CN101705213A - Preparation method of beta-glucosaccharase - Google Patents
Preparation method of beta-glucosaccharase Download PDFInfo
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- CN101705213A CN101705213A CN200910153807A CN200910153807A CN101705213A CN 101705213 A CN101705213 A CN 101705213A CN 200910153807 A CN200910153807 A CN 200910153807A CN 200910153807 A CN200910153807 A CN 200910153807A CN 101705213 A CN101705213 A CN 101705213A
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Abstract
The invention discloses a preparation method of beta-glucosaccharase. The method includes the following steps: selecting Aspergillus niger as parent strain through screening, inoculating the Aspergillus niger, the inoculum size of which is 106-108cfu/g, to bran, rice bran and ammonia chloride, wherein in a solid culture medium prepared from the bran, the rice bran and the ammonia chloride according to the weight percentage of 90-100 percent: 0-10 percent:1-3 percent, the initial water content of the culture medium is 65-75 percent, and the pH is 5.5-6.5, culturing for 48-80h at the temperature of 25-30 DEG C and detecting the activity of beta-glucosaccharase to reach 300-430U/g. The method adopts solid fermentation, uses cheap and rich subsidiary agricultural products as fermentation substrate, simultaneously optimizes the culture medium and culture conditions and achieves high yield of the beta-glucosaccharase under solid fermentation conditions, thus providing scientific basis to the industrialization development of the beta-glucosaccharase.
Description
Technical field
The present invention relates to microbial fermentation and technical field of enzyme engineering, relate in particular to a kind of preparation method of beta-glucosidase.
Background technology
Soybean is the major protein feed of livestock and poultry, occupies significant proportion in forage component, contains abundant soybean isoflavones in the soybean.People at human body, discover that at the human body soybean isoflavones anti-curing cancers, preventing cardiovascular disease, control female old aged people osteoporosis etc. are all had tangible curative effect to the research of soybean isoflavones at first.The research of soybean isoflavones in recent years begins to carry out on animal, a large amount of animal experiments show, soybean isoflavones has the growth of animal of promotion, enhancing body immunizing power, improve the animal milk performance, increase egg productivity, improve reproductive capability, promote nutrient reallocation in the animal body, improve protein synthesis efficient and improve livestock body and multiple nutrients physiological function such as lean ratio.But soybean isoflavones mainly exists with the glucosides form, accounts for the 97%-98% of total amount, and the mating type soybean isoflavones can not infiltrate intestinal epithelial cells.Must be aglycon through microbial hydrolytic, just can be absorbed and enter peripheral circulation, so most of soybean isoflavones resource can not well be utilized by animal body, has caused the waste of resource.
Discover that beta-glucosidase can be converted into the soybean isoflavones of mating type the free type aglycon with physiologically active, thereby effectively improve the bioavailability of soybean isoflavones.This enzyme is to be found in Semen Armeniacae Amarum at first, also has been purified into beta-glucosidase subsequently in microorganisms such as yeast, bacterium, fungi.But the beta-glucosidase of different sources has tangible species specificity, the different culture condition of different bacterial strains can influence the fermentation level of beta-glucosidase, tank fermentation method is adopted in the present production to beta-glucosidase simultaneously more, its application is restricted solid state fermentation always because the product beta-glucosidase yields poorly, but solid state fermentation has economic advantages than submerged fermentation, it can utilize abundant, cheap agricultural byproducts as fermentation substrate, and the fermentation crude product can be directly as the enzyme source.The present invention is dealt into the optimization of solid medium and culture condition from filtering out of bacterial classification, so that also can realize the high yield of beta-glucosidase under the solid culture condition, for the industrialized development of beta-glucosidase provides theoretical foundation.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of preparation method of beta-glucosidase is provided, but this method cost is low, the high large-scale promotion utilization of yield of enzyme.
The objective of the invention is to be achieved through the following technical solutions: a kind of preparation method of beta-glucosidase may further comprise the steps:
(1) preparation substratum: comprising:
(A) preparation inclined-plane seed culture medium: skin potato, sucrose and agar are added in the entry, through 121 ℃ of sterilization 20min, obtain the inclined-plane seed culture medium, wherein, skin potato content is 200g/l, and sucrose content is 20g/l, and agar content is 20g/l;
(B) preparation solid seed culture medium: according to 1: 1 ratio of weight wheat bran is added in the entry, stir,, obtain the solid seed culture medium through 121 ℃ of sterilization 30min;
(C) preparation solids manufacture substratum: by weight percentage 90～100%: 0～10%: 1～3% ratio is mixed wheat bran, rice bran and ammonia chloride, through 121 ℃ of sterilization 30min, obtains the solids manufacture substratum;
(2) preparation inclined-plane seed: the Aspergillus niger strain streak inoculation on the inclined-plane seed culture medium, behind 28 ℃ of constant temperature culture 5d, is got the inclined-plane seed;
(3) preparation seed: the inclined-plane seed kind that step (2) is obtained in 28 ℃ of following constant temperature culture 72h, obtains seed in the solid seed culture medium;
(4) fermentative production: the seed that step (3) is obtained is with 10
6～10
8The inoculum size of cfu/g solid medium is inoculated on the solids manufacture substratum, and adding mass percent in solid medium is its MgSO of 0.5%
47H
2O and mass percent are its KH of 1.5%
2PO
4, the initial moisture content 65～75% of adjusting substratum, PH5.5～6.5 in 25～30 ℃ of cultivation 48～80h, get beta-glucosidase.
The invention has the beneficial effects as follows: the present invention filters out the high-yield strains of beta-glucosidase, utilize abundant, cheap agricultural byproducts as fermentation substrate, product cost is low, quality is good, the enzymic activity that obtains after the optimization is very high, be applied to improve its absorption rate significantly in the dregs of beans type daily ration as fodder additives, thereby improve the economic benefit of animal rearing.
Embodiment
The preparation method of beta-glucosidase of the present invention may further comprise the steps:
One, preparation substratum: comprise,
1, preparation inclined-plane seed culture medium: skin potato, sucrose and agar are added in the entry, through 121 ℃ of sterilization 20min, obtain the inclined-plane seed culture medium, wherein, skin potato content is 200g/l, and sucrose content is 20g/l, and agar content is 20g/l;
2, preparation solid seed culture medium: according to 1: 1 ratio of weight wheat bran is added in the entry, stir,, obtain the solid seed culture medium through 121 ℃ of sterilization 30min;
3, preparation solids manufacture substratum: by weight percentage 90～100%: 0～10%: 1～3% ratio is mixed wheat bran, rice bran and ammonia chloride, through 121 ℃ of sterilization 30min, obtains the solids manufacture substratum.
Two, preparation inclined-plane seed: the Aspergillus niger strain streak inoculation on the inclined-plane of step 1 seed culture medium, behind 28 ℃ of constant temperature culture 5d, is got the inclined-plane seed, place in 4 ℃ of refrigerators and preserve, standby;
Three, preparation seed: the inclined-plane seed kind of step 2 preparation in the solid seed culture medium of step 1, in 28 ℃ of following constant temperature culture 72h, is obtained seed;
Four, fermentative production: with seed that step 3 obtained with 10
6～10
8The inoculum size of cfu/g solid medium is inoculated on the solids manufacture substratum of step 1, and adding mass percent in solid medium is its MgSO of 0.5%
47H
2O and mass percent are its KH of 1.5%
2PO
4, the initial moisture content 65～75% of adjusting substratum, PH5.5～6.5 in 25～30 ℃ of cultivation 48～80h, get beta-glucosidase, and the detection vigor reaches 300～430U/g.
Wherein, unit of enzyme U definition: catalytic production 1 μ mol/L is 1 unit of enzyme to the enzyme amount of nitro one's duty in 1min.
The invention will be further described below in conjunction with specific embodiment, and it is more obvious that purpose of the present invention and effect will become.
Embodiment 1
(1) preparation of substratum: comprise,
1. inclined-plane seed culture medium: peeling potato 200g/l, sucrose 20g/l, agar 20g/l is through 121 ℃ of sterilization 20min;
2. add wheat bran 10g in the triangular flask of solid seed culture medium: 250ml, in adding tap water, stir, through 121 ℃ of sterilization 30min with 1: 1 ratio of wheat bran weight;
3. solids manufacture substratum: the formulated solid medium 10g of 90%: 9%: 1% by weight percentage the ratio of wheat bran, rice bran and ammonia chloride of packing in the 250ml Erlenmeyer flask, through 121 ℃ of sterilization 30min;
(2) inclined-plane seed preparation: the Aspergillus niger strain streak inoculation 1. on the inclined-plane seed culture medium, behind 28 ℃ of constant temperature culture 5d, is placed preservation in 4 ℃ of refrigerators in step (1), standby;
(3) seed preparation: with step (2) inclined-plane seed in step (1) 2. in the solid seed culture medium, in 28 ℃ of following constant temperature culture 72h;
(4) fermentative production: the seed that step (3) is obtained is with 10
6The inoculum size of cfu/g solid medium is inoculated into step (1) 3. on the solids manufacture substratum, adds 0.5%MgSO in solid medium
47H
2O and 1.5%KH
2PO
4, the initial moisture content 65% of adjusting substratum, PH5.5 in 25 ℃ of cultivation 56h, gets beta-glucosidase, and the detection vigor reaches 300～430U/g.
Embodiment 2
(1) preparation of substratum: comprise,
1. inclined-plane seed culture medium: peeling potato 200g/l, sucrose 20g/l, agar 20g/l is through 121 ℃ of sterilization 20min;
2. add wheat bran 10g in the triangular flask of solid seed culture medium: 250ml, in adding tap water, stir, through 121 ℃ of sterilization 30min with 1: 1 ratio of wheat bran weight;
3. solids manufacture substratum: pack in the 250ml Erlenmeyer flask wheat bran, rice bran and ammonia chloride, the solid medium 10g that 95%: 4%: 1% by weight percentage ratio is formulated is through 121 ℃ of sterilization 30min;
(2) inclined-plane seed preparation: the Aspergillus niger strain streak inoculation 1. on the inclined-plane seed culture medium, behind 28 ℃ of constant temperature culture 5d, is placed preservation in 4 ℃ of refrigerators in step (1), standby;
(3) seed preparation: with step (2) inclined-plane seed in step (1) 2. in the solid seed culture medium, in 28 ℃ of following constant temperature culture 72h;
(4) fermentative production: the seed that step (3) is obtained is with 10
7The inoculum size of cfu/g solid medium is inoculated into step (1) 3. on the solids manufacture substratum, adds 0.5%MgSO in solid medium
47H
2O and 1.5%KH
2PO
4, the initial moisture content 70% of adjusting substratum, PH56.0 in 28 ℃ of cultivation 72h, gets beta-glucosidase, and the detection vigor reaches 300～430U/g.
Embodiment 3
(1) preparation of substratum: comprise,
1. inclined-plane seed culture medium: peeling potato 200g/l, sucrose 20g/l, agar 20g/l is through 121 ℃ of sterilization 20min;
2. add wheat bran 10g in the triangular flask of solid seed culture medium: 250ml, in adding tap water, stir, through 121 ℃ of sterilization 30min with 1: 1 ratio of wheat bran weight;
3. solids manufacture substratum: pack in the 250ml Erlenmeyer flask wheat bran and ammonia chloride by, the solid medium 10g that 98%: 2% ratio of weight percent is formulated is through 121 ℃ of sterilization 30min;
(2) inclined-plane seed preparation: the Aspergillus niger strain streak inoculation 1. on the inclined-plane seed culture medium, behind 28 ℃ of constant temperature culture 5d, is placed preservation in 4 ℃ of refrigerators in step (1), standby;
(3) seed preparation: with step (2) inclined-plane seed in step (1) 2. in the solid seed culture medium, in 28 ℃ of following constant temperature culture 72h;
(4) fermentative production: the seed that step (3) is obtained is with 10
8The inoculum size of cfu/g solid medium is inoculated into step (1) 3. on the solids manufacture substratum, adds 0.5%MgSO in solid medium
47H
2O and 1.5%KH
2PO
4, the initial moisture content 75% of adjusting substratum, PH6.5 in 30 ℃ of cultivation 80h, gets beta-glucosidase, and the detection vigor reaches 300～430U/g.
The foregoing description is used for the present invention that explains, rather than limits the invention, and in the protection domain of spirit of the present invention and claim, any modification and change to the present invention makes all fall into protection scope of the present invention.
Claims (1)
1. the preparation method of a beta-glucosidase is characterized in that, may further comprise the steps:
(1) preparation substratum: comprising:
(A) preparation inclined-plane seed culture medium: skin potato, sucrose and agar are added in the entry, through 121 ℃ of sterilization 20min, obtain the inclined-plane seed culture medium, wherein, skin potato content is 200g/l, and sucrose content is 20g/l, and agar content is 20g/l.
(B) preparation solid seed culture medium: according to 1: 1 ratio of weight wheat bran is added in the entry, stir,, obtain the solid seed culture medium through 121 ℃ of sterilization 30min.
(C) preparation solids manufacture substratum: by weight percentage 90～100%: 0～10%: 1～3% ratio is mixed wheat bran, rice bran and ammonia chloride, through 121 ℃ of sterilization 30min, obtains the solids manufacture substratum.
(2) preparation inclined-plane seed: the Aspergillus niger strain streak inoculation on the inclined-plane seed culture medium, behind 28 ℃ of constant temperature culture 5d, is got the inclined-plane seed.
(3) preparation seed: the inclined-plane seed kind that step (2) is obtained in 28 ℃ of following constant temperature culture 72h, obtains seed in the solid seed culture medium.
(4) fermentative production: the seed that step (3) is obtained is with 10
6～10
8The inoculum size of cfu/g solid medium is inoculated on the solids manufacture substratum, and adding mass percent in solid medium is its MgSO of 0.5%
47H
2O and mass percent are its KH of 1.5%
2PO
4, the initial moisture content 65～75% of adjusting substratum, PH5.5～6.5 in 25～30 ℃ of cultivation 48～80h, get beta-glucosidase.
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| CN200910153807A CN101705213A (en) | 2009-11-16 | 2009-11-16 | Preparation method of beta-glucosaccharase |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559511A (en) * | 2011-12-12 | 2012-07-11 | 中国科学院广州能源研究所 | Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea |
| CN105462846A (en) * | 2015-12-07 | 2016-04-06 | 天津科技大学 | Method for massively preparing aspergillus niger spores through solid-state fermentation method |
| CN113846077A (en) * | 2021-11-12 | 2021-12-28 | 河南省科学院生物研究所有限责任公司 | Method for preparing feed beta-mannase by solid state fermentation |
-
2009
- 2009-11-16 CN CN200910153807A patent/CN101705213A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559511A (en) * | 2011-12-12 | 2012-07-11 | 中国科学院广州能源研究所 | Hypocrea for producing mesophile ethanol-tolerant beta-glucosidase highly and application of hypocrea |
| CN105462846A (en) * | 2015-12-07 | 2016-04-06 | 天津科技大学 | Method for massively preparing aspergillus niger spores through solid-state fermentation method |
| CN113846077A (en) * | 2021-11-12 | 2021-12-28 | 河南省科学院生物研究所有限责任公司 | Method for preparing feed beta-mannase by solid state fermentation |
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Open date: 20100512 |