CN103333921A - Method for promoting high-density fermentation of Pichia pastoris by using oxygen carriers - Google Patents
Method for promoting high-density fermentation of Pichia pastoris by using oxygen carriers Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly discloses a method for promoting high-density fermentation of Pichia pastoris by using oxygen carriers. The method comprises the following step: respectively adding n-hexane, n-dodecane, Tween-80 and bean oil into a fermentation liquid when fermentation culture has been performed on Pichia pastoris for 0-3 hours, 24-27 hours, 72-75 hours and 96-98 hours, wherein the n-hexane accounts for 0.5-1% of the fermentation liquid, the n-dodecane accounts for 0.5-1% of the fermentation liquid, the Tween-80 accounts for 0.08-0.2% of the fermentation liquid, and the bean oil accounts for 0.1-0.5% of the fermentation liquid. By adding the oxygen carriers, the generation of bubbles can be reduced, the oxygen gas content in the fermentation culture system can be greatly increased, and the consumption of a defoaming agent can be reduced, thus satisfying the growth of Pichia pastoris and greatly increasing the yield of the product.
Description
Technical field
The invention belongs to the microbial fermentation technology field, relate to the fermentation process in high density of a kind of microorganism, be specifically related to a kind of method of utilizing oxygen carrier to promote the pichia spp high density fermentation.
Background technology
Pichia spp (
Pichia pastoris) be that a kind of that developed recently gets up is the eukaryotic expression system of carbon source with methyl alcohol, yeast can be modified heterologous protein, adopts when the plasmid of signal peptide is arranged, and albumen can correctly be folded and be processed, and is secreted in the substratum then.Have outside the many similar advantage to mammalian cell expression albumen, also have easy and simple to handle, nutritional requirement is low, cultivate cheap, be convenient to high density fermentation, high yield secreting, expressing foreign protein and expression product are easy to advantages such as purifying, and existing oneself successfully is used for the suitability for industrialized production of multiple protein.
But in the pichia spp fermenting process, because its demand to oxygen is very big, only by common ventilation oxygen-supplying, often can not satisfy the growth requirement of pichia spp, and become a bottleneck of restriction pichia spp high density fermentation.
Oxygen carrier (Oxygen-vectors) is a kind of and water does not dissolve each other, and is nontoxic to microorganism, the organism with higher dissolved oxygen ability.The system that it and fermented liquid form has characteristics such as the oxygen transmission speed is fast, energy consumption is low, bubble generates less, shearing force is little.
Summary of the invention
The objective of the invention is in order to overcome the defective that oxygen supply technology is difficult to capture in the existing pichia spp process of high-density fermentation, a kind of method of utilizing oxygen carrier to promote the pichia spp high density fermentation is provided, described method is simple, based on described method, can successfully realize obviously improving the purpose of expression product output.
Purpose of the present invention is achieved by following technical proposals:
A kind of method of utilizing oxygen carrier to promote the pichia spp high density fermentation comprises the steps:
S1. Pichi strain resurrection and enlarged culturing are obtained fermented bacterium;
S2. in fermented bacterium, add glycerine, preserve standby;
S3. the described fermented bacterium that has added glycerine of S2 is inserted the fermented liquid that fermention medium carries out fermentation culture with 1.0～1.2% inoculum sizes, percentage ratio meter by volume, when cultivating 0～3h, 24～27h, 72～75h, 96～98 h, add in the fermented liquid respectively and account for fermented liquid 0.5～1% normal hexane respectively, 0.5～1% n-dodecane, 0.08～0.2% tween-80 and 0.1～0.5% soya-bean oil; Cultivate after 72～75 hours, add methyl alcohol and carry out methanol induction until fermentation culture termination fermentation in 6 ~ 8 days.
Preferably, when cultivating 2～3h, 26～27h, 74～75h, 97～98 h, add in the fermented liquid respectively and account for fermented liquid 0.8～1% normal hexane respectively, 0.8～1% n-dodecane, 0.15～0.2% tween-80 and 0.3～0.5% soya-bean oil, more preferably, when cultivating 2h, 26h, 74h, 97 h, add in the fermented liquid respectively and account for fermented liquid 0.8% normal hexane, 0.8% n-dodecane, 0.15% tween-80 and 0.3% soya-bean oil respectively.
Normal hexane of the present invention, n-dodecane, tween-80 and soya-bean oil all belong to effective oxygen carrier, the present invention utilizes oxygen carrier, increase substantially the oxygen content in the pichia spp fermentation culture system, to satisfy the growth of pichia spp, and increase substantially the output of product, can reduce the generation of bubble simultaneously owing to oxygen carrier, thereby reduce the usage quantity of defoamer.And use defoamer that fermented bacterium is harmful in a large number, and product is difficult for purifying.
Described fermention medium is the BMGY liquid nutrient medium: peptone 2～2.5g, yeast extract 1～1.5g, add deionized water 70～75ml, 121～123 ℃, 20～25min autoclaving then adds 10～10.5 aseptic * GY, 10～10.5 * YNB, each 10～10.5ml of 10～10.5 * phosphate buffered saline buffer is chilled to and adds 200～205 microlitres, 500～505 * Biotin after the room temperature and namely be made into the BMGY substratum.
Trace salt solution: copper sulfate 6.0～6.5 grams, sodium iodide 0.08～0.1 gram, manganous sulfate 3.0～3.5 grams, Sodium orthomolybdate 0.2～0.25 gram, boric acid 0.02～0.025 gram, cobalt chloride 0.5～0.55 gram, zinc chloride 20.0～20.5 grams, ferrous sulfate 65.0～65.5 grams, vitamin H 0.2～0.25 gram, sulfuric acid 5.0～5.5ml adds deionized water to 1～1.1 L, room temperature storage behind the filtration sterilization.
Preferably, the addition of the described glycerine of step S2 determines according to 12～15% of fermented bacterium volume, and more preferably, the addition of the described glycerine of step S2 determines according to 12～15% of fermented bacterium volume,
Preferably, the addition manner of the described methyl alcohol of step S3 is for adding the methyl alcohol that contains 10～12ml/L trace salt solution with fed-batch mode, and initial interpolation concentration is 0.1～0.3%/h; Along with the increase of yeast to the adaptability utilization of methyl alcohol carbon source and the growth of bacterium number, improve methyl alcohol stream rate of acceleration with 0.3～0.5%/h, 0.5～0.7%/h, 0.7～0.9%/h gradient gradually, to final concentration be 1～2%/h.
Preferably, in the process of the described fermentation culture of step S3, mixing speed is 300～320rpm, and ventilation is 1:0.8～1vvm, and DO is 40～80% in control.
Preferably, the described pichia spp of step S1 is pichia spp GS115, pichia spp KM71 or pichia spp SMD1168.
Preferably, the concrete steps that the described Pichi strain of step S1 brings back to life and enlarged culturing obtains fermented bacterium are:
S1. the pichia spp bacterial classification is inoculated on the seed flat-plate solid substratum, line separates, cultivate unicellular bacterium colony; Concrete culture condition is: under 28～32 ℃ of temperature, cultivate cultivated in 12～16 hours unicellular bacterium colony;
S2. choose unicellular bacterium colony, be seeded on the seed inclined-plane solid medium, cultivate and grow thalline; Concrete culture condition is: cultivated 12～16 hours under 28～32 ℃ of temperature, grow thalline;
S3. thalline is inoculated in the bacterial classification liquid nutrient medium with 1～2% inoculum size, cultivated 16～20 hours under 28～32 ℃, 200～250rpm condition, be cultured to OD
600Be 1.0～1.2, stop to cultivate that gained bacterium liquid is fermented bacterium; Wherein, the described seed flat-plate solid of S1 substratum is the YPD solid medium; The described seed of S2 inclined-plane solid medium is YPD inclined-plane solid medium; YPD solid medium moiety is: 20～25g peptone, 10～12g yeast extract, 900～950 ml deionized waters, 121～123 ℃, 25～30 minutes autoclave sterilizations, the cooling back adds glucose solution and 14～18 g agar of 9～10g/L of filtration sterilization.
The described bacterial classification liquid nutrient medium of S3 is the YPD liquid nutrient medium, YPD liquid nutrient medium moiety is: 20～25g peptone, 10～12g yeast extract, deionized water 900～950 ml, 121～123 ℃, 25～30 minutes autoclave sterilizations, the cooling back adds the glucose solution of 9～10g/L of filtration sterilization.
Compared with prior art, the present invention has following beneficial effect:
Use by oxygen carrier, can reduce the generation of bubble, reduce the usage quantity of defoamer, and increase substantially the dissolved efficiency of oxygen, even DO in vigorous period also can reach more than 40% in pichia spp fermentation growth, and conventional pichia spp fermentation growth DO in vigorous period usually has only 10 ~ 20%.Therefore, the art of this patent is utilized oxygen carrier, increases substantially the oxygen content in the fermentation culture system, reduces the usage quantity of defoamer, satisfying the growth of pichia spp, and increases substantially the output of product.
Embodiment
Below in conjunction with embodiment the present invention is made further elaborating, but embodiment does not do any type of restriction to the present invention.
Embodiment 1
S1. original starting strain is fermented bacterium GS-115 (commercialization Pichi strain) through the bacterium liquid that brings back to life and enlarged culturing obtains;
Resurrection and the enlarged culturing of the described original starting strain of step S1 may further comprise the steps:
S11. picking one ring thalline from the original bacterial classification is inoculated on the dull and stereotyped YPD solid medium of seed, and line separates, under 28 ℃ of temperature, cultivate cultivated in 12 hours unicellular bacterium colony;
S12. choose the described unicellular bacterium colony of step S11, be seeded on the seed inclined-plane YPD solid medium, under 28 ℃ of temperature, cultivated 12 hours, grow thalline;
S13. the described thalline of step S12 is inoculated in the bacterial classification YPD liquid nutrient medium with 1% inoculum size, shaking table is cultured to OD
600=1.0, stop to cultivate, gained bacterium liquid is fermented bacterium; It is to cultivate 16 hours under 28 ℃, 200rpm condition that described shaking table is cultivated; Described bacterial classification liquid nutrient medium consists of: the YPD liquid nutrient medium: the 20g peptone, and 10g yeast extract deionized water 900 ml, 121 ℃, 30 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 10g/L.
The YPD solid medium: the 20g peptone, 10g yeast extract deionized water 900 ml, 121 ℃, 30 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 10g/L, 15 g agar.
The BMGY liquid nutrient medium: peptone 2g, yeast extract 1g adds deionized water 70ml, 121 ℃, the 20min autoclaving then adds 10 aseptic * GY, 10 * YNB, each 10ml of 10 * phosphate buffered saline buffer is chilled to and adds 500 * Biotin, 200 microlitres after the room temperature and namely be made into the BMGY substratum.
Trace salt solution: copper sulfate 6.0 grams, sodium iodide 0.08 gram, manganous sulfate 3.0 grams, Sodium orthomolybdate 0.2 gram, boric acid 0.02 gram, cobalt chloride 0.5 gram, zinc chloride 20.0 grams, ferrous sulfate 65.0 grams, vitamin H 0.2 gram, sulfuric acid 5.0ml adds deionized water to 1 liter, room temperature storage behind the filtration sterilization.
S2. in step S1 gained fermented bacterium, add glycerine, be positioned under-18 ℃ of temperature preserve standby; The addition of glycerine is preferably determined according to 12% of fermented bacterium volume.
S3. adopt 50 liters of fermentor tanks, 35 liters of fermention mediums of interior Sheng, the described fermented bacterium that has added glycerine of step S2 is inserted fermention medium BMGY with 1.0% inoculum size, carry out fermentation culture, percentage ratio meter by volume, the mode that adds with stream when cultivating 0h, 24h, 72h, 96 h is added in the fermented liquid and is accounted for fermented liquid 0.5% normal hexane respectively, 0.5% n-dodecane, 0.08% tween-80 and 0.1% soya-bean oil respectively; Cultivate after 72 hours, the mode that adds with stream adds methyl alcohol and carries out methanol induction and stopped fermentation in 6 days until fermentation culture; Wherein, stream adds pure methyl alcohol (the trace salt solution that contains 10ml/L), and initial concentration is 0.1%/h; Along with the increase that yeast is grown to adaptability utilization and the bacterium number of methyl alcohol carbon source, gradient improves methyl alcohol stream rate of acceleration (0.3%/h, 0.5%/h, 0.7%/h) gradually, to final concentration 1%/h.
In above-mentioned fermentation culture process, mixing speed is preferably 300rpm, and ventilation is preferably 1:0.8vvm, and DO is 40% in control;
Embodiment 2
S1. original starting strain is fermented bacterium GS-115 (commercialization Pichi strain) through the bacterium liquid that brings back to life and enlarged culturing obtains;
Resurrection and the enlarged culturing of the described original starting strain of step S1 may further comprise the steps:
S11. picking one ring thalline from the original bacterial classification is inoculated on the dull and stereotyped YPD solid medium of seed, and line separates, under 29 ℃ of temperature, cultivate cultivated in 13 hours unicellular bacterium colony;
S12. choose the described unicellular bacterium colony of step S11, be seeded on the seed inclined-plane YPD solid medium, under 29 ℃ of temperature, cultivated 13 hours, grow thalline;
S13. the described thalline of step S12 is inoculated in the bacterial classification YPD liquid nutrient medium with 1.2% inoculum size, shaking table is cultured to OD
600=1.05, stop to cultivate, gained bacterium liquid is fermented bacterium; It is to cultivate 17 hours under 29 ℃, 210rpm condition that described shaking table is cultivated.Described bacterial classification liquid nutrient medium consists of: the YPD liquid nutrient medium: the 21g peptone, and 10.5g yeast extract deionized water 910 ml, 121.5 ℃, 26 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 9.5g/L.
The YPD solid medium: the 21g peptone, 10.5g yeast extract deionized water 910 ml, 121.5 ℃, 26 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 9.5g/L.14.5 g agar.
BMGY liquid nutrient medium: peptone 2.1g, yeast extract 1.1g, add deionized water 71ml, 121.5 ℃, the 21min autoclaving then adds 10.1 aseptic * GY, 10.1 * YNB, 10.1 each 10.1ml of * phosphate buffered saline buffer is chilled to and adds 501 * Biotin, 201 microlitres after the room temperature and namely be made into the BMGY substratum.
Trace salt solution: copper sulfate 6.1 grams, sodium iodide 0.09 gram, manganous sulfate 3.1 grams, Sodium orthomolybdate 0.21 gram, boric acid 0.021 gram, cobalt chloride 0.51 gram, zinc chloride 20.1 grams, ferrous sulfate 65.1 grams, vitamin H 0.21 gram, sulfuric acid 5.1ml adds deionized water to 1.02 liter, room temperature storage behind the filtration sterilization.
S2. in step S1 gained fermented bacterium, add glycerine, be positioned under-18.5 ℃ of temperature preserve standby; The addition of glycerine is preferably determined according to 13% of fermented bacterium volume.
S3. adopt 100 liters of fermentor tanks, 50 liters of fermention mediums of interior Sheng, the fermented bacterium that step S2 has been added glycerine inserts fermention medium BMGY with 1.05% inoculum size, percentage ratio meter by volume, the mode that adds with stream when cultivating 0.5h, 25h, 73h, 97 h is added in the fermented liquid and is accounted for fermented liquid 0.6% normal hexane, 0.6% n-dodecane, 0.09% tween-80 and 0.2% soya-bean oil respectively, cultivate after 73 hours, the mode that adds with stream adds methyl alcohol and carries out methanol induction and stopped fermentation in 7 days until fermentation culture; Wherein, stream adds pure methyl alcohol (the trace salt solution that contains 11ml/L), and initial concentration is 0.15%/h, along with the increase of yeast to the adaptability utilization of methyl alcohol carbon source and the growth of bacterium number, gradient improves methyl alcohol stream rate of acceleration (0.4%/h, 0.6%/h, 0.75%/h) gradually, to final concentration 1.2%/h.
In above-mentioned fermentation culture process, mixing speed is preferably 310rpm, and ventilation is preferably 1:0.9vvm, and DO is 50% in control;
Embodiment 3
S1. original starting strain is fermented bacterium GS-115 (commercialization Pichi strain) through the bacterium liquid that brings back to life and enlarged culturing obtains;
Resurrection and the enlarged culturing of the described original starting strain of step S1 may further comprise the steps:
S11. picking one ring thalline from the original bacterial classification is inoculated on the dull and stereotyped YPD solid medium of seed, and line separates, under 30 ℃ of temperature, cultivate cultivated in 14 hours unicellular bacterium colony;
S12. choose the described unicellular bacterium colony of step S11, be seeded on the seed inclined-plane YPD solid medium, under 30 ℃ of temperature, cultivated 14 hours, grow thalline;
S13. the described thalline of step S12 is inoculated in the bacterial classification YPD liquid nutrient medium with 1.6% inoculum size, shaking table is cultured to OD
600=1.15, stop to cultivate, gained bacterium liquid is fermented bacterium;
It is to cultivate 18 hours under 30 ℃, 220rpm condition that described shaking table is cultivated.
Described bacterial classification liquid nutrient medium consists of: the YPD liquid nutrient medium: the 23g peptone, and 11g yeast extract deionized water 920 ml, 122 ℃, 27 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 9.6g/L.
The YPD solid medium: the 223g peptone, 11g yeast extract deionized water 920 ml, 122 ℃, 27 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 9.6g/L, 16 g agar.
BMGY liquid nutrient medium: peptone 2.4g, yeast extract 1.4g, add deionized water 72ml, 122 ℃, the 23min autoclaving then adds 10.4 aseptic * GY, 10.4 * YNB, 10.4 each 10.4ml of * phosphate buffered saline buffer is chilled to and adds 504 * Biotin, 204 microlitres after the room temperature and namely be made into the BMGY substratum.
Trace salt solution: copper sulfate 6.4 grams, sodium iodide 0.09 gram, manganous sulfate 3.4 grams, Sodium orthomolybdate 0.24 gram, boric acid 0.024 gram, cobalt chloride 0.54 gram, zinc chloride 20.4 grams, ferrous sulfate 65.4 grams, vitamin H 0.24 gram, sulfuric acid 5.4ml adds deionized water to 1.08 liter, room temperature storage behind the filtration sterilization.
S2. in step S1 gained fermented bacterium, add glycerine, be positioned under-20 ℃ of temperature preserve standby; The addition of glycerine is preferably determined according to 14% of fermented bacterium volume.
S3. adopt 100 liters of fermentor tanks, 60 liters of fermention mediums of interior Sheng, the fermented bacterium that step S2 has been added glycerine inserts fermention medium BMGY with 1.15% inoculum size and carries out fermentation culture, percentage ratio meter by volume, the mode that adds with stream when cultivating 2h, 26h, 74h, 97 h is added 0.8% normal hexane that accounts for fermented liquid in fermented liquid respectively, 0.8% n-dodecane, 0.15% tween-80 and 0.3% soya-bean oil; Cultivate after 74 hours, the mode that adds with stream adds methyl alcohol and carries out methanol induction and stopped fermentation in 8 days until fermentation culture; Wherein, stream adds pure methyl alcohol (the trace salt solution that contains 11ml/L), and initial concentration is 0.25%/h.Along with the increase that yeast is grown to adaptability utilization and the bacterium number of methyl alcohol carbon source, gradient improves methyl alcohol stream rate of acceleration (0.45%/h, 0.65%/h, 0.85%/h) gradually, to final concentration 1.8%/h.
In above-mentioned fermentation culture process, mixing speed is preferably 315rpm, and ventilation is preferably 1:0.95vvm, and DO is 75% in control.
Embodiment 4
S1. original starting strain is fermented bacterium GS-115 (commercialization Pichi strain) through the bacterium liquid that brings back to life and enlarged culturing obtains;
Resurrection and the enlarged culturing of the described original starting strain of step S1 may further comprise the steps:
S11. picking one ring thalline from the original bacterial classification is inoculated on the dull and stereotyped YPD solid medium of seed, and line separates, under 32 ℃ of temperature, cultivate cultivated in 16 hours unicellular bacterium colony;
S12. choose the described unicellular bacterium colony of step S11, be seeded on the seed inclined-plane YPD solid medium, under 32 ℃ of temperature, cultivated 16 hours, grow thalline;
S13. the described thalline of step S12 is inoculated in the bacterial classification YPD liquid nutrient medium with 2% inoculum size, shaking table is cultured to OD
600=1.2, stop to cultivate, gained bacterium liquid is fermented bacterium; It is to cultivate 20 hours under 32 ℃, 250rpm condition that described shaking table is cultivated.
Described bacterial classification liquid nutrient medium consists of: the YPD liquid nutrient medium: the 25g peptone, and 12g yeast extract deionized water 950 ml, 123 ℃, 30 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 10g/L.
YPD solid medium: YPD liquid nutrient medium: the 25g peptone, 12g yeast extract deionized water 950 ml, 123 ℃, 30 minutes autoclave sterilizations, the cooling back adds the glucose solution of the filtration sterilization of 10g/L, 18 g agar.
BMGY liquid nutrient medium: peptone 2.5g, yeast extract 1.5g, add deionized water 75ml, 123 ℃, the 25min autoclaving then adds 10.5 aseptic * GY, 10.5 * YNB, 10.5 each 10.5ml of * phosphate buffered saline buffer is chilled to and adds 505 * Biotin, 205 microlitres after the room temperature and namely be made into the BMGY substratum.
Trace salt solution: copper sulfate 6.5 grams, sodium iodide 0.1 gram, manganous sulfate 3.5 grams, Sodium orthomolybdate 0.25 gram, boric acid 0.025 gram, cobalt chloride 0.55 gram, zinc chloride 20.5 grams, ferrous sulfate 65.5 grams, vitamin H 0.25 gram, sulfuric acid 5.5ml adds deionized water to 1.1 liter, room temperature storage behind the filtration sterilization.
S2. in step S1 gained fermented bacterium, add glycerine, be positioned under-20 ℃ of temperature preserve standby; The addition of glycerine is preferably determined according to 15% of fermented bacterium volume.
S3. adopt 100 liters of fermentor tanks, 70 liters of fermention mediums of interior Sheng, the fermented bacterium that step S2 has been added glycerine inserts fermention medium BMGY with 1.2% inoculum size and carries out fermentation culture, percentage ratio meter by volume, the mode that adds with stream when cultivating 3h, 27h, 75h, 98 h is added in the fermented liquid and is accounted for fermented liquid 1% normal hexane respectively, 1% n-dodecane, 0.2% tween-80 and 0.5% soya-bean oil; Cultivate after 75 hours, the mode that adds with stream adds methyl alcohol and carries out methanol induction and stopped fermentation in 8 days until fermentation culture; Wherein, stream adds pure methyl alcohol (the trace salt solution that contains 12ml/L), and initial concentration is 0.3%/h, along with the increase of yeast to the adaptability utilization of methyl alcohol carbon source and the growth of bacterium number, gradient improves methyl alcohol stream rate of acceleration (0.5%/h, 0.7%/h, 0.9%/h) gradually, to final concentration 2%/h.
In above-mentioned fermentation culture process, mixing speed is preferably 320rpm, and ventilation is preferably 1:1vvm, and DO is 80% in control.
Embodiment 5 utilizes the oxygen carrier technology to improve the expression amount of product
The engineering bacteria GSl15/pPIC9K-PBD1 that expresses PBD1 (antibacterial peptide) is made up by Microbiological Lab of Agricultural University Of South China and preserves, and this recombinant yeast pichia pastoris methyl alcohol utilizes phenotype to be mut
+Adopt the embodiment of the invention 1～4 described method and ordinary method to carry out fermentative processing (step process such as slant activation, shaking table cultivation, fermentor cultivation) respectively, the product after the fermentation is quantitative with HPLC, and measurement result is shown in Table 1.
Table 1 utilizes the effect comparison of oxygen carrier fermentation
As known from Table 1, through the product of oxygen carrier method of the present invention fermentation, at thalli growth stage and product generation phase, DO value all significantly is better than the DO value of conventional not usefulness oxygen carrier, and production concentration is increased to 468mg/L by 168, and production concentration increases substantially.
In sum, utilize the technology of the present invention can increase substantially the unit volume amount of oxygen, the corresponding concentration that also improves product, this will be highly advantageous to and reduce the cost of fermentation enterprise, also can improve the utilization ratio of unit volume fermentor tank.
Claims (9)
1. a method of utilizing oxygen carrier to promote the pichia spp high density fermentation is characterized in that, comprises the steps:
S1. Pichi strain resurrection, enlarged culturing are obtained fermented bacterium;
S2. in fermented bacterium, add glycerine, preserve standby;
S3. the described fermented bacterium that has added glycerine of S2 is carried out fermentation culture with 1.0～1.2% inoculum sizes access fermention medium and get fermented liquid; Percentage ratio meter by volume adds accounting for fermented liquid 0.5～1% normal hexane respectively, 0.5～1% n-dodecane, 0.08～0.2% tween-80 and 0.1～0.5% soya-bean oil respectively in the fermented liquid when cultivating 0～3h, 24～27h, 72～75h, 96～98 h; Cultivate after 72～75 hours, add methyl alcohol and carry out methanol induction until fermentation culture termination fermentation in 6 ~ 8 days.
2. according to the described method of claim 1, it is characterized in that, when cultivating 2～3h, 26～27h, 74～75h, 97～98 h, add in the fermented liquid respectively and account for fermented liquid 0.8～1% normal hexane, 0.8～1% n-dodecane, 0.15～0.2% tween-80 and 0.3～0.5% soya-bean oil respectively.
3. according to the described method of claim 2, it is characterized in that, when cultivating 2h, 26h, 74h, 97 h, add accounting for fermented liquid 0.8% normal hexane, 0.8% n-dodecane, 0.15% tween-80 and 0.3% soya-bean oil respectively respectively in the fermented liquid.
4. according to the described method of claim 1, it is characterized in that the addition of the described glycerine of step S2 is determined according to 12～15% of fermented bacterium volume.
5. according to the described method of claim 1, it is characterized in that the concrete steps that the described Pichi strain resurrection of step S1 and enlarged culturing obtain fermented bacterium are:
S1. the pichia spp bacterial classification is inoculated on the seed flat-plate solid substratum, line separates, cultivate unicellular bacterium colony;
S2. choose unicellular bacterium colony, be seeded on the seed inclined-plane solid medium, cultivate and grow thalline;
S3. thalline is inoculated in the bacterial classification liquid nutrient medium with 1～2% inoculum size, cultivated 16～20 hours under 28～32 ℃, 200～250rpm condition, be cultured to OD
600Be 1.0～1.2, stop to cultivate that gained bacterium liquid is fermented bacterium.
6. according to the described method of claim 5, it is characterized in that described seed flat-plate solid substratum is the YPD solid medium; Seed inclined-plane solid medium is YPD inclined-plane solid medium; The bacterial classification liquid nutrient medium is the YPD liquid nutrient medium.
7. according to the described method of claim 1, it is characterized in that the addition manner of the described methyl alcohol of step S3 is for adding the methyl alcohol that contains 10～12ml/L trace salt solution with fed-batch mode, initial interpolation concentration is 0.1～0.3%/h; Along with the increase of yeast to the adaptability utilization of methyl alcohol carbon source and the growth of bacterium number, improve methyl alcohol stream rate of acceleration with 0.3～0.5%/h, 0.5～0.7%/h, 0.7～0.9%/h gradient gradually, to final concentration be 1～2%/h.
8. according to the described method of claim 1, it is characterized in that in the process of the described fermentation culture of step S3, mixing speed is 300～320rpm, ventilation is 1:0.8～1vvm, and DO is 40～80% in control.
9. according to the described method of claim 1, it is characterized in that the described pichia spp of step S1 is pichia spp GS115, pichia spp KM71 or pichia spp SMD1168.
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Cited By (3)
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|---|---|---|---|---|
| CN103820520A (en) * | 2014-03-06 | 2014-05-28 | 浙江皇冠科技有限公司 | High-yield natural astaxanthin fermentation method |
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| CN108841903A (en) * | 2018-06-29 | 2018-11-20 | 董骅 | A kind of method of strain fermentation production biosurfactant |
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| CN103820520A (en) * | 2014-03-06 | 2014-05-28 | 浙江皇冠科技有限公司 | High-yield natural astaxanthin fermentation method |
| CN106191203A (en) * | 2016-07-23 | 2016-12-07 | 安徽乙地生态科技有限公司 | A kind of selective medium cultivated for trichoderma harzianum |
| CN108841903A (en) * | 2018-06-29 | 2018-11-20 | 董骅 | A kind of method of strain fermentation production biosurfactant |
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