CN104212853A - Culture medium for producing nema rhzomorph by streptomyces fermentation and cultivation method - Google Patents
Culture medium for producing nema rhzomorph by streptomyces fermentation and cultivation method Download PDFInfo
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Abstract
The invention relates to a culture medium for producing nema rhzomorph by streptomyces fermentation and a cultivation method. The fermentation unit of the nema rhzomorph can be improved, the fermentation cycle can be shortened, the fermentation cost can be reduced, the effect on primary and auxiliary materials caused by the environment is reduced to the maximal extent, and adequate supply of the primary and auxiliary materials is ensured by using the culture medium formula and the fermentation control process provided by the invention, thereby achieving stable and efficient production of the nema rhzomorph.
Description
Technical field
The invention belongs to fermentation technical field, particularly relate to novel culture medium and cultural method that a kind of streptomycete fermentation produces how horse rhzomorph.
Background technology
How horse rhzomorph is the same with ivermectin, and being a kind of wide spectrum, efficient, Novel macrocyclic lactone expelling parasite microbiotic of being widely used in veterinary clinic, is a member in milbemycin family.At present, the fermentative production of domestic how horse rhzomorph adopts second order fermentation pattern, the subject matter which exists have following some:
1 how horse rhzomorph fermentation level is lower, and its fermentation unit is lower than 3000u/ml.
Containing glucose in the substratum of 2 domestic fermentative production how horse rhzomorph, through high-temperature sterilization, easily cause part glucose carbonization, reduce total sugar content, affect fermented liquid quality.
3 cost of raw how needed for horse rhzomorph fermentative production are higher, and how conventional in horse rhzomorph substratum organic nitrogen source peptone market value particularly higher, thus improve fermentation costs, reduces the market competitiveness.
The fermentation culture cycle of 4 domestic how horse rhzomorphs is longer, generally at more than 260h, causes energy consumption higher.
Summary of the invention
Object of the present invention is just the defect overcoming above-mentioned prior art, there is provided a kind of zymotechnique simple, effective raising fermentation unit, reduce supplementary material to greatest extent to the impact of fermentation unit simultaneously, reduce production cost, and supplementary material source is not affected by environment, ensures that it is in liberal supply, realize how horse rhzomorph is stablized, the novel culture medium of High-efficient Production.
Another object of the present invention is to provide the cultural method utilizing above-mentioned substratum to produce how horse rhzomorph.
Technical scheme taked for achieving the above object is:
Streptomycete fermentation produces a substratum for how horse rhzomorph, it is characterized in that comprising seed culture medium and fermention medium, wherein
Consisting of of described seed culture medium: soya-bean oil or Semen Maydis oil 5 ~ 10ml/L, maltose 10 ~ 15g/L, low temperature soybean cake powder 15 ~ 20g/L, groundnut meal 10 ~ 15g/L, corn steep liquor 5 ~ 9ml/L, Zein powder 5 ~ 10g/L, ammonium sulfate 0.5 ~ 1g/L, light calcium carbonate 1 ~ 5g/L;
Consisting of of described fermention medium: soya-bean oil or Semen Maydis oil 8 ~ 12ml/L, maltose 10 ~ 14g/L, low temperature soybean cake powder 25 ~ 30g/L, groundnut meal 15 ~ 20g/L, corn steep liquor 10 ~ 15ml/L, Zein powder 10 ~ 15g/L, ammonium sulfate 0.5 ~ 1g/L, light calcium carbonate 1 ~ 5g/L, potassium primary phosphate 0.5 ~ 0.9g/L, sodium-chlor 0.3 ~ 0.7g/L, magnesium sulfate 0.03 ~ 0.06g/L, ferrous sulfate 0.1 ~ 0.5g/L, polyoxypropylene glyceryl 0.3 ~ 0.4g/L, maltin 0.002 ~ 0.004g/L.
Utilize above-mentioned substratum to produce a cultural method for how horse rhzomorph, it is characterized in that its processing step is:
1) seed culture: first seed culture medium sterilizing is cooled to 25 ~ 30 DEG C, and uses sterile air pressurize, then under flame protection, by cultured strepto-starter bottle fermented liquid according to 0.2 ~ 0.3L/m
3inoculum size access seed culture medium in carry out seed culture, to culture transferring when cell concentration 20 ~ 30%, pH value 7 ~ 8, incubation time 40 ~ 50h, culture transferring ratio control is at 0.8 ~ 1m
3/ m
3;
2) fermentation culture: first by fermention medium sterilizing, be cooled to 20 ~ 27 DEG C, and use sterile air pressurize, then cultured seed liquor is moved into fermention medium and carry out fermentation culture, stop fermentation to when cell concentration 35 ~ 40%, total reducing sugar < 5mg/100ml, fatty < 0.8g/L, amino nitrogen 10 ~ 20mg/100ml, chemical titer > 3800u/mL, pH5 ~ 6, incubation time 230 ~ 240h.
3, according to cultural method according to claim 2, it is characterized in that the specification of quality of the seed culture medium after described sterilizing: amino nitrogen 40 ~ 60mg/100ml, molten phosphorus 50 ~ 100ug/ml, total reducing sugar 15 ~ 25mg/100ml, pH7 ~ 8;
The specification of quality of the fermention medium after sterilizing: amino nitrogen 60 ~ 80mg/100ml, molten phosphorus 100 ~ 150ug/ml, total reducing sugar 30 ~ 40 mg/100ml, pH6 ~ 7.
Described cultured strepto-starter bottle fermented liquid specification of quality is: pH6 ~ 8; Cell concentration 10 ~ 30%; Microscopy is without miscellaneous bacteria; Fermentation unit 800 ~ 1500u/ml.
Described seed culture condition is: tank pressure 0.03 ~ 0.05MPa; Tank temperature 28 ~ 33 DEG C; Air flow quantity: 0 ~ 5h, adopts pneumatic blending to replace mechanical stirring; 6h ~ culture transferring: 30 ~ 50m
3/ h; Mixing speed 60 ~ 80r/min.
Described fermentation culture conditions is:
The initial pH of a substratum: after fermention medium sterilizing, pH controls 6 ~ 7;
B temperature: adopt alternating temperature Controlling Technology,
0 ~ 60h: culture temperature 29 ~ 30 DEG C,
61 ~ 200h: culture temperature 31 ~ 32 DEG C,
201h ~ fermentation ends: culture temperature 30 ~ 31 DEG C;
C mixing speed: adopt frequency conversion mixing control technique,
0 ~ 60h: rotating speed controls at 100r/min,
61 ~ 200h: rotating speed controls at 120r/min,
201h ~ fermentation ends: rotating speed controls at 90r/min;
D pressure-controlling: tank pressure 0.05 ~ 0.06MPa;
E pH controls: in fermenting process, pH controls 5 ~ 7;
F air flow quantity: 0 ~ 60h:100 ~ 150m
3/ h; 61 ~ 200h:200 ~ 300m
3/ h; 201h ~ fermentation ends: 150 ~ 200m
3/ h;
H dissolved oxygen controls:
Before fermentation in 60h, do not control dissolved oxygen;
60 ~ 100h, dissolved oxygen controls more than 60%;
101 ~ 200h, dissolved oxygen controls more than 30%;
201 ~ fermentation ends: dissolved oxygen controls more than 40%.
Adopt stream addition to carry out feed supplement during the fermentation, feed supplement comprises repairing, moisturizing, benefit ammonium sulfate, mends sugar and pH control, wherein
A oil-supplementing process controls:
Before fermentation, 60h need not carry out repairing,
Fermentation time, at 61 ~ 200h, when lipid content is lower than 1g/L, fills into soya-bean oil or Semen Maydis oil, and control lipid content is 1 ~ 1.5g/L, detects fermented liquid lipid content every 6 ~ 8h,
Fermentation time, in 201h ~ fermentation ends, when lipid content is lower than 3%, fills into soya-bean oil or Semen Maydis oil, and control lipid content is 0.5 ~ 0.8g/L, detects fermented liquid lipid content every 6 ~ 8h;
B moisturizing technology controlling and process:
Before fermentation, 60h need not carry out moisturizing,
Fermentation time, at 61 ~ 200h, when cell concentration is higher than 50%, adds aqua sterilisa, requires to control fermented liquid cell concentration 40 ~ 45%, detects fermented liquid cell concentration every 6 ~ 8h,
Fermentation time, in 201h ~ fermentation ends, when cell concentration is higher than 40%, adds aqua sterilisa, requires to control fermented liquid cell concentration 35 ~ 40%, detects fermented liquid cell concentration every 6 ~ 8h;
C fermenting process pH Controlling Technology:
Before fermentation, 60h, pH do not control,
Fermentation time is at 61 ~ 200h, if pH < 6.0, adds the sodium hydroxide of 10 ~ 20%, regulates pH to 6 ~ 7; If pH > 7, add the ammoniumsulphate soln of 30%, regulate pH to 6 ~ 7;
Fermentation time is in 201 ~ fermentation ends, if pH < 6.5, adds the sodium hydroxide of 10 ~ 20%, regulates pH control to 5 ~ 6; If pH > 6, add the ammoniumsulphate soln of 30%, regulate pH to 5 ~ 6;
D fills into maltose:
60h before fermentation, total sugar content does not control,
During fermentation 61h to 200h: total sugar content < 10mg/100ml, fill into sterilized maltose solution, the content of total reducing sugar is controlled at 10 ~ 15mg/100ml.
201h is to fermentation ends in fermentation: during total sugar content < 3mg/100ml, fill into sterilized maltose solution, the content of total reducing sugar is controlled at 3 ~ 5mg/100ml.
Add maltin in above-mentioned benefit sugar technique, its concentration controls 0.001 ~ 0.003%.
E mends ammonium sulfate
How, in horse rhzomorph fermentation culture process, fill into the ammoniumsulphate soln of 30% respectively at 60h, 150h and 220h, its consumption controls at 0.1 ~ 0.3% of fermentating liquid volume.
Technical superiority of the present invention is embodied in:
1 the present invention confirms seed culture medium, fermentation culture carbon source, nitrogenous source and best proportion compatibility.
2 the present invention have carried out detailed elaboration to the technique of seed culture, fermentation culture, confirm zymotechnique and the controling parameters of how horse rhzomorph the best.By fermentative production of the present invention how horse rhzomorph, finally make fermentation unit reach more than 3500u/ml, the production cycle is no more than 240h, compares with domestic technique, and fermentation unit improves 16.7%, and the production cycle decreases more than 20h.
3 the present invention are applicable to mass-producing fermentative production how horse rhzomorph, can meet the Production requirement of annual output 50 tons how horse rhzomorph.
Use maltose in 4 substratum, avoid occurring glucose carbonization phenomenon.
specific implementation method
Be explained the present invention with example below, it should be understood that example is for illustration of the present invention instead of limitation of the present invention.Scope of the present invention and core content are determined according to claims.
The bacterial classification of following embodiment selects Chinese cassia tree ground streptomycete: the bacterial classification source of production and application is female bottle fermented liquid.Female bottle fermented liquid specification of quality is: pH6 ~ 8; Cell concentration 10 ~ 30%; Microscopy is without miscellaneous bacteria; Fermentation unit 800 ~ 1500u/ml.
embodiment 1
seed culture medium: seeding tank volume 1.5m
3, effective volume is 1m
3.
Seed culture medium: soya-bean oil 5L, maltose 10kg, low temperature soybean cake powder 15kg, groundnut meal 10kg, corn steep liquor 5L, Zein powder 5kg, ammonium sulfate 0.5kg, light calcium carbonate 1kg.
Seed culture medium sterilizing is also cooled to 25 DEG C, and uses sterile air pressurize, and the quality of the seed culture medium after sterilizing is amino nitrogen 41mg/100ml, molten phosphorus 50ug/ml, total reducing sugar 15mg/100ml, pH7.8.Then, under flame protection, cultured strepto-starter bottle fermented liquid is cultivated according in the inoculum size access seed culture medium of 0.2L, tank pressure 0.03 ~ 0.05MPa; Tank temperature 28 DEG C; Air flow quantity: 0 ~ 5h, adopts pneumatic blending to replace mechanical stirring; 6h ~ culture transferring: 30m
3/ h; Mixing speed 60r/min.Seed culture terminates, cell concentration 20%, pH value 7.9, incubation time 40h.
fermention medium: seeding tank volume 15m
3, effective volume is 10m
3.
Fermention medium: soya-bean oil 80L, maltose 100kg, low temperature soybean cake powder 250kg, groundnut meal 150kg, corn steep liquor 100L, Zein powder 100kg, ammonium sulfate 5kg, light calcium carbonate 10kg, potassium primary phosphate 5kg, sodium-chlor 3kg, magnesium sulfate 0.3 kg, ferrous sulfate 1kg, polyoxypropylene glyceryl 3kg, maltin 0.02kg.
First by fermention medium sterilizing, be cooled to 20 DEG C, and use sterile air pressurize, the quality of the fermention medium after sterilizing is amino nitrogen 61mg/100ml, molten phosphorus 102ug/ml, total reducing sugar 31mg/100ml, pH6.9.Cultured seed liquor is moved into fermention medium and carries out fermentation culture, culture transferring amount is 0.8m
3.Fermentation culture terminates, cell concentration 35%, total reducing sugar 3.1mg/100ml, fatty 0.2g/L, amino nitrogen 11mg/100ml, chemical titer 3967u/mL, pH5.1, incubation time 230h.
embodiment 2
seed culture medium: seeding tank volume 1.5m
3, effective volume is 1m
3.
Seed culture medium: Semen Maydis oil 6L, maltose 11kg, low temperature soybean cake powder 16kg, groundnut meal 11kg, corn steep liquor 6L, Zein powder 6kg, ammonium sulfate 0.6kg, light calcium carbonate 2kg.
Seed culture medium sterilizing is also cooled to 26 DEG C, and uses sterile air pressurize, and the quality of the seed culture medium after sterilizing is amino nitrogen 45mg/100ml, molten phosphorus 60ug/ml, total reducing sugar 17mg/100ml, pH7.7.Then, under flame protection, cultured strepto-starter bottle fermented liquid is cultivated according in the inoculum size access seed culture medium of 0.22L, tank pressure 0.03 ~ 0.05MPa; Tank temperature 29 DEG C; Air flow quantity: 0 ~ 5h, adopts pneumatic blending to replace mechanical stirring; 6h ~ culture transferring: 35m
3/ h; Mixing speed 65r/min.Seed culture terminates, cell concentration 22%, pH value 7.8, incubation time 42h.
fermention medium: seeding tank volume 15m
3, effective volume is 10m
3.
Fermention medium: Semen Maydis oil 90L, maltose 110kg, low temperature soybean cake powder 260kg, groundnut meal 160kg, corn steep liquor 110L, Zein powder 110kg, ammonium sulfate 6kg, light calcium carbonate 20kg, potassium primary phosphate 6kg, sodium-chlor 4kg, magnesium sulfate 0.4kg, ferrous sulfate 2kg, polyoxypropylene glyceryl 3.2kg, maltin 0.025kg.
First by fermention medium sterilizing, be cooled to 22 DEG C, and use sterile air pressurize, the quality of the fermention medium after sterilizing is amino nitrogen 66mg/100ml, molten phosphorus 112ug/ml, total reducing sugar 33mg/100ml, pH6.8.Cultured seed liquor is moved into fermention medium and carries out fermentation culture, culture transferring amount is 0.85m
3.Fermentation culture terminates, cell concentration 36%, total reducing sugar 3.5g/100ml, fatty 0.3g/L, amino nitrogen 13mg/100ml, chemical titer 4032u/mL, pH5.3, incubation time 232h.
embodiment 3
seed culture medium: seeding tank volume 1.5m
3, effective volume is 1m
3.
Seed culture medium: soya-bean oil 7L, maltose 12kg, low temperature soybean cake powder 17kg, groundnut meal 12kg, corn steep liquor 7L, Zein powder 7kg, ammonium sulfate 0.7kg, light calcium carbonate 3kg.
Seed culture medium sterilizing is also cooled to 27 DEG C, and uses sterile air pressurize, and the quality of the seed culture medium after sterilizing is amino nitrogen 51mg/100ml, molten phosphorus 71ug/ml, total reducing sugar 20mg/100ml, pH7.5.Then, under flame protection, cultured strepto-starter bottle fermented liquid is cultivated according in the inoculum size access seed culture medium of 0.25L, tank pressure 0.03 ~ 0.05MPa; Tank temperature 30 DEG C; Air flow quantity: 0 ~ 5h, adopts pneumatic blending to replace mechanical stirring; 6h ~ culture transferring: 40m
3/ h; Mixing speed 70r/min.Seed culture terminates, cell concentration 25%, pH value 7.5, incubation time 45h.
fermention medium: seeding tank volume 15m
3, effective volume is 10m
3.
Fermention medium: soya-bean oil 100L, maltose 120kg, low temperature soybean cake powder 270kg, groundnut meal 170kg, corn steep liquor 130L, Zein powder 130kg, ammonium sulfate 7.5kg, light calcium carbonate 30kg, potassium primary phosphate 7kg, sodium-chlor 5kg, magnesium sulfate 0.45kg, ferrous sulfate 3kg, polyoxypropylene glyceryl 3.5kg, maltin 0.03kg.
First by fermention medium sterilizing, be cooled to 23 DEG C, and use sterile air pressurize, the quality of the fermention medium after sterilizing is amino nitrogen 71mg/100ml, molten phosphorus 126ug/ml, total reducing sugar 35mg/100ml, pH6.6.Cultured seed liquor is moved into fermention medium and carries out fermentation culture, culture transferring amount is 0.9m
3.Fermentation culture terminates, cell concentration 37%, total reducing sugar 3.9g/100ml, fatty 0.4g/L, amino nitrogen 15mg/100ml, chemical titer 4157u/mL, pH5.5, incubation time 235h.
embodiment 4
seed culture medium: seeding tank volume 1.5m
3, effective volume is 1m
3.
Seed culture medium: Semen Maydis oil 9L, maltose 14kg, low temperature soybean cake powder 19kg, groundnut meal 14kg, corn steep liquor 8L, Zein powder 9kg, ammonium sulfate 0.9kg, light calcium carbonate 4kg.
Seed culture medium sterilizing is also cooled to 29 DEG C, and uses sterile air pressurize, and the quality of the seed culture medium after sterilizing is amino nitrogen 56mg/100ml, molten phosphorus 90ug/ml, total reducing sugar 23mg/100ml, pH7.3.Then, under flame protection, cultured strepto-starter bottle fermented liquid is cultivated according in the inoculum size access seed culture medium of 0.28L, tank pressure 0.03 ~ 0.05MPa; Tank temperature 32 DEG C; Air flow quantity: 0 ~ 5h, adopts pneumatic blending to replace mechanical stirring; 6h ~ culture transferring: 45m
3/ h; Mixing speed 75r/min.Seed culture terminates, cell concentration 28%, pH value 7.3, incubation time 48h.
fermention medium: seeding tank volume 15m
3, effective volume is 10m
3.
Fermention medium: Semen Maydis oil 110L, maltose 130kg, low temperature soybean cake powder 280kg, groundnut meal 180kg, corn steep liquor 140L, Zein powder 140kg, ammonium sulfate 9kg, light calcium carbonate 40kg, potassium primary phosphate 7kg, sodium-chlor 5kg, magnesium sulfate 0.5kg, ferrous sulfate 4kg, polyoxypropylene glyceryl 3.5kg, maltin 0.03kg.
First by fermention medium sterilizing, be cooled to 25 DEG C, and use sterile air pressurize, the quality of the fermention medium after sterilizing is amino nitrogen 75mg/100ml, molten phosphorus 140ug/ml, total reducing sugar 38mg/100ml, pH6.3.Cultured seed liquor is moved into fermention medium and carries out fermentation culture, culture transferring amount is 0.95m
3.Fermentation culture terminates, cell concentration 38%, total reducing sugar 4.2g/100ml, fatty 0.6g/L, amino nitrogen 17mg/100ml, chemical titer 4108u/mL, pH5.7, incubation time 237h.
embodiment 5
seed culture medium: seeding tank volume 1.5m
3, effective volume is 1m
3.
Seed culture medium: soya-bean oil 10L, maltose 15kg, low temperature soybean cake powder 20kg, groundnut meal 15kg, corn steep liquor 9L, Zein powder 10kg, ammonium sulfate 1kg, light calcium carbonate 5kg.
Seed culture medium sterilizing is also cooled to 30 DEG C, and uses sterile air pressurize, and the quality of the seed culture medium after sterilizing is amino nitrogen 60mg/100ml, molten phosphorus 99ug/ml, total reducing sugar 25mg/100ml, pH7.1.Then, under flame protection, cultured strepto-starter bottle fermented liquid is cultivated according in the inoculum size access seed culture medium of 0.3L, tank pressure 0.03 ~ 0.05MPa; Tank temperature 33 DEG C; Air flow quantity: 0 ~ 5h, adopts pneumatic blending to replace mechanical stirring; 6h ~ culture transferring: 50m
3/ h; Mixing speed 80r/min.Seed culture terminates, cell concentration 30%, pH value 7.1, incubation time 50h.
fermention medium: seeding tank volume 15m
3, effective volume is 10m
3.
Fermention medium: Semen Maydis oil 120L, maltose 140kg, low temperature soybean cake powder 300kg, groundnut meal 200kg, corn steep liquor 150L, Zein powder 150kg, ammonium sulfate 10kg, light calcium carbonate 50kg, potassium primary phosphate 9kg, sodium-chlor 7kg, magnesium sulfate 0.6kg, ferrous sulfate 5kg, polyoxypropylene glyceryl 4kg, maltin 0.04kg.
First by fermention medium sterilizing, be cooled to 27 DEG C, and use sterile air pressurize, the quality of the fermention medium after sterilizing is amino nitrogen 79mg/100ml, molten phosphorus 150ug/ml, total reducing sugar 40mg/100ml, pH6.1.Cultured seed liquor is moved into fermention medium and carries out fermentation culture, culture transferring amount is 1m
3.Fermentation culture terminates, cell concentration 40%, total reducing sugar 4.9g/100ml, fatty 0.8g/L, amino nitrogen 20mg/100ml, chemical titer 3873u/mL, pH5.9, incubation time 240h.
In the fermenting process of above-described embodiment 1-5, according to detection case, adopt stream addition to carry out feed supplement, feed supplement comprises repairing, moisturizing, benefit ammonium sulfate, mends sugar and pH control, wherein
A oil-supplementing process controls:
Before fermentation, 60h need not carry out repairing,
Fermentation time, at 61 ~ 200h, when lipid content is lower than 1g/L, fills into soya-bean oil or Semen Maydis oil, and control lipid content is 1 ~ 1.5g/L, detects fermented liquid lipid content every 6 ~ 8h,
Fermentation time, in 201h ~ fermentation ends, when lipid content is lower than 3%, fills into soya-bean oil or Semen Maydis oil, and control lipid content is 0.5 ~ 0.8g/L, detects fermented liquid lipid content every 6 ~ 8h;
B moisturizing technology controlling and process:
Before fermentation, 60h need not carry out moisturizing,
Fermentation time, at 61 ~ 200h, when cell concentration is higher than 50%, adds aqua sterilisa, requires to control fermented liquid cell concentration 40 ~ 45%, detects fermented liquid cell concentration every 6 ~ 8h,
Fermentation time, in 201h ~ fermentation ends, when cell concentration is higher than 40%, adds aqua sterilisa, requires to control fermented liquid cell concentration 35 ~ 40%, detects fermented liquid cell concentration every 6 ~ 8h;
C fermenting process pH Controlling Technology:
Before fermentation, 60h, pH do not control,
Fermentation time is at 61 ~ 200h, if pH < 6.0, adds the sodium hydroxide of 10 ~ 20%, regulates pH to 6 ~ 7; If pH > 7, add the ammoniumsulphate soln of 30%, regulate pH to 6 ~ 7;
Fermentation time is in 201 ~ fermentation ends, if pH < 6.5, adds the sodium hydroxide of 10 ~ 20%, regulates pH control to 5 ~ 6; If pH > 6, add the ammoniumsulphate soln of 30%, regulate pH to 5 ~ 6;
D fills into maltose:
60h before fermentation, total sugar content does not control,
During fermentation 61h to 200h: total sugar content < 10mg/100ml, fill into sterilized maltose solution, the content of total reducing sugar is controlled at 10 ~ 15mg/100ml.
201h is to fermentation ends in fermentation: during total sugar content < 3mg/100ml, fill into sterilized maltose solution, the content of total reducing sugar is controlled at 3 ~ 5mg/100ml.
Add maltin in above-mentioned benefit sugar technique, its concentration controls 0.001 ~ 0.003%.
E mends ammonium sulfate
How, in horse rhzomorph fermentation culture process, fill into the ammoniumsulphate soln of 30% respectively at 60h, 150h and 220h, its consumption controls at 0.1 ~ 0.3% of fermentating liquid volume.
Claims (7)
1. streptomycete fermentation produces a substratum for how horse rhzomorph, it is characterized in that comprising seed culture medium and fermention medium, wherein
Consisting of of described seed culture medium: soya-bean oil or Semen Maydis oil 5 ~ 10ml/L, maltose 10 ~ 15g/L, low temperature soybean cake powder 15 ~ 20g/L, groundnut meal 10 ~ 15g/L, corn steep liquor 5 ~ 9ml/L, Zein powder 5 ~ 10g/L, ammonium sulfate 0.5 ~ 1g/L, light calcium carbonate 1 ~ 5g/L;
Consisting of of described fermention medium: soya-bean oil or Semen Maydis oil 8 ~ 12ml/L, maltose 10 ~ 14g/L, low temperature soybean cake powder 25 ~ 30g/L, groundnut meal 15 ~ 20g/L, corn steep liquor 10 ~ 15ml/L, Zein powder 10 ~ 15g/L, ammonium sulfate 0.5 ~ 1g/L, light calcium carbonate 1 ~ 5g/L, potassium primary phosphate 0.5 ~ 0.9g/L, sodium-chlor 0.3 ~ 0.7g/L, magnesium sulfate 0.03 ~ 0.06g/L, ferrous sulfate 0.1 ~ 0.5g/L, polyoxypropylene glyceryl 0.3 ~ 0.4g/L, maltin 0.002 ~ 0.004g/L.
2. utilize substratum described in claim 1 to produce a cultural method for how horse rhzomorph, it is characterized in that its processing step is:
1) seed culture: first seed culture medium sterilizing is cooled to 25 ~ 30 DEG C, and uses sterile air pressurize, then under flame protection, by cultured strepto-starter bottle fermented liquid according to 0.2 ~ 0.3L/m
3inoculum size access seed culture medium in carry out seed culture, to culture transferring when cell concentration 20 ~ 30%, pH value 7 ~ 8, incubation time 40 ~ 50h, culture transferring ratio control is at 0.8 ~ 1m
3/ m
3;
2) fermentation culture: first by fermention medium sterilizing, be cooled to 20 ~ 27 DEG C, and use sterile air pressurize, then cultured seed liquor is moved into fermention medium and carry out fermentation culture, stop fermentation to when cell concentration 35 ~ 40%, total reducing sugar < 5mg/100ml, fatty < 0.8g/L, amino nitrogen 10 ~ 20mg/100ml, chemical titer > 3800u/mL, pH5 ~ 6, incubation time 230 ~ 240h.
3. according to cultural method according to claim 2, it is characterized in that the specification of quality of the seed culture medium after described sterilizing: amino nitrogen 40 ~ 60mg/100ml, molten phosphorus 50 ~ 100ug/ml, total reducing sugar 15 ~ 25mg/100ml, pH7 ~ 8;
The specification of quality of the fermention medium after sterilizing: amino nitrogen 60 ~ 80mg/100ml, molten phosphorus 100 ~ 150ug/ml, total reducing sugar 30 ~ 40 mg/100ml, pH6 ~ 7.
4., according to cultural method according to claim 2, it is characterized in that described cultured strepto-starter bottle fermented liquid specification of quality is: pH6 ~ 8; Cell concentration 10 ~ 30%; Microscopy is without miscellaneous bacteria; Fermentation unit 800 ~ 1500u/ml.
5., according to cultural method according to claim 2, it is characterized in that described seed culture condition is: tank pressure 0.03 ~ 0.05MPa; Tank temperature 28 ~ 33 DEG C; Air flow quantity: 0 ~ 5h, adopts pneumatic blending to replace mechanical stirring; 6h ~ culture transferring: 30 ~ 50m
3/ h; Mixing speed 60 ~ 80r/min.
6., according to cultural method according to claim 2, it is characterized in that described fermentation culture conditions is:
The initial pH of a substratum: after fermention medium sterilizing, pH controls 6 ~ 7;
B temperature: adopt alternating temperature Controlling Technology,
0 ~ 60h: culture temperature 29 ~ 30 DEG C,
61 ~ 200h: culture temperature 31 ~ 32 DEG C,
201h ~ fermentation ends: culture temperature 30 ~ 31 DEG C;
C mixing speed: adopt frequency conversion mixing control technique,
0 ~ 60h: rotating speed controls at 100r/min,
61 ~ 200h: rotating speed controls at 120r/min,
201h ~ fermentation ends: rotating speed controls at 90r/min;
D pressure-controlling: tank pressure 0.05 ~ 0.06MPa;
E pH controls: in fermenting process, pH controls 5 ~ 7;
F air flow quantity: 0 ~ 60h:100 ~ 150m
3/ h; 61 ~ 200h:200 ~ 300m
3/ h; 201h ~ fermentation ends: 150 ~ 200m
3/ h;
H dissolved oxygen controls:
Before fermentation in 60h, do not control dissolved oxygen;
60 ~ 100h, dissolved oxygen controls more than 60%;
101 ~ 200h, dissolved oxygen controls more than 30%;
201 ~ fermentation ends: dissolved oxygen controls more than 40%.
7. according to cultural method according to claim 2, it is characterized in that adopting stream addition to carry out feed supplement during the fermentation, feed supplement comprises repairing, moisturizing, benefit ammonium sulfate, mends sugar and pH control, wherein
A oil-supplementing process controls:
Before fermentation, 60h need not carry out repairing,
Fermentation time, at 61 ~ 200h, when lipid content is lower than 1g/L, fills into soya-bean oil or Semen Maydis oil, and control lipid content is 1 ~ 1.5g/L, detects fermented liquid lipid content every 6 ~ 8h,
Fermentation time, in 201h ~ fermentation ends, when lipid content is lower than 3%, fills into soya-bean oil or Semen Maydis oil, and control lipid content is 0.5 ~ 0.8g/L, detects fermented liquid lipid content every 6 ~ 8h;
B moisturizing technology controlling and process:
Before fermentation, 60h need not carry out moisturizing,
Fermentation time, at 61 ~ 200h, when cell concentration is higher than 50%, adds aqua sterilisa, requires to control fermented liquid cell concentration 40 ~ 45%, detects fermented liquid cell concentration every 6 ~ 8h,
Fermentation time, in 201h ~ fermentation ends, when cell concentration is higher than 40%, adds aqua sterilisa, requires to control fermented liquid cell concentration 35 ~ 40%, detects fermented liquid cell concentration every 6 ~ 8h;
C fermenting process pH Controlling Technology:
Before fermentation, 60h, pH do not control,
Fermentation time is at 61 ~ 200h, if pH < 6.0, adds the sodium hydroxide of 10 ~ 20%, regulates pH to 6 ~ 7; If pH > 7, add the ammoniumsulphate soln of 30%, regulate pH to 6 ~ 7;
Fermentation time is in 201 ~ fermentation ends, if pH < 6.5, adds the sodium hydroxide of 10 ~ 20%, regulates pH control to 5 ~ 6; If pH > 6, add the ammoniumsulphate soln of 30%, regulate pH to 5 ~ 6;
D fills into maltose:
60h before fermentation, total sugar content does not control,
During fermentation 61h to 200h: total sugar content < 10mg/100ml, fill into sterilized maltose solution, the content of total reducing sugar is controlled at 10 ~ 15mg/100ml.
201h is to fermentation ends in fermentation: during total sugar content < 3mg/100ml, fill into sterilized maltose solution, the content of total reducing sugar is controlled at 3 ~ 5mg/100ml.
Add maltin in above-mentioned benefit sugar technique, its concentration controls 0.001 ~ 0.003%.
E mends ammonium sulfate
How, in horse rhzomorph fermentation culture process, fill into the ammoniumsulphate soln of 30% respectively at 60h, 150h and 220h, its consumption controls at 0.1 ~ 0.3% of fermentating liquid volume.
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| CN107267569A (en) * | 2017-07-25 | 2017-10-20 | 宁夏泰瑞制药股份有限公司 | A kind of production of utilization streptomycete fermentation how the culture medium of horse rhzomorph |
| CN110387390A (en) * | 2019-06-27 | 2019-10-29 | 华东理工大学青岛创新研究院 | Fermentation medium and zymotechnique |
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