CN1417328A - Solid fermentation prepn process and application of keratinase - Google Patents
Solid fermentation prepn process and application of keratinase Download PDFInfo
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- CN1417328A CN1417328A CN01133424A CN01133424A CN1417328A CN 1417328 A CN1417328 A CN 1417328A CN 01133424 A CN01133424 A CN 01133424A CN 01133424 A CN01133424 A CN 01133424A CN 1417328 A CN1417328 A CN 1417328A
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Abstract
The present invention discloses a solid fermentation preparation process and application of keratinase. The preparation process includes the following steps: conventional inoculation; test and salt culture in solid culture medium; engrafting the strain to sporogenous solid culture medium and culture at 25-32 deg.C for 3-5 days until generating spore; inoculating the strain spore to solid culture medium while controlling the C/N ratio in 2-5 to 1-3.5, adding phsphate and maintaining humidify 30-70%, temperature 25-32 deg.c and natural pH value to produce keratinase in 40-120 hr. The product is used as feed additive. The present invention has high enzyme activity and is easy to apply in production.
Description
Technical field
The present invention relates to the Keratin sulfate biodegradation technique, specifically the solid-state fermentation preparation method of M-Zyme and application thereof.
Background technology
Protein is the important substance of the modern animal husbandry development of restriction.Keratin sulfate is abundant in china natural resources, is a kind of ideal animal proteinum, and its protein content is up to more than 95%.Traditional Keratin sulfate hydrolysis method is mainly High Temperature High Pressure boiling or acid hydrolysis, belongs to the physical chemistry treatment process, and its processing condition harshness easily damages temperature-sensitive amino acid, and the animal use rate is low, and causes environmental pollution.Yet biological degradation is a development trend, utilizes M-Zyme to decompose Keratin sulfate, the amino acid transformation efficiency increases substantially, and can solve above-mentioned drawback effectively, and effect is remarkable, and improve China based on the situation that vegetable-protein and animal proteinum lack, keep the intravital amino acid balance of animal.
The patent No. is 4,908, (C.M.Williams.1990.U.S.Patent 4 for 220 United States Patent (USP), 908,220) disclose a kind of bacterium M-Zyme hydrolysis of keratin method of using, it prepares M-Zyme by liquid fermenting, produces the feather product of amino acid or hydrolysis as fodder additives with keratin material), specifically: adopting bacterium (bacillus Bacillus licheniformis) is bacterial classification, and the molecular weight of its M-Zyme is 33kDa; 50 ℃ of optimum temperatures; Optimal pH 7.5; Liquid submerged fermentation, fermentation period 4 days belongs to biodegradation technique, and main purpose is the processing of environment waste.But it is complicated that it adopts liquid fermenting to prepare M-Zyme technology, and its M-Zyme vigor is desirable not enough.
Summary of the invention
The solid-state fermentation preparation method and the application thereof that the invention provides and a kind ofly produce that enzyme activity is higher, technology is simple, is easy to the M-Zyme of production application.
To achieve these goals, technical scheme of the present invention is to operate as follows: 1) adopt conventional microbial inoculant method inoculation: the test tube slant is cultivated, and adopts the Cha Shi solid medium; Move and connect: the bacterial strain of above-mentioned growth is moved to receive produce in the spore solid medium, 20～32 ℃, cultivated 3～5 days, produce to spore; It is characterized in that: be bacterial classification with the fungi in the step 1), step 2) described bacterial strain spore is added in the solid medium by 0.5～10% inoculum size, control carbon: the ratio of nitrogen is 2～5: in 1～3.5 scope, add inorganic phosphorus, its content is 5～8 μ mol/g butts, keeps humidity 30～70%, 25～32 ℃ of temperature, nature pH value, the M-Zyme generation time is 40～120 hours;
The inoculation dosage that described bacterial strain spore adds in the solid medium is 1～5%; Described maintenance humidity is 50～60%; Carbon in the described solid medium is cavings or wheat bran; Nitrogen in the described solid medium is wheat bran or feather meal; Described inorganic phosphorus is dipotassium hydrogen phosphate and potassium primary phosphate, and both weight ratios are 1: 1～1.5; Described fungi strain is the aspergillus terricola in the Aspergillus; Described M-Zyme generation time is preferably 60～90 hours; M-Zyme generation time the best is 72 hours.
By the application of the M-Zyme of described method preparation be with described M-Zyme culture directly as fodder additives, add in albumen or the Keratin sulfate feed, addition is 2～10% of a feed; Or, adding ammonium sulfate precipitation with described M-Zyme culture flooding, control ammonium sulfate degree of saturation is 30～60%, centrifugal drying, make crude zyme preparation as fodder additives, described crude zyme preparation is added in albumen or the Keratin sulfate feed, addition is 0.5 of feed～5 ‰.
The present invention has following advantage:
1. be easy to production application.M-Zyme of the present invention is made with the solid state fermentation mode, be easy to cultivate breeding, produce, the bacterial strain strong stress resistance that is adopted, shifted to an earlier date 30 hours than the liquid submerged fermentation time, production technique is easy, and need not to make with extra care, only through lixiviate, the step of saltouing, getting thick enzyme can use, and also can directly use, and is easy to promote.
2. have high enzymatic productivity and degradation rate.Adopt the present invention that keratic degraded is not applied high temperature, high pressure, belong to biological degradation, degradation rate is higher, and the external digestion rate reaches 89%; Have the active and temperature of the substrate hydrolysis of wide spectrum, pH tolerance.Producing enzyme activity is 4000 units/gram songs; The external digestion rate reaches 89%.Molecular weight is 39Kda; 50 ℃ of optimum temperatures; Optimal pH 8.0.
3. have broad application prospects.The M-Zyme that the present invention produces can be used as fodder additives, adjusts the structure of feedstuff protein, makes its nutrition near animal proteinum under the situation that keeps amino acid balance, has improved feed conversion rate, has increased the price of deed; In the commerciality of animal is raised, produce a large amount of feathers, hair substrate, the accumulation of these materials causes environmental pollution and becomes the hotbed that various pathogenic agent are grown, and M-Zyme and generation bacterium thereof will become the good biotechnology instrument that contains keratic refuse valueization that makes.In addition, M-Zyme can improve the permeability of external preparation for skin medicine in scleroprotein, reach the purpose of treatment tetter such as onychomycosis, the research M-Zyme, has important value to disclosing the dermatophytid infection molecular mechanism, M-Zyme can promote the nursing of makeup deep layer, helps active factor to see through skin barrier, dispels the unnecessary cutin of skin because its M-Zyme has the effect that recovers skin elasticity.
Embodiment
Embodiment 1
The preparation of M-Zyme is operated as follows: 1) be bacterial classification with the aspergillus fungi, aspergillus terricola (Aspergillus terricola) by name, purchase Beijing institute of microbiology in the Chinese Academy of Sciences, described aspergillus terricola, morphological specificity is: bacterial strain is on czapek's solution, the aerial hyphae well-grown, flocculence has projection, surface white, back side color is asked prolongation at any time by the light brown brown that becomes, and spore is a brown; Adopt conventional microbial inoculant method inoculation: the test tube slant is cultivated, and adopts the Cha Shi solid medium; Move and connect (enzyme production): the bacterial strain of above-mentioned growth is moved to receive produce in the spore solid medium, 28 ℃, cultivated 3 days, produce to spore; Described product spore substratum adopts 20% potato diffusion juice 1000ml, glucose 10g, and potassium primary phosphate 3g, sal epsom 1.5g, the VITMAIN B1 trace, agar 20g is put a filter paper bar at each slant tube; 2) described bacterial strain spore is added in the solid medium by 3% inoculum size, carbon in the described solid medium is cavings, and nitrogen is wheat bran, and phosphorus is dipotassium hydrogen phosphate, control carbon: the ratio of nitrogen is 4: 3, add inorganic phosphorus, its content is 6 μ mol/g butts, and wherein dipotassium hydrogen phosphate and potassium primary phosphate weight ratio are 1: 1, keep humidity 60%, 28 ℃ of temperature, pH is the nature value, is that M-Zyme produced the peak in 72 hours; Directly as fodder additives, addition is 5% of a feed with the M-Zyme that produces.
Decomposition principle of the present invention is: the present invention does not apply high temperature, high pressure, and Keratin sulfate is converted into amino acid, and its degradation process is a biochemical reaction process.
Result parameter: shifted to an earlier date 30 hours than the liquid submerged fermentation time in the prior art, producing enzyme activity is 4000 units/gram songs; The external digestion rate reaches 89%.Molecular weight is 39Kda; 50 ℃ of optimum temperatures; Optimal pH 8.0; Fermentation period 3 days is solid state fermentation; Adopt M-Zyme of the present invention as fodder additives, have the active and temperature of the substrate hydrolysis of wide spectrum, pH tolerance.
The feedstuff protein of China is many based on vegetable-protein, and contents of essential amino acids is lower, easily causes amino acid whose imbalance.Modal Keratin sulfate is a feather meal, approaches animal proteinum.As the by product of commercial bird processing, crude protein in its composition (CP) content sometimes can be up to 97% generally more than 80%.Gelucystine Cys content is higher in the feather meal, is generally 4～5%, and far above other plant animal proteins, beta-keratin is rich in L-glutamic acid Gly, L-Ala Ala and Serine Ser etc. especially.The M-Zyme that the present invention produces can be used as fodder additives, adjusts the structure of feedstuff protein, makes its nutrition near animal proteinum under the situation that keeps amino acid balance, has improved feed conversion rate, has increased the price of deed; Its feather substrate enzymolysis aminoacids content variation (condition: 40 ℃, water bath heat preservation 6 hours) see Table 1.
Table 1 feather substrate enzymolysis aminoacids content changes (mgL
-1) amino acid A B amino acid A B amino acid A BASP asparagus fern 87.4 85.3 CYS Guangs 18 12 PHE phenylpropyl alcohols 138 49THR 92 39.4 VAL that revive tie 195 78 LYS and rely different bright 127 54 HIS of 37 33SER silks, 165 122 MET eggs, 30 24 NH3 ammonia, 72 66GLY paddy, 185 62 ILE to organize smart 51 12ALA, the third 117 17 TYR junket of bright 175 67 ARG of sweet 105 40 LEU of 7 6GLU 45 12 PRO dried meat 26 19
Wherein: A is the enzymolysis feather sample, and B is the feather sample
In the commerciality of animal is raised, produce a large amount of feathers, hair substrate, the accumulation of these materials causes environmental pollution and becomes the hotbed that various pathogenic agent are grown.And M-Zyme and generation bacterium thereof will become the good biotechnology instrument that contains keratic refuse valueization that makes.In addition, M-Zyme can improve the permeability of external preparation for skin medicine in scleroprotein, reaches the purpose of treatment tetter such as onychomycosis.The research M-Zyme, has important value to disclosing the dermatophytid infection molecular mechanism, M-Zyme can promote the nursing of makeup deep layer, helps active factor to see through skin barrier, dispels the unnecessary cutin of skin because its M-Zyme has the effect that recovers skin elasticity.
Embodiment 2
Difference from Example 1 is:
The solid fermentation step 2 of M-Zyme) in described bacterial strain spore is added in the solid medium by 1% inoculum size, carbon is cavings in the described solid medium, nitrogen is peptone, phosphorus is potassium primary phosphate and dipotassium hydrogen phosphate (both weight ratios are 1: 1.2), and control carbon: the ratio of nitrogen is 2: 1, and the inorganic phosphorus add-on is 8 μ mol/g butts, keep humidity 30%, 25 ℃ of temperature, pH is the nature value, M-Zyme produced in 60 hours; M-Zyme culture flooding with producing adds ammonium sulfate precipitation, and control ammonium sulfate degree of saturation is 30%, and centrifugal drying is made crude zyme preparation, as fodder additives, adds in the Keratin sulfate feed again, and addition is 2 ‰ of a feed.
Present embodiment is that the example weightening finish the results are shown in Table 2 to raise growing and fattening pigs.
Table 2 adopts M-Zyme respectively to organize day weight gain, food consumption result as fodder additives
| Project | Control group | Test group |
| Raise fate | ????30 | ????30 |
| Raise an array | ????4 | ????4 |
| Average starting weight kg/ head | ????19.83 | ????19.57 |
| The heavy kg/ head in average end | ????35.13 | ????35.90 |
| Average daily gain g | ????510.00 | ????544 |
| Average feed consumption kg | ????36.20 | ????36.70 |
| Average day feed consumption kg | ????1.21 | ????1.22 |
Adopt the present invention to prepare M-Zyme as fodder additives, gaining effect is remarkable.
Embodiment 3
Difference from Example 1 is: M-Zyme solid fermentation preparation process 2) described bacterial strain spore is added in the solid medium by 5% inoculum size, carbon is wheat bran in the described solid medium, nitrogen is feather meal, control carbon: the ratio of nitrogen is 5: 4, adds inorganic phosphorus 0.5 μ mol/g butt, and wherein dipotassium hydrogen phosphate and potassium primary phosphate weight ratio are 1: 1.5, keep humidity 70%, 32 ℃ of temperature, PH is the nature value, M-Zyme produced in 90 hours; The M-Zyme that produces is soaked in water, adds ammonium sulfate precipitation, the control degree of saturation is 60%, makes crude zyme preparation, as fodder additives, adds in the Keratin sulfate feed again, and addition is 5 ‰ of a feed, and it is raised the growing and fattening pigs test effect and sees Table 3.
Table 3 adopts M-Zyme respectively to organize day weight gain, food consumption result as fodder additives
| Project | Control group | Test group |
| Raise fate | ????30 | ????30 |
| Raise an array | ????4 | ????4 |
| Average starting weight kg/ head | ????19.83 | ????19.62 |
| The heavy kg/ head in average end | ????35.13 | ????35.07 |
| Average daily gain g | ????510.00 | ????548 |
| Average feed consumption kg | ????36.20 | ????37.2 |
| Average day feed consumption kg | ????1.21 | ????1.24 |
Claims (10)
1. the solid-state fermentation preparation method of a M-Zyme, operation as follows: 1) adopt conventional microbial inoculant method inoculation: the test tube slant is cultivated, and adopts the Cha Shi solid medium; Move and connect: the bacterial strain of above-mentioned growth is moved to receive produce in the spore solid medium, 20～32 ℃, cultivated 3～5 days, produce to spore; It is characterized in that: be bacterial classification with the fungi in the step 1), step 2) described bacterial strain spore is added in the solid medium by 0.5～10% inoculum size, control carbon: the ratio of nitrogen is 2～5: in 1～3.5 scope, add inorganic phosphorus, its content is 5～8 μ mol/g butts, keeps humidity 30～70%, 25～32 ℃ of temperature, nature pH value, the M-Zyme generation time is 40～120 hours.
2. according to the solid-state fermentation preparation method of the described M-Zyme of claim 1, it is characterized in that: the inoculation dosage that described bacterial strain spore adds in the solid medium is 1～5%.
3. according to the solid-state fermentation preparation method of the described M-Zyme of claim 1, it is characterized in that: described maintenance humidity is 50～60%.
4. according to the solid-state fermentation preparation method of the described M-Zyme of claim 1, it is characterized in that: the carbon in the described solid medium is cavings or wheat bran; Nitrogen is wheat bran or feather meal.
5. according to the solid-state fermentation preparation method of the described M-Zyme of claim 1, it is characterized in that: described inorganic phosphorus is dipotassium hydrogen phosphate and potassium primary phosphate, and both weight ratios are 1: 1～1.5.
6. according to the solid-state fermentation preparation method of the described M-Zyme of claim 1, it is characterized in that: described fungi strain is the aspergillus terricola in the Aspergillus.
7. according to the solid-state fermentation preparation method of the described M-Zyme of claim 1, it is characterized in that: described M-Zyme generation time is 60～90 hours.
8. according to the solid-state fermentation preparation method of the described M-Zyme of claim 1, it is characterized in that: it is 72 hours that described M-Zyme produces Best Times.
9. application according to the M-Zyme of the described method preparation of claim 1, it is characterized in that: described M-Zyme culture directly as fodder additives, is added in albumen or the Keratin sulfate feed, and addition is 2～10% of a feed.
10. application according to the M-Zyme of the described method preparation of claim 1, it is characterized in that: with described M-Zyme culture flooding, add ammonium sulfate precipitation, control ammonium sulfate degree of saturation is 30～60%, centrifugal drying, make crude zyme preparation as fodder additives, described crude zyme preparation is added in albumen or the Keratin sulfate feed, addition is 0.5 of feed～5 ‰.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011182674A (en) * | 2010-03-05 | 2011-09-22 | Kunihiko Watabe | Keratinase and method for producing the same |
| CN107022537A (en) * | 2017-03-03 | 2017-08-08 | 陈燕芳 | A kind of keratinase compound and preparation method and application |
-
2001
- 2001-11-09 CN CN01133424A patent/CN1417328A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011182674A (en) * | 2010-03-05 | 2011-09-22 | Kunihiko Watabe | Keratinase and method for producing the same |
| CN107022537A (en) * | 2017-03-03 | 2017-08-08 | 陈燕芳 | A kind of keratinase compound and preparation method and application |
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