CN110093334A - A method of Nattokinase is prepared using soybean and semen sojae atricolor fermentation - Google Patents
A method of Nattokinase is prepared using soybean and semen sojae atricolor fermentation Download PDFInfo
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- CN110093334A CN110093334A CN201910379884.9A CN201910379884A CN110093334A CN 110093334 A CN110093334 A CN 110093334A CN 201910379884 A CN201910379884 A CN 201910379884A CN 110093334 A CN110093334 A CN 110093334A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21062—Subtilisin (3.4.21.62)
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Abstract
The invention discloses a kind of methods for preparing Nattokinase using soybean and semen sojae atricolor fermentation, including soybean and semen sojae atricolor pretreatment, the preparation of solid fermentation culture medium, boiling, the preparation of Bacillus natto seed liquor, inoculation fermentation, after-ripening, isolate and purify: the proportion of soybean and semen sojae atricolor is soybean: semen sojae atricolor=5:1-1:1, cleaning and dipping be 20-25 DEG C immersion 8-10 hours, broken to use crusher cracked soybeans, degree of fragmentation is that each soybean is broken into 5-10 piece.The present invention has the characteristics that simple process, process are short, at low cost, is suitble to large, medium and small different scales bean product processing enterprise.
Description
Technical field
The present invention relates to Nattokinase manufacture technology field, specially a kind of utilization soybean and semen sojae atricolor fermentation prepare natto and swash
The method of enzyme.
Background technique
The separation purifying technique of Nattokinase is frequently with traditional method of purifying protein, such as: organic solvent deposit, salt
The purification process such as analysis, centrifugation, gel chromatography, hydrophobic chromatography, ion-exchange chromatography.Natto is isolated and purified from solid fermentation natto
The technique of a kind of prior art of kinases are as follows: physiological saline extracts Nattokinase, and it is full 25% that ammonium sulfate (sulfate of ammoniac ammonium) is added
Lower overnight with degree, then at 4 DEG C, 7000 revs/min are centrifuged 40 minutes, collect 5Butyl-Toyoperal drainage column on supernatant, are received
Collection Peak Activity carries out the ammonium sulfate precipitation of saturation degree 25% again, takes supernatant to carry out the ammonium sulfate precipitation of saturation degree 60%, then
Upper CM- Toyopearl ion exchange column collects Peak Activity upper Sephadex-G50 again, collects Peak Activity and dialysed overnight.It adopts
Nattokinase is manufactured with above-mentioned technique, yield is low, and the product finally obtained is seldom；It is thus it is proposed that a kind of using soybean and black
The method that beans fermentation prepares Nattokinase, with to solve the above problem.
Summary of the invention
The present invention, which provides a kind of method for preparing Nattokinase using soybean and semen sojae atricolor fermentation, can effectively solve above-mentioned back
The problems in scape technology.
To achieve the above object, the invention provides the following technical scheme: a kind of prepare natto using soybean and semen sojae atricolor fermentation
The method of kinases, including soybean and semen sojae atricolor pretreatment, the preparation of solid fermentation culture medium, boiling, the preparation of Bacillus natto seed liquor, inoculation
Fermentation, isolates and purifies after-ripening:
A. the soybean and semen sojae atricolor pretreatment are as follows: soybean and semen sojae atricolor are broken with certain proportion mixing, cleaning and dipping, appropriateness；
B. the solid fermentation culture medium preparation are as follows: fermentation medium quality group becomes: pretreated soybean and semen sojae atricolor
85% -90%, glucose 10%-15%, magnesium sulfate 0.5%- 5%, calcium chloride 0.3%-3%, pH value 7.1-7.5；
C. the boiling are as follows: use cooker boiling；
D. the Bacillus natto seed liquor preparation are as follows: slant medium is prepared: agar 16g/L, peptone 8g/L, powdered beef
3g/L, sodium chloride 2.5g/L, 121 DEG C sterilize 20 minutes；Seed liquid culture medium is prepared: peptone 8g/L, beef extract 6g/L, chlorination
Sodium 2.5g/L, glucose 12g/L, yeast extract 6g/L, 121 DEG C sterilize 20 minutes；
E. the inoculation fermentation are as follows: fermentation seed liquid, 40 DEG C of the 36- -30h for 24 hours that ferment are added according to 6% ratio；
F. the after-ripening are as follows: after-ripening temperature is 4 DEG C, ripening time 18-24h；
G. described to isolate and purify are as follows: including extraction, filtering, pure heavy step.
Further, the proportion of soybean and semen sojae atricolor is soybean: semen sojae atricolor=5:1- 1:1, cleaning and dipping 20- in the step A
25 DEG C immersion 8-10 hours, be crushed use crusher cracked soybeans, degree of fragmentation be each soybean be broken into 5-10 piece.
Further, 0.08-0.1MPa vapour cooking in the step C cooker, 30- 40min.45 are cooled to after boiling
DEG C or less.
Further, in the step D prepared by Bacillus natto seed liquor: in an aseptic environment, bafillus natto strain being used
1 ring of oese picking is inoculated on slant medium, and receiving after being activated is cultivated 24-30 hours in 38 DEG C of incubators of 35-
Beans bacillus, by the bafillus natto of activation picking 1-2 ring is into seed liquid culture medium in an aseptic environment, in 35-
38 DEG C, prepare fermentation seed liquid within shaken cultivation 16 hours under the conditions of 200r/min.
Further, extraction time is 20-25h in the step G, and cold ethyl alcohol is added to saturation degree 70%-85% in alcohol precipitation.
Compared with prior art, beneficial effects of the present invention: yield rate of the present invention is high, and have simple process, process it is short,
Feature at low cost is suitble to large, medium and small different scales bean product processing enterprise.
Specific embodiment
It, below will be to embodiment party of the present invention to keep the purposes, technical schemes and advantages of embodiment of the present invention clearer
Technical solution in formula is clearly and completely described, it is clear that described embodiment is a part of embodiment party of the present invention
Formula, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making wound
Every other embodiment obtained under the premise of the property made labour, shall fall within the protection scope of the present invention.
The present invention provides a kind of method for preparing Nattokinase using soybean and semen sojae atricolor fermentation, including soybean and semen sojae atricolor are located in advance
Reason, boiling, the preparation of Bacillus natto seed liquor, inoculation fermentation, after-ripening, isolates and purifies the preparation of solid fermentation culture medium:
A. the soybean and semen sojae atricolor pretreatment are as follows: soybean and semen sojae atricolor are broken with certain proportion mixing, cleaning and dipping, appropriateness；
The proportion of soybean and semen sojae atricolor be soybean: semen sojae atricolor=5:1-1:1, cleaning and dipping be 20-25 DEG C immersions 8-10 hour, be crushed use break
Broken crusher machine soybean, degree of fragmentation are that each soybean is broken into 5-10 piece；
B. the solid fermentation culture medium preparation are as follows: fermentation medium quality group becomes: pretreated soybean and semen sojae atricolor
85% -90%, glucose 10%-15%, magnesium sulfate 0.5%-5%, calcium chloride 0.3%-3%, pH value 7.1-7.5；
C. the boiling are as follows: use cooker boiling；0.08-0.1MPa vapour cooking in cooker, 30-40min.Boiling
After be cooled to 45 DEG C or less；
D. the Bacillus natto seed liquor preparation are as follows: slant medium is prepared: agar 16g/L, peptone 8g/L, powdered beef
3g/L, sodium chloride 2.5g/L, 121 DEG C sterilize 20 minutes；Seed liquid culture medium is prepared: peptone 8g/L, beef extract 6g/L, chlorination
Sodium 2.5g/L, glucose 12g/L, yeast extract 6g/L, 121 DEG C sterilize 20 minutes；Bacillus natto seed liquor preparation: in gnotobasis
Under, bafillus natto strain is inoculated on slant medium with 1 ring of oese picking, is cultivated in 35-38 DEG C of incubator
Bafillus natto after being activated for 24-30 hours, by the bafillus natto of activation in picking 1-2 ring in an aseptic environment
Into seed liquid culture medium, fermentation seed liquid is prepared within shaken cultivation 16 hours under the conditions of 35-38 DEG C, 200r/min；
E. the inoculation fermentation are as follows: fermentation seed liquid, 40 DEG C of the 36- -30h for 24 hours that ferment are added according to 6% ratio；
F. the after-ripening are as follows: after-ripening temperature is 4 DEG C, ripening time 18-24h；
G. described to isolate and purify are as follows: including extraction, filtering, pure heavy step；Extraction time is 20-25h, and alcohol precipitation is added cold
Ethyl alcohol is to saturation degree 70%-85%.
Finally, it should be noted that being not intended to restrict the invention the foregoing is merely preferred embodiment of the invention, to the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, for those skilled in the art, still can be with
It modifies the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.It is all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in guarantor of the invention
Within the scope of shield.
Claims (5)
1. a kind of method for preparing Nattokinase using soybean and semen sojae atricolor fermentation, which is characterized in that locate in advance including soybean and semen sojae atricolor
Reason, boiling, the preparation of Bacillus natto seed liquor, inoculation fermentation, after-ripening, isolates and purifies the preparation of solid fermentation culture medium:
A. the soybean and semen sojae atricolor pretreatment are as follows: soybean and semen sojae atricolor are broken with certain proportion mixing, cleaning and dipping, appropriateness；
B. the solid fermentation culture medium preparation are as follows: fermentation medium quality group becomes: pretreated soybean and semen sojae atricolor 85%-
90%, glucose 10%-15%, magnesium sulfate 0.5%-5%, calcium chloride 0.3%-3%, pH value 7.1-7.5；
C. the boiling are as follows: use cooker boiling；
D. Bacillus natto seed liquor preparation are as follows: slant medium is prepared: agar 16g/L, peptone 8g/L, powdered beef 3g/L,
Sodium chloride 2.5g/L, 121 DEG C sterilize 20 minutes；Seed liquid culture medium is prepared: peptone 8g/L, beef extract 6g/L, sodium chloride
2.5g/L, glucose 12g/L, yeast extract 6g/L, 121 DEG C sterilize 20 minutes；
E. the inoculation fermentation are as follows: fermentation seed liquid, the 36-40 DEG C of -30h for 24 hours that ferments is added according to 6% ratio；
F. the after-ripening are as follows: after-ripening temperature is 4 DEG C, ripening time 18-24h；
G. described to isolate and purify are as follows: including extraction, filtering, pure heavy step.
2. a kind of method for preparing Nattokinase using soybean and semen sojae atricolor fermentation according to claim 1, it is characterised in that:
The proportion of soybean and semen sojae atricolor is soybean: semen sojae atricolor=5:1-1:1 in the step A, and cleaning and dipping is that 20-25 DEG C of immersion 8-10 is small
When, it is crushed and uses crusher cracked soybeans, degree of fragmentation is that each soybean is broken into 5-10 piece.
3. a kind of method for preparing Nattokinase using soybean and semen sojae atricolor fermentation according to claim 1, it is characterised in that:
0.08-0.1MPa vapour cooking in the step C cooker, 30-40min.45 DEG C or less are cooled to after boiling.
4. a kind of method for preparing Nattokinase using soybean and semen sojae atricolor fermentation according to claim 1, it is characterised in that:
In the step D prepared by Bacillus natto seed liquor: in an aseptic environment, bafillus natto strain being connect with 1 ring of oese picking
In kind to slant medium, the bafillus natto after being activated is cultivated 24-30 hours in 35-38 DEG C of incubator, will be lived
The bafillus natto of change in picking 1-2 ring in an aseptic environment into seed liquid culture medium, in 35-38 DEG C, 200r/min item
Prepare fermentation seed liquid within shaken cultivation 16 hours under part.
5. a kind of method for preparing Nattokinase using soybean and semen sojae atricolor fermentation according to claim 1, it is characterised in that:
Extraction time is 20-25h in the step G, and cold ethyl alcohol is added to saturation degree 70%-85% in alcohol precipitation.
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| CN201910379884.9A CN110093334A (en) | 2019-05-08 | 2019-05-08 | A method of Nattokinase is prepared using soybean and semen sojae atricolor fermentation |
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| CN201910379884.9A CN110093334A (en) | 2019-05-08 | 2019-05-08 | A method of Nattokinase is prepared using soybean and semen sojae atricolor fermentation |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11192085A (en) * | 1997-12-30 | 1999-07-21 | Natto Kazoku:Kk | Sod enzyme agent |
| CN102618522A (en) * | 2011-01-28 | 2012-08-01 | 湖北国力生物技术开发有限公司 | Method for industrially producing nattokinase by using chickpea |
| CN103039966A (en) * | 2011-10-14 | 2013-04-17 | 沈阳科健生物技术有限公司 | Method for separating thrombolytic substances from solid state fermented natto |
| CN103564351A (en) * | 2012-07-31 | 2014-02-12 | 黑龙江省轻工科学研究院 | Method for preparing natto from mixed beans |
| CN106085991A (en) * | 2016-07-15 | 2016-11-09 | 青岛大学 | A kind of solid fermentation prepares the method for nattokinase |
| CN108142832A (en) * | 2017-11-08 | 2018-06-12 | 珠海市微豆生物技术有限公司 | A kind of preparation method for improving black Nattokinase content and activity |
-
2019
- 2019-05-08 CN CN201910379884.9A patent/CN110093334A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11192085A (en) * | 1997-12-30 | 1999-07-21 | Natto Kazoku:Kk | Sod enzyme agent |
| CN102618522A (en) * | 2011-01-28 | 2012-08-01 | 湖北国力生物技术开发有限公司 | Method for industrially producing nattokinase by using chickpea |
| CN103039966A (en) * | 2011-10-14 | 2013-04-17 | 沈阳科健生物技术有限公司 | Method for separating thrombolytic substances from solid state fermented natto |
| CN103564351A (en) * | 2012-07-31 | 2014-02-12 | 黑龙江省轻工科学研究院 | Method for preparing natto from mixed beans |
| CN106085991A (en) * | 2016-07-15 | 2016-11-09 | 青岛大学 | A kind of solid fermentation prepares the method for nattokinase |
| CN108142832A (en) * | 2017-11-08 | 2018-06-12 | 珠海市微豆生物技术有限公司 | A kind of preparation method for improving black Nattokinase content and activity |
Non-Patent Citations (1)
| Title |
|---|
| 田青等: "调味纳豆食品开发及工艺计算探讨", 《河南工业大学学报(自然科学版)》 * |
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Effective date of registration: 20210207 Address after: 215000 No.202, tianshangjiang Road, Wuzhong Economic Development Zone, Suzhou City, Jiangsu Province Applicant after: SUZHOU JINJI FOODS Co.,Ltd. Address before: 215000 No.3, Shuangqi Road, Wuzhong Economic Development Zone, Suzhou, Jiangsu Province Applicant before: Jiangsu Boshengkang Biological Technology Co.,Ltd. |