CN101307294A - Pichia pastoris for quickly utilizing industrial methanol and application thereof - Google Patents
Pichia pastoris for quickly utilizing industrial methanol and application thereof Download PDFInfo
- Publication number
- CN101307294A CN101307294A CNA200810123749XA CN200810123749A CN101307294A CN 101307294 A CN101307294 A CN 101307294A CN A200810123749X A CNA200810123749X A CN A200810123749XA CN 200810123749 A CN200810123749 A CN 200810123749A CN 101307294 A CN101307294 A CN 101307294A
- Authority
- CN
- China
- Prior art keywords
- methyl alcohol
- pichia pastoris
- scp01
- thalline
- pichia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 title claims abstract description 147
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 13
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 19
- 241000235648 Pichia Species 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 14
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 230000001133 acceleration Effects 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 6
- 230000000996 additive effect Effects 0.000 claims description 6
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 230000000630 rising effect Effects 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 abstract description 17
- 230000004151 fermentation Effects 0.000 abstract description 17
- 108010027322 single cell proteins Proteins 0.000 abstract description 11
- 235000019750 Crude protein Nutrition 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 238000004321 preservation Methods 0.000 abstract description 4
- 239000002054 inoculum Substances 0.000 abstract description 3
- 238000004113 cell culture Methods 0.000 abstract 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 13
- 239000007788 liquid Substances 0.000 description 13
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 235000013305 food Nutrition 0.000 description 7
- 235000011187 glycerol Nutrition 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 239000002994 raw material Substances 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 244000068988 Glycine max Species 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 108010025188 Alcohol oxidase Proteins 0.000 description 3
- 235000019733 Fish meal Nutrition 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000004467 fishmeal Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- -1 pplication Species 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000235342 Saccharomycetes Species 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-L (R)-2-Hydroxy-3-(phosphonooxy)-propanal Natural products O=C[C@H](O)COP([O-])([O-])=O LXJXRIRHZLFYRP-VKHMYHEASA-L 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000235349 Ascomycota Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- LXJXRIRHZLFYRP-VKHMYHEASA-N D-glyceraldehyde 3-phosphate Chemical compound O=C[C@H](O)COP(O)(O)=O LXJXRIRHZLFYRP-VKHMYHEASA-N 0.000 description 1
- 240000005708 Eugenia stipitata Species 0.000 description 1
- 235000006149 Eugenia stipitata Nutrition 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000190932 Rhodopseudomonas Species 0.000 description 1
- 241000235344 Saccharomycetaceae Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229940036811 bone meal Drugs 0.000 description 1
- 239000002374 bone meal Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000013409 condiments Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 235000012976 tarts Nutrition 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a Pichia pastoris capable of taking industrial methanol as a unique carbon source and energy, which is classified and named as Pichia pastoris (ME-SCP 01) and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation number of the strain is CGMCC NO. 2477. The strain belongs to pichia pastoris which can rapidly utilize methanol, and can realize high-density cell culture. The strain can be used for producing single-cell protein. 1000 liter tank fermentation experiments show that: in the case of an inoculum size of 5%, the dry cell weight reached 120-130 g/l and the crude protein content reached about 50% after about 85 hours of fermentation culture.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of Pichia yeast that can utilize industrial methanol fast, utilize the method for Pichia yeast manufacture order cell protein and the application of this Pichia yeast.
Background technology
(Single-Cell Protein SCP), claims tropina or microbial proteinous again to single cell protein, is meant by mass-producing to cultivate the tropina as feed or food that non-pathogenic microorganism produces.
With the industrial methanol is that the methyl alcohol albumen that raw material production is come out is called as s-generation single cell protein, compare with native protein, its crude protein content is all higher than fish meal and soybean, contain abundant indispensable amino acid, minerals and vitamins, can the part substituted fish meal, soybean, bone meal, meat and skim-milk.And the proteic raw material of methyl alcohol is easy to get, resource is abundant relatively, do not account for the arable land, do not rely on the ocean, production is not subjected to weather influence, production rate is fast.
In April, 2008; Food and Argriculture OrganizationFAO claims; because the soil of many suitable plantation grains; be used for building city, commercial centre or even golf course in a large number; make the interior a lot of countries of global range all begin to face crisis in food; Asia, the more geographic disadvantaged countries in Africa stand in the breach, and the minimum level since 1980 has been reduced in present global grain reserves.And with grains such as soybean be the feedstuff industry of main raw material also natural be subjected to serious impact, therefore, be the methyl alcohol albumen of raw material with the industrial methanol, just be subjected to worldwide extensive concern as a kind of novel biological protein feed.
The relevant expert analyzes, and to 2010 and the year two thousand twenty, China's protein feed annual requirement was respectively 6,000 ten thousand tons and 7,200 ten thousand tons, and feed rate only is 2,200 ten thousand tons and 2,400 ten thousand tons, and insufficiency of supply-demand reaches 38,000,000 tons and 48,000,000 tons.Along with market to the albumen growth of requirement, and fish meal and soybean resource are limited, can't satisfy the demand in market, and be very favourable to the proteic development of methyl alcohol.According to scholarly forecast, China is about 300～5,000,000 tons at present to the proteic market requirement of methyl alcohol every year.
Single cell protein can be made " synthetic meat ", and is directly edible for people, can also be as foodstuff additive, in order to replenish protein or VITAMIN, mineral substance etc.Because some single cell protein has resistance of oxidation, make food be not easy to go bad, thereby be usually used in infant powder and soup stock, the condiments.The heat content of dry yeast is low, the additive of Chang Zuowei diet food.In addition, single cell protein can also improve some physicals of food, as adding live yeast in the Italian tart, can improve the thin performance of prolonging of cake.The zymic protein concentrate has significant delicate flavour, has been widely used as the freshener of food.Single cell protein has also worldwide obtained widespread use as feedstuff protein.
World wide, the proteic production fermentation time of methyl alcohol generally reached about 160 hours, and leavening temperature does not wait at 30～38 ℃, utilized this strain fermentation time at 80～90 hours, 30 ℃ of leavening temperatures have been realized the high density fermentation that temperature is relatively low, the time is short.
Summary of the invention
The objective of the invention is to filter out the Pichi strain that a strain has industrial prospect, for the production of methyl alcohol single cell protein lays the foundation at shortcomings such as methyl alcohol protein production fermentation time are long.
Another object of the present invention provides a kind of method of utilizing Pichia yeast from industrial methanol manufacture order cell protein.
A further object of the invention provides the application of this bacterium aspect feed or foodstuff additive.
Purpose of the present invention can reach by following measure:
Yeast strain of the present invention is pichia pastoris (Pichia pastoris), be numbered ME-SCP01, this bacterial strain is preserved China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on April 29th, 2008, and its preserving number is CGMCC NO:2477.
Pichia pastoris ME-SCP01 belongs to Ascomycota, Hemiascomycetes, Saccharomycetes, Saccharomycetaceae, yeast belong.Cultivate the epibasal cell majority at YPD and be oval, budding; The bacterium colony quality is even, and milky white matt, surface wettability, thickness are easily provoked.
Bacterial strain ME-SCP01 can be on minimal medium normal growth, can be with glucose, glycerine, methyl alcohol etc. as the sole carbon source and the energy.When making carbon source with glucose or glycerine, have only 1 or seldom several little peroxysome in the thalline, and with methyl alcohol during as carbon source, peroxysome almost accounts for 80% of whole cell volume, alcohol oxidase increases to the 35%-40% of total protein of cell.
Bacterial strain ME-SCP01 all can grow between 20～37 ℃, and optimum growth temperature is 27～30 ℃, and is wherein the fastest 30 ℃ growth velocity.The pH value scope of substratum is between 3.0～6.0, and the best pH of thalli growth and synthetic protein is 5.0～6.0.
It is the technology of raw material production single cell protein with the industrial methanol that the present invention provides a kind of pichia spp that utilizes simultaneously.
Methyl alcohol enters the intracellular lysosome of pichia spp, induces the expression of alcohol oxidase, and alcohol oxidase is a formaldehyde with methanol oxidation then.Part formaldehyde continues to be oxidized to formic acid, finally generates CO
2Release energy, another part formaldehyde then generates glyceraldehyde 3-phosphate by the circulation of 5 lyxulose phosphates, enters main pathways metabolism then, synthetic thalline.
Utilize the method for pichia pastoris ME-SCP01 manufacture order cell protein to be: earlier with bacterial strain ME-SCP01, under the condition of primary carbon source, breed thalline rapidly, when initial carbon source consumption will be use up, slow stream adds methyl alcohol and cultivated 70～100 hours, collect thalline, drying, pulverizing obtain single cell protein.Be specially under a small amount of glycerine or the condition of glucose as primary carbon source, breed thalline rapidly, when initial carbon source consumption will be use up, slowly stream added methyl alcohol, along with the rising of thalline to the utilization ratio of methyl alcohol, improved methyl alcohol stream rate of acceleration gradually, can reach 8-10mlh
-1L
-1About, through the cultivation of 80-90 hour (preferably), cell has been realized high-density culture, and dry cell weight can reach the 120-130 grams per liter, and crude protein content has reached 50%.By method results thalline such as centrifugal, through fluidised bed drying and pulverizer is simple promptly can be used as protein fodder after pulverizing; Through deep processing, separate the total protein that proposes in the thalline, can be used as feed or foodstuff additive, as protein additive etc.
Pichia pastoris (Pichia pastoris) is that a few can be one of microorganism of the sole carbon source and the energy with methyl alcohol.The described bacterial strain Pichia of this patent pastoris ME-SCP01 can utilize conventional industrial methanol to produce tropina, and GS115 compares with the pattern pichia spp, and growth velocity is faster, therefore has bigger commercial promise.
Biomaterial preservation information: Pichia yeast, its classification called after pichia pastoris (Pichiapastoris) ME-SCP01, (be called for short: CGMCC at China Committee for Culture Collection of Microorganisms common micro-organisms center on April 29th, 2008, address: China. Beijing. Da Tun road, Chaoyang District) preservation, its preserving number is: CGMCC NO.2477.
Embodiment
Embodiment 1: the separation of bacterial strain ME-SCP01 and culture condition
In the aerobic activated sludge of analyzing Sichuan grand safe chemical industry company limited industrial wastewater treatment system, during primary Microbial Populations in Active, on extractum carnis-protein culture medium, isolated the microorganism that many strains are subordinated to bacillus, Rhodopseudomonas and yeast belong.Utilize ability to test to the methyl alcohol of a strain pichia spp wherein, and with pichia spp GS115 in contrast.Tentative experiment shows that this pichia spp utilizes the speed of methyl alcohol concentration faster than bacterial strain GS115, that tolerate methyl alcohol higher.Therefore, utilize this pichia spp can carry out the production of single cell protein.With this bacterial strain called after ME-SCP01.
Conventional YEPD complex medium is adopted in the cultivation of bacterial strain ME-SCP01 and preservation: yeast extract, 1%, peptone, 2%, glucose, 2%.Preparation adds 2% agar during solid medium in sterilization forward direction liquid YEPD.
The optimum culturing temperature of bacterial strain ME-SCP01 is 30 ℃.During liquid culture, dress liquid 50ml in the triangular flask of 250ml, shaking speed is 200rpm.
Embodiment 2: the seed culture of bacterial strain ME-SCP01
One-level shaking table seed: YEPD substratum (it is the same to fill a prescription) is adopted in the cultivation of level liquid seed.At first bacterial classification ME-SCP01 is seeded to the 250ml triangular flask that 100ml liquid YEPD substratum is housed from the YEPD inclined-plane of preserving, 30 ℃ of shaking tables were cultivated 24-30 hour, and rotating speed is 200rpm.
Secondary seed: first order seed is forwarded to 10 liters of fermentor tanks that 6 liters of liquid nutrient mediums are housed, culture condition according to 10% inoculum size: air flow 1-2vv/m, stirring velocity 600-800rpm, incubation time 24-30 hour, 30 ℃ of culture temperature.Substratum is selected minimal medium for use, and it is as follows specifically to fill a prescription: glycerine, 40g, CaSO
4, 0.9g, K
2SO
4, 14.3g, MgSO
47H
2O, 11.8g, KOH, 3.8g, H
3PO
4(85%), 20ml, liquid microelement 20ml is settled to 1 liter with distilled water, 121 ℃ of sterilizations 30 minutes, cooling is adjusted to the pH value between the 5.0-6.0 with the ammoniacal liquor of filtration sterilization later.Trace element liquid formula: CuSO
45H
2O, 2g, NaI, 0.05g, MnSO
4H
2O, 2g, Na
2MoO
42H
2O, 0.15g, H
3BO
3, 0.02g, CoCl
2, 0.5g, ZnCl
2, 15g, FeSO
47H
2O, 20g, vitamin H, 0.2g, H
2SO
4, 3.5ml, be settled to 1 liter with distilled water, filtration sterilization.
Three grades and subsequent seed: successively the previous stage seed is pressed 10 times of fermentor tanks that amplify step by step according to 10% inoculum size access volume.Substratum, culture condition are described consistent with secondary seed.
Embodiment 3: 7 liters of jar high density fermentation tests of bacterial strain ME-SCP01
The massfraction of methyl alcohol in substratum can only remain on 1% at most, and too high methanol concentration can suppress the growth of thalline ME-SCP01 even poison its cell with poison.Therefore, for the fermentation of methyl alcohol as carbon source
Experiment, the mode that has adopted Continuous Flow to add.
Bacterial strain ME-SCP01 has been carried out the fermenting experiment of 7 liters of jars (NBS reequip jar), and liquid amount is 3.5 liters, and fermentative medium formula is consistent with secondary seed medium prescription among the embodiment 2, employing inorganic salt prescription, and primary carbon source is glycerine.Add proper quantity of defoaming agent in the fermentor tank.
Seed is prepared: the first order seed of preparing 300ml according to embodiment 2 described methods;
High density fermentation is cultivated: seed is all inserted fermentor tank, and 30 ℃ of stir culture, mixing speed is 800-1000rpm.Air flow is 1.5vvm.PH is set at 5.4, and the method that adopts stream to add ammoniacal liquor is stablized the pH value.Exhaust back (after general about 20 hours), dissolved oxygen (DO at glycerine
2) sharply rising of beginning, begin slow stream and add methyl alcohol this moment, all the time dissolved oxygen level is controlled at about 25% of maximum dissolved oxygen level.DO
2Be lower than 25%, slow down the methyl alcohol flow acceleration, be higher than 25%, suitably increase the methyl alcohol flow acceleration, the highest flow acceleration of methyl alcohol can reach 8mlh
-1L
-1After fermentation proceeded to 85 hours, methyl alcohol utilized speed obviously to slow down, and the cell growth progressively enters decline phase.
The collection of thalline and protein content detect: collect fermentation broth sample, centrifugal anhydrating, recentrifuge desalts behind the distilled water wash, with the wet thallus of centrifugal acquisition 105 ℃ of oven for drying to constant weight, take by weighing its quality, through estimation, can make the dry cell weight of fermented liquid reach 124 grams per liters by the described fermentation process of this example, realized high-density culture.
With bacterial cell disruption, measured the crude protein content of ME-SCP01 thalline with ultrasonic wave by the Xylene Brilliant Cyanine G method, through with the estimation of typical curve, the total protein content among the bacterial strain ME-SCP01 is about 50%.
Embodiment 4: 1000 liters of jar fermentation industry flow processs of bacterial strain ME-SCP01 manufacture order cell protein
Seed is prepared: according to embodiment 3 described methods, prepare 60 liters of three grades of liquid seeds.
Fermentation culture: fermentative medium formula is consistent with fermentative medium formula among the embodiment 3.
The seeding tank seed is all inserted 1000 liters of fermentor tanks, and liquid amount is 600 liters, 30 ℃ of stir culture, and mixing speed is 300-400rpm.Air flow is 1-2vvm.PH is set at 5.4, and the method that adopts stream to add ammoniacal liquor is stablized the pH value.Exhaust back stream at glycerine and add methyl alcohol, the highest flow acceleration of methyl alcohol can reach 8-10mlh
-1L
-1After fermentation proceeded to about 80-90 hour, fermentation stopped.
The methyl alcohol protein Preparation: fermented liquid is through continuous centrifuge is centrifugal concentrated automatically, with tap water washing back recentrifuge; Wet thallus dewaters through fluidised bed drying, and moisture is remained on below 10%, pulverizes moulding through pulverizer then, and packing promptly obtains feed grade methyl alcohol albumen finished product.
Because being continuously a small amount of stream, methyl alcohol adds, so in product, detect residual less than methyl alcohol.By the described fermentation process of this example the dry cell weight of fermented liquid is reached about the 120-130 grams per liter, the total protein content among the bacterial strain ME-SCP01 is about 50%.
Claims (5)
1, a kind of Pichia yeast that utilizes industrial methanol fast, its classification called after pichia pastoris (Pichia pastoris) ME-SCP01, preserve at China Committee for Culture Collection of Microorganisms common micro-organisms center, its preserving number is: CGMCC NO.2477.
2, utilize the method for the described Pichia yeast manufacture order of claim 1 cell protein, it is characterized in that earlier bacterial strain ME-SCP01, under the condition of primary carbon source, breed thalline rapidly, when initial carbon source consumption will be use up, slow stream adds methyl alcohol and cultivated 70～100 hours, collect thalline, dry, pulverizing.
3, method according to claim 2 is characterized in that when stream adds methyl alcohol along with the rising of thalline to the utilization ratio of methyl alcohol, improves methyl alcohol stream rate of acceleration gradually, is up to 8-10mlh
-1L
-1
4, method according to claim 2 is characterized in that incubation time is 80～90 hours.
5, the application of the described Pichia yeast of claim 1 aspect feed or foodstuff additive.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810123749XA CN101307294A (en) | 2008-06-02 | 2008-06-02 | Pichia pastoris for quickly utilizing industrial methanol and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200810123749XA CN101307294A (en) | 2008-06-02 | 2008-06-02 | Pichia pastoris for quickly utilizing industrial methanol and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101307294A true CN101307294A (en) | 2008-11-19 |
Family
ID=40123982
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA200810123749XA Pending CN101307294A (en) | 2008-06-02 | 2008-06-02 | Pichia pastoris for quickly utilizing industrial methanol and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101307294A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154406A (en) * | 2011-01-18 | 2011-08-17 | 东华大学 | Method for producing bacterial cellulose by taking high-concentration methanol-containing waste water as raw material |
| CN103045703A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Recycling method of methanol protein fermentation filtrate |
| CN103045494A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for efficiently converting methanol to produce single cell protein and application of pichia pastoris |
| CN103060211A (en) * | 2013-01-05 | 2013-04-24 | 义马煤业集团煤生化高科技工程有限公司 | Fermentation culture medium for producing methanol protein |
| CN103060212A (en) * | 2013-01-05 | 2013-04-24 | 义马煤业集团煤生化高科技工程有限公司 | Industrial production method for single-cell methanol protein |
| CN105176853A (en) * | 2015-10-16 | 2015-12-23 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for simultaneously producing methanol protein and xylanase and application of pichia pastoris |
| CN107177517A (en) * | 2016-03-09 | 2017-09-19 | 中国科学院微生物研究所 | Efficiently tolerance and the yeast strain using methanol and application |
| CN111359425A (en) * | 2019-09-05 | 2020-07-03 | 朱启常 | Biological material for degrading formaldehyde and preparation and use methods thereof |
| CN112646740A (en) * | 2020-11-27 | 2021-04-13 | 中国科学院天津工业生物技术研究所 | Formate single-cell protein strain MA5 and application thereof |
-
2008
- 2008-06-02 CN CNA200810123749XA patent/CN101307294A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154406A (en) * | 2011-01-18 | 2011-08-17 | 东华大学 | Method for producing bacterial cellulose by taking high-concentration methanol-containing waste water as raw material |
| CN103045703A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Recycling method of methanol protein fermentation filtrate |
| CN103045494A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for efficiently converting methanol to produce single cell protein and application of pichia pastoris |
| CN103060211A (en) * | 2013-01-05 | 2013-04-24 | 义马煤业集团煤生化高科技工程有限公司 | Fermentation culture medium for producing methanol protein |
| CN103060212A (en) * | 2013-01-05 | 2013-04-24 | 义马煤业集团煤生化高科技工程有限公司 | Industrial production method for single-cell methanol protein |
| CN103045703B (en) * | 2013-01-05 | 2014-03-12 | 义马煤业集团煤生化高科技工程有限公司 | Recycling method of methanol protein fermentation filtrate |
| CN105176853A (en) * | 2015-10-16 | 2015-12-23 | 义马煤业集团煤生化高科技工程有限公司 | Pichia pastoris for simultaneously producing methanol protein and xylanase and application of pichia pastoris |
| CN105176853B (en) * | 2015-10-16 | 2018-06-29 | 义马煤业集团煤生化高科技工程有限公司 | One plant of Pichia pastoris and its application for producing Methanol Protein and zytase simultaneously |
| CN107177517A (en) * | 2016-03-09 | 2017-09-19 | 中国科学院微生物研究所 | Efficiently tolerance and the yeast strain using methanol and application |
| CN111359425A (en) * | 2019-09-05 | 2020-07-03 | 朱启常 | Biological material for degrading formaldehyde and preparation and use methods thereof |
| CN112646740A (en) * | 2020-11-27 | 2021-04-13 | 中国科学院天津工业生物技术研究所 | Formate single-cell protein strain MA5 and application thereof |
| CN112646740B (en) * | 2020-11-27 | 2021-11-30 | 中国科学院天津工业生物技术研究所 | Formate single-cell protein strain MA5 and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101307294A (en) | Pichia pastoris for quickly utilizing industrial methanol and application thereof | |
| CN106222098B (en) | One plant of monascus strain and its application | |
| CN102796673B (en) | Feruloyl esterase production strain and method for producing feruloyl esterase by using same | |
| CN114107077B (en) | Ester-producing yeast strain and application thereof | |
| CN103045494B (en) | Pichia pastoris for efficiently converting methanol to produce single cell protein and application of pichia pastoris | |
| CN101109015A (en) | Preparation method of arachidonic acid grease | |
| CN108220175A (en) | Saccharomyces cerevisiae high-density cultivation method and its pH regulation and control methods | |
| CN101703214A (en) | Lucid Ganoderma hypra powder or Lucid Ganoderma tea and double fermentation process | |
| CN105580975A (en) | Technology for extracting seaweed protein by means of microbial fermentation method | |
| CN107287127A (en) | The production ester Pichia pastoris of one plant of resistance to lactic acid | |
| CN103265362B (en) | Compatibility and production method of pholiota nameko cultivation material | |
| Karasu-Yalcin et al. | Effects of different fermentation conditions on growth and citric acid production kinetics of two Yarrowia lipolytica strains | |
| CN103421642B (en) | Method for processing cider wine containing more ester | |
| CN109370933A (en) | A kind of co-culture method improving saccharomycete acid-fast ability | |
| CN109022227A (en) | A kind of method of ethyl acetate content in raising white spirit original wine | |
| CN100429302C (en) | Method for separating and purifying wine-brewing microzyme from black tea fungus | |
| CN103451247B (en) | A kind of arachidonic preparation method | |
| CN102987063B (en) | Organic acid animal growth regulator and preparation method thereof | |
| CN105368736A (en) | Bacillus aceticus and application of bacillus aceticus in coffee cherry peel and pulp fermentation vinegar | |
| CN110903988A (en) | Complex microbial inoculant and application thereof | |
| CN101709297A (en) | Mutagenesis screening method of arachidonic acid producing strain mortierella alpina | |
| CN116162554B (en) | Interval type mai cell strain and application thereof in replacement of protein | |
| CN105368887B (en) | A kind of fermentation manufacturing technique of epsilon-poly-L-lysine | |
| Yao et al. | Relationship between saccharifying capacity and isolation sources for strains of the Rhizopus arrhizus complex | |
| CN102860412A (en) | Co-production method for distilled spirit and livestock total nutrient feed through alfalfa fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20081119 |