CN107699557A - A kind of preparation method of high-purity D psicoses - Google Patents
A kind of preparation method of high-purity D psicoses Download PDFInfo
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- CN107699557A CN107699557A CN201711106927.3A CN201711106927A CN107699557A CN 107699557 A CN107699557 A CN 107699557A CN 201711106927 A CN201711106927 A CN 201711106927A CN 107699557 A CN107699557 A CN 107699557A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
Abstract
The present invention relates to a kind of preparation method of high-purity D psicoses, comprise the following steps：(a) D psicose epimerase crude enzyme preparations are prepared；(b) crude enzyme preparation and being fixed of glucose isomerase in step (a)；(c) prepare containing D psicoses, fructose, glucose mixed liquor；(d) mixed liquor made from step (c) is obtained into D psicoses solution and fructose, the mixed liquor of glucose through decolourizing, filtering, from handing over；(e) D psicoses solution made from step (d) passes through chromatographic isolation, concentration, crystallization or drying, and D psicoses are made.
Description
Technical field
The present invention relates to a kind of preparation method of high-purity D-Psicose, belong to technical field of bioengineering.
Background technology
D-Psicose is a kind of important rare sugar, be very little present in nature cane molasses, dried fruit,
In sugar prod, wheat and mouse thorn platymiscium.As sweetener, equivalent to the 70% of fructose, energy only has D-Psicose sugariness
The 0.3% of sucrose, heat is nearly free from after edible, is the edible sweetener of diabetes patient truly.New
Research finds, not only blood glucose will not raise after edible D-Psicose, and can also effectively regulating and controlling blood sugar, reduce taking the photograph for energy
Enter, the effect such as accumulation to slim the abdomen, its unique function and potential medical value have attracted numerous researchers'
Concern, and increasingly welcome by consumer.FDA accreditation sweeteners D-Psicose can be used as food to add within 2011
Agent uses.
Chinese patent literature CN105602879A (application number 201610051547.3) disclose one plant efficient secretion D- Ah
The engineering strain and its construction method of Lip river ketose 3- epimerases.The invention is by using by cud bacterium
The D-Psicose 3- epimerism enzyme genes rdpe structure recombination expression matter in Ruminococcus sp.5_1_39B_FAA sources
Grain pMA5-RDPE, then converts bacillus subtilis, realizes RDPE constitutive and secretive expressions in bacillus subtilis.It is logical
Cross and compare three sugared inducible promoters, obtain optimal inducible promoter PxylA, and significantly improve RDPE secretion water
It is flat.By knocking out xylose utilization genes xylAB (xylA and xylB), the xylose metabolism approach of bacillus subtilis is blocked, has been entered
One step improves RDPE secretory volume, and the optimal induced concentration of derivant xylose is reduced to 0.5% by 4.0%.Finally, adopt
With batch feeding mode, engineered strain 1A751SD-XR is evaluated in 7.5L fermentation tanks, RDPE secretion level highest
95U/mL and 2.6g/L can be reached.But the enzyme activity of the enzyme is still relatively low, the needs of actual production can not be met.
Chinese patent literature CN102869783A (application number 201080044027.0) discloses one kind by using source
In Agrobacterium tumefaciems and in the form of food security express psicose-epimerase and continuously by D-Fructose or D- grapes
The method that sugar prepares D-Psicose.
The price of current D-Psicose is higher, and its key reason is that manufacturing cost can be in any more, greatly limit its
The application of downstream food, field of health care products, product self character are difficult to effectively play.
The content of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of preparation method of high-purity D-Psicose.
Technical solution of the present invention is as follows：
A kind of preparation method of high-purity D-Psicose, comprises the following steps：
(a) the fermented culture medium fermented and cultureds of bacillus subtilis (Bacillus subtilis) BLCY-005, hair is made
Zymotic fluid, zymotic fluid are centrifuged, washing thalline, secondary centrifuging, retain sediment, and it is thick that D-Psicose epimerase is made
Enzyme preparation；
The fermentation medium, component is as follows, is mass percent：
Corn starch 3~8%, glucose 0.8~1.5%, methyl α-naphthyl acetate 0.08~0.12%, epsom salt 0.015~
0.025%, diammonium hydrogen phosphate 0.01~0.02%, manganese sulfate 0.015~0.02%, copper sulphate 0.015~0.018%, chlorination
Iron 0.08~0.12%, excess water, pH6.8~7.2；
(b) in step (a) crude enzyme preparation and glucose isomerase with 1:The ratio of (1~5) (mass ratio) is well mixed, so
In mass ratio 20%~60% ratio is added in the sodium alginate soln that mass concentration is 1%~3% afterwards, is well mixed,
Then it is added drop-wise in the calcium chloride solution that mass concentration is 1%~3%, 4 DEG C are fixed 12~36 hours；
(c) glucose solution that mass concentration is 20%~80% is prepared, by mass percentage 0.0001%~
0.0005% ratio addition cobalt chloride, is well mixed, slow transits through the pillar of addition immobilised enzymes, obtain containing D- A Luo ketone
Sugar, fructose, the mixed liquor of glucose；
(d) mixed liquor made from collection step (c), through decolourize, filter, obtained from friendship D-Psicose solution and fructose,
The mixed liquor of glucose；
(e) D-Psicose solution made from step (d) passes through chromatographic isolation, concentration, crystallization or drying, and D- A Luo are made
Ketose.
According to currently preferred, in the step (a), bacillus subtilis (Bacillus subtilis) BLCY-
005 derives from China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC No.13152, ground
Location：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
According to currently preferred, in the step (a), fermentation medium component is as follows, is weight percentage：
Corn starch 5%, glucose 1%, methyl α-naphthyl acetate 0.1%, epsom salt 0.02%, diammonium hydrogen phosphate 0.015%,
Manganese sulfate 0.018%, copper sulphate 0.016%, iron chloride 0.1%, excess water, pH6.8~7.2.
According to currently preferred, in the step (a), fermented and cultured step is as follows：
Seed is inoculated in fermentation medium by the inoculum concentration according to percent by volume 1~2%, 35~37 DEG C of temperature,
PH6.8~7.2, tank pressure 0.04~0.06Mpa, 1.8~2.2vvm of ventilating ratio, 250~400rpm of rotating speed, dissolved oxygen 30%~40%
Under conditions of, 34~38h of fermented and cultured, zymotic fluid is made.
According to currently preferred, in the step (a), washing thalline uses pH 8.0, concentration 50mmol/L Tris-
HCl buffer solutions, are then centrifuged, and retain precipitation, and crude enzyme preparation is made.
According to currently preferred, the centrifugation in the step (a) be under the conditions of 4 DEG C, 10000r/min, from
Heart 10min.
According to currently preferred, in the step (b), glucose isomerase has purchased from Novi's letter (China) biotechnology
Limit company.
According to currently preferred, in the step (d), decolorization process is as follows：
By mixed solution made from step (d), 0.5~1% ratio adds activated carbon by mass percentage, 80~85 DEG C
Stir 30~40min.
According to currently preferred, in the step (d), filtering uses plate-frame filtering, 0.2~0.4Mpa of filter pressure,
5.0~6.0t/h of water-carrying capacity.
According to currently preferred, in the step (d), from handing over, step is as follows：
By the mixed solution after decolorization filtering with the flow velocity of 3 times of resin volume/hours, pass through continuous ionic at 35~55 DEG C
Exchange system, carry out from desalination is handed over, from feed liquid light transmittance >=98% after friendship.Feed liquid after processing is as clear as crystal, free from extraneous odour.
According to currently preferred, the step (d) is also included obtained fructose, the mixed liquor of glucose and step
(c) glucose solution mixing, continues subsequent reactions.
According to currently preferred, in the step (e), chromatrographic separation step is as follows：
Chromatographic run 0.20~0.30MPa of pressure, 60~70 DEG C of temperature, water consume is than 1:(1.3~1.6), feed per hour
1.5~2.0m3, D-Psicose and fructose, the mixed liquor of glucose are collected respectively.
According to currently preferred, in the step (e), concentration uses quadruple effect falling film evaporator, vacuum 0.06-
0.09Mpa, 50~85 DEG C of feed temperature, is concentrated into the 60~75% of original volume.
According to currently preferred, in the step (e), dry for spray drying, step it is as follows：
Liquid glucose after concentration is entered in drying tower, 130~150 DEG C of EAT, starts atomizer, and liquid is dry through spraying
Dry is pulverulent solids.
According to currently preferred, in the step (e), reaction conditions for crystallization is：
Liquid glucose mass concentration 70~85%, 50~70 DEG C of temperature, the crystal seed of Solute mass 10~30% is added, stirring is
It is even, 8~16 hours are stood in 50~70 DEG C, 1 DEG C of speed slow cooling is then dropped according to 3~6h, is slowly stirred simultaneously, until
A large amount of crystal grain uniformly, regular are formed in solution, separation, D-Psicose are made.
Beneficial effect
1st, the D-Psicose epimerase and Portugal that first passage of the present invention is obtained using special fermentation medium culture
Grape sugar isomerase is by way of being made by mixing immobilised enzymes, using glucose syrup as raw material, prepares D-Psicose, glucose quilt
Glucose isomerase changes into the D-Psicose epimerase fixed simultaneously immediately after fructose and changes into D- A Luo ketone
Sugar, when preparing D-Psicose epimerase compared with other modes and being converted under the same conditions, transformation efficiency greatly improves,
Greatly reduce production cost simultaneously.
2nd, in the present invention cobalt chloride simultaneously as glucose isomerase in mixed column and D-Psicose epimerase
Activator, compared with the addition that traditional approach greatly reduces cobalt chloride, reduce the expense of subsequent treatment.
Embodiment
Technical scheme is further elaborated with reference to embodiment, but institute's protection domain of the present invention is not limited to
This.
Biological material source
Bacillus subtilis (Bacillus subtilis) BLCY-005 entrusts from Chinese microorganism strain preservation management
Member's meeting common micro-organisms center, culture presevation CGMCC No.13152；
Glucose isomerase described in embodiment is purchased from letter (China) Bioisystech Co., Ltd of Novi；
D-Psicose Enzyme activity assay method：
400ul 100g/L D-Fructoses are taken to add 100ul zymotic fluids as substrate, reacted at 55 DEG C of water bath with thermostatic control
5min, 10min terminating reactions are boiled in boiling water.Centrifuged with 10000r/min rotating speed, take supernatant to dilute 2 after centrifugation
After times, D-Psicose content is detected with high performance liquid chromatograph, with 1 μm of ol of every milliliter of zymotic fluid interior generation per minute D- Ahs
Lip river ketose is defined as an enzyme-activity unit.
D-Psicose conversion ratio defines：D-Psicose quality/substrate (D-Fructose) quality.
Conversion ratio assay method is as follows：With pH8.0,50mmol/L Tris-HCl buffer preparations, 30% (w/w's)
D-Fructose solution makees substrate, adds appropriate enzyme preparation, after 55 DEG C of water-bath oscillating reactions 3h, immediately in 100 DEG C of boiling water baths
15min, with high effective liquid chromatography for measuring D-Psicose content.
Decolourize, filter described in embodiment, from friendship, chromatographic isolation, concentration, crystallization or drying be this area conventional skill
Art means.
Embodiment 1
A kind of preparation method of high-purity D-Psicose, comprises the following steps：
(1) bacillus subtilis (Bacillus subtilis) BLCY-005 is taken, is inoculated in seed culture medium, in temperature
Seed culture 8h is carried out under conditions of 35 DEG C of degree, 220rpm, seed liquor is made；
The seed culture medium component is as follows, is weight percentage：
Peptone 1.0%, corn starch：0.5%, glycerine：1.0%, excess water, pH7.0；
(2) seed liquor made from taking step (1), is inoculated in fermentation medium according to the inoculum concentration of percent by volume 2%,
In 35 DEG C of temperature, pH7.0, tank pressure 0.05Mpa, ventilating ratio 2.0vvm, rotating speed 300rpm, under conditions of dissolved oxygen 35%, fermentation training
36h is supported, zymotic fluid is made；
The fermentation medium, component is as follows, is mass percent：
Corn starch 5%, glucose 1%, methyl α-naphthyl acetate 0.1%, epsom salt 0.02%, diammonium hydrogen phosphate 0.015%,
Manganese sulfate 0.018%, copper sulphate 0.016%, iron chloride 0.1%, excess water, pH7.0；
(3) zymotic fluid made from step (2) is taken, through centrifuging 10min under the conditions of 4 DEG C, 10000r/min, using
PH8.0, concentration 50mmol/L Tris-HCl buffer solution washing thallines, 4 DEG C, secondary centrifuging 10min under the conditions of 10000r/min,
Retain sediment, the crude enzyme preparation of D-Psicose epimerase is made；
(4) in step (3) crude enzyme preparation and glucose isomerase with 1:The ratio of 5 (mass ratioes) is well mixed, and is then pressed
The ratio of mass ratio 20% is added in the sodium alginate soln that mass concentration is 1%~3%, is well mixed, is then added drop-wise to
Mass concentration is that 4 DEG C are fixed 12~36 hours in 1%~3% calcium chloride solution；
(5) glucose solution that mass concentration is 20%~80% is prepared, by mass percentage 0.0001%~
0.0005% ratio addition cobalt chloride, is well mixed, slow transits through the pillar of addition immobilised enzymes, obtain containing D- A Luo ketone
Sugar, fructose, the mixed liquor of glucose；
(6) the ratio addition activated carbon of mixed liquor made from collection step (5), by mass percentage 0.5%, 85 DEG C of stirrings
30min is decolourized, and plate-frame filtering is then used under conditions of filter pressure 0.4Mpa, water-carrying capacity 5.0t/h, then through being obtained from friendship
D-Psicose solution and fructose, the mixed liquor of glucose, obtained fructose, the mixed liquor and the grape of step (c) of glucose
Sugar juice mixes, and continues subsequent reactions；
It is described as follows from step is handed over：
By the mixed solution after decolorization filtering with the flow velocity of 3 times of resin volume/hours, exchanged at 55 DEG C by continuous ionic
System, carry out from desalination is handed over, from feed liquid light transmittance >=98% after friendship；Feed liquid after processing is as clear as crystal, free from extraneous odour.
(7) D-Psicose solution made from step (6) is 0.06Mpa, feed liquid temperature in vacuum after chromatographic isolation
The 60% of original volume is concentrated into using quadruple effect falling film evaporator under conditions of 85 DEG C of degree, crystallization or dry, D-Psicose is made；
The chromatrographic separation step is as follows：
Chromatographic run pressure 0.30MPa, temperature 60 C, water consume is than 1:1.6,1.5m is fed per hour3, collect respectively D- Ah
Lip river ketose and fructose, the mixed liquor of glucose；
The drying is spray drying, and step is as follows：
Liquid glucose after concentration is entered in drying tower, 150 DEG C of EAT, starts atomizer, liquid is spray-dried to be
Pulverulent solids；
The crystallisation step is as follows：
Liquid glucose mass concentration 70%, temperature 70 C, the crystal seed of Solute mass 10% is added, is stirred, 8 are stood in 70 DEG C
Hour, 1 DEG C of speed slow cooling is then dropped according to 6h, is slowly stirred simultaneously, until being formed in solution a large amount of uniform, rule
Crystal grain, separation, D-Psicose is made.
After testing, D-Psicose enzyme activity is 2960, conversion ratio 35.32%.
Embodiment 2
A kind of preparation method of high-purity D-Psicose, comprises the following steps：
(1) bacillus subtilis (Bacillus subtilis) BLCY-005 is taken, is inoculated in seed culture medium, in temperature
Seed culture 8h is carried out under conditions of 35 DEG C of degree, 220rpm, seed liquor is made；
The seed culture medium component is as follows, is weight percentage：
Peptone 1.0%, corn starch：0.5%, glycerine：1.0%, excess water, pH7.0；
(2) seed liquor made from taking step (1), is inoculated in fermentation medium according to the inoculum concentration of percent by volume 2%,
In 35 DEG C of temperature, pH7.0, tank pressure 0.05Mpa, ventilating ratio 2.0vvm, rotating speed 300rpm, under conditions of dissolved oxygen 35%, fermentation training
36h is supported, zymotic fluid is made；
The fermentation medium, component is as follows, is mass percent：
Corn starch 5%, glucose 1%, methyl α-naphthyl acetate 0.1%, epsom salt 0.02%, diammonium hydrogen phosphate 0.015%,
Manganese sulfate 0.018%, copper sulphate 0.016%, iron chloride 0.1%, excess water, pH7.0；
(3) zymotic fluid made from step (2) is taken, through centrifuging 10min under the conditions of 4 DEG C, 10000r/min, using
PH8.0, concentration 50mmol/L Tris-HCl buffer solution washing thallines, 4 DEG C, secondary centrifuging 10min under the conditions of 10000r/min,
Retain sediment, the crude enzyme preparation of D-Psicose epimerase is made；
(4) in step (3) crude enzyme preparation and glucose isomerase with 1:The ratio of 1 (mass ratio) is well mixed, and is then pressed
The ratio of mass ratio 40% is added in the sodium alginate soln that mass concentration is 1%~3%, is well mixed, is then added drop-wise to
Mass concentration is that 4 DEG C are fixed 12~36 hours in 1%~3% calcium chloride solution；
(5) glucose solution that mass concentration is 20%~80% is prepared, by mass percentage 0.0001%~
0.0005% ratio addition cobalt chloride, is well mixed, slow transits through the pillar of addition immobilised enzymes, obtain containing d- A Luo ketone
Sugar, fructose, the mixed liquor of glucose；
(6) the ratio addition activated carbon of mixed liquor made from collection step (5), by mass percentage 1%, 80 DEG C of stirrings
40min is decolourized, and plate-frame filtering is then used under conditions of filter pressure 0.2Mpa, water-carrying capacity 6.0t/h, then through being obtained from friendship
D-Psicose solution and fructose, the mixed liquor of glucose, obtained fructose, the mixed liquor and the grape of step (c) of glucose
Sugar juice mixes, and continues subsequent reactions；
It is described as follows from step is handed over：
By the mixed solution after decolorization filtering with the flow velocity of 3 times of resin volume/hours, exchanged at 35 DEG C by continuous ionic
System, carry out from desalination is handed over, from feed liquid light transmittance >=98% after friendship；Feed liquid after processing is as clear as crystal, free from extraneous odour.
(7) D-Psicose solution made from step (6) is 0.09Mpa, feed liquid temperature in vacuum after chromatographic isolation
The 75% of original volume is concentrated into using quadruple effect falling film evaporator under conditions of 50 DEG C of degree, crystallization or dry, D-Psicose is made；
The chromatrographic separation step is as follows：
Chromatographic run pressure 0.20MPa, temperature 70 C, water consume is than 1:1.3,2.0m is fed per hour3, collect respectively D- Ah
Lip river ketose and fructose, the mixed liquor of glucose；
The drying is spray drying, and step is as follows：
Liquid glucose after concentration is entered in drying tower, 130 DEG C of EAT, starts atomizer, liquid is spray-dried to be
Pulverulent solids；
The crystallisation step is as follows：
Liquid glucose mass concentration 85%, temperature 50 C, the crystal seed of Solute mass 30% is added, is stirred, in 50 DEG C of standings
16 hours, 1 DEG C of speed slow cooling is then dropped according to 3h, is slowly stirred simultaneously, until forming a large amount of uniform, rules in solution
Crystal grain, separation, be made D-Psicose.
After testing, the enzyme activity of D-Psicose is 3000, conversion ratio 35.56%.
Embodiment 3
A kind of preparation method of high-purity D-Psicose, comprises the following steps：
(1) bacillus subtilis (Bacillus subtilis) BLCY-005 is taken, is inoculated in seed culture medium, in temperature
Seed culture 8h is carried out under conditions of 35 DEG C of degree, 220rpm, seed liquor is made；
The seed culture medium component is as follows, is weight percentage：
Peptone 1.0%, corn starch：0.5%, glycerine：1.0%, excess water, pH7.0；
(2) seed liquor made from taking step (1), is inoculated in fermentation medium according to the inoculum concentration of percent by volume 2%,
In 35 DEG C of temperature, pH7.0, tank pressure 0.05Mpa, ventilating ratio 2.0vvm, rotating speed 300rpm, under conditions of dissolved oxygen 35%, fermentation training
36h is supported, zymotic fluid is made；
The fermentation medium, component is as follows, is mass percent：
Corn starch 5%, glucose 1%, methyl α-naphthyl acetate 0.1%, epsom salt 0.02%, diammonium hydrogen phosphate 0.015%,
Manganese sulfate 0.018%, copper sulphate 0.016%, iron chloride 0.1%, excess water, pH7.0；
(3) zymotic fluid made from step (2) is taken, through centrifuging 10min under the conditions of 4 DEG C, 10000r/min, using
PH8.0, concentration 50mmol/L Tris-HCl buffer solution washing thallines, 4 DEG C, secondary centrifuging 10min under the conditions of 10000r/min,
Retain sediment, the crude enzyme preparation of D-Psicose epimerase is made；
(4) in step (3) crude enzyme preparation and glucose isomerase with 1:The ratio of 3 (mass ratioes) is well mixed, and is then pressed
The ratio of mass ratio 60% is added in the sodium alginate soln that mass concentration is 1%~3%, is well mixed, is then added drop-wise to
Mass concentration is that 4 DEG C are fixed 12~36 hours in 1%~3% calcium chloride solution；
(5) glucose solution that mass concentration is 20%~80% is prepared, by mass percentage 0.0001%~
0.0005% ratio addition cobalt chloride, is well mixed, slow transits through the pillar of addition immobilised enzymes, obtain containing D- A Luo ketone
Sugar, fructose, the mixed liquor of glucose；
(6) the ratio addition activated carbon of mixed liquor made from collection step (5), by mass percentage 0.8%, 82 DEG C of stirrings
35min is decolourized, and plate-frame filtering is then used under conditions of filter pressure 0.3Mpa, water-carrying capacity 5.5t/h, then through being obtained from friendship
D-Psicose solution and fructose, the mixed liquor of glucose, obtained fructose, the mixed liquor and the grape of step (c) of glucose
Sugar juice mixes, and continues subsequent reactions；
It is described as follows from step is handed over：
By the mixed solution after decolorization filtering with the flow velocity of 3 times of resin volume/hours, exchanged at 45 DEG C by continuous ionic
System, carry out from desalination is handed over, from feed liquid light transmittance >=98% after friendship；Feed liquid after processing is as clear as crystal, free from extraneous odour.
(7) D-Psicose solution made from step (6) is 0.075Mpa, feed liquid in vacuum after chromatographic isolation
The 70% of original volume is concentrated into using quadruple effect falling film evaporator under conditions of 65 DEG C of temperature, crystallization or dry, D- A Luo ketone is made
Sugar；
The chromatrographic separation step is as follows：
Chromatographic run pressure 0.25MPa, 65 DEG C of temperature, water consume is than 1:1.4,1.8m is fed per hour3, collect respectively D- Ah
Lip river ketose and fructose, the mixed liquor of glucose；
The drying is spray drying, and step is as follows：
Liquid glucose after concentration is entered in drying tower, 140 DEG C of EAT, starts atomizer, liquid is spray-dried to be
Pulverulent solids；
The crystallisation step is as follows：
Liquid glucose mass concentration 75%, temperature 60 C, the crystal seed of Solute mass 20% is added, is stirred, in 60 DEG C of standings
12 hours, 1 DEG C of speed slow cooling is then dropped according to 4h, is slowly stirred simultaneously, until forming a large amount of uniform, rules in solution
Crystal grain, separation, be made D-Psicose.
After testing, the enzyme activity of D-Psicose is 2960, conversion ratio 35.38%.
Comparative example 1
A kind of preparation method of high-purity D-Psicose as described in Example 1, difference are that the fermentation is trained
Base is supported, component is as follows, is mass percent：
Corn starch 5%, glucose 1%, ethephon (CEPHA),2-(chloroethyl) phosphonic acid 0.1%, epsom salt 0.02%, diammonium hydrogen phosphate 0.015%,
Manganese sulfate 0.018%, copper sulphate 0.016%, iron chloride 0.1%, excess water, pH7.0.
After testing, the enzyme activity of D-Psicose is 2220, conversion ratio 31.17%.
Comparative example 2
A kind of preparation method of high-purity D-Psicose, difference are as described in Example 1, glucose isomerase
The crude enzyme preparation of enzyme and obtained D-Psicose epimerase is not by the way of mixed immobilization, glucose and grape
Sugared isomery enzyme reaction, gained reaction solution and then the crude enzyme preparation reaction with obtained D-Psicose epimerase.
After testing, the enzyme activity of D-Psicose is 3020, conversion ratio 23.28%.
Interpretation of result
Embodiment is can be seen that by the data of embodiment and comparative example 1 by adding methyl α-naphthyl acetate, D-Psicose to turn
Rate improves 4% or so compared with comparative example, largely reduces D-Psicose manufacturing cost, by implementing and comparative example 2
Data can be seen that by the way of mixed immobilization enzyme compared with not using, the enzyme activity of D-Psicose there is no
Loss, and conversion ratio improves 12% or so, so the D-Psicose epimerase obtained by the present invention is more existing known
D-Psicose epimerase is more suitable for the use of mixed immobilization enzyme, and this improves conversion ratio, reduced for simplifying technique
Cost all has the advantage of highly significant.
Claims (10)
1. a kind of preparation method of high-purity D-Psicose, it is characterised in that comprise the following steps：
(a) the fermented culture medium fermented and cultureds of bacillus subtilis (Bacillus subtilis) BLCY-005, fermentation is made
Liquid, zymotic fluid are centrifuged, washing thalline, secondary centrifuging, retain sediment, and the thick enzyme of D-Psicose epimerase is made
Preparation；
The fermentation medium, component is as follows, is mass percent：
Corn starch 3~8%, glucose 0.8~1.5%, methyl α-naphthyl acetate 0.08~0.12%, epsom salt 0.015~
0.025%, diammonium hydrogen phosphate 0.01~0.02%, manganese sulfate 0.015~0.02%, copper sulphate 0.015~0.018%, chlorination
Iron 0.08~0.12%, excess water, pH6.8~7.2；
(b) in step (a) crude enzyme preparation and glucose isomerase with 1:The ratio of (1~5) (mass ratio) is well mixed, and is then pressed
The ratio of mass ratio 20%~60% is added in the sodium alginate soln that mass concentration is 1%~3%, is well mixed, then
It is added drop-wise in the calcium chloride solution that mass concentration is 1%~3%, 4 DEG C are fixed 12~36 hours；
(c) glucose solution that mass concentration is 20%~80% is prepared, by mass percentage 0.0001%~0.0005%
Ratio adds cobalt chloride, is well mixed, and slow transits through the pillar of addition immobilised enzymes, obtains containing D-Psicose, fructose, Portugal
The mixed liquor of grape sugar；
(d) mixed liquor made from collection step (c), through decolourizing, filtering, D-Psicose solution and fructose, grape being obtained from friendship
The mixed liquor of sugar；
(e) D-Psicose solution made from step (d) passes through chromatographic isolation, concentration, crystallization or drying, and D- A Luo ketone is made
Sugar.
2. preparation method as claimed in claim 1, it is characterised in that in the step (a), bacillus subtilis
(Bacillus subtilis) BLCY-005 deposit number is CGMCC No.13152.
3. preparation method as claimed in claim 1, it is characterised in that in the step (a), fermentation medium component is as follows,
It is weight percentage：
Corn starch 5%, glucose 1%, methyl α-naphthyl acetate 0.1%, epsom salt 0.02%, diammonium hydrogen phosphate 0.015%, sulfuric acid
Manganese 0.018%, copper sulphate 0.016%, iron chloride 0.1%, excess water, pH6.8~7.2.
4. preparation method as claimed in claim 1, it is characterised in that in the step (a), fermented and cultured step is as follows：
Seed is inoculated in fermentation medium by the inoculum concentration according to percent by volume 1~2%, in 35~37 DEG C of temperature, pH6.8
~7.2, tank pressure 0.04~0.06Mpa, 1.8~2.2vvm of ventilating ratio, 250~400rpm of rotating speed, the bar of dissolved oxygen 30%~40%
Under part, 34~38h of fermented and cultured, zymotic fluid is made.
5. preparation method as claimed in claim 1, it is characterised in that in the step (a), washing thalline using pH 8.0,
Concentration 50mmol/L Tris-HCl buffer solutions, are then centrifuged, and retain precipitation, and crude enzyme preparation is made.
6. preparation method as claimed in claim 1, it is characterised in that the centrifugation in the step (a) be 4 DEG C,
Under the conditions of 10000r/min, 10min is centrifuged.
7. preparation method as claimed in claim 1, it is characterised in that in the step (d), decolorization process is as follows：
By the ratio addition activated carbon of mixed solution made from step (d), by mass percentage 0.5~1%, 80~85 DEG C of stirrings
30~40min.
8. preparation method as claimed in claim 1, it is characterised in that in the step (d), filtering uses plate-frame filtering, mistake
Filtering pressure 0.2~0.4Mpa of power, 5.0~6.0t/h of water-carrying capacity；
Preferably, in the step (d), from handing over, step is as follows：
By the mixed solution after decolorization filtering with the flow velocity of 3 times of resin volume/hours, exchanged at 35~55 DEG C by continuous ionic
System, carry out from desalination is handed over, from feed liquid light transmittance >=98% after friendship；
Preferably, the step (d) is also included the mixed liquor and the glucose solution of step (c) of obtained fructose, glucose
Mixing, continues subsequent reactions.
9. preparation method as claimed in claim 1, it is characterised in that in the step (e), concentration uses quadruple effect falling film evaporation
Device, vacuum are 0.06~0.09Mpa, 50~85 DEG C of feed temperature, are concentrated into the 60~75% of original volume；
Preferably, in the step (e), chromatrographic separation step is as follows：
Chromatographic run 0.20~0.30MPa of pressure, 60~70 DEG C of temperature, water consume is than 1:(1.3~1.6), charging 1.5 per hour~
2.0m3, D-Psicose and fructose, the mixed liquor of glucose are collected respectively.
10. preparation method as claimed in claim 1, it is characterised in that in the step (e), dry as spray drying, step
It is as follows：
Liquid glucose after concentration is entered in drying tower, 130~150 DEG C of EAT, starts atomizer, liquid is spray-dried to be
Pulverulent solids；
Preferably, in the step (e), reaction conditions for crystallization is：
Liquid glucose mass concentration 70~85%, 50~70 DEG C of temperature, the crystal seed of Solute mass 10~30% is added, is stirred, in
50~70 DEG C stand 8~16 hours, and 1 DEG C of speed slow cooling is then dropped according to 3~6h, is slowly stirred simultaneously, until solution
It is middle to form a large amount of crystal grain uniformly, regular, separation, D-Psicose is made.
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| CN108866247A (en) * | 2018-09-18 | 2018-11-23 | 上海立足生物科技有限公司 | The method and apparatus that continuous large-scale separation prepares D-Psicose |
| CN109022625A (en) * | 2018-09-18 | 2018-12-18 | 上海立足生物科技有限公司 | A method of producing the D-Psicose of concentration |
| CN109022521A (en) * | 2018-09-18 | 2018-12-18 | 上海立足生物科技有限公司 | A method of D-Psicose is prepared by starch |
| CN109913438A (en) * | 2018-12-27 | 2019-06-21 | 吉林中粮生化有限公司 | A method of preparing the D-Psicose -3- epimerase of immobilization |
| WO2020057560A1 (en) * | 2018-09-18 | 2020-03-26 | 上海立足生物科技有限公司 | Process for producing d-psicose |
| CN111455003A (en) * | 2020-04-30 | 2020-07-28 | 中国科学院广州能源研究所 | Method for preparing D-psicose from microalgae |
| CN113080357A (en) * | 2021-05-17 | 2021-07-09 | 江苏赛威分离科技有限公司 | Low-calorie compound sweetener and production process thereof |
| WO2021239813A1 (en) | 2020-05-27 | 2021-12-02 | Pfeifer & Langen GmbH & Co. KG | Crystallization of allulose under reduced pressure |
| WO2022049307A1 (en) | 2020-09-07 | 2022-03-10 | Savanna Ingredients Gmbh | Extrusion process for the preparation of a solid allulose composition |
| CN114231578A (en) * | 2021-12-30 | 2022-03-25 | 保龄宝生物股份有限公司 | Method for preparing psicose by double-enzyme method |
| US11746392B2 (en) | 2020-11-23 | 2023-09-05 | Savanna Ingredients Gmbh | Drying of allulose crystals |
| WO2024047122A1 (en) | 2022-09-01 | 2024-03-07 | Savanna Ingredients Gmbh | Process for the preparation of a particulate allulose composition |
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| CN108866247A (en) * | 2018-09-18 | 2018-11-23 | 上海立足生物科技有限公司 | The method and apparatus that continuous large-scale separation prepares D-Psicose |
| CN109022625A (en) * | 2018-09-18 | 2018-12-18 | 上海立足生物科技有限公司 | A method of producing the D-Psicose of concentration |
| CN109022521A (en) * | 2018-09-18 | 2018-12-18 | 上海立足生物科技有限公司 | A method of D-Psicose is prepared by starch |
| WO2020057560A1 (en) * | 2018-09-18 | 2020-03-26 | 上海立足生物科技有限公司 | Process for producing d-psicose |
| WO2020057561A1 (en) * | 2018-09-18 | 2020-03-26 | 上海立足生物科技有限公司 | Method for preparing d-psicose from starch |
| CN109913438A (en) * | 2018-12-27 | 2019-06-21 | 吉林中粮生化有限公司 | A method of preparing the D-Psicose -3- epimerase of immobilization |
| CN111455003A (en) * | 2020-04-30 | 2020-07-28 | 中国科学院广州能源研究所 | Method for preparing D-psicose from microalgae |
| WO2021239813A1 (en) | 2020-05-27 | 2021-12-02 | Pfeifer & Langen GmbH & Co. KG | Crystallization of allulose under reduced pressure |
| WO2022049307A1 (en) | 2020-09-07 | 2022-03-10 | Savanna Ingredients Gmbh | Extrusion process for the preparation of a solid allulose composition |
| US11746392B2 (en) | 2020-11-23 | 2023-09-05 | Savanna Ingredients Gmbh | Drying of allulose crystals |
| CN113080357A (en) * | 2021-05-17 | 2021-07-09 | 江苏赛威分离科技有限公司 | Low-calorie compound sweetener and production process thereof |
| CN114231578A (en) * | 2021-12-30 | 2022-03-25 | 保龄宝生物股份有限公司 | Method for preparing psicose by double-enzyme method |
| WO2024047122A1 (en) | 2022-09-01 | 2024-03-07 | Savanna Ingredients Gmbh | Process for the preparation of a particulate allulose composition |
| WO2024047121A1 (en) | 2022-09-01 | 2024-03-07 | Savanna Ingredients Gmbh | Process for the preparation of a particulate allulose composition |
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