CN109706197A - A kind of technique of preparative separation glutamic acid and egg white icing - Google Patents
A kind of technique of preparative separation glutamic acid and egg white icing Download PDFInfo
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- CN109706197A CN109706197A CN201811210254.0A CN201811210254A CN109706197A CN 109706197 A CN109706197 A CN 109706197A CN 201811210254 A CN201811210254 A CN 201811210254A CN 109706197 A CN109706197 A CN 109706197A
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- glutamic acid
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 title claims abstract description 81
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 70
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 70
- 238000000034 method Methods 0.000 title claims abstract description 23
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 20
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 20
- 235000014103 egg white Nutrition 0.000 title claims abstract description 20
- 210000000969 egg white Anatomy 0.000 title claims abstract description 20
- 238000000926 separation method Methods 0.000 title claims abstract description 11
- 238000011218 seed culture Methods 0.000 claims abstract description 13
- 238000000855 fermentation Methods 0.000 claims description 63
- 230000004151 fermentation Effects 0.000 claims description 63
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 45
- 239000007788 liquid Substances 0.000 claims description 27
- 239000000654 additive Substances 0.000 claims description 17
- 230000000996 additive effect Effects 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 16
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 15
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 15
- 229960000367 inositol Drugs 0.000 claims description 15
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 15
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 13
- 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 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 230000003519 ventilatory effect Effects 0.000 claims description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 7
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 210000002421 cell wall Anatomy 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 102000016943 Muramidase Human genes 0.000 claims description 6
- 108010014251 Muramidase Proteins 0.000 claims description 6
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 6
- 229960000274 lysozyme Drugs 0.000 claims description 6
- 239000004325 lysozyme Substances 0.000 claims description 6
- 235000010335 lysozyme Nutrition 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000013078 crystal Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 108091005508 Acid proteases Proteins 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- 238000002425 crystallisation Methods 0.000 claims description 4
- 230000008025 crystallization Effects 0.000 claims description 4
- 238000004042 decolorization Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 4
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 4
- 238000000527 sonication Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 230000005611 electricity Effects 0.000 claims description 3
- 238000009210 therapy by ultrasound Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 235000007164 Oryza sativa Nutrition 0.000 claims 1
- 239000010977 jade Substances 0.000 claims 1
- 235000020265 peanut milk Nutrition 0.000 claims 1
- 235000009566 rice Nutrition 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 235000001014 amino acid Nutrition 0.000 abstract description 3
- 150000001413 amino acids Chemical class 0.000 abstract description 3
- 229940041514 candida albicans extract Drugs 0.000 abstract description 2
- 239000012138 yeast extract Substances 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 36
- 210000004027 cell Anatomy 0.000 description 11
- 210000000170 cell membrane Anatomy 0.000 description 11
- 239000002253 acid Substances 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000035699 permeability Effects 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 239000000908 ammonium hydroxide Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- -1 carbon dicarboxylic acids Chemical class 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000002525 ultrasonication Methods 0.000 description 3
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003610 charcoal Substances 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010073032 Grain Proteins Proteins 0.000 description 1
- KSMRODHGGIIXDV-YFKPBYRVSA-N N-acetyl-L-glutamine Chemical compound CC(=O)N[C@H](C(O)=O)CCC(N)=O KSMRODHGGIIXDV-YFKPBYRVSA-N 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 229960005488 aceglutamide Drugs 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
- 239000006052 feed supplement Substances 0.000 description 1
- 230000009123 feedback regulation Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
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- 239000003531 protein hydrolysate Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to technical field of amino acid production, disclose the technique of a kind of preparative separation glutamic acid and egg white icing comprising following steps: step 1) seed culture, step 2 fermented and cultured, step 3) prepare egg white icing, and step 4) prepares glutamic acid.The present invention obtains egg white icing while preparing glutamic acid, can substitute yeast extract use, have a extensive future.
Description
Technical field
The invention belongs to technical field of amino acid production, are related to the technique of a kind of preparative separation glutamic acid and egg white icing.
Background technique
Glutamic acid is a kind of acidic amino acid.Molecule includes two carboxyls, and chemical name is alpha-amido glutaric acid.Paddy ammonia
Acid is to find for inner Suo Xun 1856, is clear crystal, has delicate flavour, be slightly soluble in water, and is dissolved in hydrochloric acid solution, isoelectric point 3.22.
Largely it is present in grain protein, content is also more in animal brain.During the protein metabolism of glutamic acid in vivo
Critical role is accounted for, many important chemical reactions in animal, plant and microorganism are participated in.Currently, it is most common to prepare glutamic acid
Method is microbe fermentation method.
Corynebacterium glutamicum is the bacterial strain of glutamic acid fermentation, belongs to facultative aerobe, medium component and condition of culture are not
Together, product is also different.In glutamic acid fermentation process, when the change of carbon-nitrogen ratio in culture medium will affect bacterial strain proliferation and glutamic acid
Synthesis.When fermentation liquid pH is in acidity, the glutamic acid of generation can be further converted to acetyl-glutamine.Therefore, glutamic acid
Fermentation condition optimization is mainly two aspect of nutrient media components and Fermentation Process of Parameter control optimization.During the fermentation, strain from
The fermentation character of body is it some times happens that variation, causes batch indirect fermentation performance greatest differences occur.Once strain fermentation characteristic is sent out
Raw to change, thallus will decline the adaptability and acid producing ability of environmental change, show as occurring after feed supplement " only consume sugar,
Do not produce acid " the phenomenon that, final aminoglutaric acid concentration is very low, causes fermenting property unstable.The early period of glutamic acid fermentation main bacterial strain
Rapidly, middle and later periods bacterial strain growth rate slows down proliferation, but glutamic acid synthesis is accelerated, when being the key that synthesis secretion glutamic acid
Phase.
Microorganism is different under different environmental conditions, using different substrate utilization approach, purposefully to cell
Metabolic pathway is modified and is transformed, and changes the original metabolic characteristics of cell, the yield and yield of target product can be improved.When
When four carbon dicarboxylic acids are all supplied by the fixed reaction of CO2, highest theory saccharic acid conversion ratio is 81%；And when the fixed reaction of CO2
It does not work completely, four carbon dicarboxylic acids can only be supplied by glyoxalic acid, and highest theoretical yield is 54%.Glutamic acid production technology
Relative maturity is developed, the key technical indexes is aminoglutaric acid concentration 10%-12%, saccharic acid conversion ratio 55%-60%.But with
External advanced zymotechnique compares, and still has biggish room for promotion.Effectively improve conversion ratio, can save raw material at
Originally, the economic benefit of glutamic acid fermentation is promoted.Metabolic by-product when using Corynebacterium glutamicum progress glutamic acid fermentation is not
More, most important by-product is CO2.Therefore, strengthen the fixed reaction of CO2 in Corynebacterium glutamicum metabolic pathway, and thus
It allows glutamic acid to synthesize crucial enzyme system effectively, mutually to act synergistically, will be expected to improve the reclamation rate and saccharic acid conversion ratio of CO2, section
It saves cost of material, increase enterprise profit.
Glutamate producing bacterium membrane structure is special with functionally during the key of glutamic acid fermentation is fermented and cultured
Property variation, be transformed into cell membrane and be conducive to glutamic acid to film exosmosis, that is, complete from the non-accumulation type cell of glutamic acid to paddy ammonia
The transformation of acid accumulation type cell.In this way, since final product glutamic acid is constantly discharged extracellularly, prevent intracellular glutamic acid from
The concentration for causing feedback regulation is run up to, glutamic acid will be synthesized preferentially with keeping in the cell, and constantly be penetrated
Cell membrane is secreted into fermentation medium, to largely be accumulated.The substance for adjusting permeability of cell membrane is more, different
Membrane structure differs greatly between bacterial strain, thus it is not regular follow, select suitable regulator to adjust cell membrane
Permeability is also the technical issues that need to address in glutamic acid fermentation technique.
Summary of the invention
In order to overcome prior art strain fermentation conversion ratio and the defects such as glutamic acid yield is low, the invention proposes a kind of systems
Technique of the back-up from glutamic acid and egg white icing.
The present invention is achieved by the following technical solution:
A kind of technique of preparative separation glutamic acid and egg white icing comprising following steps: step 1) seed culture, step 2 fermentation
Culture, step 3) prepare egg white icing, and step 4) prepares glutamic acid.
Further, described method includes following steps:
Step 1) seed culture: Corynebacterium glutamicum is inoculated into seed culture medium, shaking table culture, obtained glutamic acid rod
Bacterium seed liquor；
Step 2 fermented and cultured: seed liquor is inoculated into the fermentor containing fermentation medium, fermentation time 48h；Fermentation
Time is divided into two stages, first stage 12h, and fermentation temperature is 32 DEG C, ventilatory capacity 0.3vvm；Second stage is 36h, hair
Ferment temperature is 32 DEG C, ventilatory capacity 0.4vvm, when second stage starts, and inositol and dimethyl formyl are added into fermentation medium
Amine, while being ultrasonically treated；In entire fermentation process, pH is controlled 5.0 by auto-feeding ammonium hydroxide, and pass through stream plus concentration
Residual sugar is controlled not less than 1.0% for the glucose solution of 100g/L；After the completion of second stage, fermentation liquid is collected；
Step 3) prepares egg white icing: being centrifuged using high-speed dish piece seperator to fermentation liquid, collects glutamic acid feed liquid and thallus
Mycoprotein is precipitated and is dried by albumen precipitation, is then added to stirred reactor, and the water that 10 times of weight are added mixes, then
Sequentially add lysozyme 10g/m3With acid protease 25g/m3, 55 DEG C of enzymolysis temperature are adjusted, sulfuric acid is added to adjust pH5.5, is delayed
Slow stirring enzymatic hydrolysis 6h, centrifugation removal cell wall, gained supernatant are concentrated by evaporation to obtain paste, and egg white icing is made in barrelling；
Step 4) prepares glutamic acid: glutamic acid feed liquid is pumped into bleacher and carries out decolorization, and addition accounts for glutamic acid material in bleacher
The powdered activated carbon of liquid quality 1-2%, controlling the temperature in bleacher is 45-50 DEG C, is concentrated to give after the 30min that decolourizes dense
Contracting liquid；Stream plus concentrate into the electric tank such as level-one, while the concentrated sulfuric acid is added and adjusts the pH 3.5 for making solution in equal electricity tank, temperature
Control successively passes through second level isoelectric point tank at 20 DEG C, by the liquid of level-one isoelectric point tank again, while concentrated sulfuric acid tune pH value is added,
Wherein, second level isoelectric point tank pH control 3.2,12 DEG C of temperature；The glutamic acid for obtaining crystallization, glutamic acid crystal is drying to obtain.
Preferably, the additive amount of the inositol accounts for the 0.6-0.8% of fermentation medium quality.
Preferably, the additive amount of the dimethylformamide accounts for the 0.3-0.4% of fermentation medium quality.
Preferably, the parameter of the ultrasonic treatment are as follows: supersonic frequency is 28 kHz, ultrasonic power density is 100W/L, super
The sonication time is 1h.
Preferably, the component of the seed culture medium are as follows: glucose 6%, corn pulp 3%, potassium dihydrogen phosphate 0.2%, seven
Water magnesium sulfate 0.02%, manganese sulfate monohydrate 0.01%.
Preferably, the component of the fermentation tank culture medium are as follows: glucose 12%, corn pulp 3%, urea 0.5%, biphosphate
Potassium 0.1%, ferrous sulfate heptahydrate 0.02%, epsom salt 0.02%.
Preferably, the additive amount of the lysozyme is 10g/m3。
Preferably, the additive amount of the acid protease is 25g/m3。
Compared with prior art, the beneficial effect that the present invention obtains mainly includes but is not limited to the following aspects:
Realize the rational proportion of each nutrient, maximum plays the acid producing ability of thallus, to improve fermentation conversion rate and produce acid；Paddy
Propylhomoserin producing strains proliferation is to maximum value, and when glutamic acid generation enzyme system forms complete, opportune moment when producing the sour phase is added suitable
Inositol and dimethylformamide can both strengthen the fixed reaction of CO2, and weaken glyoxalic acid circulation, guarantee tricarboxylic acid cycle not by
It interrupts and continually supplies α-ketoglutaric acid, by reduction of amination, largely accumulate glutamic acid, improve microbe conversion
Rate, additionally it is possible to which the enhancing for promoting permeability of cell membrane is conducive to substrate molecule and is easier to enter cell contact with biological enzyme, also have
It is secreted into conducive to glutamic acid extracellular, not only improves conversion ratio, but also increase glutamic acid yield.
Ultrasonic wave biological mechanism of action is mainly mechanical force caused by cavitation and heat effect, which may cause sky
The breakdown of the cell wall and plasma membrane of cell around steeping or reversible membrane permeability change, and this change is reversible, and cell is certainly
Body can repair wall and cell membrane and cell wall penetrability then can be improved in the breakage of film, and not influence the bioactivity of cell,
Thus ultrasound is temporary to the effect of cell membrane, and after ultrasonication terminates, permeability of cell membranes is returned to originally
State；But excessively high-intensitive ultrasonic wave will cause bacterial strain rupture death, therefore select 28 kHz of frequency, power density 100
W/L is ultrasonically treated 1 h.
The present invention is pioneeringly by CO2 is fixed and cell-permeant linking is to together, in conjunction with low intensive ultrasonication,
Conversion ratio and glutamic acid yield greatly improved.
The present invention obtains mycoprotein cream, alternative commercially available yeast powder product, thus greatly while preparing glutamic acid
Amplitude reduction fermentation costs, improve the added value of industry.
Detailed description of the invention
Fig. 1: influence of the inositol of Different adding amount to conversion ratio and aminoglutaric acid concentration；
Fig. 2: influence of the dimethylformamide of Different adding amount to conversion ratio and aminoglutaric acid concentration.
Specific embodiment
In order to make those skilled in the art better understand the technical solutions in the application, having below in conjunction with the application
The technical solution of the application is clearly and completely described in body embodiment, it is clear that described embodiment is only this Shen
Please a part of the embodiment, instead of all the embodiments.Based on the embodiment in the application, those of ordinary skill in the art are not having
Every other embodiment obtained under the premise of creative work is made, should fall within the scope of the present invention.
Embodiment 1
A kind of technique of preparative separation glutamic acid and egg white icing comprising following steps:
Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13761 is inoculated into seed culture medium,
32 DEG C, 100rpm shaking table culture 12h, obtained Corynebacterium glutamicum seed liquor；The Corynebacterium glutamicum seed culture medium
Group is divided into (being below mass percent): glucose 6%, corn pulp 3%, potassium dihydrogen phosphate 0.2%, epsom salt
0.02%, manganese sulfate monohydrate 0.01%, 115 DEG C of sterilizing 15min；
Seed liquor is inoculated into the fermentor containing fermentation medium with 8% inoculum concentration, fermentation time 48h；Fermentation time
It is divided into two stages, first stage 12h, fermentation temperature is 32 DEG C, ventilatory capacity 0.3vvm；Second stage is 36h, fermentation temperature
Degree is 32 DEG C, ventilatory capacity 0.4vvm, when second stage starts, and inositol and dimethylformamide are added into fermentation medium,
Additive amount is respectively 0.6% and 0.3%, is ultrasonically treated simultaneously, and supersonic frequency is 28 kHz, ultrasonic power density is 100W/L, super
The sonication time is 1h；In entire fermentation process, pH is controlled 5.0 by auto-feeding ammonium hydroxide, and be by stream plus concentration
The glucose solution of 100g/L, which controls residual sugar, is being not less than 1.0%；After the completion of second stage, fermentation liquid is collected；
The group of the fermentation tank culture medium is divided into (mass percent): glucose 12%, corn pulp 3%, urea 0.5%, biphosphate
Potassium 0.1%, ferrous sulfate heptahydrate 0.02%, epsom salt 0.02%；
Fermentation liquid is centrifuged using high-speed dish piece seperator, collects glutamic acid feed liquid and mycoprotein precipitating, high-speed dish piece
The revolving speed of machine separating thallus albumen is 4000r/min, and mycoprotein is dried, and is then added to stirred reactor, and be added 10
The water of times weight mixes, and is separately added into lysozyme 10g/m3Acid protease 25g/m is added in (20,000 U/g)3(100,000 U/g), adjustment
55 DEG C of enzymolysis temperature, sulfuric acid is added to adjust pH5.5, being slowly stirred enzymolysis time is 6h, and above-mentioned hydrolysate is separated with disk
Machine separation removal cell wall, gained supernatant are concentrated into 65% with triple effect plate-type evaporator, and control one imitates 78 DEG C of feeding temperature, out
43 DEG C of material temperature degree, egg white icing is made in the direct barrelling of gained paste；
Glutamic acid feed liquid is pumped into bleacher and carries out decolorization, and the powdery that addition accounts for glutamic acid feed liquid quality 1.5% in bleacher is lived
Property charcoal, control bleacher in temperature be 50 DEG C, decolourize 30min after concentration be original volume one third, the concentration parameter
Are as follows: temperature 70 C, vacuum degree are -0.1kpa；It flows to add into the electric tank such as level-one and states concentrate, while concentrated sulfuric acid adjusting is added to make
PH Deng solution in electric tank is 3.5, and temperature is controlled at 20 DEG C, by the liquid of level-one isoelectric point tank again successively by electricity such as second levels
Point tank, while concentrated sulfuric acid tune pH value is added, wherein second level isoelectric point tank pH control 3.2,12 DEG C of temperature；Obtain the paddy ammonia of crystallization
Glutamic acid crystal is drying to obtain by acid, is computed, and glutamic acid yield is 95.1%.
Embodiment 2
A kind of technique of preparative separation glutamic acid and egg white icing comprising following steps:
Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13761 is inoculated into seed culture medium,
32 DEG C, 100rpm shaking table culture for 24 hours, obtained Corynebacterium glutamicum seed liquor；The Corynebacterium glutamicum seed culture medium
Group is divided into (being below mass percent): glucose 6%, corn pulp 3%, potassium dihydrogen phosphate 0.2%, epsom salt
0.02%, manganese sulfate monohydrate 0.01%, 115 DEG C of sterilizing 15min；
Seed liquor is inoculated into the fermentor containing fermentation medium with 10% inoculum concentration, fermentation time 48h；Fermentation time
It is divided into two stages, first stage 12h, fermentation temperature is 32 DEG C, ventilatory capacity 0.4vvm；Second stage is 36h, fermentation temperature
Degree is 32 DEG C, ventilatory capacity 0.5vvm, when second stage starts, and inositol and dimethylformamide are added into fermentation medium,
Additive amount is respectively 0.8% and 0.4%, is ultrasonically treated simultaneously, and supersonic frequency is 28 kHz, ultrasonic power density is 100W/L, super
The sonication time is 1h；In entire fermentation process, pH is controlled 5.0 by auto-feeding ammonium hydroxide, and be by stream plus concentration
The glucose solution of 100g/L, which controls residual sugar, is being not less than 1.0%；After the completion of second stage, fermentation liquid is collected；
The group of the fermentation tank culture medium is divided into (mass percent): glucose 12%, corn pulp 3%, urea 0.5%, biphosphate
Potassium 0.1%, ferrous sulfate heptahydrate 0.02%, epsom salt 0.02%；
Fermentation liquid is centrifuged using high-speed dish piece seperator, collects glutamic acid feed liquid and mycoprotein precipitating, high-speed dish piece
The revolving speed of machine separating thallus albumen is 4000r/min, and mycoprotein is dried, and is then added to stirred reactor, and be added 10
The water of times weight mixes, and is separately added into lysozyme 10g/m3Acid protease 25g/m is added in (20,000 U/g)3(100,000 U/g), adjustment
55 DEG C of enzymolysis temperature, sulfuric acid is added to adjust pH5.5, being slowly stirred enzymolysis time is 6h, and above-mentioned hydrolysate is separated with disk
Machine separation removal cell wall, gained supernatant are concentrated into 65% with triple effect plate-type evaporator, and control one imitates 78 DEG C of feeding temperature, out
43 DEG C of material temperature degree, egg white icing is made in the direct barrelling of gained paste；
Glutamic acid feed liquid is pumped into bleacher and carries out decolorization, and addition accounts for the powdered activated of glutamic acid feed liquid quality 2% in bleacher
Charcoal, controlling the temperature in bleacher is 45 DEG C, and concentration is the half of original volume, the concentration parameter after the 30min that decolourizes are as follows:
Temperature 70 C, vacuum degree are -0.1kpa；It flows to add into the electric tank such as level-one and states concentrate, while concentrated sulfuric acid adjusting is added to make
The pH of solution is 3.5 in electric tank, and temperature is controlled at 20 DEG C, successively passes through second level isoelectric point again by the liquid of level-one isoelectric point tank
Tank, while concentrated sulfuric acid tune pH value is added, wherein second level isoelectric point tank pH control 3.2,12 DEG C of temperature；The glutamic acid of crystallization is obtained,
Glutamic acid crystal is drying to obtain, is computed, glutamic acid yield is 95.4%.
Embodiment 3
Influence of each factor to conversion ratio and aminoglutaric acid concentration:
Control group is set, in which:
Control group 1: not adding inositol and dimethylformamide, remaining is the same as embodiment 1；
Control group 2: not adding inositol, remaining is the same as embodiment 1；
Control group 3: not adding dimethylformamide, remaining is the same as embodiment 1；
Control group 4: not using ultrasonic treatment, remaining is the same as embodiment 1；
Experimental group is embodiment 1.
Each group conversion ratio and aminoglutaric acid concentration are shown in Table 1.
Table 1
| Group | Conversion ratio | Aminoglutaric acid concentration |
| Control group 1 | 48.21 | 10.26 |
| Control group 2 | 53.43 | 11.84 |
| Control group 3 | 55.68 | 11.55 |
| Control group 4 | 57.36 | 12.79 |
| Experimental group | 62.37 | 13.92 |
Conclusion: by three kinds of detection inositol, dimethylformamide and ultrasonic wave factors to the shadow of conversion ratio and aminoglutaric acid concentration
Discovery is rung, three kinds of factors have preferable synergy, can greatly improve conversion ratio and aminoglutaric acid concentration.
Embodiment 4
1. influence of the inositol of Different adding amount to conversion ratio and aminoglutaric acid concentration, is respectively set additive amount are as follows: 0,0.2%,
0.4%, 0.6%, 0.8,1.0%, as shown in Figure 1, conversion ratio and aminoglutaric acid concentration step up with the increase of inositol additive amount,
When increasing to 0.6%, aminoglutaric acid concentration reaches highest, continues to improve inositol additive amount, and aminoglutaric acid concentration does not obviously increase,
Conversion ratio slightly improves, and when increasing to 1.0%, conversion ratio and aminoglutaric acid concentration are declined.
2. influence of the dimethylformamide of Different adding amount to conversion ratio and aminoglutaric acid concentration, is respectively set additive amount
Are as follows: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, as shown in Fig. 2, with the increase of dimethylformamide additive amount, conversion
Rate and aminoglutaric acid concentration step up, and when increasing to 0.3%, conversion ratio highest continues raising dimethylformamide additive amount and arrives
0.4%, conversion ratio not will increase, but aminoglutaric acid concentration has a small amount of increase, continue to improve dimethylformamide additive amount, conversion
Rate and aminoglutaric acid concentration are declined；The present invention also has detected the influence of ethyl alcohol, methanol to conversion ratio and aminoglutaric acid concentration,
In this experimental system, the ethyl alcohol or methanol (within 1%) of low concentration have not significant impact conversion ratio and aminoglutaric acid concentration, mention
After high concentration, bacterial strain has certain death rate, may be related with the cell wall extent of damage, does not show in concrete outcome attached drawing.
Experimental analysis:
The difference of cell physiological metabolic activity and permeability of cell membrane is the major reason for causing transform level different.Wherein,
The present invention is conducive to substrate molecule more so that the permeability of cell membrane of thallus enhances by adding suitable dimethylformamide
It is easily accessible cell to contact with biological enzyme, and then improves conversion ratio.Suitable inositol can strengthen the fixed reaction of CO2, promote
The accumulation of glutamic acid improves fermentation conversion rate.The present invention is fixed by CO2 and cell-permeant linking is arrived together, in conjunction with low intensive
Conversion ratio and glutamic acid yield greatly improved so that the production and transportation of substance combine in ultrasonication.
Embodiment 5
Egg white icing economic benefit situation analysis prepared by the present invention:
By taking our company produces 200000 tons of glutamic acid workshops per year as an example, to after applying the present invention compared with traditional handicraft at one's duty
It analyses as follows:
By 2200 yuan/ton of mycoprotein price of separation and Extraction, annual 36000 tons of mycoprotein of sale, the costs such as dry are about
9000000 yuan, 79,200,000 yuan of income from sales, therefore 70,200,000 yuan of income can be increased newly every year；
It is about 25000 tons/year that protein hydrolysate cream, which is made, in above-mentioned thallus, and presently commercially available 22000 yuan/ton of yeast extract unit price, therefore
550,000,000 yuan of income from sales can be achieved, albumen powder per ton consumes enzyme preparation and 2000 yuan of processing cost, therefore can newly increase income every year
430,000,000 yuan of benefit.
Listed above is only best specific embodiment of the invention.It is clear that the invention is not restricted to which above embodiments, may be used also
With there are many deformations.All changes that those skilled in the art directly can export or associate from present disclosure
Shape is considered as protection scope of the present invention.
Claims (9)
1. a kind of technique of preparative separation glutamic acid and egg white icing comprising following steps: step 1) seed culture, step 2 hair
Ferment culture, step 3) prepare egg white icing, and step 4) prepares glutamic acid.
2. technique according to claim 1, which is characterized in that described method includes following steps:
Step 1) seed culture: Corynebacterium glutamicum is inoculated into seed culture medium, shaking table culture, obtained glutamic acid rod
Bacterium seed liquor；
Step 2 fermented and cultured: Corynebacterium glutamicum seed liquor is inoculated into the fermentor containing fermentation medium, when fermentation
Between be 48h；Fermentation time is divided into two stages, first stage 12h, and fermentation temperature is 32 DEG C, ventilatory capacity 0.3vvm；Second
Stage is 36h, and fermentation temperature is 32 DEG C, and when second stage starts, inositol is added into fermentation medium by ventilatory capacity 0.4vvm
And dimethylformamide, while being ultrasonically treated；After the completion of second stage, fermentation liquid is collected；
Step 3) prepares egg white icing: being centrifuged using high-speed dish piece seperator to fermentation liquid, collects glutamic acid feed liquid and thallus
Mycoprotein is precipitated and is dried by albumen precipitation, is then added to stirred reactor, and the water that 10 times of weight are added mixes, then
Lysozyme and acid protease are sequentially added, adjusts 55 DEG C of enzymolysis temperature, adds sulfuric acid to adjust pH5.5, is slowly stirred enzymatic hydrolysis
6h, centrifugation removal cell wall, gained supernatant are concentrated by evaporation to obtain paste, and egg white icing is made in barrelling；
Step 4) prepares glutamic acid: glutamic acid feed liquid is pumped into bleacher and carries out decolorization, and addition accounts for glutamic acid material in bleacher
The powdered activated carbon of liquid quality 1-2%, controlling the temperature in bleacher is 45-50 DEG C, is concentrated to give after the 30min that decolourizes dense
Contracting liquid；Stream plus concentrate into the electric tank such as level-one, while the concentrated sulfuric acid is added and adjusts the pH 3.5 for making solution in equal electricity tank, temperature
Control successively passes through second level isoelectric point tank at 20 DEG C, by the liquid of level-one isoelectric point tank again, while concentrated sulfuric acid tune pH value is added,
Wherein, second level isoelectric point tank pH control 3.2,12 DEG C of temperature；The glutamic acid for obtaining crystallization, glutamic acid crystal is drying to obtain.
3. technique according to claim 2, which is characterized in that the additive amount of the inositol accounts for fermentation medium quality
0.6-0.8%。
4. technique according to claim 2, which is characterized in that the additive amount of the dimethylformamide accounts for fermentation medium
The 0.3-0.4% of quality.
5. technique according to claim 2, which is characterized in that the parameter of the ultrasonic treatment are as follows: supersonic frequency 28
KHz, ultrasonic power density is 100W/L, sonication treatment time 1h.
6. technique according to claim 2, which is characterized in that the component of the seed culture medium are as follows: glucose 6%, jade
Rice & peanut milk 3%, potassium dihydrogen phosphate 0.2%, epsom salt 0.02%, manganese sulfate monohydrate 0.01%.
7. technique according to claim 2, which is characterized in that the component of the fermentation tank culture medium are as follows: glucose 12%,
Corn pulp 3%, urea 0.5%, potassium dihydrogen phosphate 0.1%, ferrous sulfate heptahydrate 0.02%, epsom salt 0.02%.
8. technique according to claim 2, which is characterized in that the additive amount of the lysozyme is 10g/m3。
9. technique according to claim 2, which is characterized in that the additive amount of the acid protease is 25g/m3。
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Application publication date: 20190503 |