CN105506019A - Method for industrially producing L-hydroxyproline through fermentation method - Google Patents

Method for industrially producing L-hydroxyproline through fermentation method Download PDF

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CN105506019A
CN105506019A CN201511035144.1A CN201511035144A CN105506019A CN 105506019 A CN105506019 A CN 105506019A CN 201511035144 A CN201511035144 A CN 201511035144A CN 105506019 A CN105506019 A CN 105506019A
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tank
inoculation
fermentation
pressure
oxyproline
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赵体金
林俊辉
彭久合
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TIANJIN JINGYE FINE CHEMICALS CO Ltd
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TIANJIN JINGYE FINE CHEMICALS CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/24—Proline; Hydroxyproline; Histidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Abstract

Provided is a method for industrially producing L-hydroxyproline through a fermentation method. The method comprises the steps of strain solid culture, fermentation culture, fermentation liquor extraction and purification. According to the method for industrially producing the L-hydroxyproline through the fermentation method, the L-hydroxyproline is industrially produced through a direct glucose fermentation method, the fermentation unit, yield and quality of the L-hydroxyproline is improved, the production cost is reduced, and three-waste pollution is avoided.

Description

The method of fermentation method suitability for industrialized production L-oxyproline
Technical field
The present invention relates to fermentation engineering, be specifically related to the method that microorganism directly utilizes glucose fermentation method suitability for industrialized production L-oxyproline.
Background technology
Oxyproline (Hydroxyproline, Hyp) is imino-acid, is the product after L-PROLINE hydroxylation, and its molecular formula is C 5h 9nO 3.Oxyproline is white flaky crystals or crystalline powder, micro-sweet, and fusing point is 274 DEG C, soluble in water, is slightly soluble in ethanol.Occurring in nature trans-4-oxyproline is comparatively common, trans-and 4-Hydroxyproline is found in animal collagen the earliest.Trans-4-Hydroxyproline is widely used in the aspects such as medicine, chemical industry, animal-feed, nutrition and beauty culture.
At present, the method for the production oxyproline of report mainly contains proteolysis extraction method, chemical synthesis, microbe fermentation method and enzymatic synthesis four kinds both at home and abroad, and wherein proteolysis extraction method is the industrial process generally adopted at present.But, proteolysis extraction method need through strong acid and strong base process, purification step is long, and oxyproline extraction yield is at 4%-7%, extract and raw materials cost high, not only waste large content of starting materials, and waste pollution is serious, along with the increase of current environmental stress and the rising of resource cost of material, traditional proteolysis extraction method is just gradually by market.Microbe fermentation method just develops rapidly with the technique of its environmental protection, raw material and advantage with low cost.
At present, the research of Production by Microorganism Fermentation L-oxyproline is also in laboratory stage, not yet form ripe industrialized producing technology, and the industrialized producing technology of advanced person, especially the solid culture of bacterial classification and liquid fermentation culturing method, the extracting method of fermented liquid, the purification process of concentrated solution have vital impact for the fermentation unit of L-oxyproline, yield, quality, cost and three waste discharge.
Summary of the invention
The object of the invention is to: a kind of method that fermentation method suitability for industrialized production L-oxyproline is provided, adopt the method suitability for industrialized production L-oxyproline, be conducive to the fermentation unit, yield, the quality that improve L-oxyproline, reduce production cost, avoid three-waste pollution.
Technical solution of the present invention is:
The method of fermentation method suitability for industrialized production L-oxyproline, step comprises bacterial classification solid culture, fermentation culture, broth extraction and purifying:
(1) bacterial classification solid culture: the NaOH solution of bacterial classification solid medium 2N adjusts pH to 7.0, be sub-packed in 10ml slant tube or 50ml eggplant bottle, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, after taking-up is cooled to 50 ~ 60 DEG C, adding the AMP solution of 10% in sterilisable chamber, is 50ppm to AMP final concentration, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on test tube or the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 24 ~ 48 hours, lawn is grown rear directly upper tank or is placed in 0 ~ 5 DEG C of refrigerator for subsequent use; Wherein bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation; Bacterial classification solid medium is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L; Wherein concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate;
(2) fermentation culture: be first that bacterial classification cultivates the seed obtaining maturation in liquid seed culture medium, then moves into fermentating metabolism in fermention medium and produces L-oxyproline, wherein liquid seed culture medium aqueous formulation (g/L) is: glucose 40, yeast powder 7.5, ammonium sulfate 2.0, potassium primary phosphate 7.5, Repone K 1.9, FeSO 47H 2o0.28, MgSO 47H 2o0.32, trace element (ppm): MnSO 4h 2o0.5, CoCl 26H 2o1.4, NiSO 46H 2o1.2, CuSO 47H 2o0.5, ZnSO 47H 2o0.6, VB 120, defoamer 100, AMP50, wherein fermentative medium formula (g/L) is as follows: glucose 20, yeast powder 5.0, ammonium sulfate 10, potassium primary phosphate 5.0, Repone K 1.9, MgSO 47H 2o0.32, FeSO 47H 2o0.75, trace element (ppm): MnSO 4h 2o1.0, CoCl 26H 2o1.4, NiSO 46H 2o1.4, CuSO 47H 2o1.0, ZnSO 47H 2o1.2, defoamer 200, AMP50, wherein the cultivation of mature seed comprises the following steps: 1. slack tank sterilizing: 0.15-0.20MPa, pressurize 30 minutes, 2. real tank sterilizing: liquid seed culture medium drop into seeding tank, 2.4T liquid seed culture medium joined by 4T seeding tank, adjust pH to 7.0, close cover, chuck is heated to 90 DEG C, direct steam heating to 121 DEG C, 0.1MPa pressure heat insulating 10 minutes, 3. cool: sterilizing is complete cool to 36 DEG C to be seeded, 4. inoculate: adopt pressure differential method by bacterium liquid access tank, inoculation temp 36 DEG C, pH7.0 ~ 9.0, pressure 0.1MPa, open inoculation mouth protective cover, firmly mouth is inoculated with the cotton snare soaking 95% ethanol, light cotton circle, move to and light cotton circle by including the inoculation bottle collecting the bacterium liquid got by solid culture spawn culture and inoculate by mouth, to outward winding inoculation bottle Tip protection plug, rapidly inoculation bottle syringe needle is inserted inoculation mouth, slowly open vent valve on tank, pressure regulation 0.05 ~ 0.1MPa, inoculum size 1 bottle, after inoculation terminates, be placed on inoculation mouth with the cotton ball of dipped 75% ethanol, transfer to inoculation syringe needle, screw on inoculation mouth protective cover, start to cultivate, 5. cultivate: culture temperature 36 DEG C, tank pressure 0.03MPa, air quantity 0.2 ~ 1.2VVM, pH6.5, mixing speed 200 ~ 600rpm, dissolved oxygen more than 20%, incubation time 8 ~ 24 hours, OD 600reach 10 ~ 20, final glucose content is less than 10g/L, wherein fermentation culture comprises the following steps: 1. slack tank sterilizing: 0.15-0.17MPa pressure, temperature 128 DEG C of sterilizings 30 minutes, 2. real tank sterilizing: fermention medium drops in tank, and 30T tank adds 22T substratum, close cover, chuck is preheating to 95 ~ 105 DEG C, and direct steam heating is to 121 DEG C, and pressure 0.1MPa, is incubated 10 minutes, cools to 35 DEG C, adjusts pH to 6.5, standby inoculation, 3. inoculate: adopt pressure differential method seed liquor to be pressed in tank and cultivate, 4. cultivate: culture temperature 35 DEG C, tank pressure 0.03MPa, air quantity 0.5 ~ 1.2VVM, pH6.5, mixing speed 60 ~ 300rpm, during fermentation ends, oxyproline generates concentration at 30 ~ 90g/L, sugared Nong Du≤2.0%, 5. after fermentation ends, fermented liquid is heated to 80 DEG C, is cooled to 40 DEG C immediately, is depressed into fermented liquid basin with pressurized air,
(3) broth extraction: comprise the following steps successively: first fermented liquid micro-filtration, fermented liquid pump in storage tank is squeezed in heating tank, heat and be incubated 40 ± 5 DEG C, open film filter to filter fermented liquid, micro-filtration temperature 35 ~ 45 DEG C, micro-filtration pressure 0.30Ma, micro-filtration flow velocity 80 ~ 100L/m 2/ h, membrane pore size 0.05 ~ 1.0um, film thickness 10 ~ 150um; Then, the further ultrafiltration of the filtrate after micro-filtration, ultrafiltrate temperature 40 ~ 50 DEG C, ultrafiltration pressure 1.0MPa, ultrafiltration flow velocity 30 ~ 35L/m 2/ h, membrane pore size 0.015 ~ 0.1um, film thickness 150 ~ 250um, asymmetric membrane; Finally, the further nanofiltration of purification liquid after ultrafiltration obtains concentrated solution, nanofiltration temperature 45 ~ 55 DEG C, nanofiltration pressure 0.5 ~ 3.5MPa, nanofiltration flow velocity 2.5 ~ 4.5L/m 2/ h, membrane pore size 1 ~ 5nm, film thickness 100 ~ 300um, asymmetric membrane;
(4) purifying: comprise coarse crystallization and treating process successively, wherein, coarse crystallization process is: 1. squeeze in crystallizer by concentrated solution, stirs rapidly and is cooled to 0 ~ 10 DEG C, 5 ± 3 DEG C of quiescent crystallization 4 hours; 2. by crude product centrifuge dripping, frozen water drip washing, dry 6 hours of 60 DEG C of heat circulation drying ovens, control Shui Fen≤5%, obtains crude product; Treating process is: 1. under agitation added by crude product in 60 DEG C of pure water of 20 times of weight, is warming up to 75 DEG C and dissolves completely; 2. add 769 activated carbon by 10% of crude product weight, 75 DEG C of decolourings 15 minutes, filter; 3. destainer is cooled to 0 ~ 10 DEG C through cooling coil, by volume 15% industrial acetic acid adding concentration 80%, stirs after having a large amount of crystal to separate out and stops stirring, 5 ± 3 DEG C of insulation crystallizations 4 hours, mother liquid concentration 2 ~ 3g/L at the end of crystallization; 4. crystal solution is proceeded to centrifuge, frozen water drip washing, wet-milling enters double cone dryer 80 DEG C of vacuum-dryings and obtains L-oxyproline fine work in 4 ~ 6 hours.
The present invention adopts biological fermentation process suitability for industrialized production L-oxyproline, through spawn culture, fermentation culture, broth extraction and purification procedures, is conducive to the fermentation unit, yield, the quality that improve L-oxyproline, reduces production cost, avoid three-waste pollution.
Embodiment
Below in conjunction with specific embodiment, in further detail the present invention is described.Should be understood that these embodiments just in order to demonstrate the invention, but not limit the scope of the invention by any way.
Example 1
1, bacterial classification solid culture: the NaOH solution of bacterial classification solid medium 2N adjusts pH to 7.0, be sub-packed in 10ml slant tube, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, in sterilisable chamber, the AMP solution 5ul of 10% is added after taking-up is cooled to 50 DEG C, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on test tube slant at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 24 hours, lawn directly plants female bottle after growing; Wherein bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation; Bacterial classification solid medium is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L.Wherein concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate.
2, fermentation culture: first cultivate in bacterial classification liquid medium within and obtain ripe seed, then moves into fermentating metabolism in fermention medium and produces L-oxyproline; Wherein liquid seed culture medium aqueous formulation (g/L) is: glucose 40, yeast powder 7.5, ammonium sulfate 2.0, potassium primary phosphate 7.5, Repone K 1.9, FeSO 47H 2o0.28, MgSO 47H 2o0.32, trace element (ppm): MnSO 4h 2o0.5, CoCl 26H 2o1.4, NiSO 46H 2o1.2, CuSO 47H 2o0.5, ZnSO 47H 2o0.6, VB 120, defoamer 100, AMP50.Wherein fermentative medium formula (g/L) is as follows: glucose 20, yeast powder 5.0, ammonium sulfate 10, potassium primary phosphate 5.0, Repone K 1.9, MgSO 47H 2o0.32, FeSO 47H 2o0.75, trace element (ppm): MnSO 4h 2o1.0, CoCl 26H 2o1.4, NiSO 46H 2o1.4, CuSO 47H 2o1.0, ZnSO 47H 2o1.2, defoamer 200, AMP50, wherein the cultivation of mature seed comprises the following steps: 1. slack tank sterilizing: 0.15MPa, pressurize 30 minutes, 2. real tank sterilizing: liquid seed culture medium drop into tank, 2.4T seed culture medium joined by 4T seeding tank, adjust pH to 7.0, close cover, chuck is heated to 90 DEG C, direct steam heating to 121 DEG C, 0.1MPa pressure heat insulating 10 minutes, 3. cool: sterilizing is complete cool to 36 DEG C to be seeded, 4. inoculate: adopt pressure differential method by bacterium liquid access tank, inoculation temp 36 DEG C, pH7.2, pressure 0.1MPa, open inoculation mouth protective cover, firmly mouth is inoculated with the cotton snare soaking 95% ethanol, light cotton circle, move to and light cotton circle by including the inoculation bottle collecting the bacterium liquid got by solid culture spawn culture and inoculate by mouth, to outward winding inoculation bottle Tip protection plug, rapidly inoculation bottle syringe needle is inserted inoculation mouth, slowly open vent valve on tank, pressure regulation is to 0.05MPa, inoculum size 1 bottle, after inoculation terminates, be placed on inoculation mouth with the cotton ball of dipped 75% ethanol, transfer to inoculation syringe needle, screw on inoculation mouth protective cover, start to cultivate, 5. cultivate: culture temperature 36 DEG C, tank pressure 0.03MPa, air quantity 0.2 ~ 1.0VVM, pH6..5, mixing speed 200rpm, dissolved oxygen more than 20%, incubation time 12 hours, OD 600reach 12, final glucose content 2g/L, wherein fermentation culture comprises the following steps: 1. slack tank sterilizing: 0.15MPa pressure, temperature 127 DEG C of sterilizings 30 minutes, 2. real tank sterilizing: fermention medium drops in tank, and 30T tank adds 22T substratum, close cover, chuck is preheating to 95 DEG C, and direct steam heating is to 121 DEG C, and pressure 0.1MPa, is incubated 10 minutes, cools to 35 DEG C, adjusts pH to 6.5, standby inoculation, 3. inoculate: adopt pressure differential method seed liquor to be pressed in tank and cultivate, 4. cultivate: culture temperature 35 DEG C, tank pressure 0.03MPa, air quantity 0.5 ~ 1.0VVM, pH6.5, mixing speed 180rpm, during fermentation ends, oxyproline generates concentration in 43g/L, sugared concentration 0.5%, 5. after fermentation ends, fermented liquid is heated to 80 DEG C, is cooled to 40 DEG C immediately, is depressed into fermented liquid basin with pressurized air,
3, broth extraction: first fermented liquid micro-filtration, the fermented liquid pump in storage tank is squeezed in heating tank, heats and is incubated 40 ± 5 DEG C, opens film filter and filters fermented liquid, micro-filtration temperature 37 DEG C, micro-filtration pressure 0.30Ma, micro-filtration flow velocity 85L/m 2/ h, membrane pore size 1.0um, film thickness 150um; Then, the further ultrafiltration of the filtrate after micro-filtration, ultrafiltrate temperature 40 DEG C, ultrafiltration pressure 1.0MPa, ultrafiltration flow velocity 30L/m 2/ h, membrane pore size 0.1um, film thickness 150um, asymmetric membrane; Finally, the further nanofiltration of purification liquid after ultrafiltration obtains concentrated solution, nanofiltration temperature 45 C, nanofiltration pressure 2.2MPa, nanofiltration flow velocity 3L/m 2/ h, membrane pore size 3nm, film thickness 150um, asymmetric membrane;
4, purifying: comprise coarse crystallization and treating process successively, wherein, coarse crystallization process is: 1. squeeze in crystallizer by concentrated solution, stirs rapidly and is cooled to, 5 ± 3 DEG C of quiescent crystallization 4 hours; 2. by crude product centrifuge dripping, frozen water drip washing, dry 6 hours of 60 DEG C of heat circulation drying ovens, moisture content 4%, obtains crude product, and treating process is: 1. under agitation added by crude product in 60 DEG C of pure water of 20 times of weight, is warming up to 75 DEG C and dissolves completely; 2. add 769 activated carbon by 10% of crude product weight, 75 DEG C of decolourings 15 minutes, filter; 3. destainer is cooled to 5 DEG C through cooling coil, by volume 15% industrial acetic acid adding concentration 80%, stirs after having a large amount of crystal to separate out and stops stirring, 5 ± 3 DEG C of insulation crystallizations 4 hours, mother liquid concentration 2g/L at the end of crystallization; 4. crystal solution is proceeded to centrifuge, frozen water drip washing, wet-milling enters double cone dryer 80 DEG C of vacuum-dryings and obtains L-oxyproline fine work in 4.5 hours.
Example 2
1, bacterial classification solid culture: the NaOH solution of bacterium culture medium 2N adjusts pH to 7.0, be sub-packed in 50ml eggplant bottle, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, in sterilisable chamber, the AMP solution 25ul of 10% is added after taking-up is cooled to 55 DEG C, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 38 hours, lawn is placed in 0 ~ 5 DEG C of refrigerator for subsequent use after growing; Wherein bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation; Bacterial classification solid medium is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L.Wherein concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate.
2, fermentation culture: be first cultivate in bacterial classification liquid medium within to obtain ripe seed, then move into fermentating metabolism in fermention medium and produce L-oxyproline; Wherein liquid seed culture medium aqueous formulation (g/L) is: glucose 40, yeast powder 7.5, ammonium sulfate 2.0, potassium primary phosphate 7.5, Repone K 1.9, FeSO 47H 2o0.28, MgSO 47H 2o0.32, trace element (ppm): MnSO 4h 2o0.5, CoCl 26H 2o1.4, NiSO 46H 2o1.2, CuSO 47H 2o0.5, ZnSO 47H 2o0.6, VB 120, defoamer 100, AMP50.Wherein fermentative medium formula (g/L) is as follows: glucose 20, yeast powder 5.0, ammonium sulfate 10, potassium primary phosphate 5.0, Repone K 1.9, MgSO 47H 2o0.32, FeSO 47H 2o0.75, trace element (ppm): MnSO 4h 2o1.0, CoCl 26H 2o1.4, NiSO 46H 2o1.4, CuSO 47H 2o1.0, ZnSO 47H 2o1.2, defoamer 200, AMP50, wherein the cultivation of mature seed comprises the following steps: 1. slack tank sterilizing: 0.18MPa, pressurize 30 minutes, 2. real tank sterilizing: liquid seed culture medium drop into tank, 2.4T seed culture medium joined by 4T seeding tank, adjust pH to 7.0, close cover, chuck is heated to 90 DEG C, direct steam heating to 121 DEG C, 0.1MPa pressure heat insulating 10 minutes, 3. cool: sterilizing is complete cool to 36 DEG C to be seeded, 4. inoculate: adopt pressure differential method by bacterium liquid access tank, inoculation temp 36 DEG C, pH7.8, pressure 0.1MPa, open inoculation mouth protective cover, firmly mouth is inoculated with the cotton snare soaking 95% ethanol, light cotton circle, move to and light cotton circle by including the inoculation bottle collecting the bacterium liquid got by solid culture spawn culture and inoculate by mouth, to outward winding inoculation bottle Tip protection plug, rapidly inoculation bottle syringe needle is inserted inoculation mouth, slowly open vent valve on tank, pressure regulation 0.06MPa, inoculum size 1 bottle, after inoculation terminates, be placed on inoculation mouth with the cotton ball of dipped 75% ethanol, transfer to inoculation syringe needle, screw on inoculation mouth protective cover, start to cultivate, 5. cultivate: culture temperature 36 DEG C, tank pressure 0.03MPa, air quantity 0.6 ~ 1.0VVM, pH6.5, mixing speed 300rpm, dissolved oxygen more than 20%, incubation time 14 hours, OD 600reach 16, final glucose content 4g/L, wherein fermentation culture comprises the following steps: 1. slack tank sterilizing: 0.16MPa pressure, temperature 128 DEG C of sterilizings 30 minutes, 2. real tank sterilizing: fermention medium drops in tank, and 30T tank adds 22T substratum, close cover, chuck is preheating to 100 DEG C, and direct steam heating is to 121 DEG C, and pressure 0.1MPa, is incubated 10 minutes, cools to 35 DEG C, adjusts pH to 6.5, standby inoculation, 3. inoculate: adopt pressure differential method seed liquor to be pressed in tank and cultivate, 4. cultivate: culture temperature 35 DEG C, tank pressure 0.03MPa, air quantity 0.6 ~ 1.0VVM, pH6.5, mixing speed 200rpm, during fermentation ends, oxyproline generates concentration in 48g/L, sugared concentration 0.2%, 5. after fermentation ends, fermented liquid is heated to 80 DEG C, is cooled to 40 DEG C immediately, is depressed into fermented liquid basin with pressurized air,
3, broth extraction: comprise the following steps successively: first fermented liquid micro-filtration, the fermented liquid pump in storage tank is squeezed in heating tank, heats and is incubated 40 ± 5 DEG C, open film filter to filter fermented liquid, micro-filtration temperature 40 DEG C, micro-filtration pressure 0.30Ma, micro-filtration flow velocity 90L/m 2/ h, membrane pore size 1.0um, film thickness 150um; Then, the further ultrafiltration of the filtrate after micro-filtration, ultrafiltrate temperature 45 DEG C, ultrafiltration pressure 1.0MPa, ultrafiltration flow velocity 35L/m 2/ h, membrane pore size 0.1um, film thickness 150um, asymmetric membrane; Finally, the further nanofiltration of purification liquid after ultrafiltration obtains concentrated solution, nanofiltration temperature 50 C, nanofiltration pressure 2.5MPa, nanofiltration flow velocity 3.5L/m 2/ h, membrane pore size 3nm, film thickness 150um, asymmetric membrane;
4, purifying: comprise coarse crystallization and treating process successively, wherein, coarse crystallization process is: 1. squeeze in crystallizer by concentrated solution, stir cooling rapidly, 5 ± 3 DEG C of quiescent crystallization 4 hours; 2. by crude product centrifuge dripping, frozen water drip washing, dry 6 hours of 60 DEG C of heat circulation drying ovens, moisture content 2%, obtains crude product, and treating process is: 1. under agitation added by crude product in 60 DEG C of pure water of 20 times of weight, is warming up to 75 DEG C and dissolves completely; 2. add 769 activated carbon by 10% of crude product weight, 75 DEG C of decolourings 15 minutes, filter; 3. destainer is cooled to 4 DEG C through cooling coil, by volume 15% industrial acetic acid adding concentration 80%, stirs after having a large amount of crystal to separate out and stops stirring, 5 ± 3 DEG C of insulation crystallizations 4 hours, mother liquid concentration 2g/L at the end of crystallization; 4. crystal solution is proceeded to centrifuge, frozen water drip washing, wet-milling enters double cone dryer 80 DEG C of vacuum-dryings and obtains fine work in 5 hours.
Example 3
1, bacterial classification solid culture: the NaOH solution of bacterium culture medium 2N adjusts pH to 7.0, be sub-packed in 50ml eggplant bottle, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, in sterilisable chamber, the AMP solution 25ul of 10% is added after taking-up is cooled to 60 DEG C, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 36 hours, lawn is placed in 0 ~ 5 DEG C of refrigerator for subsequent use after growing; Wherein bacterial classification derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation; Bacterial classification solid medium is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L.Wherein concentration be 10% AMP solution to be dissolved in distilled water obtained by Ampicillin Trihydrate.
2, fermentation culture: be first cultivate in bacterial classification liquid medium within to obtain ripe seed, then move into fermentating metabolism in fermention medium and produce L-oxyproline; Wherein liquid seed culture medium aqueous formulation (g/L) is: glucose 40, yeast powder 7.5, ammonium sulfate 2.0, potassium primary phosphate 7.5, Repone K 1.9, FeSO47H2O0.28, MgSO47H2O0.32, trace element (ppm): MnSO4H2O0.5, CoCl26H2O1.4, NiSO46H2O1.2, CuSO47H2O0.5, ZnSO47H2O0.6, VB120, defoamer 100, AMP50.Wherein fermentative medium formula (g/L) is as follows: glucose 20, yeast powder 5.0, ammonium sulfate 10, potassium primary phosphate 5.0, Repone K 1.9, MgSO47H2O0.32, FeSO47H2O0.75, trace element (ppm): MnSO4H2O1.0, CoCl26H2O1.4, NiSO46H2O1.4, CuSO47H2O1.0, ZnSO47H2O1.2, defoamer 200, AMP50, wherein the cultivation of mature seed comprises the following steps: 1. slack tank sterilizing: 0.18MPa, pressurize 30 minutes, 2. real tank sterilizing: liquid seed culture medium drop into tank, 2.4T seed culture medium joined by 4T seeding tank, adjust pH to 7.0, close cover, chuck is heated to 90 DEG C, direct steam heating to 121 DEG C, 0.1MPa pressure heat insulating 10 minutes, 3. cool: sterilizing is complete cool to 36 DEG C to be seeded, 4. inoculate: adopt pressure differential method by bacterium liquid access tank, inoculation temp 36 DEG C, pH8.2, pressure 0.1MPa, open inoculation mouth protective cover, firmly mouth is inoculated with the cotton snare soaking 95% ethanol, light cotton circle, move to and light cotton circle by including the inoculation bottle collecting the bacterium liquid got by solid culture spawn culture and inoculate by mouth, to outward winding inoculation bottle Tip protection plug, rapidly inoculation bottle syringe needle is inserted inoculation mouth, slowly open vent valve on tank, pressure regulation 0.08MPa, inoculum size 1 bottle, after inoculation terminates, be placed on inoculation mouth with the cotton ball of dipped 75% ethanol, transfer to inoculation syringe needle, screw on inoculation mouth protective cover, start to cultivate, 5. cultivate: culture temperature 36 DEG C, tank pressure 0.03MPa, air quantity 1VVM, pH6.5, mixing speed 400rpm, dissolved oxygen more than 20%, incubation time 18 hours, OD600 reach 18, final glucose content 1g/L, wherein fermentation culture comprises the following steps: 1. slack tank sterilizing: 0.17MPa pressure, temperature 129 DEG C of sterilizings 30 minutes, 2. real tank sterilizing: fermention medium drops in tank, and 30T tank adds 22T substratum, close cover, chuck is preheating to 105 DEG C, and direct steam heating is to 121 DEG C, and pressure 0.1MPa, is incubated 10 minutes, cools to 35 DEG C, adjusts pH to 6.5, standby inoculation, 3. inoculate: adopt pressure differential method seed liquor to be pressed in tank and cultivate, 4. cultivate: culture temperature 35 DEG C, tank pressure 0.03MPa, air quantity 0.8 ~ 1.2VVM, pH6.5, mixing speed 280rpm, during fermentation ends, oxyproline generates concentration in 52g/L, sugared concentration 0.1%, 5. after fermentation ends, fermented liquid is heated to 80 DEG C, is cooled to 40 DEG C immediately, is depressed into fermented liquid basin with pressurized air,
3, broth extraction: comprise the following steps successively: first fermented liquid micro-filtration, fermented liquid pump in storage tank is squeezed in heating tank, heat and be incubated 40 ± 5 DEG C, open film filter to filter fermented liquid, micro-filtration temperature 45 C, micro-filtration pressure 0.30Ma, micro-filtration flow velocity 100L/m2/h, membrane pore size 1.0um, film thickness 150um; Then, the further ultrafiltration of the filtrate after micro-filtration, ultrafiltrate temperature 50 DEG C, ultrafiltration pressure 1.0MPa, ultrafiltration flow velocity 35L/m2/h, membrane pore size 0.1um, film thickness 150um, asymmetric membrane; Finally, the further nanofiltration of purification liquid after ultrafiltration obtains concentrated solution, nanofiltration temperature 55 DEG C, nanofiltration pressure 2.5MPa, nanofiltration flow velocity 4.0L/m2/h, membrane pore size 3nm, film thickness 150um, asymmetric membrane;
4, purifying: comprise coarse crystallization and treating process successively, wherein, coarse crystallization process is: 1. squeeze in crystallizer by concentrated solution, stir cooling rapidly, 5 ± 3 DEG C of quiescent crystallization 4 hours; 2. by crude product centrifuge dripping, frozen water drip washing, dry 6 hours of 60 DEG C of heat circulation drying ovens, moisture content 1.8%, obtains crude product, and treating process is: 1. under agitation added by crude product in 60 DEG C of pure water of 20 times of weight, is warming up to 75 DEG C and dissolves completely; 2. add 769 activated carbon by 10% of crude product weight, 75 DEG C of decolourings 15 minutes, filter; 3. destainer is cooled to 5 DEG C through cooling coil, by volume 15% industrial acetic acid adding concentration 80%, stirs after having a large amount of crystal to separate out and stops stirring, 5 ± 3 DEG C of insulation crystallizations 4 hours, mother liquid concentration 3g/L at the end of crystallization; 4. crystal solution is proceeded to centrifuge, frozen water drip washing, wet-milling enters double cone dryer 80 DEG C of vacuum-dryings and obtains fine work in 6 hours.

Claims (6)

1. the method for fermentation method suitability for industrialized production L-oxyproline, is characterized in that, step comprises bacterial classification solid culture, fermentation culture, broth extraction and purifying:
(1) bacterial classification solid culture: the NaOH solution of bacterial classification solid medium 2N adjusts pH to 7.0, be sub-packed in 10ml slant tube or 50ml eggplant bottle, to jump a queue bag kraft paper, 121 DEG C of sterilizings 15 minutes, after taking-up is cooled to 50 ~ 60 DEG C, adding the AMP solution of 10% in sterilisable chamber, is 50ppm to AMP final concentration, shake up rear-inclined 30 ° placement, rearmounted sterilisable chamber to be solidified is for subsequent use; Be uniformly coated on test tube or the blank inclined-plane of eggplant bottle at the standardized ring bacterial classification of Bechtop transfering loop, place in the incubator of 37 ± 1.0 DEG C and cultivate 24 ~ 48 hours, lawn is grown rear directly upper tank or is placed in 0 ~ 5 DEG C of refrigerator for subsequent use;
(2) fermentation culture: be first that bacterial classification cultivates the seed obtaining maturation in liquid seed culture medium, then moves into fermentating metabolism in fermention medium and produces L-oxyproline, the cultivation of mature seed comprises the following steps: 1. slack tank sterilizing: 0.15-0.20MPa, pressurize 30 minutes, 2. real tank sterilizing: liquid seed culture medium drop into seeding tank, 2.4T liquid seed culture medium joined by 4T seeding tank, adjust pH to 7.0, close cover, chuck is heated to 90 DEG C, direct steam heating to 121 DEG C, 0.1MPa pressure heat insulating 10 minutes, 3. cool: sterilizing is complete cool to 36 DEG C to be seeded, 4. inoculate: adopt pressure differential method by bacterium liquid access tank, inoculation temp 36 DEG C, pH7.0 ~ 9.0, pressure 0.1MPa, open inoculation mouth protective cover, firmly mouth is inoculated with the cotton snare soaking 95% ethanol, light cotton circle, move to and light cotton circle by including the inoculation bottle collecting the bacterium liquid got by solid culture spawn culture and inoculate by mouth, to outward winding inoculation bottle Tip protection plug, rapidly inoculation bottle syringe needle is inserted inoculation mouth, slowly open vent valve on tank, pressure regulation 0.05 ~ 0.1MPa, inoculum size 1 bottle, after inoculation terminates, be placed on inoculation mouth with the cotton ball of dipped 75% ethanol, transfer to inoculation syringe needle, screw on inoculation mouth protective cover, start to cultivate, 5. cultivate: culture temperature 36 DEG C, tank pressure 0.03MPa, air quantity 0.2 ~ 1.2VVM, pH6.5, mixing speed 200 ~ 600rpm, dissolved oxygen more than 20%, incubation time 8 ~ 24 hours, OD 600reach 10 ~ 20, final glucose content is less than 10g/L, fermentation culture comprises the following steps: 1. slack tank sterilizing: 0.15-0.17MPa pressure, temperature 128 DEG C of sterilizings 30 minutes, 2. real tank sterilizing: fermention medium drops in tank, and 30T tank adds 22T substratum, close cover, chuck is preheating to 95 ~ 105 DEG C, and direct steam heating is to 121 DEG C, and pressure 0.1MPa, is incubated 10 minutes, cools to 35 DEG C, adjusts pH to 6.5, standby inoculation, 3. inoculate: adopt pressure differential method seed liquor to be pressed in tank and cultivate, 4. cultivate: culture temperature 35 DEG C, tank pressure 0.03MPa, air quantity 0.5 ~ 1.2VVM, pH6.5, mixing speed 60 ~ 300rpm, during fermentation ends, oxyproline generates concentration at 30 ~ 90g/L, sugared Nong Du≤2.0%, 5. after fermentation ends, fermented liquid is heated to 80 DEG C, is cooled to 40 DEG C immediately, is depressed into fermented liquid basin with pressurized air,
(3) broth extraction: comprise the following steps successively: first fermented liquid micro-filtration, fermented liquid pump in storage tank is squeezed in heating tank, heat and be incubated 40 ± 5 DEG C, open film filter to filter fermented liquid, micro-filtration temperature 35 ~ 45 DEG C, micro-filtration pressure 0.30Ma, micro-filtration flow velocity 80 ~ 100L/m 2/ h, membrane pore size 0.05 ~ 1.0um, film thickness 10 ~ 150um; Then, the further ultrafiltration of the filtrate after micro-filtration, ultrafiltrate temperature 40 ~ 50 DEG C, ultrafiltration pressure 1.0MPa, ultrafiltration flow velocity 30 ~ 35L/m 2/ h, membrane pore size 0.015 ~ 0.1um, film thickness 150 ~ 250um, asymmetric membrane; Finally, the further nanofiltration of purification liquid after ultrafiltration obtains concentrated solution, nanofiltration temperature 45 ~ 55 DEG C, nanofiltration pressure 0.5 ~ 3.5MPa, nanofiltration flow velocity 2.5 ~ 4.5L/m 2/ h, membrane pore size 1 ~ 5nm, film thickness 100 ~ 300um, asymmetric membrane;
(4) purifying: comprise coarse crystallization and treating process successively, wherein, coarse crystallization process is: 1. squeeze in crystallizer by concentrated solution, stirs rapidly and is cooled to 0 ~ 10 DEG C, 5 ± 3 DEG C of quiescent crystallization 4 hours; 2. by crude product centrifuge dripping, frozen water drip washing, dry 6 hours of 60 DEG C of heat circulation drying ovens, control Shui Fen≤5%, obtains crude product; Treating process is: 1. under agitation added by crude product in 60 DEG C of pure water of 20 times of weight, is warming up to 75 DEG C and dissolves completely; 2. add 769 activated carbon by 10% of crude product weight, 75 DEG C of decolourings 15 minutes, filter; 3. destainer is cooled to 0 ~ 10 DEG C through cooling coil, by volume 15% industrial acetic acid adding concentration 80%, stirs after having a large amount of crystal to separate out and stops stirring, 5 ± 3 DEG C of insulation crystallizations 4 hours, mother liquid concentration 2 ~ 3g/L at the end of crystallization; 4. crystal solution is proceeded to centrifuge, frozen water drip washing, wet-milling enters double cone dryer 80 DEG C of vacuum-dryings and obtains L-oxyproline fine work in 4 ~ 6 hours.
2. the method for fermentation method suitability for industrialized production L-oxyproline according to claim 1, is characterized in that, the AMP solution of 10% described in described step (1) is dissolved in distilled water obtained by Ampicillin Trihydrate.
3. the method for fermentation method suitability for industrialized production L-oxyproline according to claim 1, the bacterial classification described in step (1) derives from Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences, and bacterial strain is a strain EscherichiaColiK12W3110 mutation.
4. the method for fermentation method suitability for industrialized production L-oxyproline according to claim 1, it is characterized in that, bacterial classification solid medium described in described step (1) is made up of yeast powder, peptone, sodium-chlor, agar, AMP, and its formula is yeast powder 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, AMP50mg/L.
5. the method for fermentation method suitability for industrialized production L-oxyproline according to claim 1, it is characterized in that, the aqueous formulation (g/L) of the liquid seed culture medium described in described step (2) is: glucose 40, yeast powder 7.5, ammonium sulfate 2.0, potassium primary phosphate 7.5, Repone K 1.9, FeSO 47H 2o0.28, MgSO 47H 2o0.32, trace element (ppm): MnSO 4h 2o0.5, CoCl 26H 2o1.4, NiSO 46H 2o1.2, CuSO 47H 2o0.5, ZnSO 47H 2o0.6, VB 120, defoamer 100, AMP50.
6. the method for fermentation method suitability for industrialized production L-oxyproline according to claim 1 or 5, it is characterized in that, the fermentative medium formula (g/L) described in described step (2) is: glucose 20, yeast powder 5.0, ammonium sulfate 10, potassium primary phosphate 5.0, Repone K 1.9, MgSO 47H 2o0.32, FeSO 47H 2o0.75, trace element (ppm): MnSO 4h 2o1.0, CoCl 26H 2o1.4, NiSO 46H 2o1.4, CuSO 47H 2o1.0, ZnSO 47H 2o1.2, defoamer 200, AMP50.
CN201511035144.1A 2015-12-31 2015-12-31 Method for industrially producing L-hydroxyproline through fermentation method Pending CN105506019A (en)

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CN106543063A (en) * 2016-09-23 2017-03-29 合肥信达膜科技有限公司 A kind of proline extraction element and method
CN106977442A (en) * 2017-04-28 2017-07-25 天津科技大学 A kind of extraction process of hydroxyproline
CN107513030A (en) * 2017-10-19 2017-12-26 福建师范大学 A kind of method that L hydroxyprolines are isolated and purified in the hydroxyproline zymotic fluid from L
CN107604017A (en) * 2017-10-13 2018-01-19 广东肇庆星湖生物科技股份有限公司 A kind of method for improving hydroxyproline earlier fermentation bacteriolyze

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CN101323868A (en) * 2008-03-14 2008-12-17 江苏诚意药业有限公司 Method for industrialized production of L-tryptophan by biofermentation method
CN104278047A (en) * 2013-07-04 2015-01-14 江南大学 Method for enhancing activity of trans-4-hydroxyproline biosynthesis system containing recombinant DNA
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CN106543063A (en) * 2016-09-23 2017-03-29 合肥信达膜科技有限公司 A kind of proline extraction element and method
CN106977442A (en) * 2017-04-28 2017-07-25 天津科技大学 A kind of extraction process of hydroxyproline
CN107604017A (en) * 2017-10-13 2018-01-19 广东肇庆星湖生物科技股份有限公司 A kind of method for improving hydroxyproline earlier fermentation bacteriolyze
CN107604017B (en) * 2017-10-13 2020-10-16 广东肇庆星湖生物科技股份有限公司 Method for improving bacterial lysis in early fermentation stage of hydroxyproline
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