CN101260393A - Method for producing keratinase by liquid deep fermentation - Google Patents

Method for producing keratinase by liquid deep fermentation Download PDF

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Publication number
CN101260393A
CN101260393A CNA2008100606393A CN200810060639A CN101260393A CN 101260393 A CN101260393 A CN 101260393A CN A2008100606393 A CNA2008100606393 A CN A2008100606393A CN 200810060639 A CN200810060639 A CN 200810060639A CN 101260393 A CN101260393 A CN 101260393A
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grams
gram
milliliters
keratinase
liquid
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CN100587067C (en
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李永泉
吕龙贤
管文军
沈鈱豪
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Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The invention discloses a method for liquid submerged fermentation production of keratinase by utilization of Chryseobacterium Gleum, aiming at large-scale liquid submerged fermentation production of the keratinase. The Chrvseobacterium L99, CGMCC No.2295 pure strain separated by the laboratory is used as an initial strain, and liquid submerged fermentation production of the keratinase is realized through slant cultivation, shaking seed cultivation and fermenter cultivation. A liquid fermentation culture medium disclosed in the invention respectively consists of tragantine, sugar, glucose, caseins, yeast powder, yeast extract paste, pepetone, keratins, magnesium salts, sylvin and sodium salts. The method is suitable for liquid submerged aerated culture of the Chryseobacterium Gleum, reasonably meets the nutritive materials required by different fermentation periods of the Chryseobacterium Gleum, and improves the activity of the keratinase in fermentation broth. The method can degrade the keratin which causes environmental pollution when the keratinase is produced simultaneously, and keratin degradation products obtained have high economic value.

Description

A kind of method of producing keratinase by liquid deep fermentation
Technical field
The present invention relates to the microbial engineering field, particularly a kind of method of producing keratinase by liquid deep fermentation.
Background technology
M-Zyme can gentleness Keratin sulfate waste such as hydrolysis feather efficiently, produce feed, fertilizer.The feed saving energy, the cost that this method is produced is low, quality good, can be utilized by animal preferably.In containing keratic feed, the interpolation M-Zyme also can improve animal and absorb keratic, increases economic efficiency.Keratin sulfate can the selective hydrolysis wool, the Keratin sulfate of leather surface, is expected to replace traditional wool stripping squama and tanning process, realizes the environmental protection transformation of conventional industries.M-Zyme can improve makeup, the externally applied medicine permeability in skin, also all has a wide range of applications in industry such as medicine, makeup.The M-Zyme production technology of exploitation Cheap highly effective is protection environment and the inevitable requirement of increasing economic efficiency.
This will glue production that golden yellow bacillus liquid fermentation technology is applied to M-Zyme and still belong to the firstly at home, and result of study shows that this scheme is feasible.Sticking golden yellow bacillus is Chryseobacterium L99, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.2295.
Summary of the invention
The objective of the invention is relation, a kind of method of producing keratinase by liquid deep fermentation is provided at nutritional needs in the liquid fermenting process and thalli growth.
The method of producing keratinase by liquid deep fermentation comprises the steps:
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 18～36 hours, obtain the rejuvenation thalline in 18～37 ℃ of rejuvenation;
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated under 100～300rpm condition 18～36 hours, obtain ferment-seeded in 18～37 ℃;
3) ferment-seeded is pressed 5～15% of fermention medium volume and inserted in the fermentor tank, jar temperature 18～37 ℃, tank pressure 0.2～0.8kg/cm 2, stir speed (S.S.) 100～300rpm, air flow are 1: 0.3～2.0, fermented incubation time 18～60 hours.
Described sticking golden yellow bacillus is Chryseobacterium L99, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.2295.
The composition of described slant medium is: contain 900～1000 milliliters in NaCl5～15 grams, yeast powder or yeast extract paste 5～10 grams, peptone 5～15 grams, agar powder 10～20 grams and water in per 1000 milliliters.
The composition of described seed culture medium is: contain 900～1000 milliliters in NaCl5～15 grams, yeast powder or yeast extract paste 5～10 grams, peptone 5～15 grams and water in per 1000 milliliters.
The composition of described fermention medium is: soluble-containing starch 0.1～100 gram, sucrose 0.1～100 gram, glucose 0.1～100 gram, casein 0.1～100 gram, yeast powder or yeast extract paste 0.1～100 gram, peptone 0.1～100 gram, Keratin sulfate 0.1～200 gram, K in per 1000 milliliters 2PO 40.1～10 grams, NaH 2PO 42H 2O1～10 grams, MgCl 26H 2900～1000 milliliters in O 0.1～5 gram and water.
The beneficial effect that the present invention compared with prior art has:
1) seed culture medium provides the thalline required nutritive substance of growing fast in a short time among the present invention, cultivates can carry out submerged fermentation in about 20 hours.
2) liquid submerged aerobic fermentation base among the present invention, the nutritive ingredient reasonable ratio can guarantee the growth of sticking golden yellow bacillus L99 and produce enzyme require.The substratum material is abundant, only needs once batching in the whole batch fermentation process, once sterilization get final product, have save time, the saving of labor, reduced again because the reinforced midway chance that polluting the bacterium of mixing.
3) adopt the deep liquid aerobic fermentation technology among the present invention to carry out batch fermentation, final enzyme work can reach 100-400U/ml.The enzyme activity unit definition: 8.0,50 ℃ of pH, the 200rpm per hour blue Keratin sulfate of hydrolysis make 595nm place absorbancy increase by 0.001 enzyme amount.
4) the present invention domesticly produces the M-Zyme zymotechnique to sticking golden yellow bacillus first and studies, and experimental study and the suitability for industrialized production later for this bacterium all have very big directive significance.
5) utilized in the fermenting process of the present invention the disadvantageous Keratin sulfate of environmental protection is substrate, cost is low, can solve environmental problem again when producing M-Zyme, and the Keratin sulfate after the degraded is used for industry such as feed can produce higher economic value.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail.
Embodiment 1
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 36 hours, obtain the rejuvenation thalline in 18 ℃ of rejuvenation; The composition of slant medium is: contain 980 milliliters in NaCl10 gram, yeast powder 5 grams, peptone 5 grams, agar powder 15 grams and water in per 1000 milliliters.
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated 24 hours under the 200rpm condition, obtain ferment-seeded in 18 ℃; The composition of seed culture medium is: 980 milliliters in NaCl10 in per 1000 milliliters gram, yeast powder 5 grams, peptone 5 grams and water.
3) ferment-seeded is pressed 5% of fermention medium volume and inserted in the fermentor tank, jar temperature 18 ℃, tank pressure 0.2kg/cm 2, stir speed (S.S.) 100rpm, air flow are 1: 0.3, fermented incubation time 60 hours.The fermention medium composition is: soluble-containing starch 0.1 gram, sucrose 0.1 gram, glucose 0.1 gram, casein 0.1 gram, yeast powder 0.1 gram, peptone 0.1 gram, feather meal Keratin sulfate 20 grams, K in per 1000 milliliters 2PO 42 grams, NaH 2PO 42H 2O 4 grams, MgCl 26H 2980 milliliters in O 0.1 gram and water.
Embodiment 2
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 24 hours, obtain the rejuvenation thalline in 25 ℃ of rejuvenation; The composition of slant medium is: contain 980 milliliters in NaCl10 gram, yeast powder 5 grams, peptone 10 grams, agar powder 20 grams and water in per 1000 milliliters.
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated 18 hours under the 200rpm condition, obtain ferment-seeded in 25 ℃; The composition of seed culture medium is: 980 milliliters in NaCl10 in per 1000 milliliters gram, yeast powder 5 grams, peptone 10 grams and water.
3) ferment-seeded is pressed 6% of fermention medium volume and inserted in the fermentor tank, jar temperature 25 ℃, tank pressure 0.3kg/cm 2, stir speed (S.S.) 200rpm, air flow are 1: 0.5, fermented incubation time 36 hours.The fermention medium composition is: soluble-containing starch 0.1 gram, sucrose 60 grams, glucose 0.1 gram, casein 0.1 gram, yeast powder or yeast extract paste 0.1 gram, peptone 0.1 gram, feather meal Keratin sulfate 40 grams, K in per 1000 milliliters 2PO 44 grams, NaH 2PO 42H 2O 2 grams, MgCl 26H 2980 milliliters in O 0.2 gram and water.
Embodiment 3
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 24 hours, obtain the rejuvenation thalline in 30 ℃ of rejuvenation; The composition of slant medium is: contain 980 milliliters in NaCl12 gram, yeast powder 6 grams, peptone 12 grams, agar powder 15 grams and water in per 1000 milliliters.
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated 14 hours under the 200rpm condition, obtain ferment-seeded in 30 ℃; The composition of seed culture medium is: 980 milliliters in NaCl12 in per 1000 milliliters gram, yeast powder 6 grams, peptone 12 grams and water.
3) ferment-seeded is pressed 6% of fermention medium volume and inserted in the fermentor tank, jar temperature 30 ℃, tank pressure 0.5kg/cm 2, stir speed (S.S.) 200rpm, air flow are 1: 0.8, fermented incubation time 30 hours.The fermention medium composition is: soluble-containing starch 0.1 gram, sucrose 0.1 gram, glucose 40 grams, casein 0.1 gram, yeast powder or yeast extract paste 0.1 gram, peptone 0.1 gram, hair Keratin sulfate 40 grams, K in per 1000 milliliters 2PO 40.8 gram, NaH 2PO 42H 2O 6 grams, MgCl 26H 2980 milliliters in O 2 grams and water.
Embodiment 4
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 18 hours, obtain the rejuvenation thalline in 37 ℃ of rejuvenation; The composition of slant medium is: contain 980 milliliters in NaCl8 gram, yeast powder 5 grams, peptone 6 grams, agar powder 15 grams and water in per 1000 milliliters.
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated 12 hours under the 200rpm condition, obtain ferment-seeded in 37 ℃; The composition of seed culture medium is: 980 milliliters in NaCl8 in per 1000 milliliters gram, yeast powder 5 grams, peptone 8 grams and water.
3) ferment-seeded is pressed 6% of fermention medium volume and inserted in the fermentor tank, jar temperature 37 ℃, tank pressure 0.4kg/cm 2, stir speed (S.S.) 200rpm, air flow are 1: 1.5, fermented incubation time 24 hours.The fermention medium composition is: soluble-containing starch 0.1 gram, sucrose 0.1 gram, glucose 40 grams, casein 0.1 gram, yeast powder or yeast extract paste 0.1 gram, peptone 0.1 gram, wool keratin 40 grams, K in per 1000 milliliters 2PO 40.8 gram, NaH 2PO 42H 26 grams, MgCl 26H 2980 milliliters in O 2 grams and water.
Embodiment 5
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 24 hours, obtain the rejuvenation thalline in 30 ℃ of rejuvenation; The composition of slant medium is: contain 980 milliliters in NaCl10 gram, yeast powder 5 grams, peptone 10 grams, agar powder 15 grams and water in per 1000 milliliters.
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated 20 hours under the 200rpm condition, obtain ferment-seeded in 30 ℃; The composition of seed culture medium is: 980 milliliters in NaCl10 in per 1000 milliliters gram, yeast powder 5 grams, peptone 10 grams and water.
3) ferment-seeded is pressed 4% of fermention medium volume and inserted in the fermentor tank, jar temperature 30 ℃, tank pressure 0.5kg/cm 2, stir speed (S.S.) 300rpm, air flow are 1: 1.5, fermented incubation time 30 hours.The fermention medium composition is: soluble-containing starch 0.1 gram, sucrose 0.1 gram, glucose 0.1 gram, casein 0.1 gram, yeast powder or yeast extract paste 0.1 gram, peptone 20 grams, feather keratin 40 grams, K in per 1000 milliliters 2PO 40.8 gram, NaH 2PO 42H 2O 6 grams, MgCl 26H 2980 milliliters in O 2 grams and water.
Embodiment 6
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 24 hours, obtain the rejuvenation thalline in 30 ℃ of rejuvenation; The composition of slant medium is: contain 980 milliliters in NaCl10 gram, yeast powder 5 grams, peptone 10 grams, agar powder 20 grams and water in per 1000 milliliters.
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated 16 hours under the 200rpm condition, obtain ferment-seeded in 30 ℃; The composition of seed culture medium is: 980 milliliters in NaCl10 in per 1000 milliliters gram, yeast powder 5 grams, peptone 10 grams and water.
3) ferment-seeded is pressed 6% of fermention medium volume and inserted in the fermentor tank, jar temperature 30 ℃, tank pressure 0.5kg/cm 2, stir speed (S.S.) 250rpm, air flow are 1: 1.0, fermented incubation time 30 hours.The fermention medium composition is: soluble-containing starch 0.1 gram, sucrose 0.1 gram, glucose 20 grams, casein 0.1 gram, yeast powder or yeast extract paste 0.1 gram, peptone 20 grams, Keratin sulfate 0.1 gram, K in per 1000 milliliters 2PO 44 grams, NaH 2PO 42H 2O 6 grams, MgCl 26H 2980 milliliters in O 2 grams and water.
At last, it is also to be noted that what more than enumerate only is part specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.
The present invention can summarize with other the specific form without prejudice to spirit of the present invention and principal character.Therefore, no matter from which point, above-mentioned embodiment of the present invention all can only be thought can not limit the present invention to explanation of the present invention, claims have been pointed out scope of the present invention, and scope of the present invention is not pointed out in above-mentioned explanation, therefore, in implication suitable and any change in the scope, all should think to be included in the scope of claims with claims of the present invention.

Claims (5)

1, a kind of method of producing keratinase by liquid deep fermentation is characterized in that comprising the steps:
1) adopts the method that is coated with or rules to glue golden yellow bacillus and be inoculated on the slant medium, cultivated 18～36 hours, obtain the rejuvenation thalline in 18～37 ℃ of rejuvenation;
2) the rejuvenation thalline is inoculated in the liquid seed culture medium,, cultivated under 100～300rpm condition 18～36 hours, obtain ferment-seeded in 18～37 ℃;
3) ferment-seeded is pressed 5～15% of fermention medium volume and inserted in the fermentor tank, jar temperature 18～37 ℃, tank pressure 0.2～0.8kg/cm 2, stir speed (S.S.) 100～300rpm, air flow are 1: 0.3～2.0, fermented incubation time 18～60 hours.
2. the method for a kind of producing keratinase by liquid deep fermentation according to claim 1, it is characterized in that described sticking golden yellow bacillus is Chryseobacterium L99, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.2295.
3. the method for a kind of producing keratinase by liquid deep fermentation according to claim 1 is characterized in that the composition of described slant medium is: contain 900～1000 milliliters in NaCl5～15 grams, yeast powder or yeast extract paste 5～10 grams, peptone 5～15 grams, agar powder 10～20 grams and water in per 1000 milliliters.
4. the method for a kind of producing keratinase by liquid deep fermentation according to claim 1 is characterized in that the composition of described seed culture medium is: contain 900～1000 milliliters in NaCl5～15 grams, yeast powder or yeast extract paste 5～10 grams, peptone 5～15 grams and water in per 1000 milliliters.
5. the method for a kind of producing keratinase by liquid deep fermentation according to claim 1 is characterized in that the composition of described fermention medium is: soluble-containing starch 0.1～100 gram, sucrose 0.1～100 gram, glucose 0.1～100 gram, casein 0.1～100 gram, yeast powder or yeast extract paste 0.1～100 gram, peptone 0.1～100 gram, Keratin sulfate 0.1～200 gram, K in per 1000 milliliters 2PO 40.1～10 grams, NaH 2PO 42H 2O0.1～10 grams, MgCl 26H 2900～1000 milliliters in O 0.1～5 gram and water.
CN200810060639A 2008-04-15 2008-04-15 Method for producing keratinase by liquid deep fermentation Expired - Fee Related CN100587067C (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102433277A (en) * 2011-12-14 2012-05-02 首创爱华(天津)市政环境工程有限公司 Chryseobacterium sp. for removing phosphorus in sewage at low temperature and separation culture method
CN105567665A (en) * 2016-03-25 2016-05-11 深圳市先康达生物科技有限公司 Production method of high-efficiency keratinase

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102433277A (en) * 2011-12-14 2012-05-02 首创爱华(天津)市政环境工程有限公司 Chryseobacterium sp. for removing phosphorus in sewage at low temperature and separation culture method
CN102433277B (en) * 2011-12-14 2013-02-06 首创爱华(天津)市政环境工程有限公司 Chryseobacterium sp. for removing phosphorus in sewage at low temperature and separation culture method
CN105567665A (en) * 2016-03-25 2016-05-11 深圳市先康达生物科技有限公司 Production method of high-efficiency keratinase

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