CN106867986B - Production method of high-purity liquid glucose isomerase - Google Patents
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Abstract
The invention relates to a production method of high-purity liquid glucose isomerase, which comprises the following steps: the liquid preparation containing glucose isomerase is finally obtained through activation inoculation, continuous culture, fermentation culture and treatment of fermentation liquor, has the advantages of high activity, temperature resistance and good enzyme activity stability, is applied to the high fructose syrup industry, is suitable for intermittent production of F42 type high fructose syrup, and can be used for producing F55 type high fructose syrup by using the glucose isomerase in a one-step method. The product has lower requirements on the quality standard of the glucose syrup when producing the F42 type high fructose syrup; the cost of enzyme purchasing at one time is low; the method has the advantages of simple operation, production with addition, no need of maintenance and consideration of enzyme activity, complete realization of domestic production of the enzyme and high production flexibility.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a production method of high-purity liquid glucose isomerase.
background
in the traditional glucose isomerase production, considering the factors of low activity of fermentation enzyme, poor stability of glucose isomerase in a liquid dosage form and the like, the fermentation liquid after fermentation is generally subjected to cross-linking and enzyme activity fixation through a chemical product, and the immobilized glucose isomerase is obtained after drying, is in a solid dosage form, and is only supplied by international danish novicent and U.S. dupont. When the immobilized glucose isomerase is used, hundreds of thousands of millions of immobilized glucose isomerase can be purchased once and used after being filled in a column, glucose-fructose syrup can be produced by refining glucose liquid to reach the standard in use, the production is continuous, the quality of the glucose liquid does not reach the standard, and the glucose liquid is produced intermittently or is used and maintained improperly, so that the immobilized glucose isomerase fails or is even scrapped, and huge production loss is caused.
disclosure of Invention
In order to solve the technical problems, the invention provides a production method of high-purity liquid glucose isomerase, the glucose isomerase produced by the production method has the advantages of high activity, temperature resistance and good enzyme activity stability, is applied to the high fructose syrup industry, is suitable for intermittently producing F42 type high fructose syrup, and can be used for producing F55 type high fructose syrup by a one-step method. The product has lower requirements on the quality standard of the glucose syrup when producing the F42 type high fructose syrup; the cost of enzyme purchasing at one time is low; the method has the advantages of simple operation, production with addition, no need of maintenance and consideration of enzyme activity, complete realization of domestic production of the enzyme and high production flexibility.
The technical scheme adopted by the invention is as follows: a method for producing a high purity liquid dosage form glucose isomerase comprising the steps of:
A. Activating and inoculating: activating streptomycete, inoculating the activated streptomycete into a culture bottle, and culturing to obtain seed mother liquor;
B. And (3) continuous culture: sequentially culturing the seed mother liquor at 180-220 rpm and 30-35 deg.C for 24-28 hr, at 180-220 rpm and 30-35 deg.C for 18-36 hr, and at 150-180 rpm and 30-35 deg.C for 18-36 hr to obtain culture solution;
C. Fermentation culture: culturing the culture solution at 110-150 rpm at 30-35 deg.C for 40-55 hr to obtain fermentation broth;
D. Treating fermentation liquor: filtering the fermentation liquor, and taking the filtrate to obtain the liquid preparation containing the glucose isomerase.
The glucose isomerase produced by the production method has the advantages of high activity, temperature resistance and good enzyme activity stability.
The culture medium used in the invention is: 7% of sucrose, 3% of peptone, 1.5% of beef powder, 1% of bran, 0.5% of corn steep liquor, 0.5% of ammonium sulfate, 0.05% of magnesium sulfate, 0.05% of cobalt chloride, 0.02% of dipotassium hydrogen phosphate, 0.01% of potassium dihydrogen phosphate and the balance of purified water.
Preferably, the culture medium is 10% of sucrose, 1.5% of peptone, 2.0% of beef powder, 0.8% of bran, 1.0% of corn steep liquor, 0.35% of ammonium sulfate, 0.08% of magnesium sulfate, 0.05% of cobalt chloride, 0.02% of dipotassium hydrogen phosphate, 0.01% of potassium dihydrogen phosphate and the balance of purified water.
In order to further improve the content and the enzyme activity of glucose isomerase in the obtained liquid preparation, the preferable technical scheme is that the streptomyces is streptomyces M1033. The Streptomyces M1033 is determined by repeated experiments of the inventor of the application, and compared with the common streptomyces, the liquid preparation obtained after fermentation has higher content of glucose isomerase and higher enzyme activity and temperature tolerance.
In order to further improve the activation efficiency of streptomycete, the preferable technical scheme is that in the step A, streptomycete is taken out from a freeze-drying tube at 80-85 ℃ below zero, activated by a Charpy slant at 30-35 ℃ for 48-72 hours, and inoculated into a culture flask for culture.
in order to conveniently carry out multi-stage culture on the streptomyces, the preferable technical scheme is that in the step A, the culture bottle is a shake flask so as to conveniently carry out culture on a shaking table; in the step B, the seed mother liquor is sequentially cultured in a shake flask for 24 to 28 hours at the temperature of 30 to 35 ℃ at 180 to 220 rpm, then cultured in a first-stage seeding tank for 18 to 36 hours at the temperature of 30 to 35 ℃ at 180 to 220 rpm, cultured in a second-stage seeding tank for 18 to 36 hours at the temperature of 30 to 35 ℃ at 150 to 180 rpm to obtain a culture solution, and in the continuous culture process, different culture containers are sequentially selected from the shake flask to the first-stage seeding tank and then to the second-stage seeding tank according to different culture periods so as to facilitate the smooth operation of the whole culture process; and C, performing fermentation culture in a fermentation tank, so as to provide a stable fermentation environment.
In order to avoid the contamination of mixed bacteria and ensure the smooth operation of the whole production process, the preferred technical proposal is that the continuous culture and the fermentation culture are both carried out in the sterile environment.
In order to ensure the filtering effect, the preferable technical scheme is that the filtering treatment in the step D is as follows: adding antiseptic, diatomaceous earth, bentonite and purified water into the fermentation liquid, stirring at 15-30 deg.C for 15-30 min, standing for 1-3 hr, filtering to remove residue, micro-filtering to remove bacteria, and ultrafiltering for concentration.
The antiseptic is sodium benzoate, potassium sorbate, methyl paraben or ethyl paraben. The addition amount of the preservative relative to the fermentation liquor is different according to the difference of the preservative, for example, the addition amount of sodium benzoate is 2-3g/L, the addition amount of potassium sorbate is 1-2g/L, and the addition amount of methylparaben or ethylparaben is 200-500 ppm.
Diatomite, bentonite and purified water are added before filtration, so that flocculation is mainly achieved, subsequent filtration operation is facilitated, and the combined filtration mode of plate-frame filtration, microfiltration and spiral membrane ultrafiltration is adopted in the invention, so that slag removal and sterilization are more sufficient, and the purity of a final product is improved.
Further, the weight percentages of the diatomite, the bentonite and the purified water relative to the fermentation liquor are respectively 1-3%, 1-5% and 50-150%. According to the inventor experiment of the application, the good flocculation effect can be ensured and the reagent can be saved under the above proportion, thereby reducing the cost of the production process.
According to the working conditions and purposes of different filtering means, the inventor of the application further researches and obtains: the filtering pressure of the plate frame filtering, the microfiltration and the roll-type membrane ultrafiltration is respectively 0.05-0.25MPa, 0.15-0.35MPa and 0.30-0.50MPa, and under the pressure difference parameters, better filtering effect and filtering speed can be ensured.
In order to ensure that the final product can be stably stored for a long time, the preferable technical scheme is that in the step D, after the ultrafiltration concentration treatment, the preservative stabilization treatment is also carried out: adding salt, sodium acetate, glycerol, magnesium sulfate and cobalt chloride into the concentrated solution, wherein the volume percentages of the salt, the sodium acetate, the glycerol, the magnesium sulfate and the cobalt chloride relative to the final liquid preparation are respectively 15-20%, 5-10%, 2-5%, 0.1-0.2% and 0.05-0.1%.
Through orthogonal repeated experiments, the inventor obtains the components and the proportion of the preservative composition, and can stably store the liquid preparation containing the glucose isomerase for a long time, so that the liquid preparation is convenient for subsequent production and use.
In the step D, after the anti-corrosion stabilizing treatment, the step D is carried out, and after the inspection is qualified, the product is filled and put in storage; the standard of the test is: the enzyme activity of the liquid preparation containing glucose isomerase is greater than or equal to 5000 u/ml. It should be noted that the value of the enzyme activity is mainly determined by the concentration ratio, that is, after the fermentation is completed and the extraction is refined, the larger the ultrafiltration concentration ratio is, the higher the enzyme activity of the obtained liquid product is.
Besides enzyme activity, the following detection indexes are provided:
after the test of the test standards, the final finished product can be ensured to have better stability and can be stored for a long time.
The invention has the beneficial effects that:
The glucose isomerase produced by the liquid glucose isomerase production process has the advantages of high activity, temperature resistance and good enzyme activity stability, is applied to the high fructose syrup industry, is suitable for intermittently producing F42 high fructose syrup, and can be used for producing F55 high fructose syrup by a one-step method. The product has lower requirements on the quality standard of the glucose syrup when producing the F42 type high fructose syrup; the cost of enzyme purchasing at one time is low; the method has the advantages of simple operation, production with addition, no need of maintenance and consideration of enzyme activity, complete realization of domestic production of the enzyme and high production flexibility.
drawings
in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram showing the fructose content of different liquid preparation addition amounts in the production of glucose-fructose syrup F42 type by using crude sugar solution as a function of time;
FIG. 2 is a schematic diagram showing the fructose content over time at different temperatures in the production of glucose-fructose syrup F42 from crude sugar solution;
FIG. 3 is a schematic diagram showing the change of fructose content with time at different pH values in the production of glucose fructose 42 type F using crude sugar solution
FIG. 4 is a schematic diagram showing the fructose content of different liquid preparation addition amounts with time when refined sugar solution is used for producing F55 type fructose syrup;
FIG. 5 is a schematic diagram showing the fructose content at different temperatures over time in the production of glucose fructose 55 type F using refined sugar solution;
FIG. 6 is a schematic diagram showing the fructose content over time at different pH values in the production of glucose fructose 55 type F by using refined sugar solution.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
example 1
A method for producing a high purity liquid dosage form glucose isomerase comprising the steps of:
A. Activating and inoculating: taking out Streptomyces M1033 from a freeze-drying tube at minus 80 ℃, activating an inclined surface at 35 ℃ for 48 hours, and inoculating into a shake flask for culturing to obtain a seed mother liquor;
B. And (3) continuous culture: sequentially culturing the seed mother liquor in a shake flask at 220 r/min and 30 ℃ for 28 hours, then culturing in a first-stage seeding tank at 180 r/min and 35 ℃ for 18 hours, and culturing in a second-stage seeding tank at 180 r/min and 30 ℃ for 36 hours to obtain a culture solution;
C. Fermentation culture: culturing the culture solution in a fermentation tank at 35 deg.C for 40 hr at 110 rpm to obtain fermentation broth; the continuous culture and the fermentation culture are both carried out in an aseptic environment;
D. Treating fermentation liquor: adding sodium benzoate, diatomite, bentonite and purified water into the fermentation liquor, wherein the addition amount of the preservative is 2g/L, and the weight percentages of the diatomite, the bentonite and the purified water relative to the fermentation liquor are respectively 3%, 1% and 150%; stirring for 30 minutes at 15 ℃, standing for 1 hour, and then sequentially carrying out plate-frame filtration, microfiltration and spiral-wound membrane ultrafiltration, wherein the filtration pressures of the plate-frame filtration, the microfiltration and the spiral-wound membrane ultrafiltration are respectively 0.25MPa, 0.15MPa and 0.50 MPa; after the filtration treatment, the method also comprises the following antiseptic stabilizing treatment: adding salt, sodium acetate, glycerol, magnesium sulfate and cobalt chloride into the fermentation liquor, wherein the volume percentages of the salt, the sodium acetate, the glycerol, the magnesium sulfate and the cobalt chloride relative to the final liquid preparation are respectively 15%, 10%, 2%, 0.2% and 0.05%; and (3) performing preservative stabilization treatment to obtain a liquid preparation containing glucose isomerase, performing inspection on the liquid preparation, and filling and warehousing after the liquid preparation is qualified.
Example 2
A method for producing a high purity liquid dosage form glucose isomerase comprising the steps of:
A. activating and inoculating: taking out Streptomyces M1033 from a freeze-drying tube at the temperature of minus 85 ℃, activating an inclined surface at the temperature of 30 ℃ for 72 hours, and inoculating into a shake flask for culturing to obtain a seed mother liquor;
B. And (3) continuous culture: culturing the mother liquid in a shake flask at 35 deg.C for 24 hr at 180 rpm, culturing in a first-stage seeding tank at 30 deg.C for 36 hr at 220 rpm, and culturing in a second-stage seeding tank at 35 deg.C and 150 rpm for 18 hr to obtain culture solution;
C. Fermentation culture: culturing the culture solution in a fermentation tank at 30 ℃ for 55 hours at 150 rpm to obtain fermentation liquor; the continuous culture and the fermentation culture are both carried out in an aseptic environment;
D. Treating fermentation liquor: adding potassium sorbate, diatomite, bentonite and purified water into the fermentation liquor, wherein the addition amount of the potassium sorbate is 2g/L, and the weight percentages of the diatomite, the bentonite and the purified water relative to the fermentation liquor are respectively 1%, 5% and 50%; stirring for 15 minutes at 30 ℃, standing for 2 hours, and then sequentially carrying out plate-frame filtration, microfiltration and spiral-wound membrane ultrafiltration, wherein the filtration pressures of the plate-frame filtration, the microfiltration and the spiral-wound membrane ultrafiltration are respectively 0.05MPa, 0.35MPa and 0.30 MPa; after the filtration treatment, the method also comprises the following antiseptic stabilizing treatment: adding salt, sodium acetate, glycerol, magnesium sulfate and cobalt chloride into the fermentation liquor, wherein the volume percentages of the salt, the sodium acetate, the glycerol, the magnesium sulfate and the cobalt chloride relative to the final liquid preparation are respectively 20%, 5%, 5%, 0.1% and 0.1%; and (3) performing preservative stabilization treatment to obtain a liquid preparation containing glucose isomerase, performing inspection on the liquid preparation, and filling and warehousing after the liquid preparation is qualified.
Example 3
A method for producing a high purity liquid dosage form glucose isomerase comprising the steps of:
A. Activating and inoculating: taking out Streptomyces M1033 from a freeze-drying tube at the temperature of 82 ℃ below zero, activating the inclined surface for 60 hours at the temperature of 32 ℃, and inoculating the activated product into a shake flask for culturing to obtain a seed mother liquor;
B. And (3) continuous culture: culturing the seed mother liquor in a shake flask at 32 ℃ for 26 hours at 200 r/min in sequence, then culturing in a primary seed tank at 32 ℃ for 27 hours at 200 r/min, and culturing in a secondary seed tank at 32 ℃ at 165 r/min for 27 hours to obtain a culture solution;
C. Fermentation culture: culturing the culture solution in a fermentation tank at 32 ℃ for 47 hours at 130 r/min to obtain fermentation liquor; the continuous culture and the fermentation culture are both carried out in an aseptic environment;
D. Treating fermentation liquor: adding methyl paraben, diatomite, bentonite and purified water into the fermentation liquor, wherein the addition amount of the methyl paraben is 500ppm, and the weight percentages of the diatomite, the bentonite and the purified water relative to the fermentation liquor are respectively 2%, 3% and 100%; stirring for 22 minutes at 22 ℃, standing for 2 hours, and then sequentially carrying out plate-frame filtration, microfiltration and spiral-wound membrane ultrafiltration, wherein the filtration pressures of the plate-frame filtration, the microfiltration and the spiral-wound membrane ultrafiltration are respectively 0.15MPa, 0.25MPa and 0.4 MPa; after the filtration treatment, the method also comprises the following antiseptic stabilizing treatment: adding salt, sodium acetate, glycerol, magnesium sulfate and cobalt chloride into the fermentation liquor, wherein the volume percentages of the salt, the sodium acetate, the glycerol, the magnesium sulfate and the cobalt chloride relative to the final liquid preparation are respectively 17%, 7%, 3.5%, 0.15% and 0.07%; and (3) performing preservative stabilization treatment to obtain a liquid preparation containing glucose isomerase, performing inspection on the liquid preparation, and filling and warehousing after the liquid preparation is qualified.
experimental example 1
In this experimental example, the liquid preparations containing glucose isomerase obtained in examples 1 to 3 were tested, and the results are shown in Table 1:
The detection values of the experimental example are obtained by using a national standard GB/T23533-2009 determination method or an industry universal measurement method, wherein the preservation rate of the enzyme activity is the preservation rate of the enzyme activity after the enzyme activity is preserved for three months at room temperature of 20-25 ℃.
Table 1: test result table
| detecting items | standard of merit | Example 1 value | Example 2 value | Example 3 value | Conclusion |
| Enzyme activity | ≥5000u/ml | 5445 | 5557 | 10600 | Are all qualified |
| Preservation rate of enzyme activity | ≥85％ | 90.1％ | 92％ | 91.5％ | are all qualified |
| pH value | 5.0-7.5 | 7.11 | 7.05 | 6.95 | Are all qualified |
| Volume weight | 1.10-1.25g/ml | 1.18 | 1.20 | 1.22 | Are all qualified |
| heavy metals (in Pb) | ≤0.004 | 0.003 | 0.003 | 0.003 | Are all qualified |
| Lead (in Pb) | ≤0.001 | 0.00072 | 0.00072 | 0.0002 | are all qualified |
| arsenic (in As) | ≤0.0003 | 0.00013 | 0.00013 | 0.00007 | Are all qualified |
| Total number of colonies | less than or equal to 50000 pieces/ml | 300 | 300 | 500 | Are all qualified |
| Coliform group bacteria | Less than or equal to 30 pieces/ml | 2 | 3 | 0 | Are all qualified |
| Escherichia coli | Cannot be detected | not detected out | Not detected out | Not detected out | Are all qualified |
| Mould fungus | Less than or equal to 100 pieces/ml | 0 | 0 | 0 | Are all qualified |
| Yeast | Less than or equal to 100 pieces/ml | 0 | 0 | 0 | are all qualified |
| Aflatoxin B1 | ≤0.0000005％ | 0 | 0 | 0 | Are all qualified |
as can be seen from Table 1, the liquid preparations containing glucose isomerase obtained in examples 1 to 3 all have the advantages of high enzyme activity and good stability, which indicates that the production process provided by the invention has good stability and good quality of finished products.
Experimental example 2
In this experimental example, the use of the glucose-fructose syrup production for the liquid formulations containing glucose isomerase obtained in examples 1 and 2 is reported:
usage report 1
1. Conditions of the experiment
170kg of sugar liquid with the glucose content of more than 92 percent, the solid content of 34.8 percent and DE (dextrose equivalent) of 98.1 are taken; adjusting the pH value to about 8.1, adding 140g of magnesium sulfate heptahydrate and 32g of sodium metabisulfite, uniformly stirring, adding 550ml of the liquid preparation containing glucose isomerase obtained in example 1, heating to 65-68 ℃, after acting for 16 hours, detecting by high performance liquid chromatography, until the fructose content in the sugar solution reaches 43.19 percent and the glucose and fructose content is 94.87 percent, and finally adjusting the pH value of the sugar solution to 4.2 for enzyme deactivation.
2. Evaluation of experiments
The liquid preparation containing glucose isomerase obtained in example 3 is used for producing F42 high fructose syrup, so that the fructose content in the sugar solution can reach 43.19%, and the glucose + fructose content is 94.87%, and the requirements of the GB/T20882-2007 high fructose syrup on fructose content of 42-44% and glucose + fructose content of more than or equal to 92% are met. The liquid preparation is convenient to use, and can directly convert glucose liquid into F42 high fructose syrup by proper process control, so that the production requirement is met.
Usage report 2
1. conditions of the experiment
Taking 28 cubes of sugar solution with the glucose content of more than 93 percent, the solid content of 30 percent and DE (dextrose equivalent) of 101; adjusting the pH value to about 8.1, adding 25kg of magnesium sulfate heptahydrate and 6.1kg of sodium metabisulfite, stirring uniformly, adding 8.5L of the liquid preparation containing glucose isomerase obtained in example 2, heating to 65-68 ℃, reacting for 21 hours, detecting by high performance liquid chromatography until the fructose content in the sugar solution reaches 45.02% and the glucose and fructose content is 92.72%, and finally adjusting the pH value of the sugar solution to 4.1 for enzyme deactivation.
2. Evaluation of experiments
The liquid preparation containing glucose isomerase obtained in example 3 was used to produce F42 fructose syrup, which can make the fructose content in the sugar solution reach 45.02% and make the glucose + fructose content equal to 92.72%. The liquid preparation is convenient to use, and can directly convert glucose liquid into F42 high fructose syrup by proper process control, so that the production requirement is met.
Experimental example 3
This example is a study of the ability of the liquid preparation containing glucose isomerase obtained in example 3 to produce high fructose syrup.
Firstly, producing and preparing F42 type high fructose corn syrup by using crude sugar solution
The liquid preparation obtained in example 3 and containing glucose isomerase (enzyme activity 10600u ^ is usedml), the substrate is crude glucose solution, the solid content is 35.0 percent, MgSO4·7H2O：550-850g/M3(sugar solution), Na2S2O5：200-300g/M3(sugar solution).
1. Different liquid preparation adding amounts and fructose content changing with time
As shown in fig. 1, it can be seen from fig. 1 that as the addition amount of the liquid preparation increases, the time required for the fructose content to reach the form F42 is shorter, 30 hours are required for the liquid preparation to reach the form F42 when the addition amount of the liquid preparation is 2%, and only 18 hours are required for the liquid preparation to reach the form F42 when the addition amount of the liquid preparation is 3%.
2. the fructose content varied with time at different temperatures
as shown in FIG. 2, the higher the temperature, the higher the fructose content in the same time period, with the increase in temperature (constant temperature), indicating that the increase in temperature favors the conversion of fructose.
3. Fructose content over time at different pH
as shown in FIG. 3, the fructose content was higher at higher pH values in the same period of time as the pH value was increased (at a certain pH value).
secondly, preparing F55 type high fructose corn syrup by using refined sugar solution
Using the liquid preparation containing glucose isomerase obtained in example 3 (enzyme activity 10600u/ml), the substrate was refined glucose solution, the solid content was 35.0%, water was deionized water, MgSO4·7H2O：550-850g/M3(sugar solution), Na2S2O5：200-300g/M3(sugar solution).
1. Different liquid preparation adding amounts and fructose content changing with time
As shown in fig. 4, it can be seen from fig. 4 that the time required for the fructose content to reach form F55 is shorter as the addition amount of the liquid preparation increases, and it takes 10 hours for the liquid preparation to reach form F55 at 1% addition amount, and it takes 5 hours for the liquid preparation to reach form F55 at 2% addition amount.
2. The fructose content varied with time at different temperatures
As shown in FIG. 5, it can be seen from FIG. 5 that as the temperature is increased, the fructose content is increased at higher temperature for a certain period of time (18 hours or less), and the fructose content is slightly different when the temperature is higher than 68 ℃ for 18 hours. Indicating that the temperature increase favors the conversion of fructose over time.
3. Fructose content over time at different pH
As shown in FIG. 6, it can be seen from FIG. 6 that the fructose content of the F55 type fructose syrup is not much different between pH 8.0 and pH 8.2. At pH 8.0 compared to pH 7.8, the fructose content at pH 8.0 was higher than at pH 7.8 over the same time period. At pH 7.5, the fructose content did not reach form F55 within 20 hours.
analysis of results
1. When high-purity liquid glucose isomerase is used for producing high fructose syrup, the higher the enzyme addition amount, temperature and pH value, the more beneficial the conversion of fructose is.
2. with the time being prolonged, the fructose content is increased rapidly and shows an obvious rising trend, and after 20 hours, the fructose content is increased rapidly and gradually becomes gentle.
The advantage of using liquid formulations
1. The equipment is simple, the investment is small, and the F55 high fructose corn syrup can be produced by common sugar manufacturing equipment without purchasing expensive equipment such as a chromatographic separation column.
2. The operation is simple, the high fructose corn syrup is directly added, the production process is omitted, and the production is easier to operate.
3. the production is easier to schedule. The production can be flexibly arranged according to market conditions, and the conditions that immobilized isomerase is not easy to produce and inactivate, is difficult to protect and expensive equipment is not used are avoided.
4. The production process is simplified, the manpower, the water and electricity expenses and the production cost are saved, and the enterprise competitiveness is enhanced.
5. Is beneficial to the storage and transportation of the high fructose corn syrup, and reduces unnecessary loss of crystallization enterprises.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (1)
1. A method for producing a high-purity liquid glucose isomerase, comprising the steps of:
A. Activating and inoculating: activating streptomycete, inoculating the activated streptomycete into a culture bottle, and culturing to obtain seed mother liquor;
B. And (3) continuous culture: sequentially culturing the seed mother liquor at 180-220 rpm and 30-35 deg.C for 24-28 hr, at 180-220 rpm and 30-35 deg.C for 18-36 hr, and at 150-180 rpm and 30-35 deg.C for 18-36 hr to obtain culture solution;
C. Fermentation culture: culturing the culture solution at 110-150 rpm at 30-35 deg.C for 40-55 hr to obtain fermentation broth;
D. Treating fermentation liquor: filtering the fermentation liquor, and taking the filtrate to obtain a liquid preparation containing glucose isomerase;
The streptomyces is M1033 streptomyces;
In the step A, taking out streptomycete from a freeze-drying tube at 80-85 ℃ below zero, activating the inclined surface at 30-35 ℃ for 48-72 hours, and inoculating the streptomycete into a culture bottle for culture; the culture bottle is a shake flask; in the step B, the seed mother liquor is sequentially cultured in a shake flask for 24-28 hours at the temperature of 30-35 ℃ at the speed of 180-220 r/min, then cultured in a first-stage seeding tank for 18-36 hours at the temperature of 30-35 ℃ at the speed of 180-220 r/min, and cultured in a second-stage seeding tank for 18-36 hours at the temperature of 30-35 ℃ at the speed of 150-180 r/min to obtain a culture solution; in the step C, fermentation culture is carried out in a fermentation tank; the continuous culture and the fermentation culture are both carried out in an aseptic environment;
The filtering treatment in the step D is as follows: adding antiseptic, diatomaceous earth, bentonite and purified water into the fermentation liquor, stirring at 15-30 deg.C for 15-30 min, standing for 1-3 hr, sequentially filtering to remove residue, micro-filtering to remove bacteria, and ultrafiltering for concentration; the weight percentages of the diatomite, the bentonite and the purified water relative to the fermentation liquor are respectively 1-3%, 1-5% and 50-150%; the filtering pressure of plate-frame filtration, microfiltration and ultrafiltration is 0.05-0.25MPa, 0.15-0.35MPa and 0.30-0.50MPa respectively;
In the step D, after the ultrafiltration concentration treatment, the antiseptic stabilization treatment is also carried out: adding salt, sodium acetate, glycerol, magnesium sulfate and cobalt chloride into the concentrated solution, wherein the volume percentages of the salt, the sodium acetate, the glycerol, the magnesium sulfate and the cobalt chloride relative to the final liquid preparation are respectively 15-20%, 5-10%, 2-5%, 0.1-0.2% and 0.05-0.1%; after the anticorrosion and stabilization treatment, the inspection step is needed, and after the inspection is qualified, the raw materials are filled and put in storage; the standard of the test is: the enzyme activity of the liquid preparation containing glucose isomerase is greater than or equal to 5000 u/ml.
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| CN1050407A (en) * | 1989-10-10 | 1991-04-03 | 河北省科学院生物研究所 | Method with producing glucose isomerase with acidophilic streptomycete |
| CN106047963A (en) * | 2016-06-22 | 2016-10-26 | 浙江工商大学 | Method for producing high fructose syrup by immobilized glucose isomerase |
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| CN1050407A (en) * | 1989-10-10 | 1991-04-03 | 河北省科学院生物研究所 | Method with producing glucose isomerase with acidophilic streptomycete |
| CN106047963A (en) * | 2016-06-22 | 2016-10-26 | 浙江工商大学 | Method for producing high fructose syrup by immobilized glucose isomerase |
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