CN109355325A - The symbiosis production. art of particle threonine and granule protein - Google Patents

The symbiosis production. art of particle threonine and granule protein Download PDF

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CN109355325A
CN109355325A CN201811213884.3A CN201811213884A CN109355325A CN 109355325 A CN109355325 A CN 109355325A CN 201811213884 A CN201811213884 A CN 201811213884A CN 109355325 A CN109355325 A CN 109355325A
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threonine
fermentation
particle
granule protein
centrifugation
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赵凤良
赵兰坤
孙钦波
苏同学
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Xu Chuangao
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Xu Chuangao
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00Preparation of nitrogen-containing organic compounds
    • C12P13/04Alpha- or beta- amino acids
    • C12P13/08Lysine; Diaminopimelic acid; Threonine; Valine
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins

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Abstract

The invention belongs to technical field of amino acid production, disclose the symbiosis production. art of particle threonine and granule protein comprising following steps: step 1) fermentation, step 2 centrifugation, and step 3) prepares particle threonine, and step 4) prepares granule protein.Present invention process can be improved the content of particle threonine and granule protein, and simple process is feasible, have a extensive future.

Description

The symbiosis production. art of particle threonine and granule protein
Technical field
The invention belongs to amino acids production fields, and in particular to the symbiosis production. art of particle threonine and granule protein.
Background technique
Threonine (Threonine is abbreviated as Thr), scientific name 2 amino 3 hydroxybutyric acid, nineteen thirty-five is by W.C.Rose It separates and identifies in fibrin hydrolysate and, Meger studies its space structure within 1936, because of it Structure is similar to threose, therefore is named as threonine.Threonine belongs to aliphatic amino acid, slightly sweet, is to constitute people and dynamic plant A kind of essential amino acid of object protein, is mainly used for medicine, chemical reagent, nutrition fortifier, can strengthen dairy products, have Restore human-body fatigue, the effect of enhancing development.In recent years, with the development of economy, market continues surely threonine requirement It is fixed to increase, it is most fast one of the amino acid kind of demand growth, especially in chemistry and biochemistry, food additives, feed addition The dosage rapid development of agent etc., big substituted tryptophan and the development that becomes in addition to lysine, methionine is most rapid The third-largest amino acid.L-threonine is added in mixed feed, has the characteristics that as follows: 1. the amino acid of adjustable feed is flat Weighing apparatus promotes poultry growth;2. meat can be improved;3. the nutritive value of the low feed of amino acid digestibility can be improved;4. can reduce Feedstuff cost;Therefore in China, European Union member countries and American States, feedstuff industry has been widely used in it.
Currently, the production method of threonine mainly has fermentation method, protein Hydrolyze method and 3 kinds of chemical synthesis, microorganism Fermentation method produces threonine, because the advantages that its simple process and low cost has become current main stream approach.L-threonine it is main Production bacterial strain has Corynebacterium glutamicum, brevibacterium flavum and Escherichia coli.Threonine Fermentation technology is primarily present fermentation effect at present The defect that rate is low and purity is not up to standard.Bacterial strain is transformed to improve the yield of amino acid, early all the time by various methods in people The phase most widely used mutation breeding under the conditions of various, and with the exposition of amino acid bio metabolic pathway, it is purposive Metabolic pathway is transformed on a molecular scale and also appears its advantage gradually, in addition, the fermentation condition optimization process in middle reaches with And the reclaiming clean process in downstream is also an emphasis.
Colibacillus engineering strain is the main bacterial strain of microbe industrial fermentation production threonine, generates L- Soviet Union in fermentation While propylhomoserin, the metabolic by-products such as acetic acid, alanine, valine and arginine can be also generated, influence bacterium to a certain extent Body growth and the synthesis and accumulation of L-threonine.Wherein, the inhibitory effect of acetic acid is particularly evident, when Acetic Acid Accumulation is to certain dense When spending, the specific growth rate of thallus declines rapidly, and Product formation substantially reduces, and forms vicious circle, while foreign gene Expression is also heavily affected.Acetic Acid Accumulation is adversely affected caused by cell metabolism, is using Escherichia coli as host strain Express a very important problem of foreign protein.How the content of acetic acid is reduced, to improve biomass and threonine Yield is the emphasis that we study.Document " control of acetic acid in L-threonine fermentation process, science and technology of fermenting communication 2012 " exists During Escherichia coli fermentation prepares threonine, the generation of its by-product acetic acid is controlled by selecting suitable fermentation condition, It can reduce the generation of acetic acid, but reduce amplitude and be reduced to unobvious, the yield of threonine cannot be greatly improved, there is no industry Change the value promoted.The culture medium that patented technology " a kind of ultralow moisture content threonine production method " before applicant is recorded: Portugal Grape sugar 80g/L, corn pulp 20g/L, ammonium sulfate 2g/L, calcium carbonate 0.75g/L, KH2PO40.2g/L, K2HPO4 0.2g/L, NaCl 0.2g/L, pH value 6.5;The content of threonine can reach 10g/100ml in fermentation liquid, but the phase is dead after fermentation for bacterial strain Rate is higher, and glucose utilization is larger, also there is to be hoisted threonine yield in fermentation liquid.
Summary of the invention
In order to solve the defects of prior art, the invention proposes the symbiosis production. arts of particle threonine and granule protein. Present invention process can be improved the content of particle threonine and granule protein, and simple process is feasible, have a extensive future.
The present invention is achieved by the following technical solution:
The symbiosis production. art of particle threonine and granule protein comprising following steps: step 1) fermentation, step 2 centrifugation, step Rapid 3) to prepare particle threonine, step 4) prepares granule protein.
Further, the step 1) fermentation, includes the following steps: the colibacillus engineering that will produce threonine according to 6- 10% inoculum concentration, which is linked into the fermentor containing fermentation medium, ferments, and 30-32 DEG C of temperature, tank pressure is 0.04- 0.05MPa, ventilatory capacity 0.3-0.5vvm, revolving speed 100rpm, fermentation time culture is 36-48h, then according to 6-10%'s Inoculum concentration accesses Chlamydomonas reinhardtii, continues fermented and cultured 36-48h, stops fermentation, collects fermentation liquid.
Further, step 2 centrifugation, include the following steps: fermentation liquid first pass around disk plate centrifuge with 4000rpm is centrifuged 5min, collects supernatant liquid and precipitating.
Further, the step 3) prepares particle threonine, includes the following steps: the warp of supernatant liquid obtained by step 2 Ceramic membrane filter is crossed, filtered solution is collected, filtered solution is separated through decanter centrifuge, centrifugal speed 5000rpm, centrifugation time is 3min collects supernatant;Then pass through ultrafiltration membrance filter, collect filtered solution for the intermittent single-action condensing crystallizing pot knot of filtered solution Crystal is collected by centrifugation in crystalline substance, then 120 DEG C of dryings, compresses slabbing, then put into granulation tower, in boiling under the action of thermal current Rise state;65 DEG C of fluidized bed dryings, then through broken whole grain, pass sequentially through 20 mesh and 50 meshes, weeded out thick, meticulous particle, The granule for collecting target grain size, is packed to obtain the final product.
Further, the step 4) prepares granule protein, includes the following steps: precipitating obtained by step 2 being added to 3 In the hydrochloric acid solution of the 5M of times weight, 80 DEG C are heated to, 12h is handled under heat-retaining condition, then with disc separator with 3000rpm Revolving speed centrifugation 5min remove cell wall, collect supernatant, be condensed into paste, finally by slurry-spraying pelletizing fluidized bed drying, Obtain granule protein.
Further, the component of the fermentation medium are as follows: glucose 20-30g/L, glycerol 20-30g/L, corn pulp 20- 30g/L, ammonium sulfate 2-3g/L, potassium dihydrogen phosphate 0.2-0.3g/L, dipotassium hydrogen phosphate 0.2-0.3g/L, epsom salt 0.1- 0.2g/L, ferrous sulfate heptahydrate 0.01-0.02g/L, manganese sulfate monohydrate 0.01-0.02g/L, pH value 6.5-6.8.
The beneficial effect of starting point and acquirement that the present invention studies mainly includes but is not limited to the following aspects:
When the aerobic culture of Escherichia coli, oxygen molecule is electron transmission final receptor, and as cell is grown, oxygen consumption constantly increases Add, occur for hypoxgia, so that TCA circulation is obstructed, threonine yield is reduced, and carbon metabolism approach more flows to acetic acid way Diameter, Acetic Acid Accumulation increase sharply.
Cell concentration has a direct impact the generation of acetic acid, and earlier fermentation, cell density is not high, and oxygen demand is relatively fewer, It ferments the middle and later periods, when cell concentration increases, oxygen demand increases, and is easy to cause the too fast generation of acetic acid, when cell concentration is too low, again The synthesis of purpose product can be reduced.
Fermenting carbon source selection glucose and glycerol of the present invention, earlier fermentation, cell density is low, and oxygen-supplying amount is sufficient, large intestine bar Bacterium preferentially uses glucose as carbon source, can promote the generation of growing microorganism and threonine;It ferments the middle and later periods, glucose is consumed To the greatest extent, Escherichia coli use glycerol as carbon source at this time, since the rate that cell absorbs glycerol is lower, under the carbon flow of glycolysis Drop, to reduce the accumulation of acetic acid, while improving the yield of threonine;
The present invention can carry out non-light and work as carbon source using the acetic acid in fermentation liquid by being inoculated with Chlamydomonas reinhardtii in fermentation With, and it is more difficult use glycerol as carbon source, thus relieve to Escherichia coli produce threonine inhibiting effect, additionally it is possible to carry out micro- The photosynthesis of amount discharges oxygen, for Escherichia coli fermentation produce threonine come using.By adding Chlamydomonas reinhardtii, it is not only able to mention The yield of high threonine, and mycoprotein yield also correspondinglys increase.
Powdered threonine is prepared into graininess threonine by the present invention, can improve mobility, convenient for preservation and transport, Solubility is controlled, the quality and added value of threonine are improved;Thallus is prepared into particle mycoprotein, is convenient for preservation and fortune It is defeated, yeast powder can be substituted as nutrient media components, feed addictive is can be also used for, improve added value of product.
Figure of description
Fig. 1: the acetic acid content of each group in different time points.
Specific embodiment
Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair It is bright.Product and method of the invention is described by preferred embodiment, and related personnel can obviously not depart from this hair Product as described herein and method are modified in bright content, spirit and scope or appropriate changes and combinations, to realize and answer Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
The symbiosis production. art of particle threonine and granule protein comprising following steps:
By colibacillus engineering K12 △ dapA seed liquor, (concentration of seed liquor is 1 × 10 to step 1)8Cfu/mL) according to 10% Inoculum concentration be linked into the fermentor containing fermentation medium and ferment, 30 DEG C of temperature, tank pressure is 0.04MPa, ventilatory capacity For 0.5vvm, revolving speed 100rpm, fermentation time culture is 36h, then accesses Chlamydomonas reinhardtii (Rhein according to 10% inoculum concentration The concentration of chlamydomonas is 1 × 105Cfu/mL), continue fermented and cultured 48h, stop fermentation, collect fermentation liquid;
The component of the fermentation medium are as follows: glucose 20g/L, glycerol 20g/L, corn pulp 20g/L, ammonium sulfate 2g/L, phosphoric acid Potassium dihydrogen 0.2g/L, dipotassium hydrogen phosphate 0.2g/L, epsom salt 0.1g/L, ferrous sulfate heptahydrate 0.01g/L, sulfuric acid monohydrate Manganese 0.01g/L, pH value 6.5;
Step 2 fermentation liquid first passes around disk plate centrifuge and is centrifuged 5min with 4000rpm, collects supernatant liquid and precipitating;
Step 3) supernatant liquid is filtered by ceramic membrane (10,000 Da molecular cut off), collects filtered solution, by filtered solution through sleeping spiral shell from Scheming separation, centrifugal speed 5000rpm, centrifugation time 3min are collected supernatant (protein content is less than 0.5%);Then it passes through Ultrafiltration membrance filter is crossed, filtered solution is collected, ultrafiltration retaining molecular weight is 300Da;By the intermittent single-action condensing crystallizing of filtered solution Pot crystallization, is collected by centrifugation crystal, and then 120 DEG C of dryings to moisture content are 0.8%, compresses slabbing, then put into granulation tower, It is in fluidized state under the action of thermal current;65 DEG C of fluidized bed dryings, then through broken whole grain, 20 mesh and 50 meshes are passed sequentially through, It weeded out thick, meticulous particle, and collected the granule of target grain size, pack to obtain the final product;
Step 4) is heated to 80 DEG C, under heat-retaining condition in the hydrochloric acid solution for the 5M that precipitating is added to 3 times of weight obtained by step 2 12h is handled, cell wall is then removed with the revolving speed centrifugation 5min of 3000r/min with disc separator, supernatant is collected, is condensed into Paste obtains granule protein finally by slurry-spraying pelletizing fluidized bed drying.
Embodiment 2
The symbiosis production. art of particle threonine and granule protein comprising following steps:
By colibacillus engineering K12 △ dapA seed liquor, (concentration of seed liquor is 1 × 10 to step 1)8Cfu/mL) according to 6% Inoculum concentration, which is linked into the fermentor containing fermentation medium, ferments, and 32 DEG C of temperature, tank pressure is 0.04MPa, and ventilatory capacity is 0.4vvm, revolving speed 100rpm, fermentation time culture are 48h, then access Chlamydomonas reinhardtii (Chlamydomonas reinhardtii according to 8% inoculum concentration Concentration be 1 × 105Cfu/mL), continue fermented and cultured 48h, stop fermentation, collect fermentation liquid;
The component of the fermentation medium are as follows: glucose 30g/L, glycerol 30g/L, corn pulp 30g/L, ammonium sulfate 3g/L, phosphoric acid Potassium dihydrogen 0.3g/L, dipotassium hydrogen phosphate 0.3g/L, epsom salt 0.2g/L, ferrous sulfate heptahydrate 0.02g/L, manganese sulfate monohydrate 0.02g/L, pH value 6.8;
Step 2 fermentation liquid first passes around disk plate centrifuge and is centrifuged 5min with 4000rpm, collects supernatant liquid and precipitating;
Step 3) supernatant liquid is filtered by ceramic membrane (10,000 Da molecular cut off), collects filtered solution, by filtered solution through sleeping spiral shell from Scheming separation, centrifugal speed 5000rpm, centrifugation time 3min are collected supernatant (protein content is less than 0.5%);Then it passes through Ultrafiltration membrance filter is crossed, filtered solution is collected, ultrafiltration retaining molecular weight is 300Da;By the intermittent single-action condensing crystallizing of filtered solution Pot crystallization, is collected by centrifugation crystal, and then 120 DEG C of dryings to moisture content are 0.8%, compresses slabbing, then put into granulation tower, It is in fluidized state under the action of thermal current;65 DEG C of fluidized bed dryings, then through broken whole grain, 20 mesh and 50 meshes are passed sequentially through, It weeded out thick, meticulous particle, and collected the granule of target grain size, pack to obtain the final product;
Step 4) is heated to 80 DEG C, under heat-retaining condition in the hydrochloric acid solution for the 5M that precipitating is added to 3 times of weight obtained by step 2 12h is handled, cell wall is then removed with the revolving speed centrifugation 5min of 3000r/min with disc separator, supernatant is collected, is condensed into Paste obtains granule protein finally by slurry-spraying pelletizing fluidized bed drying.
Embodiment 3
Influence of the different factors to production amount of threonine and yield of acetic acid in present invention process:
Group is set:
Experimental group: embodiment 1;
Control group 1: not adding Chlamydomonas reinhardtii, remaining is the same as embodiment 1;
Glycerol: being replaced with the glucose of equal quality by control group 2, remaining is the same as embodiment 1;
Control group 3: not adding Chlamydomonas reinhardtii, while glycerol being replaced with to the glucose of equal quality, remaining is the same as embodiment 1.
The content of threonine and acetic acid is shown in Table 1 in each final fermentation liquid of group:
Table 1
Group Threonine g/L Acetic acid g/L
Experimental group 137.1 0.6
Control group 1 102.5 13.9
Control group 2 118.2 4.7
Control group 3 96.4 15.6
Conclusion: experimental group can utilize the acetic acid in threonine fermentation liquid by carrying out assisted fermentation processing to Chlamydomonas reinhardtii Non- light and effect are carried out as carbon source, to relieve the inhibiting effect that Escherichia coli are produced with threonine, additionally it is possible to carry out micro Photosynthesis discharge oxygen, for Escherichia coli fermentation produce threonine come using;Part glucose is substituted by glycerol simultaneously, with The consumption of glucose, Escherichia coli use glycerol as carbon source, due to cell absorb glycerol rate it is lower, reduce acetic acid Accumulation, while improving the yield of threonine, pass through each group comparative test and find, compared with control group 1-3, the present invention The production amount of threonine of experimental group is respectively increased 33.76%, 15.99%, 42.22%;And acetic acid content is only 0.6g/L, is equivalent to pair According to the 3.85% of group 1.
The present invention also has detected the yield of acetic acid of each group in different time points, by taking embodiment 1 as an example, chooses fermentation respectively Afterwards, 36h, 48h, 60h, 72h, 84h, totally 5 time points are detected, and concrete outcome is shown in Fig. 1.In experimental group, With the increase of fermentation time, acetic acid content is reduced rapidly, and is relieved the synthesis to threonine and is inhibited, to improve threonine Secretory volume;And in control group 1 and 3, due to there is no addition Chlamydomonas reinhardtii, acetic acid content is caused to continue growing;In control group 2, Due to being added to Chlamydomonas reinhardtii, so that acetic acid content gradually declines, but fall is lower than experimental group, it may be possible to because of experiment Group is added to glycerol as carbon source, and the carbon source into acetate pathway reduces, and Chlamydomonas reinhardtii is more difficult uses glycerol as carbon source, It is only capable of using acetic acid, and then causes the fall of experimental group acetic acid more obvious.
The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, though The right present invention has been described by way of example and in terms of the preferred embodiments, however, being not intended to limit the invention, any technology people for being familiar with this profession Member can make a little change or modification using the technology contents disclosed certainly without departing from the scope of the present invention, at For the equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, according to the technical essence of the invention Any simple modification, equivalent change and modification to the above embodiments, belong in the range of technical solution of the present invention.

Claims (6)

1. the symbiosis production. art of particle threonine and granule protein comprising following steps: step 1) fermentation, step 2 centrifugation, Step 3) prepares particle threonine, and step 4) prepares granule protein.
2. technique according to claim 1, which is characterized in that the step 1) fermentation includes the following steps: that Soviet Union's ammonia will be produced The colibacillus engineering of acid is linked into the fermentor containing fermentation medium according to the inoculum concentration of 6-10% to ferment, temperature 30-32 DEG C of degree, tank pressure are 0.04-0.05MPa, ventilatory capacity 0.3-0.5vvm, revolving speed 100rpm, fermentation time 36- Then 48h accesses Chlamydomonas reinhardtii according to the inoculum concentration of 6-10%, continue fermented and cultured 36-48h, stops fermentation, collects fermentation liquid.
3. technique according to claim 2, which is characterized in that the step 2 centrifugation includes the following steps: that fermentation liquid passes through It crosses disk plate centrifuge and 5min is centrifuged with 4000rpm, collect supernatant liquid and precipitating.
4. technique according to claim 3, which is characterized in that the step 3) prepares particle threonine, including walks as follows It is rapid: supernatant liquid obtained by step 2 being passed through into ceramic membrane filter, filtered solution is collected, filtered solution is centrifuged, collects supernatant;Then By ultrafiltration membrance filter, then condensing crystallizing is carried out, crystal is collected by centrifugation, then 120 DEG C of dryings, compresses slabbing, then put into and make It is in fluidized state under the action of thermal current in grain tower;65 DEG C of fluidized bed dryings, then through broken whole grain, sieving is packed to obtain the final product.
5. technique according to claim 4, which is characterized in that the step 4) prepares granule protein, includes the following steps: By in the hydrochloric acid solution for the 5M that precipitating is added to 3 times of weight obtained by step 2,80 DEG C are heated to, 12h is handled under heat-retaining condition, so Centrifugation removal cell wall afterwards, collects supernatant, is condensed into paste, finally by slurry-spraying pelletizing fluidized bed drying, obtains particle Albumen.
6. according to claim 2-5 be allowed to one described in technique, which is characterized in that the component of the fermentation medium are as follows: grape Sugared 20-30g/L, glycerol 20-30g/L, corn pulp 20-30g/L, ammonium sulfate 2-3g/L, potassium dihydrogen phosphate 0.2-0.3g/L, phosphoric acid Hydrogen dipotassium 0.2-0.3g/L, epsom salt 0.1-0.2g/L, ferrous sulfate heptahydrate 0.01-0.02g/L, manganese sulfate monohydrate 0.01-0.02g/L, pH value 6.5-6.8.
CN201811213884.3A 2018-10-18 2018-10-18 The symbiosis production. art of particle threonine and granule protein Pending CN109355325A (en)

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Application publication date: 20190219