High-conversion-rate dextrin and preparation method thereof
By combining jackfruit seed starch, ginger starch, and potato starch, and pretreating with ultrasound and microwave, the enzymatic hydrolysis parameters were optimized, solving the problems of low conversion rate and high cost in the preparation of α-cyclodextrin, and realizing the preparation of α-cyclodextrin with high efficiency and low cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-03-27
AI Technical Summary
Existing methods for preparing α-cyclodextrin suffer from low conversion rates, high costs, and high energy consumption. Furthermore, traditional enzymatic hydrolysis methods are subject to stringent conditions, hindering their industrial-scale application.
Highly active cyclodextrin glucotransferase was prepared by using a blend of jackfruit seed starch, ginger starch, and standard potato starch as starch sources and fermentation with Bacillus Y112. The enzyme hydrolysis parameters, including relative dissolved oxygen and enzyme dosage, were optimized through combined ultrasonic and microwave pretreatment before enzymatic hydrolysis and purification.
It significantly improved the conversion rate of α-cyclodextrin to 42.4%-49.1%, reduced production costs, simplified the operation process, and facilitated industrial application.
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Figure CN121737232A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the field of starch refining technology, specifically relating to a high-conversion-rate dextrin and its preparation method. Background Technology
[0002] In existing technologies, α-cyclodextrin, as an important member of the cyclodextrin family, plays a crucial role in various fields such as pharmaceuticals, food, agriculture, and cosmetics. Its unique cavity structure allows it to encapsulate various substances, improving the solubility, stability, and safety of target substances, enhancing product quality and application effects, and driving technological upgrades and product innovation in related industries. However, current α-cyclodextrin preparation methods on the market have some shortcomings.
[0003] In existing technologies, the preparation of α-cyclodextrin mainly relies on enzymatic conversion. This method utilizes cyclodextrin glucotransferase (CGT enzyme) to react with substrates such as starch or polysaccharides to generate α-cyclodextrin. Depending on whether organic solvents are added during the production process, it can be divided into organic solvent methods and solvent-free methods. While organic solvent methods can achieve higher yields of α-cyclodextrin and meet the needs of large-scale production to some extent, the organic solvents used in the process are mostly toxic reagents. These require subsequent separation and recovery through high temperature and high pressure methods, which is not only energy-intensive and dangerous, but also limits the application of α-cyclodextrin in food, pharmaceutical, and other fields with strict safety requirements. Furthermore, it also poses certain environmental hazards. Solvent-free methods, on the other hand, do not add organic solvents during the production process, representing an environmentally friendly new production method that aligns with the trend of green production and particularly meets the needs of the food, pharmaceutical, and cosmetic industries. However, this method has significant technical shortcomings. On the one hand, the conversion rate of α-cyclodextrin is low, the reaction specificity of CGT enzyme is insufficient, and its activity is easily affected by reaction conditions, resulting in low substrate utilization. On the other hand, the raw material cost is high, usually requiring potato starch as the substrate. Finding a low-cost substitute for potato starch is an urgent technical problem to be solved in dextrin production. Furthermore, product extraction is difficult during the preparation process. Due to the high water solubility of α-cyclodextrin, it is difficult to separate and purify it using conventional physical methods, further increasing production energy consumption and costs, thus hindering its industrialization. In addition, the traditional enzymatic hydrolysis method also suffers from harsh reaction conditions and long reaction times, further reducing the production efficiency and economic benefits of α-cyclodextrin.
[0004] Therefore, in order to comprehensively improve the conversion rate of α-cyclodextrin preparation, reduce production energy consumption and costs, and at the same time ensure product purity and safety, and adapt to the growing demand for α-cyclodextrin in various fields, it is now urgent to improve the existing α-cyclodextrin preparation methods to enhance production efficiency and product competitiveness. Summary of the Invention
[0005] This application aims to address the technical problems in the existing enzymatic hydrolysis method for preparing dextrin, such as low α-cyclodextrin conversion rate, insufficient specificity and activity of cyclodextrin glucotransferase, stringent reaction conditions, long reaction time, and high preparation cost due to the lack of low-cost potato starch substitutes, which restricts industrial promotion. Therefore, this application proposes a method for preparing dextrin with high conversion rate.
[0006] In order to solve the technical problems raised in this application, this application also provides a dextrin, which is prepared by the above-described method for preparing a high-conversion dextrin.
[0007] This application adopts the following scheme: a method for preparing dextrin with high conversion rate, comprising the following steps: Step 101. Add the starch source and buffer solution into the stirred tank in a mass ratio of 1:(12.4-15.5) and disperse them at room temperature and 1200rpm-1800rpm for 20min-30min to obtain reaction system A. Step 102. The reaction system A prepared in step 101 and the highly active cyclodextrin glucotransferase are sequentially added into the reaction vessel and reacted for 1.5h-3h at 30℃-35℃, 200rpm-440rpm and pH 6-8 to obtain reaction system B. Step 103. The reaction system B prepared in step 102 is subjected to resin desalting, decolorization, chromatographic purification, concentration and spray drying in sequence to obtain the dextrin product; In step 101, reaction system A undergoes a combined ultrasonic and microwave pretreatment; the starch source is obtained by compounding jackfruit seed starch, ginger starch, and standard potato starch in a mass ratio of 5:(2.1-2.4):1. In step 102, the highly active cyclodextrin glucotransferase is obtained by fermentation of Bacillus Y112; the conversion rate of α-cyclodextrin is 42.4%-49.1%.
[0008] In practice, standard potato starch was purchased from Beijing Solarbio Science & Technology Co., Ltd. Bacillus Y112 was purchased from the China Industrial Microbial Culture Collection Center (CICC), strain number CICC 1528.
[0009] In some feasible embodiments, the method for preparing the starch source includes the following steps: Step 201. Put the jackfruit seeds and ginger pieces into the grinder in sequence according to the preset target ratio, and grind them at high speed at 3000 rpm for 5 min-10 min at room temperature to obtain the raw material powder. Step 202. Filter the raw material pulverized material obtained in step 201 at room temperature and 0.8MPa-1.0MPa and collect the first filtrate. Then transfer the solid residue obtained from the filtration back to the pulverizer and pulverize it at high speed at 3000rpm for 5min-10min at room temperature to obtain the second pulverized material. Filter the second pulverized material at room temperature and 0.8MPa-1.0MPa and collect the second filtrate. Mix the first filtrate and the second filtrate to obtain the crude starch emulsion. Step 203. Transfer the crude starch emulsion prepared in step 202 to a centrifuge and centrifuge at room temperature and 5000rpm-8000rpm for 10min-15min. Repeat this process 3-5 times. Discard the supernatant, collect the precipitate and transfer it to an oven. Dry it at 40℃ to constant weight to obtain a mixture of jackfruit seed starch and ginger starch. Step 204. Mix the mixture of jackfruit seed starch and ginger starch prepared in step 203 with standard potato starch in a preset ratio to obtain the starch source.
[0010] In practice, the jackfruit seeds were purchased from the jackfruit processing plant affiliated with Nanguo Food Co., Ltd., and the seeds originated from Malaysian No. 1 (M1) jackfruit.
[0011] In some feasible embodiments, step 103, the method for preparing the highly active cyclodextrin glucotransferase includes the following steps: Step 301. Prepare seed solution: Use a sterile inoculation loop to scrape a loop of Bacillus Y112 and inoculate it into 300ml of seed solution. After culturing at 30℃ and 120rpm for 24h, the seed solution is obtained. Step 302. Fermentation: The seed liquid prepared in step 301 is inoculated into a fermenter containing the liquid to be fermented. After fermentation for 48 hours at a fermentation temperature of 27℃, 150 rpm and a relative dissolved oxygen of 30%-40%, the fermentation liquid is obtained. Step 303. The fermentation broth prepared in step 302 is subjected to sodium alginate flocculation, filtration, centrifugation, ultrafiltration membrane filtration, ethanol precipitation, and freeze drying to obtain highly active cyclodextrin glucotransferase powder.
[0012] In some feasible embodiments, in step 301, the seed solution is prepared by mixing nutrient C and deionized water at a mass ratio of 1:(10.5-12.3); By weight, the nutrient C consists of the following components: 11-13.5 parts standard potato starch, 5.4-6.6 parts peptone, 5.4-6.6 parts yeast extract, 0.5-1 part dipotassium hydrogen phosphate, 0.1-0.2 parts magnesium sulfate heptahydrate, 5-6 parts sodium chloride, and 1.12-1.33 parts anhydrous sodium carbonate.
[0013] In some feasible embodiments, in step 302, the mass ratio of the seed liquid to the fermentation liquid is (1-2):25.
[0014] In some feasible embodiments, in step 302, the fermentation broth is obtained by compounding nutrient D and deionized water at a mass ratio of 1:(11.4-12.2); by mass parts, nutrient D consists of the following components: 6.6-8.2 parts of standard potato starch, 16.2-20.3 parts of peptone, 8.2-10.1 parts of yeast extract, 0.1-0.4 parts of manganese chloride tetrahydrate, 4.5-5.5 parts of sodium chloride, and 1.33-1.66 parts of anhydrous sodium carbonate.
[0015] In some feasible embodiments, in step 101, the pretreatment of reaction system A includes the following steps: transferring reaction system A to an ultrasonic reactor and treating it for 5 min to 10 min under ultrasonic frequency of 10 kHz to 15 kHz and microwave temperature of 80 ℃ to 88 ℃.
[0016] In practice, by pretreating reaction system A with a combination of ultrasound and microwave, the crystalline structure of starch can be disrupted. The ultrasonic cavitation effect generates local high temperature and pressure, which can break the hydrogen bonds between starch molecules. The microwave thermal effect melts the crystalline regions in starch, which can effectively reduce the crystallinity of starch and eliminate enzymatic hydrolysis resistance. Secondly, by pretreating reaction system A, the dispersibility of reaction system A can be effectively improved, avoiding starch agglomeration and allowing starch molecules to be fully dispersed into single molecules / small molecule aggregates. This can effectively increase the contact area between CGT enzyme and starch source. Moreover, the viscosity of reaction system A is reduced after pretreatment, and α-cyclodextrin products diffuse rapidly, reducing the product's occupation of the enzyme active site (weakening the product inhibition effect). Furthermore, the combined ultrasound and microwave pretreatment can optimize the conformation of starch molecules, causing the α-1,4 glycosidic bonds in starch molecules to unfold, making it easier for the CGT enzyme active site to recognize and bind, and shortening the induction period of the enzymatic hydrolysis reaction.
[0017] In some feasible embodiments, in step 103, the amount of highly active cyclodextrin glucotransferase added is 52.4 U / g-55.7 U / g, based on each gram of starch source.
[0018] In some feasible embodiments, in step 101, the buffer solution is a sodium citrate solution with a concentration of 8.5 mmol / L to 10 mmol / L.
[0019] In order to solve the technical problems raised in this application, this application also provides a dextrin, which is prepared by the above-described method for preparing high-conversion dextrin.
[0020] Compared with the prior art, this application has the following beneficial effects: This application provides a method for preparing high-conversion dextrin, comprising four steps: preparation of reaction system A, pretreatment of reaction system A, enzymatic hydrolysis, and purification. By using a mixture of jackfruit seed starch, ginger starch, and standard potato starch at a mass ratio of 5:(2.1-2.4):1 as the starch source, the amount of standard potato starch used can be effectively reduced, significantly lowering the production cost of dextrin. This application, through screening the starch source, optimizing the fermentation process of Bacillus Y112, combining ultrasonic and microwave pretreatment, and strictly controlling the enzymatic hydrolysis parameters, can effectively improve the activity of cyclodextrin glucotransferase and its specificity with the starch source. During dextrin production, the conversion rate of α-cyclodextrin can be increased to 42.4%-49.1%. This application combines the advantages of low raw material cost, high enzyme activity, mild reaction conditions, short reaction cycle, high α-cyclodextrin conversion rate, simple operation, low implementation cost, and ease of promotion and implementation. Attached Figure Description
[0021] Figure 1 This is a standard curve of the concentration and absorbance of the α-cyclodextrin standard solution in Example 1 of this application; Figure 2 This is a curve showing the change of α-cyclodextrin concentration over time under different relative dissolved oxygen levels in Example 1 of this application; Figure 3 This is the curve showing the change of α-cyclodextrin concentration over time under different inoculation amounts in Example 2 of this application; Figure 4 This is the curve showing the change in α-cyclodextrin conversion rate over time under different enzyme addition amounts in Example 3 of this application; Figure 5 This is the curve showing the change in α-cyclodextrin conversion rate over time under different starch sources in Example 4 of this application; Figure 6 This is a curve showing the change in α-cyclodextrin conversion rate over time under different pretreatment processes in Example 5 of this application. Detailed Implementation
[0022] The technical solutions provided in this application will be further explained in conjunction with the descriptions in Examples 1-5.
[0023] Example 1 (1) The preparation method of highly active cyclodextrin glucotransferase includes the following steps: Step 301. Prepare seed solution: Use a sterile inoculation loop to scrape a loop of Bacillus Y112 and inoculate it into 300ml of seed solution. After culturing at 30℃ and 120rpm for 24h, the seed solution is obtained. Step 302. Fermentation: The seed liquid prepared in step 301 is inoculated into a fermenter (volume of 2L) containing 1L of fermentation liquid. After fermentation for 48h under the conditions of relative dissolved oxygen of 30%, 32%, 35%, 38%, and 40%, fermentation temperature of 27℃, stirring speed of 150rpm, and mass ratio of seed liquid to fermentation liquid of 1:25, fermentation liquid 1, fermentation liquid 2, fermentation liquid 3, fermentation liquid 4 and fermentation liquid 5 are obtained respectively. Step 303. The fermentation broth 1, fermentation broth 2, fermentation broth 3, fermentation broth 4 and fermentation broth 5 prepared in step 302 are successively subjected to sodium alginate flocculation, filtration, centrifugation, ultrafiltration membrane filtration, ethanol precipitation and freeze drying to obtain enzyme powder 1, enzyme powder 2, enzyme powder 3, enzyme powder 4 and enzyme powder 5.
[0024] The seed solution formula is as follows: The seed solution is prepared by mixing nutrient C and deionized water at a mass ratio of 1:(10.5-12.3); By weight, the nutrient C consists of the following components: 11-13.5 parts standard potato starch, 5.4-6.6 parts peptone, 5.4-6.6 parts yeast extract, 0.5-1 part dipotassium hydrogen phosphate, 0.1-0.2 parts magnesium sulfate heptahydrate, 5-6 parts sodium chloride, and 1.12-1.33 parts anhydrous sodium carbonate.
[0025] The fermentation broth is formulated as follows: the fermentation broth is obtained by compounding nutrient D and deionized water at a mass ratio of 1:(11.4-12.2); By mass, the nutrient D consists of the following components: 6.6-8.2 parts standard potato starch, 16.2-20.3 parts peptone, 8.2-10.1 parts yeast extract, 0.1-0.4 parts manganese chloride tetrahydrate, 4.5-5.5 parts sodium chloride, and 1.33-1.66 parts anhydrous sodium carbonate.
[0026] To construct a standard curve for α-cyclodextrin: Take one test tube and add 1 mL of α-cyclodextrin standard solution of varying concentrations and 1 mL of 1 mol / L hydrochloric acid solution, followed by 4 mL of methyl orange solution diluted 4 times. Shake well and let stand at room temperature for 20 min. Measure the absorbance at 507 nm. Take another test tube without α-cyclodextrin, and perform the same procedure. Plot a standard curve with α-cyclodextrin concentration on the x-axis and the difference in absorbance between the two solutions on the y-axis. The absorbance difference (ΔOD) = absorbance of blank solution - absorbance of standard solution. The standard curve is shown below. Figure 1 As shown, the α-cyclodextrin standard was purchased from Sigma-Aldrich. Figure 1 In this study, the standard equation for α-cyclodextrin concentration was: ΔOD = 0.03715 × C + 0.0001, with a correlation coefficient of R0. 2 =0.9998.
[0027] 1g of enzyme powder (1-5), 1g of potato standard starch, 18g of 10mmol / L sodium citrate, pH=8 buffer solution, and 80g of deionized water were mixed to obtain the corresponding evaluation system (1-5). The absorbance of the evaluation system (1-5) was measured every 30 minutes (referring to the process of drawing the α-cyclodextrin standard curve), and the concentration of α-cyclodextrin in each evaluation system was calculated. The corresponding α-cyclodextrin concentration change curve over time was plotted to evaluate the activity of each enzyme powder (1-5) and analyze the effect of different relative oxygen concentrations on the activity of cyclodextrin glucotransferase.
[0028] Depend on Figure 2 It can be seen that the enzyme activity of enzyme powder 4 is the highest at a relative dissolved oxygen level of 38% (peak α-cyclodextrin concentration of 4.826 mg / mL). At dissolved oxygen levels deviating from this value (30% and 40%), the enzyme activity decreases significantly, and the peak concentration drops to 3.432 mg / mL and 4.209 mg / mL, respectively.
[0029] The relative dissolved oxygen level in the fermentation broth is a key limiting factor for the production of cyclodextrin glucotransferase (CGT) by Bacillus Y112 fermentation. When the relative dissolved oxygen level is too low (≤35%), the respiratory chain of Bacillus Y112 is blocked, energy metabolism is disordered, leading to a reduction in CGT synthesis and abnormal protein folding, thus significantly reducing enzyme activity. When the relative dissolved oxygen level is too high (≥40%), reactive oxygen free radicals accumulate in the fermentation broth, oxidizing the sulfhydryl (-SH) and tyrosine residues of the enzyme protein, also resulting in loss of enzyme activity. At the same time, excessively high relative dissolved oxygen increases fermentation energy consumption and may inhibit secondary metabolism of Bacillus (CGT is a secondary metabolite). In this embodiment, by controlling the relative dissolved oxygen content of the fermentation broth at 38%, a balance is achieved between the respiratory demand of Bacillus Y112 and the synthesis efficiency of secondary metabolites. This provides sufficient energy for enzyme production while avoiding damage from reactive oxygen species, ultimately yielding highly active enzyme powder and significantly improving the conversion rate in the dextrin production process.
[0030] Example 2 Referring to Example 1, the fermentation parameters in step 302 were adjusted: under the conditions of a seed liquid to fermentation liquid mass ratio of 1:25, 1.2:25, 1.5:25, 1.8:25, and 2:25, a fixed relative dissolved oxygen level of 38%, a fixed fermentation temperature of 27°C, and a fixed stirring speed of 150 rpm, fermentation was carried out for 48 hours, resulting in fermentation liquids 6, 7, 8, 9, and 10, respectively. Enzyme powders (6-10) were then prepared according to the process in step 303. 1g of enzyme powder (6-10), 1g of potato standard starch, 18g of 10mmol / L sodium citrate buffer, and 80g of deionized water were mixed to form the corresponding evaluation system (6-10). The absorbance of the evaluation system (6-10) was measured every 30 minutes (refer to the process of drawing the α-cyclodextrin standard curve), and the concentration of α-cyclodextrin in each evaluation system was calculated. The corresponding α-cyclodextrin concentration change curve over time was plotted to evaluate the activity of each enzyme powder (6-10) and analyze the effect of different inoculum amounts on the activity of cyclodextrin glucotransferase.
[0031] Depend on Figure 3 It was found that the enzyme powder prepared at a seed culture to fermentation broth mass ratio of 1.5:25 exhibited the best activity, with a peak α-cyclodextrin concentration of 6.327 mg / mL, significantly higher than other inoculum ratios. When the inoculum ratio was below 1.5:25, the enzyme activity gradually increased with increasing seed culture inoculum; when the inoculum ratio exceeded 1.5:25, the enzyme activity began to decrease, dropping to 5.121 mg / mL at an inoculum ratio of 2:25.
[0032] When the mass ratio of seed culture to fermentation broth is too low (≤1.2:25), the initial cell mass in the fermentation system is insufficient, the lag phase is prolonged (the time for microorganisms to adapt to the environment and proliferate is increased), resulting in a shortened enzyme production phase within the 48-hour fermentation cycle and low enzyme yield. When the seed culture inoculation amount is too high (≥1.8:25), the rapid proliferation of Bacillus Y112 leads to fierce competition for nutrients (starch, peptone), premature depletion of carbon and nitrogen sources in the fermentation broth, accumulation of metabolic waste (lactic acid, ammonia), and pH fluctuations exceeding the tolerance range of microorganisms, thus inhibiting the synthesis and activity of CGT. By controlling the mass ratio of seed liquid to fermentation broth at 1.5:25, Bacillus Y112 can rapidly enter the logarithmic growth phase in the early stage of fermentation, stably produce enzymes in the middle stage of fermentation, and fully accumulate enzyme proteins in the later stage of fermentation, while maintaining a stable metabolic environment. This keeps the CGT enzyme in a highly active state and significantly improves the conversion rate in the dextrin production process.
[0033] Example 3 (1) Referring to Examples 1 and 2, the fermentation parameters in step 302 were adjusted: the seed liquid prepared in step 301 was inoculated into a fermenter (volume of 2L) containing 1L of fermentation liquid. After fermentation for 48h under the conditions of relative dissolved oxygen of 38%, fermentation temperature of 27℃, stirring speed of 150rpm, and mass ratio of seed liquid to fermentation liquid of 1.5:25, the target fermentation liquid was obtained; and the target high-activity enzyme powder was prepared according to the process in step 303.
[0034] (2) The method for preparing starch source includes the following steps. Step 201. Put jackfruit seed pieces and ginger pieces with dimensions of 2cm×2cm×2cm into a grinder in sequence according to a mass ratio of 5:2.3. Grind at high speed for 8 minutes at room temperature and 3000rpm to obtain the raw material powder. Step 202. Filter the raw material pulverized material obtained in step 201 at room temperature and 0.8 MPa and collect the first filtrate. Then transfer the solid residue obtained from the filter back to the pulverizer and pulverize it at high speed at 3000 rpm for 8 minutes at room temperature to obtain the second pulverized material. Filter the second pulverized material at room temperature and 0.8 MPa-1.0 MPa and collect the second filtrate. Mix the first filtrate and the second filtrate to obtain the crude starch emulsion. Step 203. Transfer the crude starch emulsion prepared in step 202 to a centrifuge and centrifuge at 6000 rpm for 12 min at room temperature. Repeat this process 5 times. Discard the supernatant, collect the precipitate and transfer it to an oven. Dry it at 40°C to constant weight to obtain a mixture of jackfruit seed starch and ginger starch. Step 204. Mix the mixture of jackfruit seed starch and ginger starch prepared in step 203 with standard potato starch in a preset ratio to obtain the starch source. The starch source is obtained by compounding jackfruit seed starch, ginger starch and standard potato starch in a mass ratio of 5:2.3:1.
[0035] (2) Preparation method of dextrin with high conversion rate Step 101. Add 1g of starch source and 12.5g of 10mmol / L sodium citrate buffer to a stirred tank in sequence, and disperse for 30min at room temperature and 1500rpm to obtain reaction system A. Step 102. The reaction system A prepared in step 101 and the highly active enzyme powder are sequentially added into the reaction vessel. After reacting for 3 hours under the conditions of adding 52.4 U / g, 53.5 U / g, 54.6 U / g, and 55.7 U / g of highly active enzyme powder, the reaction temperature is fixed at 33℃, the stirring speed is fixed at 200 rpm, and the pH value is fixed at 8, reaction systems B1, B2, B3, and B4 are obtained respectively. Step 103. The reaction systems B1, B2, B3 and B4 prepared in step 102 are respectively subjected to resin desalting, decolorization, chromatographic purification, concentration and spray drying to obtain dextrin 1, dextrin 2, dextrin 3 and dextrin 4. The reaction system A undergoes a combined ultrasonic and microwave pretreatment, which includes the following steps: transferring the reaction system A into an ultrasonic reactor and pretreating it for 5 minutes at an ultrasonic frequency of 15 kHz and a microwave temperature of 85 ℃.
[0036] Referring to Examples 1 and 2, the absorbance of reaction system B (1-4) was measured every 30 minutes (referring to the process of plotting the α-cyclodextrin standard curve), and the concentration of α-cyclodextrin in each reaction system B (1-4) was calculated. The amount of α-cyclodextrin produced in each reaction system and the conversion rate of α-cyclodextrin (actual amount of α-cyclodextrin produced / theoretical amount of α-cyclodextrin produced) were calculated, and the corresponding curves of α-cyclodextrin conversion rate over time were plotted to analyze the effect of the amount of highly active enzyme powder added on the α-cyclodextrin conversion rate.
[0037] The theoretical yield of α-cyclodextrin is defined as the maximum yield per unit mass of starch source under ideal conditions (no side reactions, 100% conversion of starch to α-cyclodextrin). Under ideal conditions, starch [general formula: (C6H]] 10 O5) nThe degree of polymerization (DP) is 2000 (the average degree of polymerization of biological starch is 1000-5000), and the average molecular weight of starch = DP × 162.14 = 2000 × 162.14 = 324280 g / mol. α-Cyclodextrin is a cyclic oligosaccharide composed of 6 glucose units with a molecular weight of 972.86 g / mol. Under ideal conditions, 1 mol of starch (degree of polymerization 2000) can generate 2000 / 6 ≈ 333.3 mol of α-cyclodextrin. Therefore, the theoretical amount of α-cyclodextrin produced from 1 g of starch source = (1 g / 324280 g / mol) × 333.3 mol × 972.86 g / mol ≈ 1.0 g. Depend on Figure 4 It can be seen that the reaction system with the highest B3 conversion rate (49.1% after 180 min) was when the amount of highly active enzyme powder added was 54.6 U / g. The conversion rate dropped to 42.4% (52.4 U / g) and 46.5% (55.7 U / g) when the amount of added deviated from this value.
[0038] When the amount of highly active enzyme powder added is insufficient (≤53.5U / g), the substrate (complex starch source) is not fully catalyzed and decomposed, and a large number of starch molecules are not converted into glucose units, resulting in low α-cyclodextrin production and low conversion rate. When the amount of highly active enzyme powder added is too high (≥55.7U / g), a substrate saturation effect occurs in the reaction system. The starch source concentration is fixed (1g), and excess enzyme molecules cannot bind to the substrate. Furthermore, the interactions between enzyme molecules (hydrophobic interactions, hydrogen bonds) lead to changes in the conformation of the active site, resulting in a decrease in catalytic efficiency. At the same time, excess enzyme increases production costs and has no practical application value. By controlling the addition amount of highly active enzyme powder at 54.6 U / g, the compatibility between the highly active enzyme powder and the complex starch source substrate concentration can be effectively improved, the binding efficiency of enzyme molecules and substrate can be effectively improved, the mutual inhibition between enzyme molecules can be reduced, and thus the conversion rate of α-cyclodextrin can be significantly improved.
[0039] Example 4 Referring to Example 3, some parameters in the preparation process of high-conversion dextrin were adjusted, and the starch source in step 101 was replaced with a single standard potato starch to prepare reaction system a; In step 102, reaction system a and highly active enzyme powder are sequentially added to the reaction vessel. Under the conditions of a fixed amount of highly active enzyme powder added at 54.6 U / g, a fixed reaction temperature at 33℃, a fixed stirring speed at 200 rpm, and a fixed pH value at 8, the reaction system B5 is obtained after 3 hours. Then, in step 103, reaction system B5 is prepared into dextrin 5.
[0040] Referring to Examples 1 and 2, the absorbance of reaction system B5 was measured every 30 minutes (referring to the process of plotting the α-cyclodextrin standard curve), and the concentration of α-cyclodextrin in each reaction system B5 was calculated. The actual amount of α-cyclodextrin produced in reaction system B5 and the conversion rate of α-cyclodextrin (actual amount of α-cyclodextrin produced / theoretical amount of α-cyclodextrin produced) were calculated, and the corresponding curves of α-cyclodextrin conversion rate over time were plotted to analyze the specific differences between different starch sources (compound starch sources and single standard starch sources) and high-activity enzyme powder.
[0041] Depend on Figure 5 It can be seen that the conversion rate of B3 corresponding to the compound starch source (49.1%) is significantly higher than that of B5 (43.7%) in the reaction system with single potato starch, with a total conversion rate difference of 5.4%-9.5%.
[0042] Single potato starch has high crystallinity (≈25%) and a high proportion of amylopectin (≈80%), making it difficult for CGT enzyme to penetrate the crystallized region. In the compound starch, jackfruit seed starch (30% linear chain) is easily recognized by the enzyme, while ginger starch (<10% crystallinity) reduces the overall crystallization barrier and forms a linear-low crystallinity branched complex structure, which effectively improves the specificity of the substrate and CGT enzyme, thereby significantly improving the conversion rate of α-cyclodextrin. Secondly, gingerol in ginger can regulate the microenvironment of enzymatic reactions and reduce the conformational denaturation rate of CGT enzyme; small molecule polysaccharides in jackfruit seed starch can act as catalytic primers to accelerate the cyclic closure of glucose units to generate α-cyclodextrin, thereby significantly improving the conversion rate of α-cyclodextrin. Furthermore, the different hydrolysis rates of the various components of the compound starch disperse the inhibitory effect between products (the concentrated hydrolysis products of single starch easily inhibit enzyme activity), prolong the effective catalytic time of the enzyme, and thus significantly improve the conversion rate of α-cyclodextrin.
[0043] Example 5 Referring to Example 3, some parameters in the preparation process of high-conversion dextrin were adjusted. The pretreatment process of reaction system A was cancelled. The reaction system A prepared in step 101 and the high-activity enzyme powder were directly added into the reaction vessel in sequence. After reacting for 3 hours under the conditions of fixed addition of high-activity enzyme powder of 54.6 U / g, fixed reaction temperature of 33℃, fixed stirring speed of 200 rpm and fixed pH value of 8, reaction system B6 was obtained. Then, dextrin 6 was prepared from reaction system B6 through step 103.
[0044] Referring to Examples 1 and 2, the absorbance of reaction system B6 was measured every 30 minutes (referring to the process of plotting the α-cyclodextrin standard curve), and the concentration of α-cyclodextrin in each reaction system B6 was calculated. The amount of α-cyclodextrin generated in reaction system B6 and the conversion rate of α-cyclodextrin were calculated, and the corresponding curves of α-cyclodextrin conversion rate over time were plotted to analyze the effect of the pretreatment step on the α-cyclodextrin conversion rate of reaction system A.
[0045] Depend on Figure 6 It can be seen that after the combined ultrasonic and microwave pretreatment of reaction system A, the α-cyclodextrin conversion rate of reaction system B3 (49.1%) is much higher than that of reaction system B6 (31.4%) prepared without pretreatment of reaction system A. The difference in conversion rate after 180 min is 17.7%, and the α-cyclodextrin conversion rate of reaction system B6 increases slowly throughout the process.
[0046] By pretreating reaction system A with a combination of ultrasound and microwave, the crystalline structure of starch can be disrupted. The ultrasonic cavitation effect generates localized high temperature and pressure, which can break the hydrogen bonds between starch molecules. The microwave thermal effect melts the crystalline regions in the starch, effectively reducing the crystallinity of the starch and eliminating enzymatic resistance. Secondly, pretreating reaction system A can also effectively improve its dispersibility, preventing starch agglomeration and allowing starch molecules to be fully dispersed into single molecules / small molecule aggregates. This effectively increases the contact area between CGT enzyme and starch source. Furthermore, the viscosity of reaction system A decreases after pretreatment, allowing α-cyclodextrin products to diffuse rapidly and reducing the product's occupation of the enzyme's active site (weakening the product inhibition effect), thereby significantly improving the conversion rate of α-cyclodextrin. Moreover, the combined ultrasound and microwave pretreatment can optimize the conformation of starch molecules, causing the α-1,4 glycosidic bonds in starch molecules to unwind, making it easier for the CGT enzyme's active site to recognize and bind.
[0047] In summary, this application provides a method for preparing dextrin with high α-cyclodextrin conversion rate. By optimizing the relative dissolved oxygen level (30%-40%) and the mass ratio of seed liquid to fermentation broth in the preparation process of cyclodextrin glucotransferase (CGTase), highly active cyclodextrin glucotransferase powder is obtained. By pretreating the reaction system (combined ultrasound and microwave), optimizing the enzyme addition amount (52.4 U / g-55.7 U / g), and screening the starch type (a blend of jackfruit starch, ginger starch, and potato starch), the specificity between cyclodextrin glucotransferase and starch source is improved, significantly increasing the α-cyclodextrin conversion rate during the preparation process.
[0048] Specifically, this application demonstrates that highly active enzyme powder can be prepared under conditions of 38% relative dissolved oxygen and a seed liquor to fermentation broth mass ratio of 1.5:25. Dextrin is then prepared by combining the highly active enzyme powder with a compound starch source (jackfruit seed: ginger: potato = 5:2.3:1) at an addition amount of 54.6 U / g under combined ultrasonic and microwave pretreatment. The α-cyclodextrin conversion rate reaches 49.1%, which is 12.4% higher than that of a single potato starch source (43.7%) and a system without combined ultrasonic and microwave pretreatment (31.4%), respectively. This application utilizes the synergistic effect of various process parameters in the dextrin production process to significantly improve the α-cyclodextrin conversion rate, providing important technical support and theoretical basis for the industrial production of high-value-added cyclodextrin products.
[0049] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.
Claims
1. A method for preparing dextrin with high conversion rate, characterized in that, Includes the following steps: Step 101. Add the starch source and buffer solution into the stirred tank in a mass ratio of 1:(12.4-15.5) and disperse them at room temperature and 1200rpm-1800rpm for 20min-30min to obtain reaction system A. Step 102. The reaction system A prepared in step 101 and the highly active cyclodextrin glucotransferase are sequentially added into the reaction vessel and reacted for 1.5h-3h at 30℃-35℃, 200rpm-440rpm and pH 6-8 to obtain reaction system B. Step 103. The reaction system B prepared in step 102 is subjected to resin desalting, decolorization, chromatographic purification, concentration and spray drying in sequence to obtain the dextrin product; In step 101, reaction system A undergoes a combined ultrasonic and microwave pretreatment; the starch source is obtained by compounding jackfruit seed starch, ginger starch, and standard potato starch in a mass ratio of 5:(2.1-2.4):
1. In step 102, the highly active cyclodextrin glucotransferase is obtained by fermentation of Bacillus Y112; the conversion rate of α-cyclodextrin is 42.4%-49.1%.
2. The method for preparing high-conversion dextrin according to claim 1, characterized in that, In step 101, the method for preparing the starch source includes the following steps: Step 201. Put the jackfruit seeds and ginger pieces into the grinder in sequence according to the preset target ratio, and grind them at high speed at 3000 rpm for 5 min-10 min at room temperature to obtain the raw material powder. Step 202. Filter the raw material pulverized material obtained in step 201 at room temperature and 0.8MPa-1.0MPa and collect the first filtrate. Then transfer the solid residue obtained from the filtration back to the pulverizer and pulverize it at high speed at 3000rpm for 5min-10min at room temperature to obtain the second pulverized material. Filter the second pulverized material at room temperature and 0.8MPa-1.0MPa and collect the second filtrate. Mix the first filtrate and the second filtrate to obtain the crude starch emulsion. Step 203. Transfer the crude starch emulsion prepared in step 202 to a centrifuge and centrifuge at room temperature and 5000rpm-8000rpm for 10min-15min. Repeat this process 3-5 times. Discard the supernatant, collect the precipitate and transfer it to an oven. Dry it at 40℃ to constant weight to obtain a mixture of jackfruit seed starch and ginger starch. Step 204. Mix the mixture of jackfruit seed starch and ginger starch prepared in step 203 with standard potato starch in a preset ratio to obtain the target starch source.
3. The method for preparing high-conversion dextrin according to claim 1, characterized in that, In step 103, the preparation method of the highly active cyclodextrin glucotransferase includes the following steps: Step 301. Prepare seed solution: Use a sterile inoculation loop to scrape a loop of Bacillus Y112 and inoculate it into 300ml of seed solution. After culturing at 30℃ and 120rpm for 24h, the seed solution is obtained. Step 302. Fermentation: The seed liquid prepared in step 301 is inoculated into a fermenter containing the liquid to be fermented. After fermentation for 48 hours at a fermentation temperature of 27℃, 150 rpm and a relative dissolved oxygen of 30%-40%, the fermentation liquid is obtained. Step 303. The fermentation broth prepared in step 302 is subjected to sodium alginate flocculation, filtration, centrifugation, ultrafiltration membrane filtration, ethanol precipitation, and freeze drying to obtain highly active cyclodextrin glucotransferase powder.
4. The method for preparing high-conversion dextrin according to claim 3, characterized in that, In step 301, the seed solution is prepared by mixing nutrient C and deionized water at a mass ratio of 1:(10.5-12.3); By weight, the nutrient C consists of the following components: 11-13.5 parts standard potato starch, 5.4-6.6 parts peptone, 5.4-6.6 parts yeast extract, 0.5-1 part dipotassium hydrogen phosphate, 0.1-0.2 parts magnesium sulfate heptahydrate, 5-6 parts sodium chloride, and 1.12-1.33 parts anhydrous sodium carbonate.
5. The method for preparing high-conversion dextrin according to claim 3, characterized in that, In step 302, the mass ratio of the seed liquid to the fermentation liquid is (1-2):
25.
6. The method for preparing high-conversion dextrin according to claim 3, characterized in that, In step 302, the fermentation broth is obtained by mixing nutrient D and deionized water at a mass ratio of 1:(11.4-12.2); By mass, the nutrient D consists of the following components: 6.6-8.2 parts standard potato starch, 16.2-20.3 parts peptone, 8.2-10.1 parts yeast extract, 0.1-0.4 parts manganese chloride tetrahydrate, 4.5-5.5 parts sodium chloride, and 1.33-1.66 parts anhydrous sodium carbonate.
7. The method for preparing high-conversion dextrin according to claim 1, characterized in that, In step 101, the pretreatment of reaction system A The process includes the following steps: transferring reaction system A to an ultrasonic reactor and treating it for 5-10 minutes under ultrasonic frequency of 10kHz-15kHz and microwave temperature of 80℃-88℃.
8. The method for preparing high-conversion dextrin according to claim 1, characterized in that, In step 103, the amount of highly active cyclodextrin glucotransferase added is 52.4 U / g-55.7 U / g per gram of starch source.
9. The method for preparing high-conversion dextrin according to claim 1, characterized in that, In step 101, the buffer solution is a sodium citrate solution with a concentration of 8.5 mmol / L to 10 mmol / L.
10. A dextrin, characterized in that, It is prepared by the method for preparing high-conversion dextrin according to any one of claims 1-9.