CN103045703A - Recycling method of methanol protein fermentation filtrate - Google Patents
Recycling method of methanol protein fermentation filtrate Download PDFInfo
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Abstract
The invention relates to a recycling method of a methanol protein fermentation filtrate, which effectively recycles fermentation liquid during industrial production of single-cell methanol protein and comprises the steps of standing fermentation liquid obtained during production of methanol protein by a conventional method or with pichia pastoris HGD-01, settling, centrifuging, filtering, recycling the filtrate, adding ethylene diamine tetraacetic acid with the amount being sequentially increased to the filtrate during recycling for each time, mixing the filtrate with ethylene diamine tetraacetic acid to prepare mixed liquid, and mixing the mixed liquid with inorganic salt in an inorganic salt fermentation medium to prepare a substitutive medium. According to the method, the input of inorganic salt is reduced, the waste water discharge is reduced, water resources are saved, the raw material cost is lowered, the growth of yeast cells and the yield of the methanol protein are unaffected, and the production efficiency is improved.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly a kind of method of Methanol Protein ferment filtrate recycling.
Background technology
Application of fermentation method manufacture order cell protein is as feed, have at home a lot of reports, and ripe working method is arranged, as using candiyeast, pichia spp etc. as fermented bacterium, with the single cell protein of some processed side products productions such as beet pulp, brewer's grains, oranges and tangerines waste residue, molasses.The single cell protein that obtains by unicellular organism take methyl alcohol as matrix, present production method both domestic and external mainly contains fermentation using bacteria method and saccharomycetes to make fermentation method, and the bacterium method comprises the ICI method of Britain, the Hoechst-Uhde method of Germany and the Norprotein method of Sweden; The barms working system comprises the MGC method of Japan, the IFP method of France and the Philips Petroleum method of the U.S., except ICI method suitability for industrialized production (stopping production at present), other are not industrialization all, but at present also at the early-stage for the exploitation of unicellular Methanol Protein feed in China, the cycling and reutilization problem of fermented liquid particularly, at present also without any relevant report, because fermentation is in whole unicellular Methanol Protein commercial process, step is absolutely necessary, the quantum of output of the ferment filtrate that produces therefrom is larger, fermentation producer directly drains ferment filtrate mostly, not only give environment, also so that the material that part can be utilized again in the filtrate is not effectively utilized, cause great waste.
Summary of the invention
The present invention's purpose just provides a kind of method of Methanol Protein ferment filtrate recycling, can effectively solve the cycling and reutilization problem of fermented liquid in the unicellular Methanol Protein suitability for industrialized production.
The technical scheme that the present invention solves is, will leave standstill according to a conventional method or with the fermented liquid of pichia pastoris phaff HGD-01 methanol albumen gained, and sedimentation is carried out centrifugal, filter, get filtered liquid and tropina liquid, add water in the tropina liquid, stir, spraying drying is collected albumen dry powder; Filtered liquid is recycled, difference according to the number of times of recycling, the amount of adding ethylenediamine tetraacetic acid (EDTA) (EDTA) in the each time recycling process in every liter of filtered liquid increases progressively in 0.5 g/L-1.3 g/L successively, be mixed into mixed solution with filtered liquid with ethylenediamine tetraacetic acid (EDTA) (EDTA), difference according to the number of times of recycling, in each time recycling process, with mixed solution respectively with above-mentioned steps in phosphoric acid in the BSM inorganic salt fermentative medium formula, inorganic salt, the phosphoric acid of the 10-20% of trace element solution consumption, the inorganic salt of 10-20%, the trace element solution of 10-20% and glycerine 25kg/m
3Be mixed into substitutive medium, replace the fermention medium that uses in the former methyl alcohol protein production process with substitutive medium, filtered liquid is recycled and generally can be carried out three times, and filtered liquid is not recycled for the third time; The concrete steps of described fermented liquid with pichia pastoris phaff HGD-01 methanol albumen gained are: pichia pastoris phaff HGD-01 bacterial strain is seeded on the YPD test tube slant substratum with method of scoring, cultivate to get the inclined-plane seed, the inclined-plane seed is inoculated in the shaking flask YPD liquid nutrient medium and cultivates, and obtains shake-flask seed; Shake-flask seed is inoculated in the YPD liquid nutrient medium, cultivates to get first order seed; First order seed is inoculated in the BSM inorganic salt fermention medium, cultivates to get secondary seed; Secondary seed is inoculated in the fermention medium ferments, ferment after 24 hours, begin stream and add methyl alcohol, the control methanol concentration, after 45-50 hour, the thalline weight in wet base reaches 250-300 g/L, OD
600Value reaches 42～45, namely gets fermented liquid.
The present invention has greatly reduced the input of inorganic salt, the production of each Methanol Protein, can reduce the usage quantity of the inorganic salt of 80-90%, and reduced the discharging of waste water, save water resources, reduced raw materials cost, and do not affected growth and the Methanol Protein productive rate of yeast cell, improve production efficiency, produced considerable economic benefit and social benefit.
Embodiment
Below in conjunction with practical situation the specific embodiment of the present invention is elaborated.
A kind of method of Methanol Protein ferment filtrate recycling comprises the steps:
1), shake-flask seed is cultivated:
Classification And Nomenclature be pichia pastoris phaff (
Pichia pastoris) bacterial strain of HGD-01, being preserved in Chinese Typical Representative culture collection center, deposit number is: CCTCC NO:M2012342, preservation date is: on September 12nd, 2012;
Be seeded to YPD test tube slant substratum with method of scoring (on the test tube specification 180mm * 20mm) at the bacterial strain of the upper 0.5mL pichia pastoris phaff HGD-01 with the preservation of glycerine pipe of Bechtop (instrument model SW-CJ-1FD), cultivated 48 hours, and namely got the inclined-plane seed for 30 ℃; Get 1 cultured inclined-plane seed, with the 10mL sterilized water inclined-plane seed is all washed, be inoculated in the triangular flask that 250mL YPD liquid nutrient medium is housed, bottleneck binds up with gauze with 8 layers, the shaking table shaking culture, 30 ℃ of culture temperature, shaking speed 220rpm, after cultivating about 24～30 hours, thalline OD
600Reach 3～5, obtain shake-flask seed; Described YPD test tube slant substratum is 1% yeast extract (the oxiod company product by weight percent meter, known technology, as follows), the water of 2% Tryptones (oxiod company product, known technology, as follows), 2% glucose, 2% agar and surplus mixes composition; Described YPD liquid nutrient medium is mixed by the water of 1% yeast extract, 2% Tryptones, 2% glucose and the surplus of weight percent meter and forms;
2), first order seed is cultivated:
A, first order seed is cultivated and is carried out in the fermentor tank of 50L, in the 50L tank, prepare the YPD liquid nutrient medium of 30L by the described method of step 1), open steam valve, make steam advance fermentor tank chuck layer, the YPD liquid nutrient medium is sterilized, 121 ℃ of sterilising temps, sterilization time 30min is when sterilizing to the YPD liquid nutrient medium, air filter and thief hole are sterilized, logical high-temp steam sterilizing 10min, sterilization is opened cooling water valve after finishing, make cold true water advance fermentor tank chuck layer, substratum is lowered the temperature, when the substratum temperature is down to 30 ℃, begin inoculation;
B, shake-flask seed 1000mL is inoculated in the YPD liquid nutrient medium in the 50L fermentor tank, 28～30 ℃ of temperature, rotating speed 150rpm, dissolved oxygen 40%～50%, cultivate 20～24h after, get first order seed;
3), secondary seed is cultivated:
With 3m
3BSM inorganic salt fermention medium put into 5m
3In the fermentor tank, with 2) the identical sterilising method of YPD liquid nutrient medium is sterilized to BSM inorganic salt fermention medium in the A step, after sterilization is finished, be that 35% ammoniacal liquor is transferred BSM inorganic salt fermention medium pH value to 4.8～5.0 with mass concentration, then the 30L first order seed be inoculated into 5m by pressure pipeline
3PH value is in 4.8～5.0 the BSM inorganic salt fermention medium in the fermentor tank, inspection seed quality before the inoculation, and the microscopy thalline is neat, stalwartness, the number that sprouts is at 2～3, without living contaminants, tank pressure is 0.05-0.10MPa during inoculation, during cultivation, 30～32 ℃ of culture temperature, ventilating ratio 1:1 ~ 1.5 vvm, tank pressure 0.05MPa, 24 ~ 30h is cultivated in pH5.0～5.5, gets secondary seed; Described BSM inorganic salt fermention medium is: mass concentration is 85% phosphoric acid 25kg/m
3, dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3, magnesium sulfate heptahydrate 2kg/m
3, glycerine 25kg/m
3With trace element solution 4L/m
3Add water and make, that is to say that BSM inorganic salt fermention medium is: mass concentration is that 85% phosphoric acid 25kg, dipotassium hydrogen phosphate 0.5kg, ammonium chloride 0.3kg, terra alba 0.2kg, sodium-chlor 0.5kg, vitriolate of tartar 3kg, magnesium sulfate heptahydrate 2kg, glycerine 25kg and trace element solution 4L add water to 1m
3Described trace element solution is: cupric sulfate pentahydrate 1.5g/L, potassiumiodide 0.02g/L, manganese sulfate monohydrate 0.5g/L, Sodium Molybdate Dihydrate (the extensive and profound in meaning star in Beijing chemical reagents corporation product, known technology, as follows) 0.2g/L, boric acid 0.02g/L, cobalt chloride hexahydrate (the extensive and profound in meaning star in Beijing chemical reagents corporation product, known technology, as follows) 0.3g/L, zinc chloride 20g/L, ferrous sulfate 19g/L, the vitriol oil 4mL/L of mass concentration 98%, vitamin H (claiming again vitamin H) 0.2g/L, para-amino benzoic acid 0.05g/L and calcium pantothenate 0.05g/L add water and make, and that is to say that trace element solution is: cupric sulfate pentahydrate 1.5g, potassiumiodide 0.02g, manganese sulfate monohydrate 0.5g, Sodium Molybdate Dihydrate 0.2g, boric acid 0.02g, cobalt chloride hexahydrate 0.3g, zinc chloride 20g, ferrous sulfate 19g, the vitriol oil 4mL of mass concentration 98%, vitamin H 0.2g, para-amino benzoic acid 0.05g and calcium pantothenate 0.05g add water to 1L;
4), high density fermentation:
At 50m
3Add BSM inorganic salt fermention medium 30m in the fermentor tank
3As fermention medium, sterilize with the steam more than 160 ℃, simultaneously to 50m
3The pipeline that fermentor tank links to each other is sterilized, and sterilization is down to 30～35 ℃ with the fermention medium temperature after finishing, and is that 35% ammoniacal liquor is readjusted the distribution ferment medium pH to 4.5～5.0 with mass concentration, then with secondary seed 3m
3Be inoculated into 50m
3PH ferments in 4.5～5.0 the fermention medium in the fermentor tank, leavening temperature is 30～32 ℃, air flow 1.5～2vv/m, mixing speed 80-200rpm is after 24 hours, glycerine exhausts in the substratum, dissolved oxygen begins to rise, and begins stream and adds methyl alcohol, with methyl alcohol detecting instrument on-line monitoring methanol concentration, make methanol concentration be controlled at 0.8%～1.2%, per 6 hours sampling and measuring thalline OD
600Value and weight in wet base, after 45-50 hour, the thalline weight in wet base reaches 250-300 g/L, OD
600Value reaches 42～45, and microscopy is observed and found that thalline is individual large, and content is more, the comparatively small amt that sprouts, and this moment, fermentation ends namely got fermented liquid;
5), the collection of filtered liquid:
Fermented liquid leaves standstill, sedimentation 4～8 hours, and high fermentation liquid carries out centrifugal with horizontal screw centrifuge, 3000 rev/mins of centrifuge speeds, inlet amount 5m
3/ h, centrifugal clear liquid filter with flame filter press again, filtration area 20m
2, filtration flow-rate 1.5 m
3/ h filters to get for the first time filtered liquid and bacterium cake, and the bacterium cake is put into 5m
3In the fermentation storage tank, add the clear water of bacterium cake volume 1/5, stir, enter spray-drier, regulate intake air temperature to 140～160 ℃, temperature out to 65-80 ℃, regulating feed rate is 1 ton/hour, and spraying drying is collected albumen dry powder;
6), for the first time collection of ferment filtrate
Add ethylenediamine tetraacetic acid (EDTA) (EDTA) in the filtered liquid for the first time, every liter for the first time in the filtered liquid EDTA addition be 0.5g/L, get the first mixed solution, be that 85% phosphoric acid and mass concentration are that 35% ammoniacal liquor is transferred the first pH of mixed value to 5.5～6.0 with mass concentration again, get for the first time ferment filtrate, this first time ferment filtrate why can be used to that part substitutes (be that part substitutes, because fermented liquid preparation in back the time also will add inorganic salt, only the inorganic salt consumption lack) consumption of inorganic salt in the used fermention medium when again carrying out Methanol Protein production;
7), for the second time collection of ferment filtrate
Get respectively above-mentioned 3) phosphoric acid, inorganic salt and the phosphoric acid of trace element solution consumption 10%, 10% inorganic salt, 10% trace element solution, glycerine 25kg/m in the BSM inorganic salt fermentative medium formula in the step
3With the ferment filtrate 20m first time
3Mix, add water to 30m
3, get the first substitutive medium, for subsequent use; Described inorganic salt are dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3With magnesium sulfate heptahydrate 2kg/m
3Again by above-mentioned 1), 2), 3), 4), 5), method in the step and consumption are collected for the second time filtered liquid again, wherein, in the process of collecting the filtered liquid second time, with the above-mentioned the 4th) used fermention medium replaces to the first substitutive medium in the step, collect to get for the second time filtered liquid and albumen dry powder, filtered liquid interpolation ethylenediamine tetraacetic acid (EDTA) (EDTA) is recycled for the second time, be every liter for the second time in the filtered liquid EDTA addition be 0.9g/L, get the second mixed solution, be that 85% phosphoric acid and mass concentration are that 35% ammoniacal liquor is transferred the second pH of mixed value to 5.5～6.0 with mass concentration again, get for the second time ferment filtrate, the consumption of inorganic salt in this fermention medium used when ferment filtrate can be used to carry out Methanol Protein production partly alternative next time for the second time;
8), for the third time collection of ferment filtrate
Get respectively above-mentioned 3) phosphoric acid, inorganic salt and the phosphoric acid of trace element solution consumption 15%, 15% inorganic salt, 15% trace element solution, glycerine 25kg/m in the BSM inorganic salt fermentative medium formula in the step
3With the ferment filtrate 20m second time
3Mix, add water to 30m
3, get the second substitutive medium, for subsequent use, described inorganic salt are dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3, magnesium sulfate heptahydrate 2kg/m
3Again by above-mentioned 1), 2), 3), 4), 5), method in the step and consumption are collected for the third time filtered liquid again, wherein, collect in the process of filtered liquid for the third time, with the above-mentioned the 4th) used fermention medium replaces to the second substitutive medium in the step, collect to get for the third time filtered liquid and albumen dry powder, adding for the third time ethylenediamine tetraacetic acid (EDTA) (EDTA) in the filtered liquid recycles, be every liter for the third time in the filtered liquid EDTA addition be 1.3g/L, get the 3rd mixed solution, be that 85% phosphoric acid and mass concentration are that 35% ammoniacal liquor is transferred the 3rd mixed solution with mass concentration again, pH value to 5.5～6.0, get for the third time ferment filtrate, the consumption of inorganic salt in this inorganic salt fermention medium used when ferment filtrate can be used to carry out Methanol Protein production partly alternative next time for the third time;
9), the recycling of ferment filtrate for the third time
Get respectively above-mentioned 3) phosphoric acid, inorganic salt and the phosphoric acid of trace element solution consumption 20%, 20% inorganic salt, 20% trace element solution, glycerine 25kg/m in the BSM inorganic salt fermentative medium formula in the step
3Ferment filtrate 20m for the third time
3Mix, add water to 30m
3, get the 3rd substitutive medium, for subsequent use, described inorganic salt are dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3, magnesium sulfate heptahydrate 2kg/m
3Again by above-mentioned 1), 2), 3), 4), 5), method and consumption in the step prepare albumen dry powder again, wherein, when this prepares the albumen dry powder process, with the above-mentioned the 4th) used fermention medium replaces to the 3rd substitutive medium in the step, be prepared into the 4th filtered liquid and albumen dry powder, the 4th time filtered liquid is not recycled, and can be discharged into sewage treatment plant.
The present invention relates to the problem that the microbial fermentation solution in coal biochemistry and the technical field of bioengineering repeatedly utilizes again, be specifically related to the suitability for industrialized production Methanol Protein first the fermented liquid after the fermentation ends through centrifugal, after the filtration, the filtered liquid that obtains, according to the number of times that will recycle, generally can recycle three times, the mode that increases progressively successively in recycling is each time added the ethylenediamine tetraacetic acid (EDTA) (EDTA) of 0.5-1.3g/L and is made respectively the ferment filtrate that can recycle, when again carrying out Methanol Protein production fermentation, reclaim ferment filtrate can substitute the inorganic salt of 10-20% in the former inorganic salt fermention medium, the 10-20% trace element solution, Carbon and nitrogen sources still adopts primary fermentation parameter, record through repeated multiple times experiment, repeatedly do not affect the growth of yeast cell and the productive rate of Methanol Protein fully in the fermentation in follow-up, and reduced that the input of inorganic salt and trace element solution reaches 10-20% in the inorganic salt fermention medium, also improved the Methanol Protein production technique, reduced cost, saved water resources, reduced discharge of wastewater, enhance productivity and reach 30-40%, huge economic benefit and social benefit are arranged, operating procedure is batch operation, continuous circulation operation, having solved provides a kind of and adds technology and the method that an amount of inorganic salt carry out secondary and repeatedly ferment in the fermented liquid after the fermentation ends first to the suitability for industrialized production Methanol Protein.
Claims (1)
1. the method for a Methanol Protein ferment filtrate recycling is characterized in that, comprises the steps:
1), shake-flask seed is cultivated:
Classification And Nomenclature is the bacterial strain of pichia pastoris phaff HGD-01, has been preserved in Chinese Typical Representative culture collection center, and deposit number is: CCTCC NO:M2012342;
The bacterial strain of the 0.5mL pichia pastoris phaff HGD-01 of glycerine pipe preservation is seeded on the YPD test tube slant substratum with method of scoring, cultivated 48 hours, and namely got the inclined-plane seed for 30 ℃; Get 1 cultured inclined-plane seed, with the 10mL sterilized water inclined-plane seed is all washed, be inoculated in the triangular flask that 250mL YPD liquid nutrient medium is housed, bottleneck binds up with gauze with 8 layers, the shaking table shaking culture, 30 ℃ of culture temperature, shaking speed 220rpm, after cultivating about 24～30 hours, thalline OD
600Reach 3～5, obtain shake-flask seed; Described YPD test tube slant substratum is mixed by the water of 1% yeast extract, 2% Tryptones, 2% glucose, 2% agar and the surplus of weight percent meter and forms; Described YPD liquid nutrient medium is mixed by the water of 1% yeast extract, 2% Tryptones, 2% glucose and the surplus of weight percent meter and forms;
2), first order seed is cultivated:
A, first order seed is cultivated and is carried out in the fermentor tank of 50L, in the 50L tank, prepare the YPD liquid nutrient medium of 30L by the described method of step 1), open steam valve, make steam advance fermentor tank chuck layer, the YPD liquid nutrient medium is sterilized, 121 ℃ of sterilising temps, sterilization time 30min is when sterilizing to the YPD liquid nutrient medium, air filter and thief hole are sterilized, logical high-temp steam sterilizing 10min, sterilization is opened cooling water valve after finishing, make cold true water advance fermentor tank chuck layer, substratum is lowered the temperature, when the substratum temperature is down to 30 ℃, begin inoculation;
B, shake-flask seed 1000mL is inoculated in the YPD liquid nutrient medium in the 50L fermentor tank, 28～30 ℃ of temperature, rotating speed 150rpm, dissolved oxygen 40%～50%, cultivate 20～24h after, get first order seed;
3), secondary seed is cultivated:
With 3m
3BSM inorganic salt fermention medium put into 5m
3In the fermentor tank, with 2) the identical sterilising method of YPD liquid nutrient medium is sterilized to BSM inorganic salt fermention medium in the A step, after sterilization is finished, be that 35% ammoniacal liquor is transferred BSM inorganic salt fermention medium pH value to 4.8～5.0 with mass concentration, then the 30L first order seed be inoculated into 5m by pressure pipeline
3The pH value is that tank pressure is 0.05-0.10MPa during inoculation, during cultivation in 4.8～5.0 the BSM inorganic salt fermention medium in the fermentor tank, 30～32 ℃ of culture temperature, ventilating ratio 1:1 ~ 1.5 vvm, tank pressure 0.05MPa, 24 ~ 30h is cultivated in pH5.0～5.5, gets secondary seed; Described BSM inorganic salt fermention medium is: mass concentration is 85% phosphoric acid 25kg/m
3, dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3, magnesium sulfate heptahydrate 2kg/m
3, glycerine 25kg/m
3With trace element solution 4L/m
3Add water and make, that is to say that BSM inorganic salt fermention medium is: mass concentration is that 85% phosphoric acid 25kg, dipotassium hydrogen phosphate 0.5kg, ammonium chloride 0.3kg, terra alba 0.2kg, sodium-chlor 0.5kg, vitriolate of tartar 3kg, magnesium sulfate heptahydrate 2kg, glycerine 25kg and trace element solution 4L add water to 1m
3Described trace element solution is: cupric sulfate pentahydrate 1.5g/L, potassiumiodide 0.02g/L, manganese sulfate monohydrate 0.5g/L, Sodium Molybdate Dihydrate 0.2g/L, boric acid 0.02g/L, cobalt chloride hexahydrate 0.3g/L, zinc chloride 20g/L, ferrous sulfate 19g/L, the vitriol oil 4mL/L of mass concentration 98%, vitamin H 0.2g/L, para-amino benzoic acid 0.05g/L and calcium pantothenate 0.05g/L add water and make, and that is to say that trace element solution is: cupric sulfate pentahydrate 1.5g, potassiumiodide 0.02g, manganese sulfate monohydrate 0.5g, Sodium Molybdate Dihydrate 0.2g, boric acid 0.02g, cobalt chloride hexahydrate 0.3g, zinc chloride 20g, ferrous sulfate 19g, the vitriol oil 4mL of mass concentration 98%, vitamin H 0.2g, para-amino benzoic acid 0.05g and calcium pantothenate 0.05g add water to 1L;
4), high density fermentation:
At 50m
3Add BSM inorganic salt fermention medium 30m in the fermentor tank
3As fermention medium, sterilize with the steam more than 160 ℃, simultaneously to 50m
3The pipeline that fermentor tank links to each other is sterilized, and sterilization is down to 30～35 ℃ with the fermention medium temperature after finishing, and is that 35% ammoniacal liquor is readjusted the distribution ferment medium pH to 4.5～5.0 with mass concentration, then with secondary seed 3m
3Be inoculated into 50m
3PH ferments in 4.5～5.0 the fermention medium in the fermentor tank, and leavening temperature is 30～32 ℃, air flow 1.5～2vv/m, mixing speed 80-200rpm, after 24 hours, glycerine exhausts in the substratum, and dissolved oxygen begins to rise, begin stream and add methyl alcohol, with methyl alcohol detecting instrument on-line monitoring methanol concentration, make methanol concentration be controlled at 0.8%～1.2%, 45-50 hour after, the thalline weight in wet base reaches 250-300 g/L, OD
600Value reaches 42～45, and this moment, fermentation ends namely got fermented liquid;
5), the collection of filtered liquid:
Fermented liquid leaves standstill, sedimentation 4～8 hours, and high fermentation liquid carries out centrifugal with horizontal screw centrifuge, 3000 rev/mins of centrifuge speeds, inlet amount 5m
3/ h, centrifugal clear liquid filter with flame filter press again, filtration area 20m
2, filtration flow-rate 1.5 m
3/ h filters to get for the first time filtered liquid and bacterium cake, and the bacterium cake is put into 5m
3In the fermentation storage tank, add the clear water of bacterium cake volume 1/5, stir, enter spray-drier, regulate intake air temperature to 140～160 ℃, temperature out to 65-80 ℃, regulating feed rate is 1 ton/hour, and spraying drying is collected albumen dry powder;
6), for the first time collection of ferment filtrate
Add ethylenediamine tetraacetic acid (EDTA) in the filtered liquid for the first time, every liter for the first time in the filtered liquid EDTA addition be 0.5g/L, the first mixed solution, be that 85% phosphoric acid and mass concentration are that 35% ammoniacal liquor is transferred the first pH of mixed value to 5.5～6.0 with mass concentration again, get the ferment filtrate first time;
7), for the second time collection of ferment filtrate
Get respectively above-mentioned 3) phosphoric acid, inorganic salt and the phosphoric acid of trace element solution consumption 10%, 10% inorganic salt, 10% trace element solution, glycerine 25kg/m in the BSM inorganic salt fermentative medium formula in the step
3With the ferment filtrate 20m first time
3Mix, add water to 30m
3, get the first substitutive medium, for subsequent use; Described inorganic salt are dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3With magnesium sulfate heptahydrate 2kg/m
3Again by above-mentioned 1), 2), 3), 4), 5), method in the step and consumption are collected for the second time filtered liquid again, wherein, in the process of collecting the filtered liquid second time, with the above-mentioned the 4th) used fermention medium replaces to the first substitutive medium in the step, collect to get for the second time filtered liquid and albumen dry powder, for the second time filtered liquid interpolation ethylenediamine tetraacetic acid (EDTA) is recycled, be every liter for the second time in the filtered liquid EDTA addition be 0.9g/L, get the second mixed solution, be that 85% phosphoric acid and mass concentration are that 35% ammoniacal liquor is transferred the second pH of mixed value to 5.5～6.0 with mass concentration again, get for the second time ferment filtrate;
8), for the third time collection of ferment filtrate
Get respectively above-mentioned 3) phosphoric acid, inorganic salt and the phosphoric acid of trace element solution consumption 15%, 15% inorganic salt, 15% trace element solution, glycerine 25kg/m in the BSM inorganic salt fermentative medium formula in the step
3With the ferment filtrate 20m second time
3Mix, add water to 30m
3, get the second substitutive medium, for subsequent use, described inorganic salt are dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3, magnesium sulfate heptahydrate 2kg/m
3Again by above-mentioned 1), 2), 3), 4), 5), method in the step and consumption are collected for the third time filtered liquid again, wherein, collect in the process of filtered liquid for the third time, with the above-mentioned the 4th) used fermention medium replaces to the second substitutive medium in the step, collect to get for the third time filtered liquid and albumen dry powder, adding for the third time ethylenediamine tetraacetic acid (EDTA) in the filtered liquid recycles, be every liter for the third time in the filtered liquid EDTA addition be 1.3g/L, get the 3rd mixed solution, be that 85% phosphoric acid and mass concentration are that 35% ammoniacal liquor is transferred the 3rd mixed solution with mass concentration again, pH value to 5.5～6.0 get for the third time ferment filtrate;
9), the recycling of ferment filtrate for the third time
Get respectively above-mentioned 3) phosphoric acid, inorganic salt and the phosphoric acid of trace element solution consumption 20%, 20% inorganic salt, 20% trace element solution, glycerine 25kg/m in the BSM inorganic salt fermentative medium formula in the step
3Ferment filtrate 20m for the third time
3Mix, add water to 30m
3, get the 3rd substitutive medium, for subsequent use, described inorganic salt are dipotassium hydrogen phosphate 0.5kg/m
3, ammonium chloride 0.3kg/m
3, terra alba 0.2kg/m
3, sodium-chlor 0.5kg/m
3, vitriolate of tartar 3kg/m
3, magnesium sulfate heptahydrate 2kg/m
3Again by above-mentioned 1), 2), 3), 4), 5), method and consumption in the step prepare albumen dry powder again, wherein, when this prepares the albumen dry powder process, with the above-mentioned the 4th) used fermention medium replaces to the 3rd substitutive medium in the step, be prepared into the 4th filtered liquid and albumen dry powder, the 4th time filtered liquid is discharged into sewage treatment plant.
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| CN111172046A (en) * | 2020-01-20 | 2020-05-19 | 义马煤业集团煤生化高科技工程有限公司 | Production method for extracting fiber-grade protein from microorganism |
| CN113564216A (en) * | 2021-07-20 | 2021-10-29 | 蓝科医美科学技术(吉林)有限公司 | Method for obtaining tridecapeptide through yeast fermentation and application thereof |
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| CN101307294A (en) * | 2008-06-02 | 2008-11-19 | 南京工业大学 | Pichia yeast for rapid utilizing industrial methanol and uses thereof |
| CN102517223A (en) * | 2011-12-28 | 2012-06-27 | 温州市龙泰轻工机械有限公司 | High-density fermentation process and equipment for methanol type pichia pastoris |
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| CN101307294A (en) * | 2008-06-02 | 2008-11-19 | 南京工业大学 | Pichia yeast for rapid utilizing industrial methanol and uses thereof |
| CN102517223A (en) * | 2011-12-28 | 2012-06-27 | 温州市龙泰轻工机械有限公司 | High-density fermentation process and equipment for methanol type pichia pastoris |
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| CN111172046A (en) * | 2020-01-20 | 2020-05-19 | 义马煤业集团煤生化高科技工程有限公司 | Production method for extracting fiber-grade protein from microorganism |
| CN113564216A (en) * | 2021-07-20 | 2021-10-29 | 蓝科医美科学技术(吉林)有限公司 | Method for obtaining tridecapeptide through yeast fermentation and application thereof |
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