Method for co-producing high fructose corn syrup with different purities by using high-purity crystalline fructose
阅读说明：本技术 一种高纯度结晶果糖联产不同纯度果葡糖浆的方法 (Method for co-producing high fructose corn syrup with different purities by using high-purity crystalline fructose ) 是由 李林海 董得平 黄继红 董瑜琪 周义圣 高素珍 冯军伟 杨秋霞 侯银臣 廖爱美 于 2020-12-21 设计创作，主要内容包括：本发明属于结晶果糖技术领域,具体涉及一种高纯度结晶果糖联产不同纯度果葡糖浆的方法。本发明以葡萄糖为底物,采用新型葡萄糖异构酶GIM进行异构反应,提高异构效率,缩短异构时间,经过色谱分离获得不同果糖含量的产品,并进一步通过混床精制、高温脱味、蒸发、结晶、分离、干燥等工艺以及不同阶段产物的组合和处理,获得高纯度结晶果糖、高纯度的果葡糖浆以及普通42果葡糖浆产品。该工艺最大化的利用了葡萄糖底物,并缩短工艺流程和时间,具有较好的经济效益。(The invention belongs to the technical field of crystalline fructose, and particularly relates to a method for co-producing high fructose corn syrup with different purities by using high-purity crystalline fructose. The invention takes glucose as a substrate, adopts novel glucose isomerase GIM to carry out isomerization reaction, improves isomerization efficiency, shortens isomerization time, obtains products with different fructose contents through chromatographic separation, and further obtains high-purity crystalline fructose, high-purity fructose syrup and common 42 fructose syrup products through processes of mixed bed refining, high-temperature deodorization, evaporation, crystallization, separation, drying and the like and combination and treatment of products at different stages. The process maximally utilizes the glucose substrate, shortens the process flow and time, and has good economic benefit.)
1. A method for co-producing high fructose corn syrup with different purity by using high purity crystalline fructose is characterized by comprising the following steps:
1) sugar dissolving: dissolving edible glucose in water to obtain glucose solution with the concentration of 48-52%;
2) isomerization: degassing the dissolved sugar solution, adding magnesium sulfate and sodium metabisulfite, and driving into an isomerase fixing column to obtain an F42 isomerous sugar solution with fructose content of 42-44%;
the isomerase added into the fixed column is glucose isomerase GIM, and the amino acid sequence is shown as SEQ ID NO: 3 is shown in the specification;
3) and (3) secondary decolorization: primary decoloring: adding activated carbon into the isomerized sugar solution obtained in the step 2), and performing decoloration and original heat-sensitive material removal treatment by using a plate and frame filter; and (3) secondary regulation and decoloration: cooling the primary decolorized feed liquid to 30-55 ℃, adding active carbon, adjusting the pH value to be 4.8-5.2 of the protein isoelectric point, and further decolorizing and removing heat-sensitive raw materials by using a plate and frame filter;
4) low-temperature ion exchange: cooling the decolorized isomerized sugar solution in the step 3) to 5-10 ℃, and passing through ion exchange resin in the sequence of strong acid cation → weak base anion → strong acid cation → weak base anion, wherein the conductivity of the ion exchange material is less than or equal to 3 mus/cm;
5) primary evaporation: pumping the ion exchange discharge into an evaporator for evaporation, and controlling the dry matter of the evaporation discharge to be 59-61%;
6) and (3) chromatographic separation: carrying out flash evaporation and degassing on the evaporated sugar solution, carrying out chromatographic separation, and separating an AD solution with the fructose content of more than or equal to 98%, a BD solution with the fructose content of 3-6% and a CD solution with the fructose content of 1-3%;
7) mixed bed refining: adsorbing the AD feed liquid separated in the step 6) to the negative and positive ions and the peculiar smell compounds in the feed liquid through the special mixed bed resin, purifying the syrup smell, wherein the discharge conductance is less than or equal to 1 mu s/cm, and the 5-hydroxymethyl furfural in the syrup is less than or equal to 0.0005%;
8) removing odor at high temperature: pumping the material liquid refined by the mixed bed into a deodorizing column, controlling the operation temperature to be 35-40 ℃, controlling the discharge conductance to be less than or equal to 1 mu s/cm, and controlling the 5-hydroxymethylfurfural to be less than or equal to 0.0005%;
9) and (3) evaporation: evaporating the deodorized feed liquid to obtain feed liquid with concentration of 90.0-90.3%;
10) pre-crystallization: pumping the feed liquid obtained by evaporation into a pre-crystallizer, and culturing seed crystals;
11) vertical crystallization: pumping the pre-crystallized material cultured by the pre-crystallizer into a vertical crystallizer, reducing the temperature to 22-23 ℃ at a constant speed, crystallizing for 70-80 hours, and discharging to obtain crystallized massecuite;
12) separation: separating the crystallized massecuite mother liquor; fully washing and centrifuging the fructose crystals separated from the mother liquor by using pure water at 58-62 ℃;
13) fluidized bed drying: controlling the temperature of the inlet air to be 80 ℃ and the humidity to be 20%, uniformly drying the separated material, and controlling the water content to be less than or equal to 0.1% after drying to obtain high-purity crystalline fructose;
14) liquid mixing and ion exchange: mixing the BD liquid separated in the step 6) with the mother liquid separated in the step 12), heating to 45-50 ℃, and passing through ion exchange resin in the sequence of strong acid cation → weak base anion → strong acid cation → weak base anion to control the discharge conductivity to be less than or equal to 20 mus/cm;
15) and (3) evaporating the mixed solution: evaporating the ion-exchange mixed solution obtained in the step 14), controlling the dry matter to be more than or equal to 77% after evaporation and the pH value to be 3.3-4.5, and obtaining a high-purity high fructose corn syrup product;
16) isomerizing, ion-exchanging and concentrating the CD liquid separated in the step 6) to obtain a common 42 high fructose corn syrup product.
2. The method for co-producing high fructose corn syrup with different purity according to claim 1, wherein the pH is adjusted to 7.5-8.0 in step 2), and then magnesium sulfate in an amount of 0.7-0.8kg/TDS and sodium metabisulfite in an amount of 0.2-0.3kg/TDS are added.
3. The method for co-production of high-purity crystalline fructose and high fructose syrup with different purity according to claim 1, wherein the glucose solution is pumped into an isomerase fixing column at a flow rate of 7m when the temperature of the glucose solution in the step 2) is reduced to 52-62 ℃3The temperature in the fixed column is 50-55 ℃.
4. The method for co-producing high-purity fructose syrup with different purity according to claim 1, wherein the sulfonic acid group (-SO) is carried on the styrene-divinylbenzene copolymer with the macroporous strong acid cation in the step 4)3H) The weakly basic anion is a weakly basic anion exchange resin with a polystyrene macroporous structure, and is in a free amine type.
5. The method for co-producing high fructose corn syrup with different purity according to claim 1, wherein the chromatographic separation in step 6) is performed by a Sequential Simulated Moving Bed (SSMB), and the chromatographic column is Ca-type cation exchange resin; the chromatographic separation mobile phase is water, and the column temperature of the chromatographic separation column is 62-65 ℃.
6. The method for co-producing high fructose corn syrup with different purity according to claim 1, wherein the content of fructose in the massecuite after centrifugation in step 12) is more than or equal to 99.9%, the water content is less than or equal to 4.5%, the content of other sugars is less than or equal to 0.01%, and the content of 5-hydroxy furfural is less than or equal to 0.0001%.
7. The method for co-producing high purity fructose corn syrup containing high purity crystalline fructose as claimed in claim 1, wherein in the step 16), the isomerization is carried out by adding 35-50ppm of Mg to the CD solution after adjusting the pH to 7.8-8.02+80-100ppm SO2When the glucose liquid is cooled to 52-62 ℃, an isomerase fixing column is driven to ensure that the content of discharged fructose is 42-44%;
the ion exchange is to carry out strong acid cation → weak base anion → strong acid cation → weak base anion exchange on the feed liquid in the last step once, wherein the ion exchange temperature is 45-50 ℃, and the discharge conductivity is less than or equal to 20 mu s/cm;
the concentration of the concentrated discharge material is 71.0-71.3%.
8. A fructose and glucose-fructose syrup product produced by the process of any one of claims 1-7.
The technical field is as follows:
the invention relates to the technical field of crystalline fructose, in particular to a method for co-producing high fructose corn syrup with different purities by using high-purity crystalline fructose.
Background art:
the fructose has high sweetness, fruit fragrance and low calorific value, is metabolized faster than glucose in vivo, is easy to be absorbed and utilized by the body, does not depend on insulin, has small influence on blood sugar, and is suitable for supplementing energy for patients with glucose metabolism and hepatic insufficiency. Can promote the growth and reproduction of beneficial bacteria such as bifidobacterium in human body, inhibit the growth of harmful bacteria, improve the functions and metabolism of intestines and stomach of human body, reduce blood fat, and prevent decayed teeth, thus being an ideal sweetener for foods of diabetes patients, obesity patients and children. The crystallized fructose can be used as a high-sweetness sweetener, a sweetness synergist, a humectant and the like to be applied to the food industry, and has the characteristics of high osmotic pressure, low active moisture, freezing point reduction and the like. The crystallized fructose can also be used for medicine, and the medicinal aspect requires higher purity of the crystallized fructose and lower harmful impurities such as 5-hydroxymethylfurfural and the like. According to the requirement of wide application, a new production process of the crystalline fructose with ultrahigh purity is developed, and the market demand is met by reducing the production cost.
The invention content is as follows:
in order to solve the technical problems, the invention provides a method for co-producing high fructose corn syrup with different purity by using high purity crystalline fructose.
The method for co-producing high-purity fructose and glucose syrup with different purities by using high-purity crystalline fructose comprises the following steps:
1) sugar dissolving: dissolving edible glucose in water to obtain glucose solution with the concentration of 48-52%;
preferably, the water content of the edible glucose is less than or equal to 8.0 percent, the glucose content is more than or equal to 99.9 percent, and the content of other sugars is less than or equal to 0.1 percent;
preferably, the dissolution temperature is 75-85 ℃;
2) isomerization: degassing the dissolved sugar liquor, adding magnesium sulfate and sodium metabisulfite, pumping into an isomerase fixing column, and controlling the fructose content at the outlet of the isomerase fixing column to be 42-44% by adjusting the temperature of the isomerase fixing column to obtain an F42 isomerized sugar liquor;
further, firstly adjusting the pH value to 7.5-8.0, and then adding 0.7-0.8kg/TDS of magnesium sulfate and 0.2-0.3kg/TDS of sodium metabisulfite;
further, cooling the glucose solution to 52-62 deg.C, adding isomerase fixed column at flow rate of 7m3The temperature in the fixed column is 50-55 ℃;
further, the isomerase added into the fixed column is glucose isomerase GIM, and the amino acid sequence is shown as SEQ ID NO: 3 is shown in the specification;
3) and (3) secondary decolorization: primary decoloring: adding activated carbon into the isomerized sugar solution obtained in the step 2) in proportion, and performing decoloration and thermo-sensitive protogen (protein) removal treatment by using a plate and frame filter; and (3) secondary regulation and decoloration: cooling the primary decolorized feed liquid to 30-55 deg.C, adding activated carbon in proportion, adjusting pH to protein isoelectric point 4.8-5.2, and further decolorizing and removing heat-sensitive original (protein) with plate-and-frame filter;
furthermore, the adding proportion of the secondary decolorized active carbon is 0.5-0.8 kg/TDS;
further, the primary decolorization feed liquid is cooled to 30-55 ℃ through heat exchange with cold water;
4) low-temperature ion exchange: cooling the decolorized isomerized sugar solution in the step 3) to 5-10 ℃, and passing through ion exchange resin in the sequence of strong acid cation → weak base anion → strong acid cation → weak base anion, wherein the conductivity of the ion exchange material is less than or equal to 3 mus/cm;
further, cooling the decolored isomerous sugar solution to 5-10 ℃ through heat exchange with cold water;
further, the strong acid cation is a macroporous structure styrene-divinylbenzene copolymerThe polymer has sulfonic acid group (-SO)3H) The cation exchange resin plays roles of desalting and deashing in sugar liquor;
further, the weak base anion is weak base anion exchange resin with a polystyrene macroporous structure; the resin is in a free amine type, is easy to combine with strong acid, generates a neutralization reaction with alkali during regeneration, and is restored to the free amine type; desalting and decoloring in sugar liquor to improve the transparency, taste and other properties of the product;
5) primary evaporation: pumping the ion exchange discharge into an evaporator for evaporation, and controlling the dry matter of the evaporation discharge to be 59-61%;
6) and (3) chromatographic separation: carrying out flash evaporation and degassing on the evaporated sugar solution, carrying out chromatographic separation, and separating an AD solution with the fructose content of more than or equal to 98%, a BD solution with the fructose content of 3-6% and a CD solution with the fructose content of 1-3%;
mixing the BD solution with the separated mother solution in the step 12) to produce high-purity fructose-glucose syrup; carrying out membrane concentration on the low-concentration CD solution to produce the conventional high fructose corn syrup;
further, in the step 6), the chromatographic separation mobile phase is water, the column temperature of the chromatographic separation column is 62-65 ℃, the dosage of elution water is 0.65-0.75 ton/cubic feed, and the treatment capacity is 0.6-0.7 ton/m per hour3A resin;
further, the chromatographic separation adopts a Sequential Simulated Moving Bed (SSMB), and the chromatographic column is Ca-type cation exchange resin;
7) mixed bed refining: adsorbing substances such as anions, cations, peculiar smell compounds and the like in the AD feed liquid separated in the step 6) by using special mixed-bed resin to obtain and separate, purifying the smell of syrup, wherein the discharge conductance is less than or equal to 1 mu s/cm, and the content of 5-hydroxymethylfurfural in the syrup is less than or equal to 0.0005%;
further, controlling the refining temperature of the mixed bed to be 35-40 ℃;
8) removing odor at high temperature: pumping the material liquid refined by the mixed bed into a deodorizing column, controlling the operation temperature to be 35-40 ℃, controlling the discharge conductance to be less than or equal to 1 mu s/cm, and controlling the 5-hydroxymethylfurfural to be less than or equal to 0.0005%;
further, when the discharging conductance is close to the upper limit of control, replacing a spare deodorizing column for operation, regenerating the replaced deodorizing column, and controlling the temperature of dilute alkali solution for regeneration to be 80-90 ℃;
9) and (3) evaporation: evaporating the deodorized feed liquid to obtain feed liquid with concentration of 90.0-90.3%;
10) pre-crystallization: pumping the feed liquid obtained by evaporation into a pre-crystallizer, and culturing seed crystals;
further, the pre-crystallization temperature is 40-42 ℃, and the time is 62-72 hours;
11) vertical crystallization: pumping the pre-crystallized material cultured by the pre-crystallizer into a vertical crystallizer, reducing the temperature to 22-23 ℃ at a constant speed, crystallizing for 70-80 hours, and discharging to obtain crystallized massecuite;
12) separation: transferring the crystallized massecuite to a distribution tank, and separating mother liquor by a separator; fully washing and centrifuging the fructose crystals separated from the mother liquor by using pure water at 58-62 ℃;
furthermore, the content of fructose in the massecuite after centrifugation is controlled to be more than or equal to 99.9 percent, the water content is less than or equal to 4.5 percent, the content of other sugar is less than or equal to 0.01 percent, and the content of 5-hydroxy furfural is less than or equal to 0.0001 percent;
13) fluidized bed drying: controlling the temperature of the inlet air to be 80 ℃ and the humidity to be 20%, uniformly drying the separated material, and controlling the water content to be less than or equal to 0.1% after drying to obtain high-purity crystalline fructose;
14) liquid mixing and ion exchange: mixing the BD liquid separated in the step 6) with the mother liquid separated in the step 12), heating to 45-50 ℃, and passing through ion exchange resin in the sequence of strong acid cation → weak base anion → strong acid cation → weak base anion to control the discharge conductivity to be less than or equal to 20 mus/cm;
15) and (3) evaporating the mixed solution: evaporating the ion-exchange mixed solution obtained in the step 14), controlling the dry matter to be more than or equal to 77% after evaporation and the pH value to be 3.3-4.5, and obtaining a high-purity high fructose corn syrup product;
16) isomerizing, ion-exchanging and concentrating the CD liquid separated in the step 6) to obtain a common 42 high fructose corn syrup product;
further, the isomerization is to add 35-50ppm Mg into the CD liquid after adjusting the pH value to 7.8-8.02+80-100ppm SO2Cooling the glucose solution to 52-62 deg.C, and adding isomerase fixed column to make the solution reach a certain temperatureObtaining the fructose content of 42-44%;
further, the ion exchange is to carry out strong acid cation → weak base anion → strong acid cation → weak base anion exchange on the feed liquid in the last step once, wherein the ion exchange temperature is 45-50 ℃, and the discharge conductivity is less than or equal to 20 mu s/cm;
furthermore, the discharge concentration after concentration is 71.0-71.3%.
Has the advantages that:
1. secondary decolorization
The secondary decolorization can effectively decolorize and remove protein, and simultaneously, the secondary decolorization temperature and pH adjustment can be properly combined with low-temperature ion exchange, so that the energy consumption is reduced.
2. Low temperature ion exchange
The low-temperature ion exchange operation reduces the ion exchange operation temperature (20 ℃ lower than the conventional ion exchange temperature), reduces side reactions, effectively reduces the generation of harmful substances such as 5-hydroxymethylfurfural and the like in the feed liquid which easily affect the product quality, and improves the product quality.
3. The isomeric feed liquid is refined and separated into three feed liquids of AD, BD and CD by chromatography, and glucose is utilized as much as possible.
4. By adopting the novel glucose isomerase, the improvement of the enzyme activity can greatly shorten the isomerization time, improve the efficiency and reduce the process cost.
Description of the drawings:
FIG. 1 error-prone PCR electrophoresis results.
FIG. 2 shows the restriction map of pGAPZ. alpha.C-gim.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
The invention will be further explained below by means of specific embodiments.
Example 1A Glucose Isomerase (GI) mutant
The invention provides a Glucose Isomerase (GI) mutant and a gene thereof, the invention uses an error-prone PCR technology to randomly mutate a Glucose isomerase coding Gene (GI) from Streptomyces rubiginosus to obtain a Glucose isomerase mutant gene (gim), the enzyme activity of the mutant is improved by 57% compared with the original gene, and the mutant is expressed in pichia pastoris to obtain the high-activity Glucose isomerase.
The following definitions are used in the present invention:
(1) nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
(2) Identification of glucose isomerase mutants
"amino acid substituted at original amino acid position" is used to indicate a mutated amino acid in a glucose isomerase mutant. Such as Met88Lys, the amino acid at position 88 is replaced by Met of the original glucose isomerase to Lys, and the numbering of the position corresponds to SEQ ID NO: 1, amino acid sequence number of wild-type glucose isomerase.
In the present invention, GI represents the original glucose isomerase (amino acid sequence shown in SEQ ID NO: 1), GIM represents the mutated glucose isomerase (amino acid sequence shown in SEQ ID NO: 3); gi represents the coding gene of the original glucose isomerase (shown as SEQ ID NO: 2), and gim represents the coding gene of the mutated glucose isomerase (shown as SEQ ID NO: 4).
The host cell for expressing the glucose isomerase mutant is Pichia pastoris SMD1168, and the expression vector is pGAPZ alpha C.
1. Obtaining of wild-type glucose isomerase DNA
The amino acid sequence (SEQ ID NO.1) of wild-type glucose isomerase of Streptomyces rubiginosis was searched using NCBI database, and codon optimization was performed for Escherichia coli (E.coli) to obtain a DNA sequence (SEQ ID NO. 2). Synthesizing the whole gene of SEQ ID NO.2, connecting to pUC57 vector, transferring into E.coli DH5 alpha, preparing into glycerol strain, and storing at-80 deg.C for a long time.
1 glycerol strain was inoculated into a test tube containing 5ml of Amp-resistant LB medium, cultured overnight at 37 ℃ and subjected to Plasmid mini-extraction using High Pure Plasmid Isolation Kit from Roche to obtain a wild-type glucose isomerase DNA fragment as a template for subsequent random mutagenesis.
2. Obtaining of glucose isomerase mutant Gene
(1) Random mutagenesis
Carrying out random mutation by using TaKaRa Taq PCR amplification enzyme of TaKaRa company based on an error-prone PCR technology to obtain a high-activity glucose isomerase gene;
primers were designed as follows:
upstream P1(SEQ ID No. 5):
5’-TAAGAAGGAGATATACCATGGATGAACTACCAGCCGACCCCGGA-3’
downstream P2(SEQ ID No. 6):
5’-GTGGTGGTGGTGGTGCTCGAG TTA gccacgtgcgcccagca-3’
the reaction system for amplification is as follows:
10×PCR buffer
5μL
dNTPs(2.5mmol/L each)
5μL
upstream primer P1 (10. mu. mol/L)
1.5μL
Downstream primer P2 (10. mu. mol/L)
1.5μL
25mmol/L MgCl2
11μL
5mmol/L MnCl2
5μL
Form panel
20pmol
Taq DNA polymerase
1μL
ddH2O
Make up to 50 μ L
The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 60 s; annealing at 61 ℃ for 60s, extending at 72 ℃ for 180s, and reacting for 30 cycles; keeping the temperature at 72 ℃ for 10 min; storing at 4 ℃. The PCR amplification product was detected by 1.0% agarose gel electrophoresis, and a band of about 1200bp was observed (FIG. 1). The PCR amplification product does not need to be processed, can be immediately used for vector construction, and can also be stored for a long time at the temperature of 20 ℃ below zero.
(2) Expression vector linearization
The pET-28a plasmid was linearized using the conventional restriction enzyme TaKaRa, as follows:
Nco I
5μL
Xho I
5μL
10*K buffer
10μL
0.1％BSA
10μL
pET-28a
5μg
ddH2O
make up to 100 mu L
Linearization conditions are as follows: preserving the heat for 3 hours at 37 ℃; keeping the temperature at 65 ℃ for 20 min; storing at 4 ℃. The linearized product can be used for vector construction immediately and can also be stored for a long time at the temperature of 20 ℃ below zero.
(3) Vector construction
The gim mutant expression vector library was constructed by ligating the error-prone PCR product with linearized pET-28a using Clonexpress II one-step ligase from Vazyme. To ensure sufficient storage capacity, 5 ligation reactions were performed simultaneously, totaling 100. mu.L of ligation system.
The linking system is as follows:
note that: the reaction system was prepared in an ice bath.
Reaction conditions are as follows: keeping the temperature at 37 ℃ for 30 min; keeping the temperature at 4 ℃ for 5 min.
After the reaction is finished, the product can be stored for a short time at 4 ℃ or for a long time at-20 ℃.
(4) gim mutant expression library construction
Connecting the mutant expression vector of the step (3) with products (20. mu.L/cell), and transforming the expression strain E.coli BL21 as follows:
e.coli BL21 competent cells (100. mu.L/cell) were taken out from-80 ℃, dissolved on ice, 20. mu.L of the ligation product was added to 100. mu.L of E.coli competent cells BL21 immediately after dissolution in a sterile environment, placed on ice for 30min, heat-shocked in a water bath at 42 ℃ for 90s, cooled on ice for 1.5min, added to 900. mu.L of LB medium, and pre-cultured at 37 ℃ and 200r/min for 30 min. Centrifuging at 3000rpm for 2min, discarding 600 μ L of supernatant, mixing thallus precipitate and the rest supernatant by pipette, coating each 100 μ L of concentrated bacterial liquid on LB plate containing Kan resistance, making 4 parallel groups, and culturing at 37 deg.C in constant temperature incubator upside down overnight.
Finally, 20 blocks of gim mutant expression strain libraries are obtained and coated on a flat plate, and after sealing by a sealing film, the flat plate is stored for a short time at 4 ℃.
(5) Wild type expression Strain construction
Wild type gi gene was directly amplified by general PCR, and by referring to the steps (2)/(3)/(4) in this example, wild type expression strain was constructed as a control sample for screening enzyme activity.
(6) Enzyme activity screening sample preparation
Preparing an induction culture medium:
consists of the following components: 12.0g of Tryptonen, 24.0g of Yeast extract, 2.0g of alpha-lactose, 0.5g of glucose, 17.05g of Studier salt and pH7.0 +/-0.2.
Weighing 55.55g of dehydrated culture medium powder, and heating and dissolving with 1L of deionized water; boiling for 1 min; sterilizing at 115 deg.C for 20 min.
Positive transformants were selected by plating plates from 20 pools of gim mutant expression strains. At least 2000 positive transformants (containing 1 wild-type expression strain) were picked and each positive transformant was inoculated into a new kanamycin-resistant plate for strain preservation and simultaneously inoculated into a 96-well shallow well plate containing 200. mu.L of LB liquid medium (containing 50. mu.g/mL of Kan) per well.
After the strain preservation plate is cultured overnight at 37 ℃, the strain preservation plate is sealed by a sealing film and preserved at 4 ℃.
Inoculated into a 96-well shallow plate (each plate contains 1 wild-type expression strain as a control), and cultured at 37 ℃ for 12h at 400 r/min. 50. mu.l of overnight-cultured bacterial suspension was transferred to a 96-well deep-well plate (1ml of auto-induction medium/well, Kan concentration 50. mu.g/ml), cultured at 37 ℃ and 400rpm for 4 hours, and then induced at 25 ℃ for expression for 20 hours. Centrifuging at 4000r/min at 4 deg.C for 30min, collecting thallus, standing at-80 deg.C for 2h, standing at room temperature for 1h, and repeating the above operation for 2 times. Add 70. mu.l of bacteriolysis buffer (0.5mg/ml lysozyme, 0.7U/ml Dnasel, 50mmol/L PBS, pH7.5) to each well to resuspend the thallus, transfer to a new 96-well shallow-well plate, and place in a 37 ℃ incubator for 90min to fully break the thallus. The mutant was centrifuged at 4000rpm for 30min at 4 ℃ and 10. mu.l of the supernatant enzyme solution was carefully pipetted into a new 96-well ELISA plate in preparation for the measurement of the specific activity of the mutant.
3. Glucose isomerase preliminary screening
The enzyme activity is measured by cysteine-carbazole method, each sample well of ELISA plate contains 10 μ l enzyme solution, 0.6mol D-glucose 25 μ l, 0.025mol triethanolamine-hydrochloric acid (containing 10mmol MgSO)4·7H2O) 20. mu.l of a buffer solution of pH8, and reacted at 35 ℃ for 15 minutes. The reaction was stopped by adding 5. mu.l of 50% trichloroacetic acid. Immediately adding 300 mul of 70% sulfuric acid which is cooled in an ice way, 10 mul of 2.4% cysteine-hydrochloride and 10 mul of 0.12% ethanol-carbazole bath solution, uniformly mixing, reacting for 30 minutes at 25 ℃, and detecting the absorption value A of each hole by using a 560nm wavelength in an enzyme-labeling instrument.
And (3) recording the strain number of the strain with the absorption value A larger than that of the wild type control well, re-inoculating the strain into a new photocopy plate (convenient for centralized storage) and a 96-well shallow-hole plate from the photocopy storage plate, repeating the step (6) of the step (2) to prepare the sample, re-testing the enzyme activity, and finally, performing the next round of screening on the remaining 28 mutant strains.
4. And detecting the specific activity of the glucose isomerase mutant.
28 mutant strains and 1 wild-type strain were inoculated into a test tube containing 5ml of Kan-resistant LB medium and cultured overnight at 37 ℃ with shaking at 160 rpm. Inoculating a 250mL shake flask containing 50mL Kan resistant LB culture medium according to the inoculation amount of 1%, carrying out shake culture at 37 ℃ and 200rpm until OD600 reaches 0.6, adding IPTG (final concentration is 1mmol/L), inducing for 16h at 16 ℃, centrifuging for 15min at 4 ℃ and 4000r/min, collecting thalli, suspending in 15mL precooled PBS buffer solution with pH of 7.4, crushing cells by using a low-temperature ultrahigh-pressure continuous flow cell crusher, centrifuging for 45min at 4 ℃ and 12000r/min, collecting supernatant to obtain crude enzyme solution, and then carrying out specific activity detection.
The glucose isomerase concentration was estimated by SDS-PAGE electrophoresis.
The enzyme activity is measured by cysteine-carbazole method, adding 0.6mol D-glucose 250 μ l glucose isomerase 100 μ l (equivalent to 0.2-0.8 activity unit), and 0.025mol triethanolamine-hydrochloric acid (containing 10mmol MgSO4·7H2O) 200. mu.l of a buffer solution of pH8, and reacted at 35 ℃ for 15 minutes. The reaction was stopped by adding 50. mu.l of 50% trichloroacetic acid. 3ml of 70% sulfuric acid, 100. mu.l of 2.4% cysteine-hydrochloride and 100. mu.l of 0.12% ethanol-carbazole bath solution, which are cooled in the ice, are immediately added, and after uniform mixing, the mixture is reacted at 25 ℃ for 30 minutes, and the absorption value A is measured on the quartz cup side with the wavelength of 560nm and the optical path length of 1cm on a spectrophotometer.
The amount of enzyme required to produce 1. mu.g fructose per minute in a standard reaction mixture is defined as 1 activity unit ((U), and the specific activity is expressed in terms of enzyme activity per mg glucose isomerase, i.e., U/mg.
Numbering
Specific activity
Numbering
Specific activity
Numbering
Specific activity
Numbering
Specific activity
Wild type
100
281
132
889
83
1593
86
147
91
409
93
950
112
1747
107
170
80
473
116
996
108
1794
131
217
98
488
114
1015
142
1835
146
222
141
725
88
1063
130
1977
121
226
151
822
101
1240
129
244
148
862
156
1309
103
271
126
872
123
1507
140
The detection result shows that the specific activity of the mutant 862 is the highest, and is improved by 56% compared with the wild type enzyme activity. The mutant is sequenced by using a universal primer T7/T7 ter, and the sequencing result shows that the mutant is a high-activity glucose isomerase gene gim with Met88Lys, Ala131Pro, Ala136Gly, Gly248Cys, the nucleotide sequence is shown in SEQ ID No.4, and the corresponding amino acid sequence is shown in SEQ ID No. 3.
5. Construction of glucose isomerase mutant free expression pichia pastoris recombinant bacteria
Constructing a pichia pastoris high-activity glucose isomerase free expression recombinant strain. The high-activity glucose isomerase (gim) is optimized by a codon and added with a stop codon and an EcoRI/XbaI enzyme cutting site (SEQ ID NO:7) to carry out whole gene synthesis to be connected with a pichia pastoris secretion expression vector pGAPZ alpha C, so that a high-activity glucose isomerase pichia pastoris expression vector pGAPZ alpha C-gim is constructed (enzyme cutting verification is shown in figure 2), and pichia pastoris is transformed.
(1) Preparation of linearized plasmid DNA
Before transformation of Pichia pastoris, the constructed recombinant expression plasmid pGAPZ alpha C-gim is linearized to improve the integration efficiency of the plasmid on Pichia pastoris chromosome. The linearized digestion was carried out with the restriction enzyme BspH I.
(2) Electrically transforming pichia pastoris with linearized plasmid pGAPZ alpha C-gim, identifying positive transformants and screening high-yield strains of glucose isomerase
Adding 80 mu L of pichia pastoris SMD1168 competent cells and 10 mu g of linearized DNA into a 1.5mL precooled centrifuge tube, uniformly mixing, and transferring the reaction solution into a conversion cup in an ice bath in advance;
and secondly, carrying out ice bath on a transformation cup filled with transformation liquid for 5min, and carrying out pichia pastoris electrotransformation according to parameters recommended by an electrotransformation device:
③ immediately adding 1mL of precooled 1mol/L sorbitol solution into the transformation cup after pulse, and transferring the transformation solution into a new 1.5mL centrifuge tube;
standing and culturing at 30 ℃ for 1.5h, and sucking 200 mu L of pichia pastoris SMD1168 electrotransfer liquid and coating the liquid on an MD culture medium;
fifthly, culturing at 30 ℃ until transformants appear;
sixthly, selecting a single colony of the transformant, dissolving the single colony in 10 mu L of deionized water, taking 2 mu L of bacterial liquid, adding Lyticase wall-breaking enzyme, reacting at 30 ℃ for L0min, immediately placing the reaction liquid in a refrigerator at-80 ℃ for freezing for L0min, cracking the cell wall of the yeast, and taking the released genome as a template for PCR. Positive transformants were determined using empty plasmid-transferred Pichia pastoris SMD 1168/pGAPZ. alpha.C as a control.
Seventhly, on the basis of determining positive transformants, firstly, using resistance plates containing geneticin with different concentrations to screen the transformants with high geneticin resistance, and then respectively measuring the enzyme activity of glucose isomerase of the transformants with high geneticin resistance so as to obtain the production strain SMD1168/pGAPZ alpha C-palm of the glucose isomerase.
6. Expression and preparation of glucose isomerase mutant in pichia free expression recombinant bacteria
Inoculating the recombinant strain SMD1168/pPIC9K-gim of the Pichia pastoris free expression glucose isomerase mutant into a YPD liquid culture medium, and culturing at 30 ℃ and 250r/min for 24 h. Transferring the strain into a fresh BMGY culture medium with the inoculation amount of 1%, culturing at 30 ℃ and 250r/min for 24h, centrifuging at 6000r/min for 5min to obtain thalli, and transferring the thalli into a BMMY culture medium. Culturing at 30 deg.C and 250r/min for 120 hr to obtain crude glucose isomerase enzyme solution, precipitating high activity glucose isomerase by fractional salting-out method, collecting protein precipitate, dissolving, dialyzing to remove salt, performing ion exchange chromatography and gel chromatography, and freeze drying to obtain high activity pure glucose isomerase enzyme powder.
Approximately 211mg of pure enzyme powder of glucose isomerase mutant per liter of medium was obtained.
In the same manner, about 180mg of pure enzyme powder of wild-type glucose isomerase per liter of the medium was obtained.
And (4) determining the specific enzyme activities of the original (wild type) glucose isomerase GI and the mutated glucose isomerase GIM to be 110U/mg and 173.14U/mg respectively by adopting the enzyme activity determination method in the step 4, wherein the specific enzyme activity of the mutated glucose isomerase is improved by 57 percent compared with that before mutation.
The mutated glucose isomerase GIM obtained in example 1 is used as an isomerase to produce crystalline fructose and glucose-fructose syrup in the following examples of the present invention.
Embodiment 2 method for co-production of high-purity fructose syrup by high-purity crystalline fructose
A method for co-producing high fructose corn syrup with different purity by using high purity crystalline fructose comprises the following specific steps:
1) sugar dissolving: dissolving edible glucose in water at 75 deg.C to obtain 48% glucose solution;
the water content of the edible glucose is less than or equal to 8.0 percent, the glucose content is more than or equal to 99.9 percent, and the content of other sugars is less than or equal to 0.1 percent;
2) isomerization: degassing the dissolved sugar solution, adjusting the pH to 7.5, and adding 0.7kg/TDS magnesium sulfate and 0.2kg/TDS sodium metabisulfite; when the temperature is adjusted to 52 ℃, an isomerase fixing column is driven, the temperature in the fixing column is 50 ℃, and the feeding flow of the isomerase column is 7m3The fructose content at the outlet of the isomerase fixing column is 42 percent, F42 isomerized sugar liquid is obtained, the isomerization time from feeding to discharging is 3.5 hours, which is far lower than that of the prior art;
3) and (3) secondary decolorization: primary decoloring: adding activated carbon into the isomerized sugar solution obtained in the step 2) in proportion, and performing decoloration and thermo-sensitive protogen (protein) removal treatment by using a plate and frame filter; and (3) secondary regulation and decoloration: cooling the primary decolorized feed liquid to 30 ℃ through heat exchange with cold water, adding activated carbon in proportion, adjusting the pH value to be 4.8-5.2 of the protein equivalent point, and further decolorizing and removing heat-sensitive original (protein) by using a plate and frame filter;
the adding proportion of the secondary decolorized active carbon is 0.5 kg/TDS;
4) low-temperature ion exchange: cooling the decolorized isomerous sugar solution obtained in the step 3) to 5 ℃ through heat exchange with cold water, and passing through ion exchange resin in the sequence of strong acid cation → weak base anion → strong acid cation → weak base anion, wherein the conductivity of the ion exchange material is less than or equal to 3 mus/cm;
the styrene-divinylbenzene copolymer with the strong acid cation of the macroporous structure is provided with sulfonic group (-SO)3H) The cation exchange resin of (1); the weak base anion is weak base anion exchange resin with a polystyrene macroporous structure;
5) primary evaporation: pumping the ion exchange discharge into an evaporator for evaporation, and controlling the dry matter of the evaporation discharge to be 59%;
6) and (3) chromatographic separation: carrying out flash evaporation and degassing on the evaporated sugar solution, and carrying out chromatographic separation to separate an AD solution with fructose content of 99.0%, a BD solution with fructose content of 5.5% and a CD solution with fructose content of 3.0%; the chromatographic separation adopts a Sequential Simulated Moving Bed (SSMB), and a chromatographic column is Ca-type cation exchange resin; the chromatographic separation mobile phase is water, the column temperature of the chromatographic separation column is 62 ℃, the dosage of elution water is 0.65 ton/cubic feed, and the treatment capacity is 0.6 ton/m of liquid/m per hour3A resin;
7) mixed bed refining: adsorbing substances such as anions, cations, peculiar smell compounds and the like in the AD feed liquid separated in the step 6) by using special mixed-bed resin to obtain and separate, purifying the smell of syrup, wherein the discharge conductance is less than or equal to 1 mu s/cm, and the content of 5-hydroxymethylfurfural in the syrup is less than or equal to 0.0005%;
further, controlling the refining temperature of the mixed bed to be 35 ℃;
8) removing odor at high temperature: pumping the material liquid refined by the mixed bed into a deodorizing column, controlling the operation temperature to be 35 ℃, controlling the discharge conductance to be less than or equal to 1 mu s/cm, and controlling the 5-hydroxymethylfurfural to be less than or equal to 0.0005%;
when the discharging electric conductivity is close to the upper limit of control, replacing a spare deodorizing column for operation, regenerating the replaced deodorizing column, and controlling the temperature of dilute alkali solution for regeneration to be 80 ℃;
9) and (3) evaporation: evaporating the deodorized feed liquid to obtain feed liquid with the concentration of 90.0%;
10) pre-crystallization: pumping the feed liquid obtained by evaporation into a pre-crystallizer, and culturing seed crystals at the pre-crystallization temperature of 40-42 ℃ for 62 hours;
11) vertical crystallization: pumping the pre-crystallized material cultured by the pre-crystallizer into a vertical crystallizer, cooling to 22 ℃ at a constant speed, crystallizing for 70 hours, and discharging to obtain crystallized massecuite;
12) separation: transferring the crystallized massecuite to a distribution tank, and separating mother liquor by a separator; fully washing and centrifuging the fructose crystals separated from the mother liquor by using pure water at 58 ℃; the fructose content in the massecuite obtained after centrifugation is 99.91 percent, the water content is 4.3 percent, the content of other sugar is 0.01 percent, and the content of 5-hydroxy furfural is 0.0001 percent;
13) fluidized bed drying: controlling the temperature of the inlet air to be 80 ℃ and the humidity to be 20%, uniformly drying the separated material, and controlling the water content to be less than or equal to 0.1% after drying to obtain high-purity crystalline fructose;
14) liquid mixing and ion exchange: mixing the BD liquid separated in the step 6) with the mother liquid separated in the step 12), heating to 45 ℃, and controlling the discharge conductivity to be less than or equal to 20 mus/cm through ion exchange resin according to the sequence of strong acid cations → weak base anions → strong acid cations → weak base anions;
15) and (3) evaporating the mixed solution: evaporating the ion-exchange mixed liquid obtained in the step 14), controlling the dry matter after evaporation to be 77.3 percent and the pH value to be 3.3, and obtaining a high-purity high fructose corn syrup product;
16) isomerizing, ion-exchanging and concentrating the CD liquid separated in the step 6) to obtain a common 42 high fructose corn syrup product;
the isomerization is to add 35ppm Mg into the CD liquid after adjusting the pH to 7.82+80ppm SO2,Adjusting the temperature of the glucose liquid to 52 ℃, and driving an isomerase fixing column to ensure that the content of discharged fructose is 42%;
the ion exchange is to carry out strong acid cation → weak base anion → strong acid cation → weak base anion exchange on the feed liquid in the last step once, wherein the ion exchange temperature is 45 ℃, and the discharge conductivity is less than or equal to 20 mu s/cm;
the concentration of the concentrated product was 71.0%.
Example 3 method for co-production of high purity fructose syrup from high purity crystalline fructose
A method for co-producing high fructose corn syrup with different purity by using high purity crystalline fructose comprises the following specific steps:
1) sugar dissolving: dissolving edible glucose in water at 85 deg.C to obtain 52% glucose solution;
the water content of the edible glucose is less than or equal to 8.0 percent, the glucose content is more than or equal to 99.9 percent, and the content of other sugars is less than or equal to 0.1 percent;
2) isomerization: degassing the dissolved sugar solution, adjusting the pH to 8.0, and adding 0.8kg/TDS magnesium sulfate and 0.3kg/TDS sodium pyrosulfite; when the temperature is adjusted to 62 ℃, an isomerase fixing column is driven, the temperature in the fixing column is 53 ℃, and the feeding flow of the isomerase column is 7m3The fructose content at the outlet of the isomerase fixing column is 43 percent, F42 isomerized sugar liquid is obtained, the isomerization time from feeding to discharging is 3.5 hours, which is far lower than that of the prior art;
3) and (3) secondary decolorization: primary decoloring: adding activated carbon into the isomerized sugar solution obtained in the step 2) in proportion, and performing decoloration and thermo-sensitive protogen (protein) removal treatment by using a plate and frame filter; and (3) secondary regulation and decoloration: cooling the primary decolorized feed liquid to 55 ℃ through heat exchange with cold water, adding activated carbon in proportion, adjusting the pH value to be 4.8-5.2 of the protein equivalent point, and further decolorizing and removing heat-sensitive original (protein) by using a plate and frame filter;
the adding proportion of the secondary decolorized active carbon is 0.8 kg/TDS;
4) low-temperature ion exchange: cooling the decolorized isomerous sugar solution obtained in the step 3) to 10 ℃ through heat exchange with cold water, and passing through ion exchange resin in the sequence of strong acid cation → weak base anion → strong acid cation → weak base anion, wherein the conductivity of the ion exchange material is less than or equal to 3 mus/cm;
the styrene-divinylbenzene copolymer with the strong acid cation of the macroporous structure is provided with sulfonic group (-SO)3H) The cation exchange resin of (1); the weak base anion is weak base anion exchange resin with a polystyrene macroporous structure;
5) primary evaporation: pumping the ion exchange discharge into an evaporator for evaporation, and controlling the dry matter of the evaporation discharge to be 60%;
6) and (3) chromatographic separation: carrying out flash evaporation and degassing on the evaporated sugar solution, carrying out chromatographic separation, and separating an AD solution with fructose content of 99.3%, a BD solution with fructose content of 4.0% and a CD solution with fructose content of 2.0%;
the chromatographic separation adopts a Sequential Simulated Moving Bed (SSMB), and a chromatographic column is Ca-type cation exchange resin; the chromatographic separation mobile phase is water, and the chromatogram isThe column temperature of the separation column is 65 ℃, the dosage of elution water is 0.75 ton/cubic feed, and the treatment capacity is 0.7 ton/m of liquid/m per hour3A resin;
7) mixed bed refining: adsorbing substances such as anions, cations, peculiar smell compounds and the like in the AD feed liquid separated in the step 6) by using special mixed-bed resin to obtain and separate, purifying the smell of syrup, wherein the discharge conductance is less than or equal to 1 mu s/cm, and the content of 5-hydroxymethylfurfural in the syrup is less than or equal to 0.0005%;
further, controlling the refining temperature of the mixed bed to be 40 ℃;
8) removing odor at high temperature: pumping the material liquid refined by the mixed bed into a deodorizing column, controlling the operation temperature to be 40 ℃, controlling the discharge conductance to be less than or equal to 1 mu s/cm, and controlling the 5-hydroxymethylfurfural to be less than or equal to 0.0001%;
when the discharging electric conductivity is close to the upper limit of control, replacing a spare deodorizing column for operation, regenerating the replaced deodorizing column, and controlling the temperature of dilute alkali solution for regeneration to be 90 ℃;
9) and (3) evaporation: evaporating the deodorized feed liquid to obtain feed liquid with the concentration of 90.2%;
10) pre-crystallization: pumping the feed liquid obtained by evaporation into a pre-crystallizer, and culturing seed crystals at the pre-crystallization temperature of 40-42 ℃ for 72 hours;
11) vertical crystallization: pumping the pre-crystallized material cultured by the pre-crystallizer into a vertical crystallizer, reducing the temperature to 22-23 ℃ at a constant speed, crystallizing for 80 hours, and discharging to obtain crystallized massecuite;
12) separation: transferring the crystallized massecuite to a distribution tank, and separating mother liquor by a separator; fully washing and centrifuging the fructose crystals separated from the mother liquor by using pure water at 62 ℃; the fructose content in the massecuite obtained after centrifugation is 99.95 percent, the water content is 4.1 percent, the content of other sugar is 0.01 percent, and the content of 5-hydroxy furfural is 0.0001 percent;
13) fluidized bed drying: controlling the temperature of the inlet air to be 80 ℃ and the humidity to be 20%, uniformly drying the separated material, and controlling the water content to be less than or equal to 0.1% after drying to obtain high-purity crystalline fructose;
14) liquid mixing and ion exchange: mixing the BD liquid separated in the step 6) with the mother liquid separated in the step 12), heating to 50 ℃, and controlling the discharge conductivity to be less than or equal to 20 mus/cm through ion exchange resin in the sequence of strong acid cations → weak base anions → strong acid cations → weak base anions;
15) and (3) evaporating the mixed solution: evaporating the ion-exchange mixed liquid obtained in the step 14), controlling the dry matter after evaporation to be 77.2 percent and the pH value to be 4.5, and obtaining a high-purity high fructose corn syrup product;
16) isomerizing, ion-exchanging and concentrating the CD liquid separated in the step 6) to obtain a common 42 high fructose corn syrup product;
the isomerization is to add 50ppm Mg into the CD liquid after adjusting the pH value to 8.02+100ppm SO2,Adjusting the temperature of the glucose liquid to 62 ℃, and driving an isomerase fixing column to ensure that the content of discharged fructose is 43%;
the ion exchange is to carry out strong acid cation → weak base anion → strong acid cation → weak base anion exchange on the feed liquid in the last step once, wherein the ion exchange temperature is 50 ℃, and the discharge conductivity is less than or equal to 20 mu s/cm;
the concentration of the concentrated product was 71.3%.
Example 4 method for co-production of high purity fructose syrup from high purity crystalline fructose
A method for co-producing high fructose corn syrup with different purity by using high purity crystalline fructose comprises the following specific steps:
1) sugar dissolving: dissolving edible glucose in water at 80 deg.C to obtain 50% glucose solution;
the water content of the edible glucose is less than or equal to 8.0 percent, the glucose content is more than or equal to 99.9 percent, and the content of other sugars is less than or equal to 0.1 percent;
2) isomerization: degassing the dissolved sugar solution, adjusting the pH to 7.8, and adding 0.75kg/TDS magnesium sulfate and 0.25kg/TDS sodium metabisulfite; cooling to 55 deg.C, adding isomerase fixed column at 55 deg.C, and feeding into the column at a flow rate of 7.0m3The fructose content at the outlet of the isomerase fixing column is 44%, so that F42 isomerized sugar liquid is obtained, the isomerization time from feeding to discharging is 3.5h, which is far lower than that of the prior art; (ii) a
3) And (3) secondary decolorization: primary decoloring: adding activated carbon into the isomerized sugar solution obtained in the step 2) in proportion, and performing decoloration and thermo-sensitive protogen (protein) removal treatment by using a plate and frame filter; and (3) secondary regulation and decoloration: cooling the primary decolorized feed liquid to 45 ℃ through heat exchange with cold water, adding activated carbon in proportion, adjusting the pH value to be 4.8-5.2 of the protein equivalent point, and further decolorizing and removing heat-sensitive original (protein) by using a plate and frame filter;
the adding proportion of the secondary decolorized active carbon is 0.65 kg/TDS;
4) low-temperature ion exchange: cooling the decolorized isomerous sugar solution obtained in the step 3) to 8 ℃ through heat exchange with cold water, and passing through ion exchange resin in the sequence of strong acid cation → weak base anion → strong acid cation → weak base anion, wherein the conductivity of the ion exchange material is less than or equal to 3 mus/cm;
the styrene-divinylbenzene copolymer with the strong acid cation of the macroporous structure is provided with sulfonic group (-SO)3H) The cation exchange resin of (1); the weak base anion is weak base anion exchange resin with a polystyrene macroporous structure;
5) primary evaporation: pumping the ion exchange discharge into an evaporator for evaporation, and controlling the dry matter of the evaporation discharge to be 61%;
6) and (3) chromatographic separation: carrying out flash evaporation and degassing on the evaporated sugar solution, and carrying out chromatographic separation to separate an AD solution with fructose content of 99.6%, a BD solution with fructose content of 3.0% and a CD solution with fructose content of 1.0%;
the chromatographic separation adopts a Sequential Simulated Moving Bed (SSMB), and a chromatographic column is Ca-type cation exchange resin; the chromatographic separation mobile phase is water, the column temperature of the chromatographic separation column is 62 ℃, the dosage of elution water is 0.70 ton/cubic feed, and the treatment capacity is 0.65 ton/m of liquid/m per hour3A resin;
7) mixed bed refining: adsorbing substances such as anions, cations, peculiar smell compounds and the like in the AD feed liquid separated in the step 6) by using special mixed-bed resin to obtain and separate, purifying the smell of syrup, wherein the discharge conductance is less than or equal to 1 mu s/cm, and the content of 5-hydroxymethylfurfural in the syrup is less than or equal to 0.0005%;
further, controlling the refining temperature of the mixed bed to be 37 ℃;
8) removing odor at high temperature: pumping the material liquid refined by the mixed bed into a deodorizing column, controlling the operation temperature to be 37 ℃, controlling the discharge conductance to be less than or equal to 1 mu s/cm, and controlling the 5-hydroxymethylfurfural to be less than or equal to 0.0003 percent;
when the discharging electric conductivity is close to the upper limit of control, replacing a spare deodorizing column for operation, regenerating the replaced deodorizing column, and controlling the temperature of dilute alkali solution for regeneration to be 85 ℃;
9) and (3) evaporation: evaporating the deodorized feed liquid to obtain feed liquid with the concentration of 90.3%;
10) pre-crystallization: pumping the feed liquid obtained by evaporation into a pre-crystallizer, carrying out pre-crystallization at the temperature of 40-42 ℃ for 67 hours, and culturing seed crystals;
11) vertical crystallization: pumping the pre-crystallized material cultured by the pre-crystallizer into a vertical crystallizer, reducing the temperature to 22-23 ℃ at a constant speed, crystallizing for 75 hours, and discharging to obtain crystallized massecuite;
12) separation: transferring the crystallized massecuite to a distribution tank, and separating mother liquor by a separator; fully washing and centrifuging the fructose crystals separated from the mother liquor by using pure water at 60 ℃; the fructose content in the massecuite obtained after centrifugation is 99.93 percent, the moisture content is 4.2 percent, the content of other sugar is 0.01 percent, and the content of 5-hydroxy furfural is 0.0001 percent;
13) fluidized bed drying: controlling the temperature of the inlet air to be 80 ℃ and the humidity to be 20%, uniformly drying the separated material, and controlling the water content to be less than or equal to 0.1% after drying to obtain high-purity crystalline fructose;
14) liquid mixing and ion exchange: mixing the BD liquid separated in the step 6) with the mother liquid separated in the step 12), heating to 48 ℃, and controlling the discharge conductivity to be less than or equal to 20 mus/cm through ion exchange resin according to the sequence of strong acid cations → weak base anions → strong acid cations → weak base anions;
15) and (3) evaporating the mixed solution: evaporating the ion-exchange mixed liquid obtained in the step 14), controlling the dry matter to be more than or equal to 77.1% after evaporation and the pH value to be 4.0, and obtaining a high-purity high fructose corn syrup product;
16) isomerizing, ion-exchanging and concentrating the CD liquid separated in the step 6) to obtain a common 42 high fructose corn syrup product;
the isomerization is to add 40ppm Mg into the CD liquid after adjusting the pH value to 8.02+90ppm SO2,Adjusting the temperature of the glucose liquid to 55 ℃, and driving an isomerase fixing column to ensure that the content of discharged fructose is 44%;
the ion exchange is to carry out strong acid cation → weak base anion → strong acid cation → weak base anion exchange on the feed liquid in the last step once, wherein the ion exchange temperature is 48 ℃, and the discharge conductivity is less than or equal to 20 mu s/cm;
the concentration of the concentrated product was 71.3%.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
SEQUENCE LISTING
<110> Henan flying agricultural development Co., Ltd
<120> method for co-producing high fructose corn syrup with different purity by using high purity crystalline fructose
<130> 1
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 388
<212> PRT
<213> Streptomyces rubiginosus)
<400> 1
Met Asn Tyr Gln Pro Thr Pro Glu Asp Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
Thr Val Gly Trp Gln Gly Arg Asp Pro Phe Gly Asp Ala Thr Arg Arg
20 25 30
Ala Leu Asp Pro Val Glu Ser Val Gln Arg Leu Ala Glu Leu Gly Ala
35 40 45
His Gly Val Thr Phe His Asp Asp Asp Leu Ile Pro Phe Gly Ser Ser
50 55 60
Asp Ser Glu Arg Glu Glu His Val Lys Arg Phe Arg Gln Ala Leu Asp
65 70 75 80
Asp Thr Gly Met Lys Val Pro Met Ala Thr Thr Asn Leu Phe Thr His
85 90 95
Pro Val Phe Lys Asp Gly Gly Phe Thr Ala Asn Asp Arg Asp Val Arg
100 105 110
Arg Tyr Ala Leu Arg Lys Thr Ile Arg Asn Ile Asp Leu Ala Val Glu
115 120 125
Leu Gly Ala Glu Thr Tyr Val Ala Trp Gly Gly Arg Glu Gly Ala Glu
130 135 140
Ser Gly Gly Ala Lys Asp Val Arg Asp Ala Leu Asp Arg Met Lys Glu
145 150 155 160
Ala Phe Asp Leu Leu Gly Glu Tyr Val Thr Ser Gln Gly Tyr Asp Ile
165 170 175
Arg Phe Ala Ile Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Leu
180 185 190
Leu Pro Thr Val Gly His Ala Leu Ala Phe Ile Glu Arg Leu Glu Arg
195 200 205
Pro Glu Leu Tyr Gly Val Asn Pro Glu Val Gly His Glu Gln Met Ala
210 215 220
Gly Leu Asn Phe Pro His Gly Ile Ala Gln Ala Leu Trp Ala Gly Lys
225 230 235 240
Leu Phe His Ile Asp Leu Asn Gly Gln Asn Gly Ile Lys Tyr Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ala Gly Asp Leu Arg Ala Ala Phe Trp Leu Val
260 265 270
Asp Leu Leu Glu Ser Ala Gly Tyr Ser Gly Pro Arg His Phe Asp Phe
275 280 285
Lys Pro Pro Arg Thr Glu Asp Phe Asp Gly Val Trp Ala Ser Ala Ala
290 295 300
Gly Cys Met Arg Asn Tyr Leu Ile Leu Lys Glu Arg Ala Ala Ala Phe
305 310 315 320
Arg Ala Asp Pro Glu Val Gln Glu Ala Leu Arg Ala Ser Arg Leu Asp
325 330 335
Glu Leu Ala Arg Pro Thr Ala Ala Asp Gly Leu Gln Ala Leu Leu Asp
340 345 350
Asp Arg Ser Ala Phe Glu Glu Phe Asp Val Asp Ala Ala Ala Ala Arg
355 360 365
Gly Met Ala Phe Glu Arg Leu Asp Gln Leu Ala Met Asp His Leu Leu
370 375 380
Gly Ala Arg Gly
385
<210> 2
<211> 1164
<212> DNA
<213> Artificial sequence
<400> 2
atgaactacc agccgacccc ggaagatcgc tttacttttg gcctgtggac tgtaggttgg 60
cagggtcgcg acccgttcgg cgatgctact cgtcgtgccc tggatccggt tgaatctgtg 120
caacgcctgg cggaactggg cgcacatggt gtaactttcc acgacgatga tctgatcccg 180
tttggctcca gcgactccga gcgcgaagaa cacgtgaaac gctttcgtca ggcgctggac 240
gatactggca tgaaagtccc gatggcgacg accaacctgt tcacgcaccc tgtgttcaag 300
gatggtggct tcacggctaa cgatcgtgac gttcgtcgct acgccctgcg taaaaccatt 360
cgcaacattg acctggcggt tgaactgggc gctgagacct atgttgcttg gggtggtcgt 420
gaaggtgcag aatccggtgg tgcaaaagat gtgcgtgatg ccctggatcg catgaaagaa 480
gcgttcgacc tgctgggtga atatgtcacc tctcagggtt acgatatccg ttttgctatt 540
gaaccgaaac cgaacgaacc acgtggtgac attctgctgc caaccgtagg tcacgctctg 600
gcgttcatcg agcgtctgga acgcccggaa ctgtacggtg tgaacccgga ggtcggccat 660
gagcagatgg caggtctgaa cttccctcac ggcatcgctc aggcactgtg ggctggtaaa 720
ctgttccaca ttgatctgaa cggtcagaac ggtatcaaat acgaccagga tctgcgtttc 780
ggcgctggtg atctgcgtgc agctttctgg ctggtggatc tgctggaaag cgctggttac 840
agcggtccgc gtcacttcga cttcaaaccg ccgcgtactg aagacttcga tggtgtatgg 900
gcgagcgctg cgggttgtat gcgcaattat ctgatcctga aggaacgtgc tgctgctttt 960
cgcgcggacc cggaagtaca ggaagcactg cgtgcgtctc gtctggatga gctggcgcgc 1020
cctactgctg ctgatggtct gcaggctctg ctggatgacc gctccgcttt tgaagaattc 1080
gacgtcgacg ctgccgcagc tcgtggtatg gctttcgaac gtctggatca gctggcaatg 1140
gaccatctgc tgggcgcacg tggc 1164
<210> 3
<211> 388
<212> PRT
<213> Artificial sequence
<400> 3
Met Asn Tyr Gln Pro Thr Pro Glu Asp Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
Thr Val Gly Trp Gln Gly Arg Asp Pro Phe Gly Asp Ala Thr Arg Arg
20 25 30
Ala Leu Asp Pro Val Glu Ser Val Gln Arg Leu Ala Glu Leu Gly Ala
35 40 45
His Gly Val Thr Phe His Asp Asp Asp Leu Ile Pro Phe Gly Ser Ser
50 55 60
Asp Ser Glu Arg Glu Glu His Val Lys Arg Phe Arg Gln Ala Leu Asp
65 70 75 80
Asp Thr Gly Met Lys Val Pro Lys Ala Thr Thr Asn Leu Phe Thr His
85 90 95
Pro Val Phe Lys Asp Gly Gly Phe Thr Ala Asn Asp Arg Asp Val Arg
100 105 110
Arg Tyr Ala Leu Arg Lys Thr Ile Arg Asn Ile Asp Leu Ala Val Glu
115 120 125
Leu Gly Pro Glu Thr Tyr Val Gly Trp Gly Gly Arg Glu Gly Ala Glu
130 135 140
Ser Gly Gly Ala Lys Asp Val Arg Asp Ala Leu Asp Arg Met Lys Glu
145 150 155 160
Ala Phe Asp Leu Leu Gly Glu Tyr Val Thr Ser Gln Gly Tyr Asp Ile
165 170 175
Arg Phe Ala Ile Glu Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Leu
180 185 190
Leu Pro Thr Val Gly His Ala Leu Ala Phe Ile Glu Arg Leu Glu Arg
195 200 205
Pro Glu Leu Tyr Gly Val Asn Pro Glu Val Gly His Glu Gln Met Ala
210 215 220
Gly Leu Asn Phe Pro His Gly Ile Ala Gln Ala Leu Trp Ala Gly Lys
225 230 235 240
Leu Phe His Ile Asp Leu Asn Cys Gln Asn Gly Ile Lys Tyr Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ala Gly Asp Leu Arg Ala Ala Phe Trp Leu Val
260 265 270
Asp Leu Leu Glu Ser Ala Gly Tyr Ser Gly Pro Arg His Phe Asp Phe
275 280 285
Lys Pro Pro Arg Thr Glu Asp Phe Asp Gly Val Trp Ala Ser Ala Ala
290 295 300
Gly Cys Met Arg Asn Tyr Leu Ile Leu Lys Glu Arg Ala Ala Ala Phe
305 310 315 320
Arg Ala Asp Pro Glu Val Gln Glu Ala Leu Arg Ala Ser Arg Leu Asp
325 330 335
Glu Leu Ala Arg Pro Thr Ala Ala Asp Gly Leu Gln Ala Leu Leu Asp
340 345 350
Asp Arg Ser Ala Phe Glu Glu Phe Asp Val Asp Ala Ala Ala Ala Arg
355 360 365
Gly Met Ala Phe Glu Arg Leu Asp Gln Leu Ala Met Asp His Leu Leu
370 375 380
Gly Ala Arg Gly
385
<210> 4
<211> 1164
<212> DNA
<213> Artificial sequence
<400> 4
atgaactacc agccgacccc ggaagatcgc tttacttttg gcctgtggac tgtaggttgg 60
cagggtcgcg acccgttcgg cgatgctact cgtcgtgccc tggatccggt tgaatctgtg 120
caacgcctgg cggaactggg cgcacatggt gtaactttcc acgacgatga tctgatcccg 180
tttggctcca gcgactccga gcgcgaagaa cacgtgaaac gctttcgtca ggcgctggac 240
gatactggca tgaaagtccc gaaggcgacg accaacctgt tcacgcaccc tgtgttcaag 300
gatggtggct tcacggctaa cgatcgtgac gttcgtcgct acgccctgcg taaaaccatt 360
cgcaacattg acctggcggt tgaactgggc cctgagacct atgttggttg gggtggtcgt 420
gaaggtgcag aatccggtgg tgcaaaagat gtgcgtgatg ccctggatcg catgaaagaa 480
gcgttcgacc tgctgggtga atatgtcacc tctcagggtt acgatatccg ttttgctatt 540
gaaccgaaac cgaacgaacc acgtggtgac attctgctgc caaccgtagg tcacgctctg 600
gcgttcatcg agcgtctgga acgcccggaa ctgtacggtg tgaacccgga ggtcggccat 660
gagcagatgg caggtctgaa cttccctcac ggcatcgctc aggcactgtg ggctggtaaa 720
ctgttccaca ttgatctgaa ctgtcagaac ggtatcaaat acgaccagga tctgcgtttc 780
ggcgctggtg atctgcgtgc agctttctgg ctggtggatc tgctggaaag cgctggttac 840
agcggtccgc gtcacttcga cttcaaaccg ccgcgtactg aagacttcga tggtgtatgg 900
gcgagcgctg cgggttgtat gcgcaattat ctgatcctga aggaacgtgc tgctgctttt 960
cgcgcggacc cggaagtaca ggaagcactg cgtgcgtctc gtctggatga gctggcgcgc 1020
cctactgctg ctgatggtct gcaggctctg ctggatgacc gctccgcttt tgaagaattc 1080
gacgtcgacg ctgccgcagc tcgtggtatg gctttcgaac gtctggatca gctggcaatg 1140
gaccatctgc tgggcgcacg tggc 1164
<210> 5
<211> 44
<212> DNA
<213> Artificial sequence
<400> 5
taagaaggag atataccatg gatgaactac cagccgaccc cgga 44
<210> 6
<211> 41
<212> DNA
<213> Artificial sequence
<400> 6
gtggtggtgg tggtgctcga gttagccacg tgcgcccagc a 41
<210> 7
<211> 1179
<212> DNA
<213> Artificial sequence
<400> 7
ggaattcaac tatcaaccaa ccccagaaga cagatttaca tttggactgt ggaccgtggg 60
atggcagggt agagatcctt tcggtgatgc cacaagaaga gcacttgacc cagttgaatc 120
tgttcaaaga ttagccgaat tgggagccca cggagttact ttccatgacg atgacctaat 180
tccttttggc agtagtgact ctgagaggga ggaacatgtc aagagattta gacaagctct 240
tgacgatact ggtatgaaag tccctaaggc aaccacgaat ttgtttactc atcctgtttt 300
caaagatggt ggatttaccg caaatgatag agatgtgagg aggtatgctc tgagaaaaac 360
tatcagaaac atcgatctgg cagtcgaatt gggtccagaa acctacgttg gctggggtgg 420
aagagaggga gcagagtcag gtggtgccaa agatgtcaga gatgctctgg atcgaatgaa 480
ggaagccttt gacctgctag gagagtacgt cacctctcag ggttatgaca tcagattcgc 540
tatagaacca aaacctaatg aaccaagagg tgacatttta ttacccacag ttggtcacgc 600
tttagccttt attgaacgtt tggaaagacc tgaattgtat ggagtgaatc ctgaagttgg 660
tcacgaacag atggcaggac tgaactttcc acatggaatc gctcaggccc tgtgggccgg 720
taaattattt catattgacc tgaattgtca aaacggtatc aaatacgatc aggatttaag 780
attcggtgca ggtgacttga gagctgcttt ttggcttgtt gacttattgg aatccgcagg 840
ttactcagga cctagacact ttgacttcaa acctcccaga actgaagatt ttgatggagt 900
ttgggcttct gctgccggct gcatgagaaa ctacttgata ttgaaggaga gagcagcagc 960
ctttagggca gacccagagg tgcaagaggc tttgagagca tccaggttag atgagttggc 1020
tagaccaaca gcagcagacg gattacaggc acttttggat gacagatctg cattcgagga 1080
gtttgatgtg gatgctgctg ccgctcgtgg aatggctttc gagagactag atcagctagc 1140
tatggatcat ctgctgggcg ctagaggcta atctagagc 1179
- 上一篇：一种医用注射器针头装配设备
- 下一篇：低压注塑自动挂皮设备