CN102618522A - Method for industrially producing nattokinase by using chickpea - Google Patents
Method for industrially producing nattokinase by using chickpea Download PDFInfo
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- CN102618522A CN102618522A CN2011100346542A CN201110034654A CN102618522A CN 102618522 A CN102618522 A CN 102618522A CN 2011100346542 A CN2011100346542 A CN 2011100346542A CN 201110034654 A CN201110034654 A CN 201110034654A CN 102618522 A CN102618522 A CN 102618522A
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- garbanzo
- chickpea
- nattokinase
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Abstract
The invention relates to a method for industrially producing nattokinase by using chickpea, which belongs to the biological field. The invention discloses the method for industrially producing nattokinase by using chickpea. According to the invention, high-yield strain fermented by a nattokinase solid is inoculated in the chickpea and a nutrient solution for fermenting and producing nattokinase, the method comprises the steps of crushing, immersing, disinfecting, inoculating, fermenting, ripening, extracting, separating, freeze-drying and the like. The method for producing nattokinase by fermenting chickpea, the enzyme activity of a crude enzyme liquid can reach 3270IU/g, the enzyme activity after extraction and separation is 35000IU/g, compared with the nattokinase produced by a traditional fermentation method which takes soybean as a raw material, the nattokinase produced by using chickpea has the advantages of high enzyme activity, short fermentation period, low ammoniacal smell and good mouthfeel. According to the invention, methods of cross flow nanofiltration and alcohol precipitation are used for extracting and separating nattokinase, the production technology is simple and is benefit for industrial production, simultaneously, advantages of more abundant nutrition and natural antioxidation of the chickpea are kept.
Description
Technical field
The present invention relates to adopt garbanzo is the technology of raw material production Subtilisin NAT solid fermentation, extraction separation and the prescription that nutritive medium is soaked in fermentation, belongs to biological technical field.
Background technology
(Cicer arietinum, Chickpea) the slave spy by name of the Uygur nationality, Nuo Hu carry garbanzo, another name chicken beans, brain bean or pea and peach beans etc.Belonging to vetch family, is the rare variety in the leguminous plants, and because of its unique outlook, similar olecranon and be called as garbanzo is a Uygur medicine Chinese medicinal materials commonly used.Garbanzo originates from western part of Asia and area, the Near East, in world legume crop, occupies the 3rd, and planting in more than 40 countries is one of edible legume crops that cultivated area is bigger in the world.
Garbanzo is rich in 18 seed amino acids (wherein the content of 8 seed amino acids of needed by human is very similar with the human body requirements ratio) and the nutritive substances such as protein, food fibre, trace element and VITAMINs of needed by human body, is described as " flower of nutrition, the king of bean or pea ".The protein contnt of garbanzo is close with soy proteinaceous content; Lipid content is lower than soybean; It is 13.3 times of soybean that robust fibre contains; Total acidity is lower than soybean, not peracid or alkali excessively, and its ability of regulating the acid of human body is superior to other beans, can alleviate the burden of internal organs to a certain extent; The albumen of garbanzo and polypeptide have the characteristics of natural anti-oxidation.Garbanzo all has good herbal cuisine and dietary function to mellitus, hyperlipidemia, hypertension, cardiovascular and cerebrovascular diseases, tuberculosis, maldigestion, dermatosis etc.; Can be used for daily bread and nutrition-fortifying agent; To enhancing human immune, delay senility, beautifying face and moistering lotion and children ' s intelligence development be all of great advantage, so garbanzo is described as " Long life bean " again.
Subtilisin NAT (Nattokinase; NK) be a kind of Tryase that in the natto fermenting process, produces by natto Bacillus subtilus (Bacillus subtilis natto); Significantly the inside and outside thrombus of dissolve body has very strong fibrinolytic, not only can directly act on fibrinolytic protein; And can also activate Profibrinolysin in the body, thereby increase the amount and the effect of endogenous plasmin.It has, and security is good, and effect is rapid, and cost is low, can be used for Development of New Generation thrombolytics or protective foods by advantages such as fermentation using bacteria productions, is a kind of very potential new oral thrombolytic drug, has very wide prospect.
Because Subtilisin NAT has the advantage of using in the market that thrombolytic drug did not have, the enzyme activity and the production efficiency that improve the units of product of Subtilisin NAT so have great importance to the popularization of Subtilisin NAT.On the mode of fermentation, the present mode of production of Subtilisin NAT mainly is to be main with fermentation.Do not have at present also temporarily the patent of utilizing garbanzo fermentative prodn Subtilisin NAT, have only one piece and be entitled as the article of " pre-test of garbanzo fermentative prodn natto ", the Subtilisin NAT enzyme of the garbanzo fermentation crude enzyme liquid of its report is lived and is 915IU/g; Other about the patent of utilizing soy bean solid fermentative prodn Subtilisin NAT in, the patent No. is 20081023117.2 patent, the enzyme of its lyophilized powder is lived to about 5500IU/g; The patent No. is 200710164632.1 patent, and the enzyme of its lyophilized powder is lived and is 5000IU/g; The enzyme of Subtilisin NAT crude enzyme liquid is lived and is 2250IU/g in the article " solid fermentation of Subtilisin NAT, separation and purification and applied research ".The activity of the Subtilisin NAT of reporting in these patents or the article is all not high, and how to produce natto and Subtilisin NAT with soybean as the traditional zymotic mode of fermentation raw material.The characteristics of this mode be operate easier, technology is simple.But live not high and shortcoming such as natto fermentation ammonia stink for the raw material production Subtilisin NAT exists enzyme with the soybean.It is that raw material replaces soybean to ferment that the present invention adopts garbanzo, the Subtilisin NAT enzyme of not only producing high, fermentation period weak point alive, and reduced the ammonia stink that fermentation produces, and having improved mouthfeel, more should be accepted by people.In addition, the bean dregs in the production technique of the present invention are capable of using, and its nutritive ingredient is superior to Semen sojae atricolor small peptide in the little peptide of production garbanzo, and also has the characteristics of natural anti-oxidation, effect such as have the body immunity of raising, delay senility.Made full use of " waste " when producing Subtilisin NAT, improved utilization ratio of raw materials, for this technology has increased the certain economic added value.The present invention simultaneously adopts the method extraction separation Subtilisin NAT of cross-flow nanofiltration and alcohol precipitation, has avoided the height in the salt analysis method to ooze effect, has reduced the loss of zymoprotein, the suitability for industrialized production of Subtilisin NAT preferably.
Summary of the invention
The object of the present invention is to provide a kind of method that adopts garbanzo suitability for industrialized production Subtilisin NAT; Select for use garbanzo to replace traditional soybean fermentative prodn Subtilisin NAT, provide a kind of produce enzyme live high, fermentation period is short, the ammonia flavor is low, comprehensive nutrition, production technique that mouthfeel is good.Adopt the solid fermentation superior strain H-13 of Subtilisin NAT, form through fermentation, after-ripening, extraction separation, freeze-drying in access garbanzo and the nutritive medium.
Technical scheme of the present invention is:
1, solid fermentation
(1) seed culture fluid and culture condition: with 1% glucose, 2% soy peptone, 0.3%KH
2PO
4, 0.1%K
2HPO
4, 0.04%MgSO
4, 0.02%CaCl
2Be seed culture medium, culture temperature is 35-38 ℃, and initial pH is 7.2-7.4, and liquid amount is the 20-25mL/100mL triangular flask, and inoculum size is 2-3%, and shaking speed is 120-150r/min, and fermentation period is 18-24h.
(2) pulverize: the garbanzo of selected mature and plump cleans up the back and becomes 5-10 purpose particle with mechanical disintegration.
(3) soak: preparation nutrition soak solution, its composition is the glucose of 0.1-0.2g/mL and the lactose of 0.1-0.2g/mL.The ratio of garbanzo and nutrition soak solution is 1: 1.0-1.5, and use the NaOH adjust pH to be 7.1-7.5, soak 12-24h down at 36-45 ℃.Feed 100-110 ℃ of high-temperature steam sterilization 20min then.
(4) solid fermentation: treat that temperature drops to about 40 ℃, insert bacterial classification, inoculum size is 8-16%; In the solid fermentation pond, olecranon bean powder thickness is layered on 10-20cm; Initial moisture content is 50%-60%, and initial pH is 7.1-7.5, and culture temperature is 36-42 ℃; Relative humidity is 60%-75%, and fermentation time is 18-24h.After the fermentation, 4 ℃ of after-ripening 12-24h.
2, extraction separation
(1) lixiviate: the product after the fermentation is in 1: mixing in the saline water of the ratio adding 0.9% of 5-10, lixiviate 1-24h.Again through Plate Filtration, what obtain is the solid fermentation crude enzyme liquid.Adopt 4 described methods to detect enzyme and live, and record.
(2) cross-flow nanofiltration: with the cross-flow nano filtering process it is concentrated, under force, take away the retentate on the face, and collect retentate.
(3) alcohol precipitation: with above-mentioned retentate, be transferred to setting tank, add cold ethanol, preserve down for 4 ℃ and spend the night abandoning supernatant, collecting precipitation to the 75%-80% saturation ratio.
3, lyophilize: collect above-mentioned being deposited in-40 ℃ vacuum lyophilization 24h, promptly get the Subtilisin NAT lyophilized powder.Adopt 4 described methods to detect enzyme and live, and record.
4, the mensuration of natto kinase activity: also improve the preparation fibrin plate with reference to the Astrup method.On the flat board of new preparation, place 37 ℃ of incubators to cultivate 18h urokinase (UK) standard substance of different concns (100,80,60,40,20,10IU/mL) point sample.Measure the perpendicular diameter of solusphere, calculate the solusphere area, the production standard curve.The crude enzyme liquid 10 μ L points of taking the preparation of equivalent natto are dull and stereotyped, put 37 ℃ of insulation 18h.Take out the back and measure the solusphere area, in order to the expression fibrinolytic.Area according to the perpendicular diameter of institute's test sample article is calculated utilizes typical curve to try to achieve corresponding enzymic activity.
Record the work of crude enzyme liquid enzyme and be stabilized in about 3270IU/g, the enzyme work that records behind the extraction separation is stabilized in about 35000IU/g.The enzyme that enzyme work is reported than existing patent is lived high, and the ammonia stink is low, has improved mouthfeel, more should be accepted by people.In addition, the bean dregs in this production technique are capable of using, and its nutritive ingredient not only is superior to Semen sojae atricolor small peptide in the little peptide of production garbanzo, and also has the characteristics of natural anti-oxidation, effect such as have the body immunity of raising, delay senility.Made full use of " waste " when producing Subtilisin NAT, improved utilization ratio of raw materials, for this technology has increased the certain economic added value.Technology of the present invention is simple simultaneously, has reduced the loss of zymoprotein, the suitability for industrialized production of Subtilisin NAT preferably.
Embodiment:
Case study on implementation 1:
1, solid fermentation
Get the glucose of 1kg and the lactose of 1kg, join the zero(ppm) water of 1000L, be mixed with the nutrition soak solution.The garbanzo machinery of 500kg is worn into 5 purpose particles, add the nutrition soak solution at present in 1: 1 ratio, adjust pH to 7.2,37 ℃ are soaked 24h down, feed 110 ℃ of high-temperature steams sterilization 20min then.Temperature is reduced to about 40 ℃, inserts 8% bacillus natto, and olecranon bean powder thickness is at 10cm in the solid fermentation pond, and initial moisture content is controlled at 50%, and initial pH is 7.2, and culture temperature is 37 ℃, and relative humidity is 75%, and fermentation time is 24h.After the fermentation, 4 ℃ of after-ripening 12h.
2, extraction separation
Product after the fermentation adds mixing in 0.9% the saline water in 1: 10 ratio, lixiviate 12h, and through Plate Filtration, what obtain is the solid fermentation crude enzyme liquid.Concentrate through the cross-flow nanofiltration, under force, take away the retentate on the face, collect retentate.It is transferred to setting tank, adds ethanol to 75% saturation ratio, preserve down for 4 ℃ and spend the night abandoning supernatant, collecting precipitation.
3, lyophilize: above-mentioned substance in-40 ℃ of lyophilize 24h, is promptly got the Subtilisin NAT lyophilized powder.
Case study on implementation 2:
1, solid fermentation
Get the glucose of 1kg and the lactose of 1kg, join the zero(ppm) water of 1000L, be mixed with the nutrition soak solution.The garbanzo machinery of 500kg is worn into 8 purpose particles, add the nutrition soak solution at present in 1: 1.2 ratio, adjust pH to 7.2,40 ℃ are soaked 18h down, feed 110 ℃ of high-temperature steams sterilization 20min then.Temperature is reduced to about 40 ℃, inserts 10% bacillus natto, and olecranon bean powder thickness is at 15cm in the solid fermentation pond, and initial moisture content is controlled at 55%, and initial pH is 7.2, and culture temperature is 37 ℃, and relative humidity is 75%, and fermentation time is 24h.After the fermentation, 4 ℃ of after-ripening 12h.
2, extraction separation
Product after the fermentation adds mixing in 0.9% the saline water in 1: 10 ratio, lixiviate 12h, and through Plate Filtration, what obtain is the solid fermentation crude enzyme liquid.Concentrate through the cross-flow nanofiltration, under force, take away the retentate on the face, collect retentate.It is transferred to setting tank, adds ethanol to 75% saturation ratio, preserve down for 4 ℃ and spend the night abandoning supernatant, collecting precipitation.
3, lyophilize: above-mentioned substance in-40 ℃ of lyophilize 24h, is promptly got the Subtilisin NAT lyophilized powder.
Case study on implementation 3:
1, solid fermentation
Get the glucose of 1kg and the lactose of 1kg, join the zero(ppm) water of 1000L, be mixed with the nutrition soak solution.Existing the garbanzo machinery of 500kg is worn into 10 purpose particles, add the nutrition soak solution, transfer pH to soak 12h down up to 7.2,45 ℃, feed 110 ℃ of high-temperature steams 20min that sterilize then by 1: 1.5 ratio.Temperature is reduced to about 40 ℃, inserts 12% bacillus natto, and olecranon bean powder thickness is at 20cm in the solid fermentation pond, and initial moisture content is controlled at 60%, and initial pH is 7.2, and culture temperature is 37 ℃, and relative humidity is 75%, and fermentation time is 24h.After the fermentation, 4 ℃ of after-ripening 12h.
2, extraction separation
Product after the fermentation adds mixing in 0.9% the saline water in 1: 10 ratio, lixiviate 12h, and through Plate Filtration, what obtain is the solid fermentation crude enzyme liquid.Concentrate through the cross-flow nanofiltration, under force, take away the retentate on the face, collect retentate.It is transferred to setting tank, adds ethanol to 75% saturation ratio, preserve down for 4 ℃ and spend the night abandoning supernatant, collecting precipitation.
3, lyophilize: above-mentioned substance in-40 ℃ of lyophilize 24h, is promptly got the Subtilisin NAT lyophilized powder.
The foregoing description is the embodiment that fits of the present invention, is not to be used for limiting practical range of the present invention, so all equivalences of being done with the described structure of claim of the present invention, characteristic and principle thereof change or modify, all should be included within the claim of the present invention.
Claims (9)
1. method that adopts garbanzo suitability for industrialized production Subtilisin NAT, it is characterized in that adopting garbanzo be raw material replace soybean and in addition the nutrition soak solution produce Subtilisin NAT.Its process comprises: pulverizing, immersion, sterilization, inoculation, fermentation, after-ripening, extraction separation, freeze-drying.
2. garbanzo according to claim 1, (Cicer arietinum Chickpea) originates in Xinjiang to its characteristic, comprises two kind: Ka Buli and Di Xi in garbanzo.
3. according to the said pulverizing of claim 1, it is characterized in that the selected garbanzo of removing mature and plump, clean up the back and become 5-10 purpose particle with mechanical disintegration.
4. nutrition soak solution according to claim 1, it is characterized in that the composition of nutrition soak solution is: 0.1-0.2g/mL glucose and 0.1-0.2g/mL lactose, ratio are garbanzo: nutrition soak solution=1: 1.0-1.5, the pH value is 7.1-7.5.Soaking temperature is 36-45 ℃, and soak time is 12-24h.
5. sterilization according to claim 1 is characterized in that: feed high-temperature steam and sterilize, controlled temperature is at 100-110 ℃, and sterilization time is 20-30min.
6. solid fermentation according to claim 1; It is characterized in that: the olecranon bean powder thickness in the solid fermentation pond inserts 8-16% Subtilisin NAT superior strain, initial moisture content 50%-60% at 10-20cm; Initial pH is 7.1-7.5; Culture temperature is 36-42 ℃, and relative humidity is 60%-75%, and fermentation time is 18-24h.
7. after-ripening according to claim 1 is characterized in that: the temperature of after-ripening is 4 ℃, and the time is 12-24h.
8. extraction separation according to claim 1 is characterized in that: comprise steps such as lixiviate, Plate Filtration, cross-flow nanofiltration, alcohol precipitation.Wherein extraction time is 1-24h, adds cold ethanol to saturation ratio in the alcohol precipitation step to 75%-80%.
9. according to the described superior strain of claim 6, it is characterized in that bacterial strain uses therefor is natto solid fermentation superior strain H-13, this bacterial strain belongs to bacillus subtilis Pseudomonas subso natto (Bacillus subtilis natto).
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205409A (en) * | 2013-04-28 | 2013-07-17 | 武汉真福医药科技发展有限公司 | Method for preparing high-activity nattokinase by taking black beans as raw material |
| CN103849610A (en) * | 2012-12-04 | 2014-06-11 | 天津市宝恒生物科技有限公司 | Method for preparing nattokinase |
| CN104152429A (en) * | 2013-05-15 | 2014-11-19 | 亚宝药业集团股份有限公司 | Screening and liquid fermentation method of high-yield bacillus subtilis strain of nattokinase |
| CN105309872A (en) * | 2015-07-03 | 2016-02-10 | 王军 | Preparing method for bacillus natto powder |
| CN106387676A (en) * | 2016-09-19 | 2017-02-15 | 江苏师范大学 | Preparation method of active natto powder |
| CN106957833A (en) * | 2017-05-10 | 2017-07-18 | 林峰 | A kind of high enzymatic activity chick-pea kinases liquid and preparation method thereof |
| CN107099487A (en) * | 2017-06-27 | 2017-08-29 | 南京工业大学 | A kind of bacillus subtilis of hypersecretion Nattokinase and its application |
| CN110093334A (en) * | 2019-05-08 | 2019-08-06 | 江苏博晟康生物科技有限公司 | A method of Nattokinase is prepared using soybean and semen sojae atricolor fermentation |
-
2011
- 2011-01-28 CN CN2011100346542A patent/CN102618522A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103849610A (en) * | 2012-12-04 | 2014-06-11 | 天津市宝恒生物科技有限公司 | Method for preparing nattokinase |
| CN103205409A (en) * | 2013-04-28 | 2013-07-17 | 武汉真福医药科技发展有限公司 | Method for preparing high-activity nattokinase by taking black beans as raw material |
| CN103205409B (en) * | 2013-04-28 | 2014-12-03 | 武汉真福医药科技发展有限公司 | Method for preparing high-activity nattokinase by taking black beans as raw material |
| CN104152429A (en) * | 2013-05-15 | 2014-11-19 | 亚宝药业集团股份有限公司 | Screening and liquid fermentation method of high-yield bacillus subtilis strain of nattokinase |
| CN105309872A (en) * | 2015-07-03 | 2016-02-10 | 王军 | Preparing method for bacillus natto powder |
| CN106387676A (en) * | 2016-09-19 | 2017-02-15 | 江苏师范大学 | Preparation method of active natto powder |
| CN106957833A (en) * | 2017-05-10 | 2017-07-18 | 林峰 | A kind of high enzymatic activity chick-pea kinases liquid and preparation method thereof |
| CN107099487A (en) * | 2017-06-27 | 2017-08-29 | 南京工业大学 | A kind of bacillus subtilis of hypersecretion Nattokinase and its application |
| CN107099487B (en) * | 2017-06-27 | 2020-07-24 | 南京工业大学 | Bacillus subtilis with high nattokinase secretion and application thereof |
| CN110093334A (en) * | 2019-05-08 | 2019-08-06 | 江苏博晟康生物科技有限公司 | A method of Nattokinase is prepared using soybean and semen sojae atricolor fermentation |
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Application publication date: 20120801 |