CN105567665A - Production method of high-efficiency keratinase - Google Patents
Production method of high-efficiency keratinase Download PDFInfo
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- CN105567665A CN105567665A CN201610174787.2A CN201610174787A CN105567665A CN 105567665 A CN105567665 A CN 105567665A CN 201610174787 A CN201610174787 A CN 201610174787A CN 105567665 A CN105567665 A CN 105567665A
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- powder
- zyme
- production method
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- rejuvenation
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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
Abstract
The invention discloses a production method of high-efficiency keratinase. The method comprises the following steps: inoculating Leuconostoc mesenteroides into a slant culture medium by a coating or scribing process, and carrying out rejuvenation culture at 18-37 DEG C for 18-36 hours to obtain rejuvenated cells; inoculating the rejuvenated cells into a liquid seed culture medium, and culturing at 18-37 DEG C under the condition of 100-300 rpm for 18-36 hours to obtain a fermentation seed; and inoculating the fermentation seed, which accounts for 5-15 vol% of the fermentation culture medium, into a fermentation tank, and fermenting and culturing for 18-60 hours to obtain the high-efficiency keratinase, wherein the tank temperature is 18-37 DEG C, the tank pressure is 0.2-0.8 kg/cm<2>, the stirring rate is 100-300 rpm and the ventilatory capacity is 1:(0.3-2.0). The prepared keratinase is widely used in the fields of tanning unhairing and keratin hydrolysis.
Description
Technical field
The present invention relates to a kind of M-Zyme production method, particularly the efficient M-Zyme production method of one.
Background technology
M-Zyme (Keratinase) is that one can the keratic enzyme of selective degradation, is produced by multiple-microorganisms such as bacterium, actinomycetes and fungies.Feather is as Poultry farming and to butcher the by product annual output of industry huge, and Amino acid and protein content enriches, and is potential fine protein resource.The Appropriate application of feather can reduce the pollution of waste to environment on the one hand, can raise the raising that section's protein is applied to livestock and poultry as a kind of simultaneously.Utilize in feather process in tradition and mainly adopt acid and alkali hydrolysis.The methods such as thermal destruction, there is environmental pollution problem in the former, the latter is larger to energy consumption, and can destroy partial amino-acid, reduces the nutritive value of product.Other common Keratin sulfate are the hair of animal, and as ox hair, wool and human hair etc., outside this kind of keratin protein part is processed as commercial materials, all the other are many as refuse process, also result in the pollution of environment and the waste of protein resource.M-Zyme can make keratin degrading be polypeptide and amino acid, both can be used for the production of agricultural fertilizer, can be used as again the feedstuff protein source of livestock and poultry; M-Zyme is the important invasion and attack factor of dermatophytes, and its research in skin diseases treatment needs to excavate further.In addition, M-Zyme " can digest " toxalbumin causing mad cow disease and mankind's Keyashi's syndrome, thus can be applicable to the purification of medical treatment and laboratory apparatus; It also can be used for leather depilation tanning, the cosmetics such as preparation profit dew, bath soap, shampoo and depilatory cream.
Summary of the invention
The present invention improves a kind of efficient M-Zyme production method; taurine is selected to promote biological metabolism; Microcrystalline Cellulose makes Leuconostoc mesenteroides be adsorbed onto on slant medium; Sharpleaf Galangal Fruit increases sticky Chryseobacterium sp stomach invigorating; supplement VITMAIN B1, Lin Suanna Vitamin B2 Sodium Phosphate, vitamins C, vitamin-E and multiple amino acids needed for microorganism growth, lipid acid, white gourd powder increases microorganism excretion, is convenient to microbial reproduction; and Rhizoma amorphophalli powder absorbs moisture, be convenient to sticky Chryseobacterium sp Growth and Reproduction and obtain efficient M-Zyme.
A kind of efficient M-Zyme production method, is characterized in that comprising the steps:
1) adopt the method for coating or line to be inoculated on slant medium by Leuconostoc mesenteroides, cultivate 18 ~ 36 hours in 18 ~ 37 DEG C of rejuvenation, obtain rejuvenation thalline;
2) rejuvenation thalline is inoculated in liquid seed culture medium, in 18 ~ 37 DEG C, cultivates 18 ~ 36 hours under 100 ~ 300rpm condition, obtain ferment-seeded;
3) ferment-seeded is pressed in 5 ~ 15% access fermentor tanks of fermention medium volume, tank temperature 18 ~ 37 DEG C, tank pressure 0.2 ~ 0.8kg/cm
2, stir speed (S.S.) 100 ~ 300rpm, air flow is 1: 0.3 ~ 2.0, fermented incubation time 18 ~ 60 hours, and after the centrifugal 15min of 12000rpm, collecting precipitation thing, water redissolves after solution, adopts the ammonium sulfate process of concentration 35%, obtains this M-Zyme after salt precipitation.
The composition of described every 1000ml slant medium is: salt 6 ~ 14g, yeast powder 5 ~ 10g, peptone 5 ~ 15g, agar powder 10 ~ 19g, taurine 5-12g, Microcrystalline Cellulose 1 ~ 2g, surplus are water; The composition of described every 1000ml seed culture medium is: salt 3 ~ 8g, yeast powder 5 ~ 10g, peptone 5 ~ 15g, agar powder 10 ~ 19g, Sharpleaf Galangal Fruit 2 ~ 3g, white gourd powder 1 ~ 2g, Rhizoma amorphophalli powder 1 ~ 3g, and surplus is water; The composition of described every 1000ml fermention medium is: ox hair powder Keratin 10 ~ 15g, glucose 0.1 ~ 0.7g, yeast powder 0.1 ~ 0.8g, peptone 0.1g, calcium chloride 0.1g, and all the other are water.
Advantage of the present invention:
(1) taurine can promote nutrient metabolism, thus improves sticky Chryseobacterium sp resistance of oxidation, enhancing body immunizing power; And taurine participates in
neuroendocrineregulate, strengthen sticky Chryseobacterium sp myocardial contraction;
(2) Microcrystalline Cellulose makes Leuconostoc mesenteroides be adsorbed onto on slant medium;
(3) Sharpleaf Galangal Fruit is containing volatile oil, Sharpleaf Galangal Fruit ketone, VITMAIN B1, Lin Suanna Vitamin B2 Sodium Phosphate, vitamins C, vitamin-E and multiple amino acids, lipid acid etc., and Sharpleaf Galangal Fruit increases Leuconostoc mesenteroides stomach invigorating;
(4) white gourd powder increases microorganism excretion, is convenient to microbial reproduction;
(5) Rhizoma amorphophalli powder absorbs moisture, is convenient to Leuconostoc mesenteroides Growth and Reproduction;
(6) Leuconostoc mesenteroides of the present invention is bought in Ming Rui bio tech ltd, Shanghai;
(7) sodium laurylsulfonate increases the dispersion of Microcrystalline Cellulose, improves the reproductive efficiency of sticky Chryseobacterium sp, and Sodium Benzoate and wilkinite increase the dispersion of sodium laurylsulfonate;
(8) each producer of raw material that the present invention is used all can be practical, as long as main component is similar or identical.
Embodiment
Embodiment is introduced below by citing
Embodiment one
1) adopt the method for coating or line to be inoculated on slant medium by Leuconostoc mesenteroides, cultivate 18 hours in 18 DEG C of rejuvenation, obtain rejuvenation thalline; The composition of described every 1000ml slant medium is: salt 6g, yeast powder 5g, peptone 5g, agar powder 10g, taurine 5g, Microcrystalline Cellulose 1g, water are 975g;
2) rejuvenation thalline is inoculated in liquid seed culture medium, in 18 DEG C, cultivates 18 hours under 100rpm condition, obtain ferment-seeded; The composition of described every 1000ml seed culture medium is: salt 3g, yeast powder 5g, peptone 5g, agar powder 10g, Sharpleaf Galangal Fruit 2g, white gourd powder 1g, Rhizoma amorphophalli powder 1g, and water is 981g;
3) ferment-seeded is pressed in 5% access fermentor tank of fermention medium volume, tank temperature 18 DEG C, tank pressure 0.2kg/cm
2, stir speed (S.S.) 100rpm, air flow is 1: 0.3, fermented incubation time 18 hours, after the centrifugal 15min of 12000rpm, collecting precipitation thing, water redissolves after solution, adopts the ammonium sulfate process of concentration 35%, obtains this M-Zyme after salt precipitation; The composition of described every 1000ml fermention medium is: ox hair powder Keratin 10 g, glucose 0.1g, yeast powder 0.1g, peptone 0.1g, calcium chloride 0.1g, water is 995g.
Embodiment two
1) adopt the method for coating or line to be inoculated on slant medium by Leuconostoc mesenteroides, cultivate 36 hours in 37 DEG C of rejuvenation, obtain rejuvenation thalline; The composition of described every 1000ml slant medium is: salt 14g, yeast powder 10g, peptone 15g, agar powder 19g, taurine 12g, sodium laurylsulfonate 2g, Sodium Benzoate 1g, Microcrystalline Cellulose 2g, wilkinite 0.5g, water 950g;
2) rejuvenation thalline is inoculated in liquid seed culture medium, in 37 DEG C, cultivates 36 hours under 300rpm condition, obtain ferment-seeded; The composition of described every 1000ml seed culture medium is: salt 8g, yeast powder 10g, peptone 15g, agar powder 19g, Sharpleaf Galangal Fruit 3g, white gourd powder 2g, Rhizoma amorphophalli powder 3g, and water is 956g;
3) ferment-seeded is pressed in 15% access fermentor tank of fermention medium volume, tank temperature 37 DEG C, tank pressure 0.8kg/cm
2, stir speed (S.S.) 300rpm, air flow is 1: 2.0, and fermented incubation time 60 hours, obtains this M-Zyme; The composition of described every 1000ml fermention medium is: ox hair powder keratin 15 g, glucose 0.7g, yeast powder 0.8g, peptone 0.1g, calcium chloride 0.1g, water is 992g.
Embodiment three
1) adopt the method for coating or line to be inoculated on slant medium by Leuconostoc mesenteroides, cultivate 27 hours in 28 DEG C of rejuvenation, obtain rejuvenation thalline; The composition of described every 1000ml slant medium is: salt 10g, yeast powder 7.5g, peptone 10g, agar powder 15g, taurine 9g, Microcrystalline Cellulose 1.5g, water are 962g;
2) rejuvenation thalline is inoculated in liquid seed culture medium, in 28 DEG C, cultivates 27 hours under 200rpm condition, obtain ferment-seeded; The composition of described every 1000ml seed culture medium is: salt 5.5g, yeast powder 7.5g, peptone 10g, agar powder 14.5g, Sharpleaf Galangal Fruit 2.5g, white gourd powder 1.5g, Rhizoma amorphophalli powder 2g, and surplus is water 968g;
3) ferment-seeded is pressed in 10% access fermentor tank of fermention medium volume, tank temperature 28 DEG C, tank pressure 0.5kg/cm
2, stir speed (S.S.) 200rpm, air flow is 1: 1.2, fermented incubation time 39 hours, after the centrifugal 15min of 12000rpm, collecting precipitation thing, water redissolves after solution, adopts the ammonium sulfate process of concentration 35%, obtains this M-Zyme after salt precipitation; The composition of described every 1000ml fermention medium is: ox hair powder Keratin 1 2.5g, glucose 0.4g, yeast powder 0.45g, peptone 0.1g, calcium chloride 0.1g, water is 991g.
This M-Zyme is detected according to the external enzymolysis profile test of the detection method of patent 201410148157.9,
The different M-Zyme preparation of table 1 is on the impact of protein digestibility in dregs of beans
| Sample | Crude protein digestibility |
| Blank | 0.45 |
| Embodiment one | 4.98 |
| Embodiment two | 5.36 |
| Embodiment three | 6.23 |
As can be seen from Table 1, M-Zyme preparation of the present invention effectively can improve the digestibility of albumen in dregs of beans, wherein M-Zyme preparation having the greatest impact to protein digestibility described in embodiment 2, the digestibility of albumen in dregs of beans is made to improve 4.91%, more be conducive to the absorption of cultivated animals to albumen, thus the addition of protein raw materials in feed can be reduced, reduce production cost.
Embodiment two M-Zyme is used for process hides depilation, finds that M-Zyme consumption is made a living 0.2% of tare weight amount, the hair of ox-hide just can be made to come off.
Claims (4)
1. an efficient M-Zyme production method, is characterized in that comprising the steps:
Adopt the method for coating or line to be inoculated on slant medium by Leuconostoc mesenteroides, cultivate 18 ~ 36 hours in 18 ~ 37 DEG C of rejuvenation, obtain rejuvenation thalline;
2) rejuvenation thalline is inoculated in liquid seed culture medium, in 18 ~ 37 DEG C, cultivates 18 ~ 36 hours under 100 ~ 300rpm condition, obtain ferment-seeded;
3) ferment-seeded is pressed in 5 ~ 15% access fermentor tanks of fermention medium volume, tank temperature 18 ~ 37 DEG C, tank pressure 0.2 ~ 0.8kg/cm
2, stir speed (S.S.) 100 ~ 300rpm, air flow is 1: 0.3 ~ 2.0, and fermented incubation time 18 ~ 60 hours, obtains this M-Zyme.
2. the efficient M-Zyme production method of one according to claim 1, is characterized in that the composition of described every 1000ml slant medium is: salt 6 ~ 14g, yeast powder 5 ~ 10g, peptone 5 ~ 15g, agar powder 10 ~ 19g, taurine 5-12g, Microcrystalline Cellulose 1 ~ 2g, surplus are water.
3. the efficient M-Zyme production method of one according to claim 1, it is characterized in that the composition of described every 1000ml seed culture medium is: salt 3 ~ 8g, yeast powder 5 ~ 10g, peptone 5 ~ 15g, agar powder 10 ~ 19g, Sharpleaf Galangal Fruit 2 ~ 3g, white gourd powder 1 ~ 2g, Rhizoma amorphophalli powder 1 ~ 3g, surplus is water.
4. the efficient M-Zyme production method of one according to claim 1, it is characterized in that the composition of described every 1000ml fermention medium is: ox hair powder Keratin 10 ~ 15g, glucose 0.1 ~ 0.7g, yeast powder 0.1 ~ 0.8g, peptone 0.1g, calcium chloride 0.1g, all the other are water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106258478A (en) * | 2016-07-29 | 2017-01-04 | 四川省农业科学院土壤肥料研究所 | Utilize Morchella esculenta (L.) Pers nutrient bag that stalk fermentation substrate makes and preparation method thereof |
| CN111424026A (en) * | 2020-04-22 | 2020-07-17 | 江南大学 | Method for producing keratinase |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106258478A (en) * | 2016-07-29 | 2017-01-04 | 四川省农业科学院土壤肥料研究所 | Utilize Morchella esculenta (L.) Pers nutrient bag that stalk fermentation substrate makes and preparation method thereof |
| CN111424026A (en) * | 2020-04-22 | 2020-07-17 | 江南大学 | Method for producing keratinase |
| CN111424026B (en) * | 2020-04-22 | 2022-05-24 | 江南大学 | Method for producing keratinase |
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Application publication date: 20160511 |