles of concentration is 25 times, approximately obtains the 0.2L concentrated broth.
4, allotment is filtered
Add the beta-schardinger dextrin-(content of beta-schardinger dextrin-is about 2% in the final products) of about 4g in the concentrated solution, keep about 40 ℃ and stirred 1-2 hour, cyclodextrin is dissolved fully.
Take by weighing the protein sugar of 0.6g, the Mel of 3g respectively according to weight proportion, 0.02g sodium benzoate, the sodium citrate of 0.3g, the xylitol of 4g, 0.5g lecithin, join in the nattokinase concentrated solution, after in material-compound tank, mixing, carry out pre-filtering, to remove the impurity in the mixed liquor.
5, fill
According to the requirement of sterile working, be in the ampere bottle of 10ml to the went out capacity of bacterium of ultraviolet with liquid medicine filling.
6, sterilization
Irradiation sterilization is adopted in sterilization, and radioactive source is Co60, and irradiation dose is 6.5Kgy, to kill the microorganism in the mixed liquor, prolongs the shelf-life of product.
This product outward appearance is colourless bright liquid, sour and sweet palatability, the about 3200IU/ of fibrinolytic props up, nutritious, the lecithin that adds can play the effect of Synergistic to nattokinase, this product has added functional oligomeric polysaccharide simultaneously, can not only effectively prevent and treat cardiovascular and cerebrovascular disease, also is the good health product of diabetics.Can preserve more than 8 months under the oral liquid product room temperature of present embodiment.
Embodiment 3
1, fermentation
Test tube strains → flat board activation → inoculation → seed liquor fermentation → production liquid fermentation
Plating medium wherein, seed fermentation culture medium are LB (Luria-Bertani) culture medium, and its prescription is: antibacterial culturing tryptone 10g/L, antibacterial culturing yeast extract 5g/L, NaCl10g/L; Producing fermentation medium is soy peptone 2.5%, sucrose 6.0%, KH
2PO
40.2%, K
2HPO
40.4%, MgSO
40.05%, CaCl
20.02%.
The seed liquor fermentation temperature is 37 ℃, stops when fermenting to OD660=0.4-0.8;
Get 2% seed liquor and be inoculated into 5L and produce fermentation liquid and carry out enzymatic production, fermentation temperature 37-38 ℃, fermentation period 30h, pH7, fermentation stops fermentation during to the viscosity filament that produces a large amount of whites.
2, fermentation liquor treatment
After fermentation finishes, carry out low-speed centrifugal earlier, remove thalline and impurity in the fermentation liquid, rotating speed is 4000rpm;
Supernatant after centrifugal adopts sand rod filter to take off charcoal and handles, and removes the bitter taste of fermentation liquid.
3, concentrate
Fermentation liquid carries out ultrafiltration and concentration through taking off after charcoal is handled, and the ultrafilter membrane molecular cut off is 200000, ultrafiltration pressure 0.3Mpa, and the ultrafiltration temperature is a room temperature, cycles of concentration is 10 times, obtains about 0.5L fermentation concentrated solution.
4, allotment is filtered
Add hydroxyl third-beta-schardinger dextrin-of 15g in the concentrated solution, keep about 40 ℃ and stirred 1-2 hour, cyclodextrin is dissolved fully.
Add hawthorn extract (extraction and adding method are with embodiment 1) again, addition is 25% of an above-mentioned concentrated solution quality, take by weighing the sucrose of 40g respectively according to weight proportion, 0.05g sodium benzoate, the citric acid of 1g, the VE of 1g, the VC of 1g adds the people in the nattokinase concentrated solution, after in material-compound tank, mixing, carry out pre-filtering, to remove the impurity in the mixed liquor.
5, fill
According to the requirement of sterile working, liquid medicine filling gone out the ampere bottle of capacity 10ml of bacterium to ultraviolet.
6, sterilization
Irradiation sterilization is adopted in sterilization, and radioactive source is Co60, and irradiation dose is 8Kgy, to kill the microorganism in the mixed liquor, prolongs the shelf-life of product.
This product outward appearance is erythroid bright liquid, and is nutritious, sour and sweet palatability, and the about 1800IU/ of fibrinolytic props up, and can effectively prevent and treat cardiovascular and cerebrovascular disease, and can remove intravital harmful free radical, plays slow down aging, salubrious effect.
The mouthfeel test
50 experimenters (random choose) give different samples (interpolation of auxiliary and condiment is identical) respectively and taste, and add up their reflection, and the result is as follows:
| The sample mouthfeel | Concentrated solution (does not add cyclodextrin and hawthorn extract | Add 2% cyclodextrin, do not add hawthorn extract (embodiment 2) | 2% cyclodextrin and 25% hawthorn extract (embodiment 3) |
| Substantially there is not salty, bitterness | 2 people | 16 people | 21 people |
| Weak salty, bitterness | 9 people | 18 people | 17 people |
| Salty, bitterness is heavier | 29 people | 6 people | 2 people |
As seen, the fermentation liquid that adds after 1% cyclodextrin is allocated can be accepted by majority, and after adding hawthorn extract again, mouthfeel more is significantly improved.
The adding of Fructus Crataegi water extract is to the influence of thrombolytic effect:
Synergy with fibrin plate method checking Fructus Crataegi water extract: according to the experimental establishment shown in the following table, respectively from ready 1#, 2#, draw 50 μ l in the 3# sample, be added to containing in thrombin and the fibrinogenic fibrin plate of preparing in advance, under 37 ℃, left standstill 18 hours observing response, add up corresponding solusphere diameter, calculate corresponding solusphere area.Experimental result finds that 2# is obviously big by about 22% than the solusphere area of 1#, and the not dissolving of the blood clotting of 3# sample in contrast illustrates that there are synergy in nattokinase and Fructus Crataegi water extract on thrombolytic effect.
Nattokinase and the experiment of Fructus Crataegi water extract synergy
| Sample | 1# nattokinase fermentation liquid 20ml+ sterilized water 5ml | 2# nattokinase fermentation liquid 20ml+ Fructus Crataegi water extract 5ml | 3# Fructus Crataegi water extract 1ml |
| Solusphere area (mm 2) | 316.45 | 386.24 | Do not dissolve |
Claims (10)
1, a kind of natto kinase oral liquid is prepared from by the fermentation liquid of the bacillus subtilis that produces nattokinase, wherein contains cyclodextrin or the cyclodextrin derivative of 0.5-3%, and the nattokinase fibrinolytic in every milliliter of oral liquid is 100-400IU.
2, the described natto kinase oral liquid of claim 1, its fermentation liquid by bacillus natto is prepared from.
3, the described natto kinase oral liquid of claim 1 wherein also contains 15-30% Fructus Crataegi water extract.
4, a kind of manufacture method of natto kinase oral liquid may further comprise the steps:
(1) utilize the bacillus subtilis that produces nattokinase to prepare the nattokinase fermentation liquid;
(2) centrifugal with the rotating speed of 3000-5000rpm, remove thalline and impurity in the fermentation liquid;
(3) supernatant after centrifugal adopts sand rod filter to take off charcoal and handles;
(4) be concentrated into predetermined concentration being lower than under 60 ℃ the temperature, add cyclodextrin or cyclodextrin derivative then, its content is the 0.5-3% of oral liquid, stirs 1-2 hour being lower than 60 ℃, and allocates; With
(5) be lower than under 60 ℃ concentrated solution enforcement microfiltration sterilization or irradiation sterilization.
5, the manufacture method of the described natto kinase oral liquid of claim 4 wherein, uses bacillus natto to carry out the fermenting process of preparing seed liquor, ferment to OD=0.3-1.0, carry out liquid submerged fermentation, fermentation temperature 25-40 ℃ with the inoculum concentration of 1.0-2.0%, fermentation period 12-35 hour, pH was 6-8.
6, the manufacture method of the described natto kinase oral liquid of claim 4, wherein, described simmer down to room temperature ultrafiltration and concentration, the ultrafilter membrane molecular cut off is 50000-200000, ultrafiltration pressure 0.1-0.3Mpa.
7, the manufacture method of the described natto kinase oral liquid of claim 4, wherein, microfiltration degerming, micro-filtration membrane aperture 0.05-0.45 μ m, sterile filling are afterwards adopted in described sterilization.
8, the manufacture method of the described natto kinase oral liquid of claim 4, wherein, irradiation sterilization is adopted in described sterilization, and irradiation dose is 1-12Kgy.
9, the manufacture method of the described natto kinase oral liquid of claim 8, wherein, the radioactive source of described irradiation sterilization is Co60, irradiation dose is 3-8Kgy.
10, the manufacture method of the described natto kinase oral liquid of claim 4, wherein, in adding the also deployed concentrated solution of cyclodextrin or derivatives thereof, add the Fructus Crataegi water extract, the preparation method of described Fructus Crataegi water extract comprises and will add the water stirring and leaching 12-24 hour after the haw berry grinding, supernatant is collected in centrifugalize, and this Fructus Crataegi water extract accounts for the 15-30% of this oral liquid weight in the oral liquid of making.
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| WO2009132575A1 (en) * | 2008-04-28 | 2009-11-05 | 赖文育 | Nattokinase powders with super high activity and producing method thereof |
| CN102578584A (en) * | 2011-01-12 | 2012-07-18 | 松尾理 | Food for preventing thrombotic diseases |
| TWI381049B (en) * | 2010-07-08 | 2013-01-01 | Meiho Inst Of Technology | Liquid medium for bacillus subtilis and method for manufacturing of the liquid medium |
| CN103602651A (en) * | 2013-11-06 | 2014-02-26 | 北京燕京啤酒股份有限公司 | Nattokinase production method |
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