CN115595317B - Immobilized beta-fructofuranosidase and method for preparing lactosucrose by using immobilized beta-fructofuranosidase - Google Patents
Immobilized beta-fructofuranosidase and method for preparing lactosucrose by using immobilized beta-fructofuranosidase Download PDFInfo
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- C12Y302/01026—Beta-fructofuranosidase (3.2.1.26), i.e. invertase
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Abstract
The invention discloses immobilized beta-fructofuranosidase and a method for preparing lactosucrose by using the immobilized beta-fructofuranosidase, and belongs to the technical field of lactosucrose preparation. Adding a mixed solution obtained by mixing chitosan, polyquaternium and acetic acid into a carbonate solidification solution to prepare modified chitosan microspheres; adding tripolyphosphate into the modified chitosan microsphere sodium hydroxide dispersion liquid to perform a crosslinking reaction to obtain macroporous chitosan microspheres; mixing the beta-fructofuranosidase liquid with the macroporous chitosan microspheres, and oscillating at low temperature to obtain the immobilized beta-fructofuranosidase. According to the invention, the polyquaternium is utilized to develop the pores of chitosan, the tripolyphosphate is crosslinked with the chitosan microspheres, the mechanical strength of the microspheres is improved, the fixing strength and the pH stability of the chitosan microspheres to beta-fructofuranosidase are improved, the material flow is facilitated, the stability of the enzyme is enhanced, the reuse rate and the use period of the enzyme are improved, the production cost is reduced, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of lactosucrose preparation, and particularly relates to immobilized beta-fructofuranosidase and a method for preparing lactosucrose by using the same.
Background
The lactosucrose is low-degree polymerized sugar formed by connecting 3-9 monosaccharides through glycosidic bonds, and is polymerized by three monomers, namely beta-D-galactoside, alpha-D-glucoside and beta-D-fructofuranoside. The lactosucrose as functional food additive has the functions of proliferating bifidobacteria, regulating intestinal micro-ecology, improving intestinal immunity, keeping moisture, and having high stability to acid and heat, and is widely popular in the industries of food, pharmacy, daily chemicals, feed and the like.
The preparation of lactosucrose mainly takes sucrose and lactose as raw materials, and enzyme synthesis is carried out by utilizing enzymes capable of producing lactosucrose, such as levansucrase, beta-galactosidase or beta-fructofuranosidase. The enzyme specificity for producing the lactosucrose is not high, the enzyme source influence is high, in addition, the enzymatic reaction yield is low, the enzyme cost is high, and the enzyme is the main reason for restricting the production efficiency of the lactosucrose.
Immobilized enzymes have been known for a long time and can be used for solving the problems of instability and short half-life period of the enzymes. The enzyme can be immobilized by using the modified material, so that the constraint conditions of the enzyme on pH, temperature and the like can be improved, the enzyme catalysis efficiency is obviously influenced, the reaction rate is increased, and the recovered enzyme can be repeatedly utilized.
Disclosure of Invention
Aiming at the problems of poor immobilization effect and low enzyme catalytic activity of the immobilized beta-fructofuranosidase in the prior art, the invention provides the immobilized beta-fructofuranosidase and the method for preparing lactosucrose by using the immobilized beta-fructofuranosidase.
The invention is realized by the following technical scheme:
an immobilized beta-fructofuranosidase is prepared by the following method:
(1) Adding a mixed solution obtained by mixing chitosan, polyquaternium and acetic acid into a carbonate solidification solution to prepare modified chitosan microspheres;
(2) Dispersing the modified chitosan microspheres prepared in the step (1) in a sodium hydroxide solution to obtain a modified chitosan microsphere sodium hydroxide dispersion solution, then adding tripolyphosphate into the modified chitosan microsphere sodium hydroxide dispersion solution to perform a crosslinking reaction, and obtaining macroporous chitosan microspheres after the reaction is finished;
(3) Mixing the beta-fructofuranosidase liquid with the macroporous chitosan microspheres, and oscillating at low temperature to obtain the immobilized beta-fructofuranosidase.
Furthermore, the concentration of the chitosan in the mixed solution in the step (1) is 5 to 8wt%, the concentration of the polyquaternium is 5 to 8wt%, and the concentration of the acetic acid is 5 to 5.2 wt%.
Further, the concentration of the sodium hydroxide solution in the step (2) is 1mol/L, and the mass concentration of the modified chitosan microspheres in the modified chitosan microsphere sodium hydroxide dispersion liquid is 60-70%.
Further, the concentration of the tripolyphosphate in the modified chitosan microsphere sodium hydroxide dispersion liquid after the tripolyphosphate is added in the step (2) is 1wt%.
Further, the reaction condition for preparing the modified chitosan microspheres in the step (1) is heating in water bath at 60-70 ℃ for 7-10h; in the step (2), the crosslinking reaction condition is heating in water bath at 50-60 ℃ for 5-10h; the low-temperature oscillation temperature in the step (3) is 8-10 ℃, and the time is 10-20h.
Furthermore, the polyquaternium is one of polyquaternium-6, polyquaternium-7 and polyquaternium-10.
Further, in the step (3), the mass ratio of the dry enzyme in the beta-fructofuranosidase liquid to the macroporous chitosan microspheres is 30mg:1g of the total weight of the composition.
The method for preparing lactosucrose by immobilized beta-fructofuranosidase specifically comprises the following steps:
(1) Filling the immobilized beta-fructofuranosidase in a column type packed bed;
(2) Dissolving sucrose and lactose in PBS buffer solution to prepare mixed solution, and allowing the mixed solution to pass through a column type packed bed at the flow rate of 1.0V column volume/h, wherein the reaction temperature is 40 ℃, so as to obtain lactosucrose mixed solution;
the total mass volume concentration of the sucrose and the lactose in the mixed solution is 60 percent;
(3) And refining the lactulose oligosaccharide mixed solution to obtain the lactulose oligosaccharide.
Further, the mass ratio of the sucrose to the lactose in the step (2) is 1; the pH of the PBS buffer was 7.0.
Further, the refining step in the step (3) comprises high-concentration decolorization, continuous ion-exchange desalting, chromatographic separation and vacuum drying in sequence.
Advantageous effects
(1) According to the invention, the polyquaternium is used to develop the pores of chitosan, the tripolyphosphate is crosslinked with the chitosan microspheres, the mechanical strength of the microspheres is improved, the fixing strength of the chitosan microspheres to beta-fructofuranosidase is improved, the pH stability is obviously improved, the stability of the enzyme is enhanced while the material flow is facilitated, the reuse rate or the use period of the enzyme is improved, the production cost is reduced, and the application prospect is wide;
(2) The immobilized beta-fructofuranosidase prepared by the invention is used for preparing lactosucrose without high-temperature enzyme deactivation, so that the energy is saved, the pigment generated by feed liquid is less, the addition of activated carbon is less, the addition of chemical reagents during pH control is reduced, and the production efficiency is obviously improved.
Drawings
FIG. 1 is a diagram showing the enzyme activity analysis of the immobilized beta-fructofuranosidase prepared in example 1 under different pH conditions.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
Example 1
Preparation of immobilized beta-fructofuranosidase:
(1) Preparing modified chitosan microspheres: dripping a mixed solution containing 5wt% of chitosan, 5wt% of polyquaternium-6 and 5wt% of acetic acid into 0.01 mol/L sodium carbonate solidification solution, continuously stirring, heating in a water bath at 70 ℃, standing for 7h to solidify the chitosan into microspheres, and washing the microspheres for three times by using an ethanol water solution (80% ethanol) to obtain modified chitosan microspheres;
(2) Crosslinking the chitosan microspheres: uniformly dispersing the modified chitosan microspheres obtained in the step (1) in a 1mol/L NaOH solution to obtain a modified chitosan microsphere sodium hydroxide dispersion solution, wherein the mass concentration of the chitosan microspheres in the modified chitosan microsphere sodium hydroxide dispersion solution is 60%, then adding sodium tripolyphosphate (the concentration of the sodium tripolyphosphate in the modified chitosan microsphere sodium hydroxide dispersion solution is 1 wt%), carrying out water bath at 50 ℃ and shaking for 5 hours to carry out a crosslinking reaction, washing the solid substances after the reaction is finished with RO water, and obtaining macroporous chitosan microspheres which are stored in the RO water for later use;
(3) Immobilization of the enzyme: dissolving beta-fructofuranosidase in PBS buffer (pH = 7.0), mixing with macroporous chitosan microspheres at a ratio of 30mg (dry enzyme)/g, shaking and mixing at 10 ℃ for 10h, standing at 4 ℃ for 24h, and washing with PBS for three times to obtain immobilized beta-fructofuranosidase.
Example 2
Preparation of immobilized beta-fructofuranosidase:
(1) Preparing modified chitosan microspheres: dripping a mixed solution containing 6wt% of chitosan, 6wt% of polyquaternium-7 and 5.1 wt% of acetic acid into 0.01 mol/L sodium carbonate solidification solution, continuously stirring, heating in a water bath at 60 ℃, standing for 10h to solidify the chitosan into microspheres, and washing the microspheres for three times by using an ethanol water solution (80% ethanol) to obtain modified chitosan microspheres;
(2) Crosslinking the chitosan microspheres: uniformly dispersing the modified chitosan microspheres in the step (1) in a 1mol/L NaOH solution to obtain a modified chitosan microsphere sodium hydroxide dispersion solution, wherein the mass concentration of the chitosan microspheres in the modified chitosan microsphere sodium hydroxide dispersion solution is 70%, then adding sodium tripolyphosphate (the concentration of the sodium tripolyphosphate in the modified chitosan microsphere sodium hydroxide dispersion solution is 1 wt%), carrying out water bath at 60 ℃ and shaking for 10 hours to carry out crosslinking reaction, washing the solid substances with RO water after the reaction is finished to obtain macroporous chitosan microspheres, and storing the macroporous chitosan microspheres in the RO water for later use;
(3) Immobilization of the enzyme: dissolving beta-fructofuranosidase in PBS buffer solution (pH = 7.0), mixing with macroporous chitosan microspheres at a ratio of 30mg/g, shaking and mixing at a low temperature of 8 ℃ for 10h, standing at a temperature of 4 ℃ for 24h, and washing with PBS for three times to obtain immobilized beta-fructofuranosidase.
Example 3
Preparation of immobilized beta-fructofuranosidase:
(1) Preparing modified chitosan microspheres: dripping a mixed solution containing 8wt% of chitosan, 8wt% of polyquaternium-10 and 5.2% of acetic acid into 0.01 mol/L sodium carbonate solidification solution, continuously stirring, heating in water bath at 80 ℃, standing for 10h to solidify the chitosan into microspheres, and washing the microspheres with ethanol aqueous solution (80% ethanol) for three times to obtain modified chitosan microspheres;
(2) Crosslinking the chitosan microspheres: uniformly dispersing the modified chitosan microspheres in the step (1) in a 1mol/L NaOH solution to obtain a modified chitosan microsphere sodium hydroxide dispersion solution, wherein the mass concentration of the chitosan microspheres in the modified chitosan microsphere sodium hydroxide dispersion solution is 70%, then adding sodium tripolyphosphate (the concentration of the sodium tripolyphosphate in the modified chitosan microsphere sodium hydroxide dispersion solution is 1 wt%), carrying out water bath at 60 ℃ and shaking for 10 hours to carry out crosslinking reaction, washing the solid substances with RO water after the reaction is finished to obtain macroporous chitosan microspheres, and storing the macroporous chitosan microspheres in the RO water for later use;
(3) Immobilization of the enzyme: dissolving beta-fructofuranosidase in PBS buffer (pH = 7.0), mixing with macroporous chitosan microspheres at a ratio of 30mg (dry enzyme)/g, shaking and mixing at 10 ℃ for 20h, standing at 4 ℃ for 24h, and washing with PBS for three times to obtain immobilized beta-fructofuranosidase.
Example 4
A method for preparing lactosucrose using the immobilized β -fructofuranosidase prepared in example 1:
(1) Filling the immobilized beta-fructofuranosidase-chitosan microspheres in a column type packed bed, wherein the filling amount is 90% of the column volume;
(2) Dissolving sucrose and lactose (the mass ratio of sucrose to lactose is 1;
(3) And (3) high-concentration decolorization: adding active carbon into the lactulose oligosaccharide mixed solution prepared in the step (2) according to 0.5wt% of dry basis, fully and uniformly mixing, preserving the heat at 55-60 ℃ for 40min, filtering to remove the active carbon, wherein the light transmittance of the product reaches 98%;
(4) Desalting of continuous ion-exchange traveling materials: the decolorized feed liquid is treated by continuous ion exchange resin, and the electric conductivity is reduced to less than or equal to 15 mu s/cm;
(5) And (3) chromatographic separation: concentrating the desalted feed liquid by vacuum evaporation until the sugar concentration is 50%, performing six-column mode sequential intermittent operation purification and separation by using potassium chromatographic resin, wherein the treatment capacity is 0.05 kg dry basis/(cubic meter per hour), the feed-water ratio is 1.5, the temperature is 65 ℃, the discharge purity is more than or equal to 98%, collecting the lactosucrose liquid, and concentrating;
(6) And (3) vacuum drying: and (4) putting the concentrated liquid into vacuum belt type drying at the drying temperature of 50 ℃ to obtain the lactosucrose.
Comparative example 1
Preparation of immobilized beta-fructofuranosidase:
(1) Preparing modified chitosan microspheres: dripping a mixed solution containing 5wt% of chitosan, 5wt% of ammonium carbonate and 5wt% of acetic acid into 0.01 mol/L sodium carbonate coagulating liquid, continuously stirring, heating in a water bath at 70 ℃, standing for 7h to coagulate the chitosan into microspheres, and washing the precipitate with an ethanol water solution (80% ethanol) for three times to obtain modified chitosan microspheres;
(2) Crosslinking the chitosan microspheres: uniformly dispersing the chitosan microspheres in the step (1) in 1mol/L NaOH solution, then adding sodium tripolyphosphate (the concentration of the sodium tripolyphosphate in the dispersion liquid is 1 wt%), carrying out water bath at 50 ℃ and shaking for 5 hours, washing the solid substances after the reaction is finished with RO water to obtain macroporous chitosan microspheres, and storing the macroporous chitosan microspheres in the RO water for later use;
(3) Immobilization of the enzyme: dissolving beta-fructofuranosidase in PBS buffer (pH = 7.0), mixing with macroporous chitosan microspheres at a ratio of 30mg (dry enzyme)/g, shaking and mixing at 10 ℃ for 10h, standing at 4 ℃ for 24h, and washing with PBS for three times to obtain immobilized beta-fructofuranosidase.
Comparative example 2
(1) Preparing modified chitosan microspheres: dripping a mixed solution containing 5wt% of chitosan, 5wt% of polyquaternium and 5wt% of acetic acid into 0.01 mol/L sodium carbonate coagulating liquid, continuously stirring, heating in a water bath at 70 ℃, standing for 7h to coagulate the chitosan into microspheres, and washing the precipitate with an ethanol water solution (80% ethanol) for three times to prepare the modified chitosan microspheres;
(2) Crosslinking the chitosan microspheres: uniformly dispersing the chitosan microspheres in the step (1) in 1mol/L NaOH solution, then adding glutaraldehyde (the concentration of the glutaraldehyde in the dispersion solution is 1 wt%), carrying out water bath at 50 ℃ and shaking for 5 hours, washing the solid substances after the reaction is finished with RO water to obtain macroporous chitosan microspheres, and storing the macroporous chitosan microspheres in the RO water for later use;
(3) Immobilization of the enzyme: dissolving beta-fructofuranosidase in PBS buffer (pH = 7.0), mixing with macroporous chitosan microspheres at a ratio of 30mg (dry enzyme)/g, shaking and mixing at 10 ℃ for 10h, standing at 4 ℃ for 24h, and washing with PBS for three times to obtain immobilized beta-fructofuranosidase.
Performance analysis of immobilized beta-fructofuranosidase
(1) Analysis of immobilization efficiency of immobilized beta-fructofuranosidase:
the immobilization efficiency (the amount of enzyme immobilized on the carrier as a percentage of the total amount of free enzyme added under the same conditions) of the immobilized β -fructofuranosidase prepared in examples 1 to 3 and comparative examples 1 to 2 was measured, and the results are shown in the following index 1:
TABLE 1 analysis results of immobilization efficiency of immobilized beta-fructofuranosidase
As can be seen from Table 1, the chitosan microspheres are modified by the polyquaternium and crosslinked by the tripolyphosphoric acid, so that the fixation strength of beta-fructofuranosidase is enhanced while the enzyme and binding sites are increased, and the immobilization efficiency is improved.
(2) Analysis of pH stability of immobilized beta-fructofuranosidase
The pH stability of the immobilized β -fructofuranosidase prepared in example 1 was analyzed by taking the β -fructofuranosidase immobilized enzyme, soaking it in phosphate buffer solutions with different pH (pH =5, 6, 7, 8, 9) for a certain period of time, then measuring the enzyme activity with a mixed solution of sucrose and lactose, taking the enzyme activity of the immobilized enzyme not soaked in the phosphate buffer solution as 100%, and calculating the remaining enzyme activity of the immobilized enzyme after the treatment. The results are shown in FIG. 1. As shown in figure 1, when the pH value of the beta-fructofuranosidase immobilized enzyme is 6, the enzyme activity is more than 85 percent; when the pH value is 7, the enzyme activity is 100 percent; at pH 8, the enzyme activity was 95%.
(3) Reuse rate of immobilized beta-fructofuranosidase
Measuring the repeated use times of the immobilized beta-fructofuranosidase by using a batch reaction, taking a phosphate buffer solution (pH = 7) with the total mass volume concentration of 60% of sucrose and lactose (the mass ratio of the sucrose to the lactose is 1) as a substrate, adding 10% (w/w) of the immobilized beta-fructofuranosidase, reacting at 40 ℃, adding and stirring, measuring the content of low-polymerized lactulose in the mixed solution after reacting for 3 hours, and measuring the conversion rate; then, filtering to obtain immobilized enzyme, adding a new phosphate buffer (pH = 7) of sucrose and lactose with a total mass volume concentration of 60% (mass ratio of sucrose to lactose is 1) to perform a second reaction, measuring the content of lactosucrose after the reaction is performed for 3h, and measuring the conversion rate; the above steps are repeated for 50 rounds of reaction. The conversion rates of the immobilized beta-fructofuranosidase obtained in examples 1 to 3 and comparative examples 1 to 2 were measured, and the results are shown in Table 2 below;
(4) Half-life test: the half-life of the immobilized β -fructofuranosidase prepared in examples 1 to 3 and comparative examples 1 to 2 was tested by filling the immobilized β -fructofuranosidase into a column-type packed bed, optimizing the reaction conditions and parameters, controlling the temperature of an insulating jacket at 50 ℃, passing a phosphate buffer (pH = 7) having a total mass volume concentration of 60% sucrose and lactose (the mass ratio of sucrose to lactose is 1) through the packed bed at a flow rate of 1.0V column volume/h, and testing the results as shown in table 2 below.
TABLE 2 reuse rates of immobilized beta-fructofuranosidases
Claims (8)
1. An immobilized beta-fructofuranosidase prepared by the following method:
(1) Adding a mixed solution obtained by mixing chitosan, polyquaternium and acetic acid into a carbonate solidification solution to prepare modified chitosan microspheres;
the polyquaternium is one of polyquaternium-6, polyquaternium-7 and polyquaternium-10;
(2) Dispersing the modified chitosan microspheres prepared in the step (1) in a sodium hydroxide solution to obtain a modified chitosan microsphere sodium hydroxide dispersion solution, then adding tripolyphosphate into the modified chitosan microsphere sodium hydroxide dispersion solution to perform a crosslinking reaction, and obtaining macroporous chitosan microspheres after the reaction is finished;
(3) Mixing the beta-fructofuranosidase liquid with the macroporous chitosan microspheres, and oscillating at low temperature to obtain immobilized beta-fructofuranosidase;
the reaction condition for preparing the modified chitosan microspheres in the step (1) is heating in water bath at 60-70 ℃ for 7-10h; in the step (2), the crosslinking reaction condition is heating in water bath at 50-60 ℃ for 5-10h; the low-temperature oscillation temperature in the step (3) is 8-10 ℃, and the time is 10-20h.
2. The immobilized β -fructofuranosidase according to claim 1, wherein the concentration of chitosan in the mixed solution of step (1) is 5 to 8wt%, the concentration of polyquaternium is 5 to 8wt%, and the concentration of acetic acid is 5 to 5.2 wt%.
3. The immobilized beta-fructofuranosidase according to claim 1, wherein the concentration of the sodium hydroxide solution in step (2) is 1mol/L, and the mass concentration of the modified chitosan microspheres in the modified chitosan microsphere sodium hydroxide dispersion is 60-70%.
4. The immobilized beta-fructofuranosidase according to claim 1, wherein the concentration of tripolyphosphate in the sodium hydroxide dispersion of the modified chitosan microspheres after tripolyphosphate addition in step (2) is 1.0wt%.
5. The immobilized beta-fructofuranosidase according to claim 1, wherein the mass ratio of dry enzyme to macroporous chitosan microspheres in the beta-fructofuranosidase liquid in step (3) is 30mg:1g.
6. A method for preparing lactosucrose by using the immobilized beta-fructofuranosidase according to any one of claims 1 to 5, which is characterized by comprising the following steps:
(1) Filling the immobilized beta-fructofuranosidase in a column type packed bed;
(2) Dissolving sucrose and lactose in PBS buffer solution to prepare mixed solution, and allowing the mixed solution to pass through a column type packed bed at the flow rate of 1.0V column volume/h, wherein the reaction temperature is 40 ℃, so as to obtain lactosucrose mixed solution;
the total mass volume concentration of the sucrose and the lactose in the mixed solution is 60 percent;
(3) And refining the lactulose oligosaccharide mixed solution to obtain the lactulose oligosaccharide.
7. The method for preparing lactosucrose using immobilized β -fructofuranosidase according to claim 6, wherein the mass ratio of sucrose to lactose in step (2) is 1; the pH of the PBS buffer was 7.0.
8. The method for preparing lactosucrose using immobilized β -fructofuranosidase according to claim 6, wherein the refining step in step (3) comprises high concentration decolorization, continuous ion desalting, chromatographic separation, and vacuum drying.
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