Process for improving sugar conversion rate of corn starch

Abstract

The invention belongs to the technical field of biology, and discloses a process for improving the sugar conversion rate of corn starch, which comprises the following steps: adjusting the DE value of liquefied liquid to 14-16 and the pH value to 4-5, cooling to 55-65 DEG C, adding 100-300 units of saccharifying enzyme into each gram of starch, carrying out saccharification reaction under sealed heat preservation conditions, controlling the saccharification reaction time to be 6-24 hours, and carrying out solid-liquid separation to obtain the corn starch sugar conversion rate. And after the saccharification reaction is completed, raising the temperature of the feed liquid to 80-90 DEG C, carrying out heat preservation and enzyme deactivation for 5-10 minutes to inactivate the saccharifying enzyme, and terminating the reaction. According to the method, saccharification conditions are further optimized, and the sugar conversion rate is increased.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a process for improving the sugar conversion rate of corn starch. Background Technology

[0002] In recent years, with the technological development of the starch sugar industry, increased production, and decreased material consumption, the cost of starch sugar production has dropped significantly, leading to a gradual expansion of the starch sugar market and its becoming an important supplement to the sugar market. Corn, due to its wide planting area, high yield, low price, and high starch content, has become the preferred raw material for starch sugar production enterprises. The saccharification process is a crucial step in the corn starch sugar production process. Saccharification involves the hydrolysis of starch milk into small-molecule dextrins by amylase, followed by further hydrolysis into monosaccharides under the action of glucoamylase. This process plays a decisive role in the composition and quality of the final product. Currently, the starch sugar saccharification process still faces problems such as slow saccharification rate, low final yield, and complex and unstable composition of the saccharification solution.

[0003] Monosodium glutamate (MSG) produced by fermentation requires the hydrolysis of starch into glucose before it can be used for fermentation. Starch saccharification primarily employs a two-enzyme method. The starch slurry is first slurried and mixed with other ingredients, then liquefied before entering the saccharification stage. At a specific temperature, the liquefied dextrin and oligosaccharides are further hydrolyzed into glucose in the saccharification tank. During saccharification, specific temperature, pH, and cycle times must be controlled, and filtration is performed. The filtered glucose is then used for fermentation. The parameters that need to be controlled during the conversion of starch slurry into glucose are crucial. Experiments are needed to determine the optimal control parameters to ensure a more complete conversion of starch slurry into glucose, providing sufficient and high-quality raw materials for fermentation. The DE value of the saccharification substrate, saccharification temperature, saccharification pH, and enzyme dosage are closely related to the final saccharification product. Existing technologies have conducted extensive research on saccharification conditions, as exemplified below.

[0004] Reference 1: "Study on co-immobilization of amylase and saccharifying enzyme in diatomaceous earth, Feed Engineering, 2006" used saccharifying enzyme and α-amylase as a dual-enzyme system, and diatomaceous earth as a carrier to simultaneously immobilize the two enzymes by adsorption. The optimal co-immobilization conditions were diatomaceous earth (g): saccharifying enzyme (U): amylase (U) = 1:100:160, pH 4.5–5.0, temperature 5–15℃, and citrate buffer. The properties of the co-immobilized enzymes were: optimal pH 5.0; optimal temperature 55℃; Michaelis constant Km = 16.830 mg / ml; maximum reaction rate Vmax = 1.500 mg ( / ml·min), and good stability.

[0005] Reference 2: "Study on co-immobilization of α-amylase and saccharifying enzyme, Chinese Journal of Biochemistry and Molecular Biology" used cellulose as a carrier and diazotization to simultaneously immobilize saccharifying enzyme and α-amylase, determined the optimal conditions for co-immobilization, studied the properties of the co-immobilized enzyme, and found that the co-immobilized enzyme could exert a better synergistic effect than the immobilized single enzyme, and could hydrolyze starch into glucose in one step at a lower temperature. The half-life of the co-immobilized enzyme at 30℃ could reach 920 hours.

[0006] Reference 3, "Optimization of the process conditions for the synergistic enzymatic hydrolysis of potato starch by α-amylase and saccharifying enzyme, Journal of Northwest A&F University (Natural Science Edition)," explored the process conditions for the synergistic enzymatic hydrolysis of potato starch by α-amylase and saccharifying enzyme, providing a reference for reducing the cost of biodiesel production from microalgae. The synergistic enzymatic hydrolysis of potato starch by α-amylase and saccharifying enzyme was employed. Using glucose content as the determining index, four influencing factors were selected: reaction temperature, substrate concentration, enzyme dosage (m(α-amylase):m(saccharifying enzyme) = 3:1), and reaction time. An orthogonal experiment was conducted to determine the optimal enzymatic hydrolysis process conditions.

[0007] The existing technology still needs further optimization of the saccharification conditions. Summary of the Invention

[0008] To reduce the content of impurities in corn starch saccharification products and improve productivity, this invention investigates the effects of different influencing factors (saccharification pH, saccharification temperature, enzyme dosage, and substrate DE value) on saccharification efficiency during the corn starch saccharification stage. The dynamic changes in the DE value of the saccharification solution under different conditions are measured, and the sugar composition of the final saccharified products is analyzed. The corn starch sugar yield and by-product content are calculated to determine the optimal conditions. This invention is beneficial for improving the efficiency of large-scale corn starch saccharification production and provides a theoretical basis for improving product quality.

[0009] The present invention is achieved through the following technical solution.

[0010] A process for improving the sugar conversion rate of corn starch includes the following steps: Adjust the DE value of the liquefied liquid to 14-16 and the pH to 4-5, cool it to 55-65℃, add saccharifying enzyme at a rate of 100-300 units per gram of starch, and carry out the saccharification reaction under sealed and insulated conditions. Control the saccharification reaction time to 6-24 hours. After the saccharification reaction is completed, raise the temperature of the liquid to 80-90℃ and insulate it for 5-10 minutes to inactivate the saccharifying enzyme and terminate the reaction.

[0011] Preferably, the liquefied liquid has a DE value of 15 and a pH of 4.5.

[0012] Preferably, the saccharifying enzyme is added at a rate of 200 units per gram of starch.

[0013] Preferably, the chemical reaction time is controlled at 12 hours.

[0014] Preferably, the enzyme inactivation time is 10 minutes.

[0015] Preferably, the liquefied liquid is prepared according to the following method: Corn starch and deionized water were mixed in a certain proportion to prepare a 30% (w / v) suspension. Then, gelatinization was carried out by heating to 85°C and maintaining for 5 minutes. The pH was then adjusted to 6.5 and the temperature was 65°C. α-Amylase was added at a ratio of 10-30 U / g starch. The mixture was stirred to ensure uniform dispersion of the enzyme. The reaction was carried out at 65°C for 60 minutes. Then, the enzyme was inactivated at 105°C for 10 minutes to obtain a liquefied solution.

[0016] Preferably, the α-amylase is a mesophilic α-amylase.

[0017] Preferably, the mesophilic α-amylase is added at a ratio of 20 U / g starch. Attached Figure Description

[0018] Figure 1 The effect of temperature on the activity of saccharifying enzymes.

[0019] Figure 2 Effect of pH on DE value of saccharification.

[0020] Figure 3 The effect of enzyme dosage on the DE value of saccharification.

[0021] Figure 4 The effect of DE value of saccharification substrate on saccharification.

[0022] Figure 5 The effect of DE value of saccharification substrate on saccharification. Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0024] Example 1 Preparation of saccharified liquid: Corn starch and deionized water were mixed in a specific ratio to prepare a 30% (w / v) suspension. The mixture was stirred thoroughly to prevent clumping and then pretreated for gelatinization by heating to 85°C and maintaining the temperature for 5 minutes. This allowed the starch granules to absorb water, swell, and rupture, thus accelerating subsequent enzymatic hydrolysis. Next, the pH and temperature were adjusted. The optimal pH for mesophilic α-amylase was set at 6.5, and the optimal temperature at 65°C. The dosage was calculated based on enzyme activity units (U / g), typically 20 U / g of starch. The mixture was stirred to ensure uniform enzyme dispersion and reacted at 65°C for 60 minutes. Then, the enzyme was inactivated at 105°C for 10 minutes to obtain a liquefied solution.

[0025] To determine the reaction endpoint, take a small amount of the reaction solution and add iodine solution. If the solution turns blue, brownish-red, or yellow, it indicates that the starch has been fully liquefied, thus terminating the reaction.

[0026] In response to the problems existing in the current process, the optimal experimental conditions for saccharifying enzymes were studied using the liquefied liquid obtained above, taking into account the following factors.

[0027] Experimental design scheme: (1) The activity of saccharifying enzyme was measured at different temperatures (40, 50, 55, 60, 65, 70, and 75°C). One unit of enzyme activity was defined as the amount of glucose produced by hydrolyzing soluble starch in 1 hour at 40°C and pH 4.6 using 1 mL of enzyme solution or 1 g of enzyme powder. This was expressed as U / mL (or U / g). The optimal temperature for saccharifying enzyme was thus determined.

[0028] (2) To investigate the effect of pH on the saccharification reaction, the DE value of the final saccharification was measured at different pH values ​​(3.5, 4.0, 4.5, 5.0, and 5.5) within a 60-hour saccharification time. The measurements were taken three times every 6 hours, and the average value was used to determine the optimal feeding time.

[0029] (3) By setting the enzyme addition amount to 100, 150, 200, and 250 U / g under the optimal enzyme reaction temperature and pH conditions, the change in saccharification DE value during the saccharification time was studied. The value was measured three times every 6 hours, and the average value was taken. Thus, the optimal enzyme addition amount was determined.

[0030] (4) Under optimal pH and enzyme addition conditions, the changes in the DE value of the saccharification raw material solution with initial DE values ​​of 10, 12, 15, 18, and 20 were measured during the saccharification time. The measurements were taken three times every 6 hours, and the average value was taken. The initial DE value of the saccharification substrate was thus determined.

[0031] (5) Based on the experimental results of (1)-(4), determine the parameter range, select three factors: saccharification pH, enzyme dosage, and substrate DE value, and use the DE value of the saccharified product as a reference index for saccharification effect in L9(3) 4 ) 3-factor 3-level orthogonal design.

[0032] Experimental results: 1. Determination of the optimal temperature for saccharifying enzymes Saccharifying enzymes, as biological agents, can accelerate the hydrolysis of starch. Their catalytic rate is closely related to the reaction temperature; enzyme activity is lower at low temperatures, while excessively high temperatures can lead to enzyme inactivation. Controlling the temperature within the optimal range during the saccharification reaction helps reduce saccharification time and enzyme consumption. Figure 1 This graph shows the trend of saccharifying enzyme activity as a function of temperature. As temperature gradually increases, enzyme activity increases positively, reaching its peak at 60℃ (95.80%). Excessively high temperatures damage the enzyme protein structure, causing a rapid decline in activity. After reaching 70℃, enzyme activity is less than 25% of its initial value, and at 75℃, the enzyme is essentially inactive. Above the enzyme's optimum temperature, activity decreases drastically until inactivation occurs, and this process is irreversible. Therefore, using 60℃ as the optimum reaction temperature is beneficial for the saccharification reaction.

[0033] 2. Effect of pH on glycation value Optimal pH optimization experiments were conducted at a temperature of 60℃, an initial DE value of 15 for the saccharification solution, and an enzyme addition of 250 U / g. Similar to temperature, the pH of the reaction environment affects enzyme activity and the affinity between the enzyme and the substrate. The changes and trends of the DE value throughout the saccharification process at different pH values ​​are shown below. Figure 2 As shown, at pH 4.5, the saccharification value increased most rapidly in the first 6 hours. As the reaction time increased, the DE value of the saccharified products gradually stabilized, with the final DE value of the saccharified solution reaching 95.6. At pH 3.5, the saccharification efficiency was too slow; the DE value of the saccharified solution only reached its peak after 48 hours, with a DE value of only 84.3. This indicates that an acidic environment with excessively low pH significantly reduces the enzymatic hydrolysis rate and efficiency of the saccharifying enzyme. In contrast, at pH 4.5, the overall saccharification reaction efficiency remained at a high level, making it the most suitable condition.

[0034] 3. Effect of enzyme dosage on glycation value The amount of enzyme added directly affects the length of the saccharification cycle. Based on previous research findings, and under optimal conditions of a reaction temperature of 60℃, a pH of 4.5, and an initial DE value of 15 for the substrate, the dynamic changes in the DE value of the saccharification products with saccharification time under different enzyme addition amounts were investigated. Figure 3As shown, when the enzyme dosage was 50 U / g and 100 U / g, the DE value increased slowly, the saccharification time was long, and the DE values ​​at the saccharification endpoint were only 82.1 and 87.4, indicating that the saccharification reaction was not sufficient and the enzyme dosage was too low. With increasing enzyme dosage, 200 U / g and 250 U / g showed the best saccharification effect. After 18 hours of saccharification, the substrate DE value reached equilibrium, and the difference between the two groups was not significant. The final saccharification value reached approximately 95.5. Increasing the enzyme dosage can accelerate the enzyme reaction to some extent. As shown in the figure, the reaction rate is directly proportional to the enzyme dosage in the first 6 hours of reaction. However, it also intensifies the complex reactions of the enzyme, ultimately affecting the yield and increasing costs in industrial production. Therefore, considering all factors, an enzyme dosage of 200 U / g is preferable without affecting the final product DE value.

[0035] 4. Determination of the optimal DE value for saccharified substrates To investigate the effects of initial DE values ​​of different saccharification substrates on the saccharification process and products, this study measured the changes in DE values ​​of products from five saccharification substrates with different DE values ​​(10, 12, 15, 18, and 20) after 60 hours of saccharification. The changes in DE values ​​of the saccharide solutions in the five experimental groups throughout the saccharification process are shown below. Figure 4 As shown in the figure, the higher the initial DE value before 12 hours of reaction, the faster the rate of increase. After 36 hours, the DE values ​​of each group gradually stabilized. The DE10 and DE12 groups had the lowest final product glucose equivalents, at 85.3 and 87.5, respectively, while the DE15 and DE18 groups had the highest, at 95.2 and 93.3, respectively. Therefore, it can be concluded that the initial DE value of the saccharification substrate is preferably in the range of 15-18, with DE15 being optimal, which is conducive to the binding of enzyme and substrate. This may be due to the different viscosities of the raw materials. If the DE value is too high, the raw material molecules in the saccharification solution are smaller, which is not conducive to the formation of complex structures between the saccharifying enzyme and the substrate, thus affecting the catalytic efficiency.

[0036] 5. Optimization analysis of saccharification parameters Based on previous experimental results, an orthogonal experiment was conducted using three parameters: pH value, enzyme dosage, and substrate DE value. The results are shown in Table 1 below. Range analysis revealed that, for the saccharification process, the order of influence of each factor on the saccharification reaction is: pH value (A) > enzyme dosage (B) > initial substrate DE value (C). The orthogonal experiment using k-value comparisons yielded the ideal process conditions as A²B²C², meaning that at pH 4.5, an enzyme dosage of 250 U / g, and an initial substrate DE value of 15, the saccharification reaction is most complete, resulting in the highest product DE value. This is consistent with previous measurements; under optimal conditions, the DE value of the saccharified product can reach over 95. Furthermore, when adjusting the pH, it is advisable to control it at its upper limit. This helps reduce the crosslinking load, decreases acid usage, thus saving costs, and also prevents the final saccharification acidity from being too low, which would affect the quality of the saccharified product solution.

[0037] Table 1. Results and analysis of orthogonal saccharification.

[0038] 6. Viscosity analysis of saccharification products In the industrial production of corn starch saccharide, the DE value of the substrate after liquefaction is usually controlled between 12 and 18, as this viscosity facilitates the saccharification reaction. To investigate the effect of optimized saccharification on product viscosity, this study measured the changes in saccharification viscosity under optimal substrate conditions at five different DE values ​​(10, 12, 15, 18, and 20). Figure 5 As shown in the figure, the initial DE value is negatively correlated with the viscosity of the sugar solution. The initial viscosity of the DE10 group reached 486 cp, while that of the DE20 group was only 103 cp. The higher the DE value, the shorter the saccharification time for the substrate to reach equilibrium. This is because a higher initial DE value results in lower sugar solution viscosity, increasing the probability of collision between the saccharifying enzyme and the substrate, thus improving saccharification efficiency. However, too high a DE value will produce many ultrashort-chain sugar molecules that are difficult to be hydrolyzed by the saccharifying enzyme during the liquefaction stage. On the other hand, sugar solutions with low DE values ​​have higher system viscosity and poorer water flow during saccharification, resulting in stronger sedimentation and thus poor enzymatic hydrolysis efficiency, increasing the difficulty of saccharification and affecting the yield of various products. In the figure, saccharified solutions with DE values ​​of 15-18 showed moderate system viscosity during saccharification and could reach the viscosity equilibrium point relatively quickly, indicating that the orthogonal optimization results were relatively ideal.

[0039] 7. Composition analysis of saccharification products To verify whether different DE values ​​of saccharification raw materials affect the yield and composition of the final product, the composition of the final products of five experimental groups was analyzed under optimal saccharification conditions. The results are shown in Table 2 below. The product composition in the saccharification solution includes glucose, maltose, maltotriose, and maltotetraose. Oligosaccharides with more than four sugars and other miscellaneous sugars are represented as "other". In terms of composition, the glucose content (DX value) of each group was above 90%, with DE10 and DE12 groups having DX values ​​of only 91.89% and 92.50%, respectively, and DE15 group having the highest DX value of 96.86%. Substrates with lower DE values ​​showed slower saccharification rates, higher miscellaneous sugar formation rates, and more complex compositional diversity. Furthermore, with increasing substrate concentration, the yields of glucose and maltose did not change significantly, but the content of miscellaneous sugars and other substances decreased. Combined with previous results, this may be because the viscosity of the saccharification solution with different DE values ​​affected the enzymatic effect. In summary, the saccharification solution with a DE value of 15 had the lowest content of miscellaneous sugars in its final product and the highest product yield. Currently, in industrial sugar production, approximately 35% initial starch milk is commonly used as raw material, with an average product DX value of around 90%. Through optimization in this project, the raw material concentration can be increased to 40%, and the product DE value can reach over 95. At the same time, the generation of miscellaneous sugar components is reduced, and the final DX value can reach 96.86%, thus indicating that the optimization of saccharification conditions has a good effect.

[0040] Table 2 Effect of DE value of saccharified substrate on product composition

[0041] in conclusion Using liquefied liquid as raw material, the effects of different influencing factors on saccharification efficiency during the corn starch saccharification reaction stage were studied, and the final saccharification products were analyzed. Orthogonal optimization results of the saccharification reaction showed that the order of influence of factors affecting the saccharification reaction was: pH value > enzyme dosage > substrate DE value. The optimal saccharification condition combination was A2B2C2, i.e., temperature 60℃, saccharification pH 4.5, enzyme dosage 200 U / g, and substrate DE value of 15. Under these conditions, the viscosity of the saccharification liquid was moderate, which was more conducive to the full enzymatic hydrolysis reaction and suitable as a reaction substrate. Analysis of the composition of the final saccharification products revealed that substrates with low DE values ​​not only had slower reaction rates but also lower saccharification product conversion rates. The substrate with a DE value of 15 had the highest final product yield, with a DX value of 96.86%, and generated fewer miscellaneous sugars, indicating a good optimization effect for the saccharification reaction, higher than the average level of current industrial sugar production in China.

[0042] The above examples are merely preferred embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A process for improving the sugar conversion rate of corn starch, comprising the following steps: Adjust the DE value of the liquefied liquid to 14-16 and the pH to 4-5, cool it to 55-65℃, add saccharifying enzyme at a rate of 100-300 units per gram of starch, and carry out the saccharification reaction under sealed and insulated conditions. Control the saccharification reaction time to 6-24 hours. After the saccharification reaction is completed, raise the temperature of the liquid to 80-90℃ and insulate it for 5-10 minutes to inactivate the saccharifying enzyme and terminate the reaction.

2. The process according to claim 1, characterized in that, The liquefied liquid has a DE value of 15 and a pH of 4.

5.

3. The process according to claim 1, characterized in that, The saccharifying enzyme is added at a rate of 200 units per gram of starch.

4. The process according to claim 1, characterized in that, The chemical reaction time is controlled at 12 hours.

5. The process according to claim 1, characterized in that, The enzyme inactivation time is 10 minutes.

6. The process according to any one of claims 1-5, characterized in that, The liquefied liquid is prepared according to the following method: Corn starch and deionized water were mixed in a certain proportion to prepare a 30% (w / v) suspension. Then, gelatinization was carried out by heating to 85°C and maintaining for 5 minutes. The pH was then adjusted to 6.5 and the temperature was 65°C. α-Amylase was added at a ratio of 10-30 U / g starch. The mixture was stirred to ensure uniform dispersion of the enzyme. The reaction was carried out at 65°C for 60 minutes. Then, the enzyme was inactivated at 105°C for 10 minutes to obtain a liquefied solution.

7. The process according to claim 6, characterized in that, The α-amylase is a mesophilic α-amylase.

8. The process according to claim 7, characterized in that, The mesophilic α-amylase was added at a ratio of 20 U / g starch.

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