CN105349599A - Trehalose production method - Google Patents
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- CN105349599A CN105349599A CN201410416316.9A CN201410416316A CN105349599A CN 105349599 A CN105349599 A CN 105349599A CN 201410416316 A CN201410416316 A CN 201410416316A CN 105349599 A CN105349599 A CN 105349599A
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 title claims abstract description 123
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 title claims abstract description 123
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 title claims abstract description 122
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 40
- 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 abstract description 76
- 239000008103 glucose Substances 0.000 claims abstract description 74
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims abstract description 46
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- 238000004587 chromatography analysis Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 27
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- 238000000605 extraction Methods 0.000 description 14
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Abstract
The invention provides a trehalose production method. The method of the invention comprises the following steps: an acceptor containing maltose is provided; secondly, dry acidophilus trehalose synthase is provided to directly convert part of maltose in the acceptor into trehalose; thirdly, glucoamylase is provided to convert maltose, which hasn't been converted by the dry acidophilus trehalose synthase, into glucose so as to obtain a reaction mixture; and finally, trehalose and glucose in the reaction mixture are separated by simulated moving bed chromatography, so as to obtain high-purity trehalose.
Description
Technical field
The invention relates to a kind of trehalose production method, also relate to a kind of trehalose production method.
Background technology
Trehalose (trehalose, namely α-D-glucopyranosyl-α-D-glucopyranoside) is the non-reduced disaccharide that a kind of occurring in nature exists, for being formed with 1,1-glycosidic link bond by two glucose molecules.Although trehalose has three kinds of mutarotation isomers to exist, i.e. α, α-1,1-, α, β-1,1-and β, β-1,1-trehalose, but only have α, α-1, the form of 1-trehalose is distributed widely in nature and comprises bacterium, yeast, Mushrooms, insect, in the varied organisms such as invertebrates and plant.
Trehalose (trehalose) can be widely used in food-processing, medicine, biochemical product, cosmetic industry.Such as trehalose can be applicable on makeup, as the wetting Agent for Printing Inks composition of skin care products.In food-processing, because trehalose has in moderate, the water-soluble high and low cariogenicity of sugariness, agent of low hygroscopicity and processing storage process characteristics such as stablizing, therefore can be applicable to need through the food and drink of heat treated, desiccating food products, frozen product, preserved fruit, candy, beverage, leavened food etc.
The mode of production of early stage trehalose is the cultivation by yeast, is more directly extracted by yeast, but due to production cost too high, be difficult to widespread use.The a large amount of production development of follow-up industry goes out enzyme two-phase method, with low unit price starch for raw material manufactures trehalose.
Summary of the invention
The embodiment of the present invention provides a kind of trehalose production method.The method comprises provides comprise maltose one to be subject to matter.Then, provide dry acidophilic bacteria TreP (Picrophilustorridustrehalosesynthase is called for short PTTS) that this is converted into trehalose by the maltose of part in matter.Glucoamylase is provided the maltose not having dried acidophilic bacteria TreP to transform to be converted into glucose and to obtain reaction mixture.Also with simulated moving bed chromatography method, the trehalose in this reaction mixture and glucose are separated.And this simulated moving bed chromatography method comprises: (i) providing package is simulation moving-bed containing at least three sections, it moves mutually by one and a stationary phase formed, wherein this moves and comprises punching mutually and wash liquid, this stationary-phase particle size inside has hole, this three section is sequentially first, second and third section, there is first, second and third phase respectively to flow rate ratio m1, m2 and m3, this moves and flows through between this three section in the same direction in this is simulation moving-bed, and this stationary phase moves mutually simulate movement in the opposite direction relative to this; (ii) this reaction mixture is injected this simulation moving-bed second and the 3rd between section, glucose in this reaction mixture and trehalose have the first delay constant and second respectively and are detained constant, this the first delay constant is greater than this second delay constant, and the hole of this stationary phase can cause trehalose and glucose to move according to friction-motion speed; (iii) this first-phase of this first section should be greater than this first delay constant, with holomorphosis stationary phase to flow rate ratio m1; And this second and third relative velocity ratio m2 and m3 of (iv) this second and third section should between this first delay constant and this second delay constant, glucose is made to move to this second section, trehalose moves to the 3rd section, with separating glucose and trehalose.
According to the embodiment of the present invention, this first, second and third section respectively comprises two endowels, three endowels and three endowels, and in every endowel, filler particles inside has this stationary phase of this hole.
According to the embodiment of the present invention, when this second-phase of this second section is X-axis to flow rate ratio m2 and this third phase of the 3rd section is Y-axis to flow rate ratio m3, theoretical according to triangle, this second-phase should to fall within flow rate ratio m3 flow rate ratio m2 and this third phase and first be detained constant and this second is detained in the oblique line block of constant institute corral by this.
According to the embodiment of the present invention, when this reaction mixture inject this simulation moving-bed after, this moves and drives this reaction mixture to move in the hole of those tubing strings mutually, because this first delay constant is greater than this second delay constant, therefore, glucose is less than trehalose in this simulation moving-bed translational speed in this simulation moving-bed translational speed, the glucose time of flowing out needed for those tubing strings is greater than the time needed for trehalose, glucose is made to move to this first and second section, trehalose moves to the 3rd section, with separating glucose and trehalose; Again because the relative velocity m1 of the first section is greater than the first delay constant, so glucose is identical with moving phase at the flow direction of the first section, also can make stationary phase holomorphosis simultaneously.
According to the embodiment of the present invention, this first delay constant is 0.135, and this second delay constant is 0.015, and this porosity of this stationary-phase particle size inside is 0.40.
According to the embodiment of the present invention, be 100 % by weight with this by the maltose weight in matter, maltose is transformed the trehalose into about 67 % by weight by dry acidophilic bacteria TreP, and remaining maltose is transformed the glucose into about 33 % by weight by glucoamylase, this is made to change into this reaction mixture comprising trehalose and glucose by the maltose in matter.
According to the embodiment of the present invention, the purity of the trehalose be separated is more than or equal to 98 % by weight.
According to the embodiment of the present invention, this simulation moving-bed also comprising after the 4th section is connected to the 3rd section washs liquid to reclaim circulating flushing.The purity of the trehalose be separated is more than or equal to 99 % by weight.
According to the embodiment of the present invention, this simulation moving-bed pre-treatment section that also comprises removes impurity.
For the present invention can be become apparent, special embodiment below, and coordinate accompanying drawing to be described in detail below.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet of the trehalose production method of one embodiment of the invention;
What Fig. 2 illustrated trehalose, maltose and glucose runs through curve synoptic diagram;
Fig. 3 illustrates the operational condition coordinate diagram of simulated moving bed chromatography method according to solute separable in triangle balanced;
Fig. 4 illustrates the tubing string configuration schematic diagram of the post simulated moving bed chromatography of three sections;
The operational condition coordinate diagram of separable trehalose and glucose in the volume Vee formation shape theory of dead angle is adjusted under Fig. 5 illustrates embodiment condition;
Fig. 6 illustrates the tubing string configuration schematic diagram of the post simulated moving bed chromatography of four sections;
What Fig. 7 illustrated experiment purity data and computer simulation data compares schematic diagram;
Fig. 8 illustrates the tubing string configuration schematic diagram of In-Situ Cleaning-simulation moving-bed (CIP-SMB) chromatography;
Fig. 9 illustrates that application In-Situ Cleaning-simulated moving bed chromatography method is separated the conceptual schematic view of three kinds of compositions;
Figure 10 illustrates the separable operational condition coordinate diagram of In-Situ Cleaning-simulated moving bed chromatography method according to triangle balanced.
Embodiment
The mode of production trehalose of the present invention comprises the dry acidophilic bacteria TreP of use thermophilic (Picrophilustorridustrehalosesynthase, be called for short PTTS), PTTS is a heat resistant type TreP, can enzyme reaction be carried out for maltose and transform generation trehalose, this enzymatic conversion rate up to about 67%, and still can remain about 33% unreacted maltose and very micro-glucose after its enzyme reaction.
After aforementioned enzyme reaction, still the trehalose of generation must be further purified, mainly trehalose and maltose will be separated in its purge process.But because trehalose and maltose belong to disaccharide, and be not easy to utilize ion to reject chromatography (ionexclusionchromatography) and be separated, therefore separation and purification poor effect.
In response in this, the mode of production trehalose of the present invention then also comprises the maltose utilizing glucoamylase (glucoamylase) to continue aforementioned enzyme reaction is residual and is degraded to glucose, then it is further purified separate targets required in trehalose process is that trehalose is relative to glucose, it belongs to being separated of monose and disaccharide, and easily utilizing ion exchange resin to reject chromatography with ion will separate both it.
The mode of production trehalose of the present invention mainly utilizes specific heat resistant type TreP, after converting maltose to trehalose, convert unreacted maltose to glucose further with glucoamylase, then with continous way ion-exchange-resin process, glucose is separated with trehalose.
In addition, the mode of production trehalose of the present invention also comprises use resinbed and analyses method, particularly simulated moving bed chromatography method (simulatedmovingbedchromatography, be called for short SMBC) carries out being separated of trehalose and glucose.
Below enumerate embodiment so that details or the condition of production method of the present invention to be described, and be not used to limit scope.Shown accompanying drawing is that schematic diagram is only drawn for convenience of explanation, and not representative limits the method, conditioned disjunction device etc. of its reality.
Embodiment 1: two benches enzymatic conversion
The mode of production trehalose of the present invention adopts two benches enzymatic conversion mode, first maltose is changed into (trehalose and maltose), be translated into again (trehalose and glucose), carry out separation and purification more afterwards, and directly obtain high purity trehalose.
Dry acidophilic bacteria TreP (PTTS) of the tool thermotolerance that this case utilizes, its come from a kind of height addicted to acid, addicted to the dry acidophilic bacteria (Picrophilustorridus) of Archimycetes of high-temperature aerobic (see Cabib, E., Leloir, L.F.J.Biol.Chem.231, pp259-275,1958).This kind of PTTS (Picrophilustorridus bacterium TreP) has more superior heat impedance and resistance to acid.The TreP used in the present invention because can simultaneously heat resistanceheat resistant with antiacid, can increase the danger by polluting in the solubleness of matter and minimizing production process, the reliability of raising production trehalose method and superiority.Also because the TreP used in production method of the present invention, can utilize and easily obtain and not expensive parent material: maltose work to manufacture trehalose by matter, not only can be saved cost and can also improve industrial economic worth.
Dry acidophilic bacteria TreP (Picrophilustorridustrehalosesynthase used in the present invention, be called for short PTTS), shown by patent No. TWI328611 be by gene recombination mode the Picrophilustorridus bacterium TreP protein with function that produces, this protein has the activity that bond in catalytic molecular rearranges, α 1 can be made, the maltose of 4-bond becomes the trehalose of α 1,1-bond, and vice versa.For the nucleotide sequence (the sequence numbering SEQID:2 described in Patent Case TWI328611) manufacturing Picrophilustorridus bacterium TreP used in the present invention or the carrier etc. comprising this sequence of encoding, refer to the description of aforementioned patent case TWI328611, this invention full content and its sequence table etc. are all incorporated as bibliography of the present invention.
Picrophilustorridus bacterium TreP (PTTS) used in the present invention in high temperature and sour environment, can remain high activity and stability.The maltose (with the gross weight of former maltose for 100%) of about 67% can be transformed into trehalose by the Picrophilustorridus bacterium TreP used in the present invention (as 20 ~ 30 DEG C) under preferably service temperature in 12 ~ 24 hours.Also therefore, the stability of trehalose production method of the present invention and specificity high.
PTTS used in the present invention is high by matter specificity, only acts on maltose, to other carbohydrates as glucose, Fructus Hordei Germinatus oligose, alga oligosaccharide, sucrose and lactose etc. do not act on, so inoperative to the carbohydrate of other kind existing in raw material.Due to maltose low price, pole is suitable for suitability for industrialized production trehalose.
Maltose under aforementioned enzyme reaction remains is arranged in pairs or groups glucoamylase (glucoamylase) again, maltose can be transformed into glucose completely (see Lee by described immobilized glucoamylase at 30 DEG C in 1.5 hours, D.D., Lee, G.K. with Reilly, P.J., BiotechnologyandBioengineering, 22 (1980), pp1-17).
The mode of production trehalose of the present invention is connected two kinds of enzyme reactions, can transform the maltose of 100 weight parts into about the trehalose of 67 weight parts and the glucose of about 33 weight parts at low temperatures or under room temperature.
Fig. 1 is the schematic flow sheet of the trehalose production method of one embodiment of the invention.Please refer to Fig. 1, in the present embodiment, trehalose production method comprises provides one by matter, and should comprise maltose (step S102) by matter.Then, provide Picrophilustorridus bacterium TreP (PTTS) that this is converted into trehalose (step S104) by the maltosyl moiety in matter.There is provided glucoamylase that the maltose be not converted (maltose do not reacted with PTTS) is converted into glucose (step S106).Then, with simulated moving bed chromatography method, the trehalose in reaction mixture (mainly comprising trehalose and glucose) and glucose are separated (step S108).
Embodiment 2: screening ion exchange resin
Applicable ion exchange resin is filtered out, to carry out the separation of trehalose/glucose in the embodiment of the present invention.First fill DiaionUBK555 (Hform) resin among size 1.0x25.0cm tubing string, and after washing liquid using water as punching, maltose, trehalose and glucose analysis sample are provided, the relative concentration then obtaining maltose, trehalose and glucose moving phase in section runs through curve (breakthroughcurve) in the residence time, what Fig. 2 illustrated trehalose, maltose and glucose runs through curve synoptic diagram, run through graphic representation see Fig. 2, wherein C/Co is relative concentration.In Fig. 2 visible maltose and trehalose run through Similar Broken Line, both equal anelasticities are very low, therefore not easily separated.But the anelasticity of glucose is higher than trehalose, be therefore easy to both separation.Td person is indicated for not using maltose concentration reaction (response) during tubing string in figure.
After a solute starts to inject a packed bed, it, in the response of packed bed outlet, can be expressed as:
C is adsorption density, c
ffor input concentration, ε
efor the interparticle voidage of solid adsorbent, ρ
pfor solid adsorbent density, q is adsorbate in whole adsorptive capacitys of solids external surface and inner void, v be moving phase at the interparticle flow velocity of solid adsorbent, D
mwith E
dfor solutes accumulation coefficient and eddy diffusivity, D
m+ E
dbe referred to as again axial diffusion coefficient or Axial dispersion coefficient (axialdispersioncoefficient), z and t then represents axial coordinate and the time of adsorption bed, and K is the delay constant of solute.If maltose is used as and is not detained composition, then the delay constant of trehalose and glucose is 0.015 and 0.135, and the porosity of bed body is 0.40.
Embodiment 3: trehalose and glucose analysis method
Application HPLC/RI carries out analysis glucose and trehalose, and using and analyzing tubing string is SHISEIDOCAPCELLPAKNH2UG80 (0.46x25.0cm, 5 μm), and with the ACN/H of 75/25 ratio
2o is moving phase, analyzes at the flow velocity of 1.5mL/min.Its integral area of the sample of different concns as table 1, its calibration curve be respectively following listed by:
Table 1
Embodiment 4: trehalose is separated with glucose
Suppose the simulated moving bed chromatography method (simulatedmovingbedchromatography comprising four sections, be called for short SMBC), it is by a stationary phase (Stationaryphase, be called for short SP) and one move phase (Mobilephase, be called for short MP) relative flowing between this four section, with the material in separating mixture.This stationary phase is filled in several tubing strings of each section, and this moves and flows in the same direction in this tubing string, and changes the feed entrance point of this mixture by an opening for feed shifter, to simulate the relative flow direction that this stationary phase and this move phase.After this mixture enters chromatography tubing string (charging), the substance A that this mixture comprises and B can be detained by this stationary phase respectively according to the Henry's constant H of each material (or being detained constant K) or move phase shift with this, and then each substance A of isolated or purified and B.Because the delay constant rejecting chromatography can't change along with concentration, therefore according to triangle theory (" OptimizationofaSMBbasedonanapproximatedLangmuirModel " AIChEJ.48,2240-2246) defined, for being separated A and B with simulated moving bed chromatography method, then in the middle of each section, the relative volumetric flow rates of its liquid and solid must meet following condition:
M
1> K
a; K
b< m
2< K
a; K
b< m
3< K
a; m
4< K
bformula (3)
Wherein K
awith K
bbe then the delay constant of composition A and composition B, and m
jfor the ratio of mobile phase volume relative velocity and solid volume relative velocity in j section, be defined as:
Q
jfor liquid is at the flow velocity of jth section, t
swfor tubing string switching time, V
cfor empty tube column volume, ε is tubing string total porosity, V
dfor the dead angle volume of each endowel.
Fig. 3 illustrates the operational condition coordinate diagram of simulated moving bed chromatography method according to solute separable in triangle balanced, as shown in Figure 3, if with the m of the second section
2for transverse axis, the m of the 3rd section
3for the longitudinal axis, then the operational condition that can be separated completely is just in time seated in trilateral, that is the trilateral of separable operating restraint for this reason in coordinate diagram.In vertex of a triangle, then there is best separating effect and separation efficiency.Except second and the 3rd the relative velocity demand fulfillment of section be seated except the condition in trilateral, first and the 4th the relative velocity of section also must meet the condition of formula (3) simultaneously.
Suppose for three section simulated moving bed chromatography methods, the method comprises: (i) providing package is simulation moving-bed containing at least three sections, it moves mutually by one and a stationary phase formed, wherein this stationary-phase particle size inside has hole, this three section is sequentially first, second and third section, there is relative velocity ratio m1, m2 and a m3 respectively, this moves and flows through between this three section in the same direction in this is simulation moving-bed, and this stationary phase moves mutually simulate movement in the opposite direction relative to this; (ii) providing the mixture with solute A and B to inject, this is simulation moving-bed, and this composition A and B has respectively and is detained constant K
aand K
band K
abe greater than K
b, the hole of this stationary phase can cause composition A, B to advance with different in flow rate; (iii) the relative velocity ratio m1 of this first section should be greater than K
a, relative velocity ratio m2 and m3 of this second and third section should between K
aand K
bbetween, make this composition A move to this first and second section, this composition B moves to the 3rd section, with separated component A and B.
Accordingly, the present invention uses the SMBC carrying UBK555 (Hform) ion exchange resin, has carried out the confirmation of separable operational condition.Fig. 4 illustrates the tubing string configuration schematic diagram of the post simulated moving bed chromatography of three sections, and its tubing string configuration is formed by being divided into 8 tubing strings of 2 pipe/3 pipe/3 pipes.As Fig. 4, wherein the first section is in series by two endowels, and the second section and the 3rd section are in series by three endowels.The major function of the second section and the 3rd section is carrying out being separated of trehalose and glucose, and the first section is then carrying out regeneration of ion-exchange resin.The diameter of each endowel is 4.2cm and length is 23cm.
See Fig. 4, trehalose (200g/L) and glucose (100g/L) aqueous solution (charging A/B) are by being located at second and the 3rd between section (between the 5th and the 6th tubing string, in figure between C5/C6) opening for feed with the flow velocity of 0.7mL/min inject SMBC, 40mL/min pure water then from the 1st tubing string (C1) inject.At the same time, between first and second section (between the 2nd and the 3rd tubing string, in figure between C2/C3) extraction liquid exit end then measure and draw the liquid glucose (B of extraction liquid shown in Fig. 4) of 25mL/min, and allowing unnecessary liquid glucose (15.7mL/min) export (the remaining liquid exit end of extraction) outflow from the 8th tubing string, unnecessary liquid glucose is remaining liquid A shown in Fig. 4.After the above-mentioned mode of use operates for some time, as 10.75 minutes, just by all outlets and entrance, switch toward next endowel simultaneously.After continuing one section of same time again, again by all gangways next endowel of migration, the switching tubing string so continued, just can simulate the left-hand of solid along Fig. 4 to movement, and forms the behavior with liquid countercurrent flow.The embodiment of the present invention tests different switching time, help the operational condition confirming to be applicable to being separated trehalose and glucose, and adjust that dead angle volume is empty tube column volume 8.7%, the separable operation bar obtained to make experiment meets the triangle balanced representated by formula (3) completely.Table 2 experimental result that under disclosing previous embodiment condition, different tubing string obtains switching time.Table 2 shows switching time between 10.5 ~ 11.25, and the purity of extraction liquid and remaining liquid can close to more than 90%.The operational condition coordinate diagram of separable trehalose and glucose in the volume Vee formation shape theory of dead angle is adjusted under Fig. 5 illustrates embodiment condition, Fig. 5 represents (m2, m3) phase plane, and according to formula (3) trilateral on coordinate plane representated by triangle balanced.Under the present embodiment condition, suppose that dead angle volume is after 8.7% of tubing string simultaneously, under the present embodiment 5 experiment conditions second and the 3rd the coordinate position of relative velocity in (m2, m3) phase plane of section also indicate the circle become in Fig. 5 simultaneously.Can confirm that from Fig. 5 switching time is between 10.5 ~ 11.25, the separable trehalose of the flow conditions set by the present embodiment and glucose.Meanwhile, also by vertex of a triangle, the optimum operation condition separating glucose and trehalose can be understood.
Lower list 2 show needle is to different tubing string gained switching time separating experiment result.
Table 2
The purity defined in table 2 and the rate of recovery, be defined as follows respectively:
Extraction liquid (extract) purity:
Formula (5)
Remaining liquid (raffinate) purity:
Formula (6)
Wherein C represents concentration, and its subscript represents solute A and B, and its subscript E and R then represents the concentration at the remaining liquid end of extraction liquid exit end and extraction, and P represents purity, and its subscript E and R then represents the purity at the remaining liquid end of extraction liquid exit end and extraction.B is strong anelasticity composition, solute component B and glucose in this embodiment, and composition A is weak anelasticity composition, i.e. trehalose.In table 2 upon handover between when being with 10.75 or 10.5 minutes, the purity comprising trehalose in so-called remaining liquid is quite high, up to 0.984 or 1.0, namely directly can obtain the trehalose of purity more than 98%, met volume production purity demand.Similarly, the purity of gained glucose also can up to 93 ~ 94wt%.
Then be defined as follows respectively in the rate of recovery of the remaining liquid end of extraction and the outlet of extraction liquid exit end:
Wherein Q represents volumetric flow rate, and C represents concentration, and its subscript represents solute A and B, and its subscript E and R then represents at the remaining liquid end (raffinate end) of extraction liquid exit end (extract and end) and extraction, Y
awith Y
bthen divide and represent the trehalose rate of recovery and the glucose rate of recovery.Be it seems by table 2 data, under suitable switching time condition, the rate of recovery of trehalose or glucose is all rather good.The water used also can make the aqueous solution of below pH=3 into, to improve the steady chemical structure of sugar, also can at high temperature operate, such as 70 DEG C, to avoid breed bacteria.
Embodiment 5: adopt four sections to have the SMBC of loop design to solve dilution problem
The SMBC that the present invention has tested confirmation three sections in example 4 can effectively be separated trehalose and glucose, applies triangle balanced simultaneously and confirms the operating parameters of separable trehalose and glucose, but its result shows trehalose and glucose has and diluted.Fig. 6 illustrates the tubing string configuration schematic diagram of the post simulated moving bed chromatography of four sections, 10 tubing strings that its tubing string configuration manages/3 pipe/2 pipes by 2 pipe/3 are formed, adopt the SMBC of four sections can recycle a large amount of water, therefore can reduce input concentration by the situation of diluting.The SMBC of Fig. 6 adds after the 4th section is connected to the 3rd section, liquid exit end more than extraction is made to be arranged in (between Fig. 6 the 8th and the 9th tubing string) between the 3rd and the 4th section, the main function of 4th section is that liquid is washed in punching to be cleaned up completely, then recycles.
It is very fast that experiment of the present invention supposes that the matter of SMBC passes speed further, collocation simulation software carries out the simulation of table 2 experimental result above, what Fig. 7 illustrated experiment purity data and computer simulation data compares schematic diagram, simulates the data obtained and tests with table 2 and comparing of the data obtained be shown in Fig. 7.Its result display the simulated data representated by solid line and the experimental data representated by square mutually rather identical.According to the parameter that simulation software confirms, again simulate for four section designs of Fig. 6 again, its result confirms after 2 tubing strings of increase the 4th section, be that the switching time of 14mL/min and 10.75 minute is under condition reclaiming flow velocity, wash liquid with charging and punching and be respectively 0.7 and 4.0mL/min, and extract end and raffinate end be respectively 3.0 with the setting of 1.7mL/min, the trehalose of purity 99.3% and the glucose of 92.5% can be obtained.If represent the situation of diluting with D/F (velocity ratio of liquid and charging is washed in punching), then, compared with embodiment 4, the dilute strength of embodiment 5 has reduced 10 times.
The present invention carries out the simulation of volume production equipment further, and the tubing string adopted is diameter 4.0m and is highly 1.5m, ignores dead angle volume simultaneously.Be then that the switching time of 50.4 tons/hour (T/hr) and 8.75 minutes is under condition reclaiming flow velocity, wash liquid with charging and punching and be respectively 2.52 and 14.4T/hr, and extract end and raffinate end be respectively 10.8 with the setting of 6.12T/hr, the trehalose of more than purity 99.9wt% and the glucose of 95.8wt% can be obtained.Confirm with the design of Fig. 6, the target producing about 4000 tonnes of trehaloses and 2000 tonnes of glucose per year can be reached.
Embodiment 6: adopt In-Situ Cleaning-simulation moving-bed (CIP-SMB) chromatography of on-line cleaning function to promote glucose purity
In the middle of the product utilizing enzyme to carry out decomposing, often because of impurity contained in by product or raw material, cause the existence of impurity.Therefore, for a SMBC, the reaction solution after enzyme reaction, except the filtration of necessity, also must consider that in reaction solution, solvability impurity is on the impact of SMBC.These impurity general all belong to acid or the aldehyde of molecular weight, and therefore its anelasticity is all taller than glucose.Therefore, while separation trehalose and glucose, most impurity all can also flow with glucose and go out.Except this can be contained impurity glucose more further other to be separated with SMBC except, the SMBC with on-line cleaning function also can be utilized to carry out the function of Impurity removal.
Fig. 8 illustrates the tubing string configuration schematic diagram of In-Situ Cleaning-simulation moving-bed (CIP-SMB) chromatography, suppose that the delay intensity of A/B/C tri-kinds of compositions in tubular column system is sequentially composition A< composition B< composition C, then the design of Fig. 8 can make three kinds of compositions effectively be separated.Fig. 8 shows In-Situ Cleaning-simulation moving-bed (CIP-SMB) chromatography schematic diagram, for 8 tubing strings SMB and to set configuration be 1-3/1/3/0, being separated of composition B and A is identical with the method for traditional SMB, but the composition C of strong anelasticity, then taken to the 0th section (pre-treatment section), carried out desorption with the strong desorbing agent of another kind simultaneously.Recycling punching is subsequently washed after agent is soaked and is got back in SMB system.In operational design, the separation of composition A/B must meet formula (3).But because the anelasticity of composition C is stronger than composition B, so the composition C of flowing of turning left is easy to along with composition B is extracting the outlet outflow end, and pollute B product.For a real moving-bed (truemovingbed is called for short TMB), such pollution also cannot be avoided, but for SMB, as long as utilize the concept of single tubing string, also can the product that end obtains pure composition B extracting.Pollute to extract in order to composition C will be avoided and end, operationally need to be controlled especially.Fig. 9 describe how to utilize the concept of single tubing string to avoid composition C from extract end flow out.
Fig. 9 illustrates that application In-Situ Cleaning-simulated moving bed chromatography method is separated the conceptual schematic view of three kinds of compositions.After A/B/C tri-kinds of composition chargings, composition C is washed by punching the 3rd section that the right is taken in agent, blacking part in Fig. 9, then when next switching time, it is the second section that the position of this tubing string is adjusted to, then the switching continued through once is later again by migration first section.As long as the number of switching time, flow velocity and each section tubing string can make the part of blacking can not end and flow out from extracting, composition C just can be allowed completely to flow out from the 0th section.Therefore the operation of the second section and the 3rd section still must meet following restricted condition:
N in formula
2it is the tubing string number of the second section.
Except second and the 3rd section restriction except, the operation of the 0th section also will meet following two conditions:
K in formula
c orepresent the Henry's constant of the composition C of the 0th section, m
orepresent the relative velocity of the 0th section.Because use different rushing to wash agent, so K
c owith K
cnot identical.
Figure 10 illustrates the separable operational condition coordinate diagram of In-Situ Cleaning-simulated moving bed chromatography method according to triangle balanced.The design of this embodiment is at traditional (m
2, m
3) coordinate plane is drawn, then as shown in Figure 10, the separable operational condition region of figure intermediate cam shape Regional Representative.Right-angle triangle Regional Representative in Figure 10 can be separated the operational condition of the two composition of A/B, the inequality of the thick line representative formula (9) in Figure 10.Owing to must meet formula (3) and formula (9) simultaneously, so the operational condition of separated component A/B/C completely the K of figure can be arranged in
athe delta-shaped region that-P-Q is linked to be, and P point also represents best operational condition.So, K
ait is larger that-P-Q surrounds leg-of-mutton scope, then the efficiency of SMBC is higher.Therefore, the tubing string number n of the second section in the design of SMBC
2little.
Trehalose production method of the present invention is separated different carbohydrate by application simulation moving-bed chromatography, not only can directly obtain high purity trehalose with or glucose, also have more economic benefit by material (maltose) at a low price because use.
In addition, trehalose production method of the present invention, with simulation moving-bed separation carbohydrate, can be purified into trehalose with continous way feeding manner, contributes to improving Purifing Trehalose output.
Last it is noted that above each embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to foregoing embodiments to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein some or all of technical characteristic; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the scope of various embodiments of the present invention technical scheme.
Claims (10)
1. a trehalose production method, is characterized in that, comprising:
There is provided one by matter, and maltose should be comprised by matter;
There is provided dry acidophilic bacteria TreP that this is converted into trehalose by the maltose of part in matter;
Glucoamylase is provided the maltose not having dried acidophilic bacteria TreP to transform to be converted into glucose and to obtain reaction mixture; And
With simulated moving bed chromatography method, the trehalose in this reaction mixture and glucose are separated, wherein this simulated moving bed chromatography method comprises: (i) providing package is simulation moving-bed containing at least three sections, it moves mutually by one and a stationary phase formed, wherein this moves and comprises punching mutually and wash liquid, this stationary-phase particle size inside has hole, this three section is sequentially first, second and third section, have first respectively, second with third phase to flow rate ratio m1, m2 and m3, this moves and flows through between this three section in the same direction in this is simulation moving-bed, this stationary phase moves mutually simulate movement in the opposite direction relative to this, (ii) this reaction mixture is injected this simulation moving-bed second and the 3rd between section, glucose in this reaction mixture and trehalose have the first delay constant and second respectively and are detained constant, this the first delay constant is greater than this second delay constant, and the hole of this stationary phase can cause trehalose and glucose to move according to friction-motion speed, (iii) this first-phase of this first section should be greater than this first delay constant, with holomorphosis stationary phase to flow rate ratio m1, and this second and third relative velocity ratio m2 and m3 of (iv) this second and third section should between this first delay constant and this second delay constant, glucose is made to move to this second section, trehalose moves to the 3rd section, with separating glucose and trehalose.
2. trehalose production method according to claim 1, is characterized in that, this first, second and third section respectively comprises two endowels, three endowels and three endowels, and in every endowel, filler particles inside has this stationary phase of this hole.
3. trehalose production method according to claim 1, it is characterized in that, when this second-phase of this second section is X-axis to flow rate ratio m2 and this third phase of the 3rd section is Y-axis to flow rate ratio m3, theoretical according to triangle, this second-phase should to fall within flow rate ratio m3 flow rate ratio m2 and this third phase and first be detained constant and this second is detained in the block of constant institute corral by this.
4. trehalose production method according to claim 2, it is characterized in that, when this reaction mixture inject this simulation moving-bed after, this moves and drives this reaction mixture to move in the hole of those tubing strings mutually, because this first delay constant is greater than this second delay constant, therefore, glucose is less than trehalose in this simulation moving-bed translational speed in this simulation moving-bed translational speed, the glucose time of flowing out needed for those tubing strings is greater than the time needed for trehalose, glucose is made to move to this first and second section, trehalose moves to the 3rd section, with separating glucose and trehalose.
5. trehalose production method according to claim 4, is characterized in that, this first delay constant is 0.135, and this second delay constant is 0.015, and this porosity of this stationary-phase particle size inside is 0.40.
6. trehalose production method according to claim 1, it is characterized in that, be 100 % by weight with this by the maltose weight in matter, maltose is transformed the trehalose into about 67 % by weight by dry acidophilic bacteria TreP, and remaining maltose is transformed the glucose into about 33 % by weight by glucoamylase, this is made to change into this reaction mixture comprising trehalose and glucose by the maltose in matter.
7. trehalose production method according to claim 1, is characterized in that, the purity of the trehalose be separated is more than or equal to 98 % by weight.
8. trehalose production method according to claim 1, is characterized in that, this simulation moving-bed also comprising after the 4th section is connected to the 3rd section washs liquid to reclaim circulating flushing.
9. trehalose production method according to claim 8, is characterized in that, the purity of the trehalose be separated is more than or equal to 99 % by weight.
10. trehalose production method according to claim 8, is characterized in that, this simulation moving-bed pre-treatment section that also comprises removes impurity.
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