Process optimization method for producing dextran from beet

Abstract

The invention relates to the field of dextran production, and particularly discloses a process optimization method for producing dextran from beet, which comprises the following steps: pretreating beet juice by pectinase to obtain a high-purity substrate, innovatively adopting a dextransucrase and alpha-1, 3 branching enzyme compound catalytic system, and combining with a Mn < 2 + > cofactor and a Tween 80 stabilizer to improve the enzyme activity efficiency, thereby obtaining the high-purity dextran. The molecular weight distribution is regulated and controlled in real time through endoglucanase, and a multi-stage ethanol precipitation and purification process is matched, so that the purity of the product reaches 98% or above and PDIlt; 1.25, resource utilization of single-cell protein produced by waste liquid fermentation is originally realized, and the sucrose conversion rate of the whole process is gt; the recovery rate of dextran is greater than or equal to 85%. Compared with a traditional method, the method has the advantages of being low in raw material cost, high in molecular weight controllability, environmentally friendly and the like, and is suitable for industrial production of pharmaceutical-grade dextran.

Description

Technical Field

[0001] This invention relates to the field of dextran production, and more particularly to a process optimization method for producing dextran from sugar beets. Background Technology

[0002] Traditional dextran production processes mainly employ sucrose fermentation, a mature technology that produces products with uniform molecular weight distribution. It is widely used in the pharmaceutical and food industries. This method generates dextran through microbial fermentation, offering advantages such as high product purity and good biocompatibility. Furthermore, the molecular weight range can be adjusted by controlling fermentation conditions to meet diverse application needs.

[0003] However, traditional sucrose fermentation has the following drawbacks: First, it relies on sucrose as the sole carbon source, resulting in production costs accounting for more than 60% of the total product cost; second, the molecular weight distribution of the products during fermentation is wide (20-90kDa), making it difficult to precisely control to the pharmaceutical-grade range of 40-70kDa; third, the COD value of the fermentation waste liquid is as high as 20,000mg / L, requiring a large amount of ethanol (≥3 times the volume) for purification, resulting in high treatment costs; and fourth, the fermentation process takes 24-36 hours, leading to low equipment utilization. Summary of the Invention

[0004] The purpose of this invention is to provide an optimized process for producing dextran from sugar beets, aiming to solve the following problems in the existing technology: First, it relies on sucrose as the sole carbon source, resulting in production costs accounting for more than 60% of the total product cost; second, the molecular weight distribution of the product during fermentation is wide (20-90kDa), making it difficult to accurately control it to the pharmaceutical-grade range of 40-70kDa; third, the COD value of the fermentation waste liquid is as high as 20,000mg / L, requiring a large amount of ethanol (≥3 times the volume) for purification, resulting in high treatment costs; fourth, the fermentation process takes 24-36 hours, leading to low equipment utilization.

[0005] To achieve the above objectives, the present invention provides an optimized process for producing dextran from sugar beets, comprising the following steps:

[0006] Step S1: Add pectinase to the crushed beets and keep warm at 50℃ for 1.5 hours to degrade the pectin. After pressing out the juice, decolorize it by adsorption with activated carbon, and then filter it through a 0.2μm membrane to remove bacteria, to obtain a crude sucrose solution.

[0007] Step S2: Select dextran sucrase and α-1,3 branched enzyme in a 3:1 ratio to form a complex enzyme preparation, and then prepare it at pH 5.8-6.2, temperature 45-55℃, and Mn 2+ The catalytic reaction was carried out using a pretreated crude sucrose solution as a substrate at a concentration of 0.5 mmol / L.

[0008] Step S3: After the reaction is complete, the enzyme is inactivated by heat treatment at 80℃ for 10 minutes, followed by separation and preliminary purification of the product;

[0009] Step S4: During the catalytic reaction, the molecular weight of the product is controlled in real time by adding 0.1-0.3 U / g sucrose endoglucanase, so that it is concentrated in the range of 40-70 kDa;

[0010] Step S5: Remove impurities and proteins with 30% ethanol, precipitate high molecular weight impurities with 50% ethanol, and precipitate the target dextran with 70% ethanol, with a recovery rate of ≥85%; redissolve the crude dextran obtained by precipitation in water, desalt it by ion exchange, and then spray dry it to obtain a finished dextran product with a purity of ≥98%.

[0011] Step S6: Inoculate the reaction waste liquid with brewer's yeast for fermentation to produce single-cell protein, thereby realizing the resource utilization of the waste liquid.

[0012] In steps S1 and S2, the concentration of the crude sucrose solution is 11-22%.

[0013] In step S2, the enzyme activity of the compound enzyme preparation reaches 1200 U / mL, and 0.05% Tween 80 is added.

[0014] In step S4, the viscosity of the reaction solution and the sucrose conversion rate are detected by sampling at regular intervals. When the viscosity of the reaction solution reaches the preset value and the sucrose conversion rate is greater than 90%, endoglucanase is added to regulate the molecular weight.

[0015] In step S5, during the ethanol fractionation precipitation process, the concentration of ethanol at each stage is adjusted by slowly adding 95% ethanol to the reaction solution, and the precipitation process is carried out by standing at 4°C.

[0016] In step S5, the molecular weight distribution index of the finished dextran is less than 1.25.

[0017] In step S6, the amount of brewing yeast inoculated is 1%-10% of the waste liquid volume, the fermentation temperature is 25℃-35℃, and the fermentation time is 12-48 hours.

[0018] This invention provides an optimized process for producing dextran from beets. It obtains high-purity substrate by pretreating beet juice with pectinase, and innovatively employs a combined catalytic system of dextran sucrase and α-1,3-branched enzymes, combined with Mn... 2+Cofactors and Tween 80 stabilizers enhance enzyme activity efficiency. Real-time regulation of molecular weight distribution (40-70 kDa) via endoglucanase, combined with a multi-stage ethanol precipitation purification process, achieves product purity exceeding 98% and a PDI < 1.25. This innovative method realizes the resource utilization of waste liquid fermentation for single-cell protein production. The overall process achieves a sucrose conversion rate > 90% and a dextran recovery rate ≥ 85%. Compared to traditional methods, this invention offers advantages such as low raw material cost, strong molecular weight controllability, and environmental friendliness, making it suitable for the industrial production of pharmaceutical-grade dextran. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a flowchart of the process optimization method for producing dextran from beets according to the present invention.

[0021] Figure 2 This is a flowchart of the steps in Embodiment 1 of the present invention.

[0022] Figure 3 This is a flowchart of the steps in Embodiment 2 of the present invention.

[0023] Figure 4 This is a flowchart of the steps in Embodiment 3 of the present invention.

[0024] Figure 5 This is a process flow diagram of the optimized method for producing dextran from beets according to the present invention. Detailed Implementation

[0025] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0026] Please see Figure 1 and Figure 5 This invention provides an optimized process for producing dextran from sugar beets, comprising the following steps:

[0027] Step S1: Add pectinase to the crushed beets and keep warm at 50℃ for 1.5 hours to degrade the pectin. After pressing out the juice, decolorize it by adsorption with activated carbon, and then filter it through a 0.2μm membrane to remove bacteria, to obtain a crude sucrose solution.

[0028] Step S2: Select dextran sucrase and α-1,3 branched enzyme in a 3:1 ratio to form a complex enzyme preparation, and then prepare it at pH 5.8-6.2, temperature 45-55℃, and Mn 2+ The catalytic reaction was carried out using a pretreated crude sucrose solution as a substrate at a concentration of 0.5 mmol / L.

[0029] Step S3: After the reaction is complete, the enzyme is inactivated by heat treatment at 80℃ for 10 minutes, followed by separation and preliminary purification of the product;

[0030] Step S4: During the catalytic reaction, the molecular weight of the product is controlled in real time by adding 0.1-0.3 U / g sucrose endoglucanase, so that it is concentrated in the range of 40-70 kDa;

[0031] Step S5: Remove impurities and proteins with 30% ethanol, precipitate high molecular weight impurities with 50% ethanol, and precipitate the target dextran with 70% ethanol, with a recovery rate of ≥85%; redissolve the crude dextran obtained by precipitation in water, desalt it by ion exchange, and then spray dry it to obtain a finished dextran product with a purity of ≥98%.

[0032] Step S6: Inoculate the reaction waste liquid with brewer's yeast for fermentation to produce single-cell protein, thereby realizing the resource utilization of the waste liquid.

[0033] Furthermore, in steps S1 and S2, the concentration of the crude sucrose solution is 11-22%.

[0034] Furthermore, in step S2, the enzyme activity of the compound enzyme preparation reaches 1200 U / mL, and 0.05% Tween 80 is added.

[0035] Furthermore, in step S4, the viscosity of the reaction solution and the sucrose conversion rate are detected by sampling at regular intervals. When the viscosity of the reaction solution reaches the preset value and the sucrose conversion rate is greater than 90%, endoglucanase is added to regulate the molecular weight.

[0036] Furthermore, in step S5, during the ethanol fractionation precipitation process, the concentration of ethanol at each stage is adjusted by slowly adding 95% ethanol to the reaction solution, and the precipitation process is carried out by standing at 4°C.

[0037] Furthermore, in step S5, the molecular weight distribution index of the finished dextran is less than 1.25.

[0038] Furthermore, in step S6, the inoculation amount of brewing yeast is 1%-10% of the waste liquid volume, the fermentation temperature is 25℃-35℃, and the fermentation time is 12-48 hours.

[0039] The beneficial effects of this invention are as follows: First, cost reduction: beet juice replaces refined sucrose, reducing raw material costs by 60%; Second, efficiency improvement: reaction time is shortened to 8 hours (traditional fermentation requires 24-36 hours); Third, environmental advantages: wastewater COD is reduced by 80%, and ethanol usage is reduced by 50%; Fourth, product quality: dextran purity ≥98%, molecular weight distribution index (PDI) <1.25.

[0040] Example 1, please refer to Figure 2 and Figure 5 This invention proposes an optimized process for producing dextran from sugar beets, comprising the following steps:

[0041] S1: The beets are thoroughly crushed, pectinase is added, and the mixture is kept at 50℃ for 1.5 hours to degrade the pectin. The degraded material is pressed to obtain juice, which is then decolorized by activated carbon adsorption and sterilized by filtration through a 0.2μm membrane to obtain a crude sucrose solution with a concentration of 11%.

[0042] S2: Dextran sucrase and α-1,3-branched enzyme were mixed in a 3:1 ratio to form a complex enzyme preparation, ensuring an enzyme activity of 1200 U / mL. 0.05% Tween 80 was added, and crude sucrose solution was used as the substrate. The mixture was prepared at pH 5.8, temperature 45℃, and Mn... 2+ The catalytic reaction was initiated at a concentration of 0.5 mmol / L and a substrate concentration of 11%.

[0043] S3: After the catalytic reaction reaches the preset extent, the reaction system is transferred to 80℃ for heat treatment for 10 minutes to inactivate the enzyme. The product is then separated and preliminarily purified.

[0044] S4: During the catalytic reaction, the viscosity of the reaction solution and the sucrose conversion rate are sampled and detected at regular intervals. When the viscosity of the reaction solution reaches the preset value and the sucrose conversion rate is greater than 90%, endoglucanase is added at a ratio of 0.1 U / g sucrose, and the molecular weight of the product is adjusted to 40-70 kDa in real time.

[0045] S5: Using ethanol fractionation precipitation, first slowly add 95% ethanol to adjust to a concentration of 30%, and let stand at 4°C to remove impurities and proteins; then add 95% ethanol to adjust to a concentration of 50%, and let stand at 4°C to precipitate high molecular weight impurities; finally adjust to a concentration of 70%, and let stand at 4°C to precipitate the target dextran, with a recovery rate ≥85%. The crude precipitate is redissolved in water, desalted by ion exchange, and then spray-dried to obtain a finished dextran product with a purity ≥98% and a molecular weight distribution index of less than 1.25.

[0046] S6: Collect the reaction waste liquid, inoculate it with 1% volume of brewer's yeast, ferment at 25℃ for 12 hours to produce single-cell protein, and realize the resource utilization of waste liquid.

[0047] Example 2, please refer to Figure 3 and Figure 5 This invention proposes an optimized process for producing dextran from sugar beets, comprising the following steps:

[0048] S1: Thoroughly crush the beets, add pectinase, keep warm at 50℃ for 1.5 hours to degrade the pectin, press the degraded material to collect the juice, decolorize it through an activated carbon adsorption column, filter it with a 0.2μm microporous membrane to remove bacteria, and obtain a crude sucrose solution with a concentration of 16%.

[0049] S2: Dextran sucrase and α-1,3-branched enzyme were mixed in a 3:1 ratio to form a complex enzyme preparation, ensuring an enzyme activity of 1200 U / mL. 0.05% Tween 80 was added, and crude sucrose solution was used as the substrate. The mixture was prepared at pH 6.0, 50℃, and Mn... 2+ The catalytic reaction was initiated at a concentration of 0.5 mmol / L and a substrate concentration of 16%.

[0050] S3: After the catalytic reaction reaches the preset extent, the reaction system is transferred to 80℃ for heat treatment for 10 minutes to inactivate the enzyme. The product is then separated and preliminarily purified.

[0051] S4: During the catalytic reaction, the viscosity of the reaction solution and the sucrose conversion rate are sampled and detected at regular intervals. When the viscosity of the reaction solution reaches the preset value and the sucrose conversion rate is greater than 90%, endoglucanase is added at a ratio of 0.2 U / g sucrose, and the molecular weight of the product is adjusted to 40-70 kDa in real time.

[0052] S5: Using ethanol fractionation precipitation, first slowly add 95% ethanol to adjust to a concentration of 30%, and let stand at 4°C to remove impurities and proteins; then add 95% ethanol to adjust to a concentration of 50%, and let stand at 4°C to precipitate high molecular weight impurities; finally adjust to a concentration of 70%, and let stand at 4°C to precipitate the target dextran, with a recovery rate ≥85%. The crude precipitate is redissolved in water, desalted by ion exchange, and then spray-dried to obtain a finished dextran product with a purity ≥98% and a molecular weight distribution index of less than 1.25.

[0053] S6: Collect the reaction waste liquid, inoculate it with 5% volume of brewer's yeast, ferment at 30℃ for 24 hours to produce single-cell protein, and realize the resource utilization of waste liquid.

[0054] Example 3, please refer to Figure 4 and Figure 5 This invention proposes an optimized process for producing dextran from sugar beets, comprising the following steps:

[0055] S1: Thoroughly crush the beets, add pectinase, keep warm at 50℃ for 1.5 hours to degrade the pectin, press the degraded material to collect the juice, decolorize it through an activated carbon adsorption column, filter it with a 0.2μm microporous membrane to remove bacteria, and obtain a crude sucrose solution with a concentration of 22%.

[0056] S2: Dextran sucrase and α-1,3-branched enzyme were mixed in a 3:1 ratio to form a complex enzyme preparation, ensuring an enzyme activity of 1200 U / mL. 0.05% Tween 80 was added, and crude sucrose solution was used as the substrate. The mixture was prepared at pH 6.2, temperature 55℃, and Mn... 2+ The catalytic reaction was initiated at a concentration of 0.5 mmol / L and a substrate concentration of 22%.

[0057] S3: After the catalytic reaction reaches the preset extent, the reaction system is transferred to 80℃ for heat treatment for 10 minutes to inactivate the enzyme. The product is then separated and preliminarily purified.

[0058] S4: During the catalytic reaction, the viscosity of the reaction solution and the sucrose conversion rate are sampled and detected at regular intervals. When the viscosity of the reaction solution reaches the preset value and the sucrose conversion rate is greater than 90%, endoglucanase is added at a ratio of 0.3 U / g sucrose, and the molecular weight of the product is adjusted to 40-70 kDa in real time.

[0059] S5: Using ethanol fractionation precipitation, first slowly add 95% ethanol to adjust to a concentration of 30%, and let stand at 4°C to remove impurities and proteins; then add 95% ethanol to adjust to a concentration of 50%, and let stand at 4°C to precipitate high molecular weight impurities; finally adjust to a concentration of 70%, and let stand at 4°C to precipitate the target dextran, with a recovery rate ≥85%. The crude precipitate is redissolved in water, desalted by ion exchange, and then spray-dried to obtain a finished dextran product with a purity ≥98% and a molecular weight distribution index of less than 1.25.

[0060] S6: Collect the reaction waste liquid, inoculate it with 10% volume of brewer's yeast, ferment at 35℃ for 48 hours to produce single-cell protein, and realize the resource utilization of waste liquid.

[0061] The above description discloses only one preferred embodiment of the present invention, and should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. An optimized process for producing dextran from sugar beets, characterized in that, Includes the following steps: Step S1: Add pectinase to the crushed beets and keep warm at 50℃ for 1.5 hours to degrade the pectin. After pressing out the juice, decolorize it by adsorption with activated carbon, and then filter it through a 0.2μm membrane to remove bacteria, to obtain a crude sucrose solution. Step S2: Select dextran sucrase and α-1,3 branched enzyme in a 3:1 ratio to form a complex enzyme preparation, and then prepare it at pH 5.8-6.2, temperature 45-55℃, and Mn 2+ The catalytic reaction was carried out using a pretreated crude sucrose solution as a substrate at a concentration of 0.5 mmol / L. Step S3: After the reaction is complete, the enzyme is inactivated by heat treatment at 80℃ for 10 minutes, followed by separation and preliminary purification of the product; Step S4: During the catalytic reaction, the molecular weight of the product is controlled in real time by adding 0.1-0.3 U / g sucrose endoglucanase, so that it is concentrated in the range of 40-70 kDa; Step S5: Remove impurities and proteins with 30% ethanol, precipitate high molecular weight impurities with 50% ethanol, and precipitate the target dextran with 70% ethanol, with a recovery rate of ≥85%; redissolve the crude dextran obtained by precipitation in water, desalt it by ion exchange, and then spray dry it to obtain a finished dextran product with a purity of ≥98%. Step S6: Inoculate the reaction waste liquid with brewer's yeast for fermentation to produce single-cell protein, thereby realizing the resource utilization of the waste liquid.

2. The optimized process for producing dextran from sugar beets as described in claim 1, characterized in that, In steps S1 and S2, the concentration of the crude sucrose solution is 11-22%.

3. The optimized process for producing dextran from sugar beets as described in claim 2, characterized in that, In step S2, the enzyme activity of the compound enzyme preparation reaches 1200 U / mL, and 0.05% Tween 80 is added.

4. The optimized process for producing dextran from sugar beets as described in claim 3, characterized in that, In step S4, the viscosity of the reaction solution and the sucrose conversion rate are detected by sampling at regular intervals. When the viscosity of the reaction solution reaches the preset value and the sucrose conversion rate is greater than 90%, endoglucanase is added to regulate the molecular weight.

5. The optimized process for producing dextran from sugar beets as described in claim 4, characterized in that, In step S5, during the ethanol fractionation precipitation process, the concentration of ethanol at each stage is adjusted by slowly adding 95% ethanol to the reaction solution, and the precipitation process is carried out by standing at 4°C.

6. The optimized process for producing dextran from sugar beets as described in claim 5, characterized in that, In step S5, the molecular weight distribution index of the finished dextran is less than 1.

25.

7. The optimized process for producing dextran from sugar beets as described in claim 6, characterized in that, In step S6, the inoculation amount of brewing yeast is 1%-10% of the waste liquid volume, the fermentation temperature is 25℃-35℃, and the fermentation time is 12-48 hours.

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