Method for preparing sucralose using biological enzyme

Abstract

Provided in the present invention is a method for preparing sucralose using a biological enzyme, which belongs to the technical field of biocatalysis. The method refers to a method for removing the acetyl group at the 6-position in sucralose-6-acetate in an enzymatic reaction system by controlling enzymatic reaction conditions to obtain a sucralose product. According to the present invention, based on a study on the enzymatic hydrolysis mechanism of sucralose-6-acetate, efficient sucralose-6-acetate hydrolases are selected. The enzyme-to-substrate ratio, reaction temperature, and pH are optimized by reducing the inhibitory effect of reaction substrates and products on the enzyme, thereby greatly improving the hydrolysis efficiency of sucralose-6-acetate. In addition, in the present invention, pure water is used to replace a 0.15 mol / L PBS buffer system, and a 5 L tank is used to achieve repeated use of enzyme over 120 batches, greatly reducing the cost of starting materials. The operation is simple, the reaction is complete, and the industrialized and large-scale production can be easily implemented.

Description

A method for preparing sucralose using biological enzymes Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a method for preparing sucralose using biological enzymes. Background Technology

[0002] Sucralose (TGS) is a highly concentrated sweetener. Developed and patented in 1976 by Tyrell & Co. in collaboration with the University of London, it entered the market in 1988. It is the only functional sweetener made from sucrose, achieving a sweetness approximately 600 times (400-800 times) that of sucrose. Sucralose is characterized by being calorie-free, highly sweet, having a pure sweet taste, and being very safe, making it one of the most ideal sweeteners.

[0003] The deacetylation of sucralose-6-ethyl ester to sucralose is one of the key steps in sucralose preparation. This deacetylation of sucralose-6-ethyl ester to sucralose is generally achieved chemically. While methods exist to achieve high conversion rates and continuous deacetylation, these require methanol-based reactants, placing high demands on the construction standards of subsequent production facilities and increasing safety and production costs. Furthermore, chemical methods easily generate byproducts, requiring complex separation and purification processes to obtain relatively pure sucralose, resulting in high production costs. Enzymes, as highly efficient biocatalysts, are mostly proteins produced by living cells. Enzyme catalysis is mild, occurring at room temperature and pressure. Enzyme-catalyzed reactions are called enzyme-catalyzed reactions and are typically 10 times faster than their non-catalyzed counterparts. 3 -10 7 Enzyme kinetics, or enzyme reaction kinetics for short, mainly studies the relationship between the rate of an enzyme-catalyzed reaction and the concentration of the substrate (i.e., reactant) and other factors. The choice of temperature, pH, enzyme concentration, substrate concentration, inhibitors, activators, etc., in enzyme-catalyzed reactions has a significant impact on the reaction results and efficiency. Enzymatic catalytic synthesis can significantly avoid these problems; therefore, selecting appropriate biological enzyme-catalyzed reaction conditions is crucial.

[0004] Currently, although there are some research reports on the production of sucralose through the biological catalytic hydrolysis of sucralose-6-ethyl ester, using free and immobilized lipases, esterases, and proteolytic enzymes, as well as immobilized Bacillus subtilis catalyzing the synthesis of sucralose, Chinese patent CN108315372A discloses an enzymatic method for removing the acetyl group from sucralose-6-ethyl ester. This invention utilizes an organic solvent system to enzymatically catalyze the removal of the acetyl group from sucralose-6-ethyl ester to obtain sucralose. This invention offers advantages such as mild reaction conditions, good reaction selectivity, a single product, high conversion rate, and product safety, providing a new and feasible route for the biological preparation of sucralose.

[0005] The existing technology, Bioconversion of sucralose-6-acetate to sucralose using immobilized microbial cells, discloses a scheme for the bioconversion of sucralose-6-acetate to the artificial sweetener sucralose using strains of *Arthrobacter* (ABL) and *Bacillus subtilis* (RRL-1789) isolated in India. The bioconversion process involves the use of whole-cell microorganisms, immobilized whole-cell microorganisms, and immobilized whole-cell bioreactors. In an aqueous system using green technology, the immobilized whole-cell packed-bed reactor exhibits a superior bioconversion process and eliminates the need for purification of the final product. The final sucralose bioproduct can be directly concentrated under vacuum to obtain a white crystalline powder.

[0006] The aforementioned prior art has explored the process of deacetylating sucralose-6-ethyl ester in organic and aqueous phase systems, respectively.

[0007] However, existing bio-enzymatic synthesis processes for sucralose still have the following drawbacks: First, the catalytic process is complex, resulting in high separation costs; second, the enzyme dosage is large, the reaction time is long, and the synthesis efficiency is low; third, batch reproducibility is poor, failing to meet the needs of industrial production; and fourth, the enzyme's cycle life is short, leading to high overall costs. These limitations restrict the widespread use of enzymatic methods.

[0008] Therefore, it is essential to develop a bio-enzymatic method for preparing sucralose that is simple, has a short cycle time, low cost, high product yield, and is easy to industrialize and scale up.

[0009] Summary of the Invention

[0010] Based on the shortcomings of existing technologies, such as low yield, high production cost, cumbersome synthesis process, and long production cycle, this invention aims to provide a method for the efficient preparation of sucralose using biological enzymes. This method is simple, has a short cycle, low cost, and high product yield, and is easy to industrialize and scale up.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A method for preparing sucralose using biological enzymes, wherein the method refers to the method of removing the acetyl group at the 6-position of sucralose-6-ethyl ester by controlling the enzyme-catalyzed reaction conditions in an enzyme-catalyzed reaction system to obtain the sucralose product;

[0013] The bioenzyme is selected from one or more of alkaline protease, Novozymes PJN05576-Alcalase, immobilized lipase, and immobilized deacetylesterase.

[0014] The enzymatic reaction system includes a biological enzyme and sucralose-6-ethyl ester.

[0015] The enzyme-catalyzed reaction system also includes sucralose; the mass fraction of sucralose in the enzyme-catalyzed reaction system is 1%-20%, preferably less than 10%.

[0016] The mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1%-40%; preferably 5-20%, and particularly preferably 10-20%.

[0017] The enzyme-catalyzed reaction system also includes ammonium acetate, wherein the mass fraction of ammonium acetate in the enzyme-catalyzed reaction system is 0%-10%, preferably less than 4%.

[0018] The enzyme-to-enzyme ratio of the biological enzyme and sucralose-6-ethyl ester is 7.5-120:1, preferably 15-60:1, and particularly preferably 15-30:1.

[0019] The enzymatic reaction conditions include temperature and pH; the temperature is 30-60℃, preferably 46℃; the pH is 6.6-7.7, preferably 7.0-7.8; and particularly preferably 7.0-7.4.

[0020] A method for preparing sucralose using a biological enzyme includes the following steps:

[0021] Step 1: Dissolve the sucralose-6-ethyl ester substrate in pure water or PBS buffer to obtain mixture 1;

[0022] Step 2: Add the biological enzyme to the mixture to obtain mixture 2;

[0023] Step 3: Treat mixture 2 and carry out the reaction.

[0024] In step 3, the mixture is treated by adding sucralose and ammonium acetate to mixture 2 to obtain mixture 3; the reaction temperature is 30-60 degrees Celsius and the pH value is 6.6-8.2.

[0025] In step 2, the bioenzyme is selected from one or more of the following: Connecticut alkaline protease, serine endopeptide protease, immobilized lipase, and immobilized deacetylesterase.

[0026] As some preferred embodiments, the specific steps of the above method are as follows: Sucralose-6-ethyl ester is dissolved in pure water / 0.15M PBS buffer (pH 7.0) and ultrasonically heated until clear to obtain mixture 1; a biological enzyme is added to mixture 1 and the pH is adjusted to obtain mixture 2; sucralose and ammonium acetate are added to mixture 2 to obtain mixture 3; mixture 3 is placed in a shaker, the temperature is set at 30℃, the rotation speed at 220 rpm, and the reaction time is 2 hours. After the reaction is completed, a sample is taken, diluted 4 times with pure methanol, and filtered through a 0.22 μm organic filter membrane to obtain a sucralose liquid phase detection sample.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] This invention investigates the enzymatic mechanism of sucralose-6-ethyl ester hydrolysis, screens for highly efficient sucralose-6-ethyl ester hydrolases, and modifies the inhibitory effects of the substrate and product on the enzyme by adding the product and ammonium acetate. Optimizing the enzyme-to-substrate ratio, reaction temperature, and pH significantly improves the hydrolysis conversion rate of sucralose-6-ethyl ester. Furthermore, this invention allows the use of pure water instead of the 0.15 mol / L PBS buffer system and enables 120 batches of enzyme to be recycled using a 5L tank, greatly reducing raw material costs. The operation is simple, the reaction is thorough, and it is easily scalable for industrial-scale production. Attached Figure Description

[0029] 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 from these drawings without creative effort.

[0030] Figure 1. Effect of different concentrations of sucralose-6-ethyl ester on the conversion rate of alkaline protease enzymatic reaction;

[0031] Figure 2. Effect of different sucralose concentrations on the conversion rate of alkaline protease enzymatic reaction;

[0032] Figure 3. Effect of different ammonium acetate concentrations on the conversion rate of alkaline protease enzymatic reaction;

[0033] Figure 4. Effect of different reaction pH on the conversion rate of alkaline protease enzymatic reaction;

[0034] Figure 5. Effect of different reaction temperatures on the conversion rate of alkaline protease enzymatic reaction;

[0035] Figure 6. Effect of different pH adjusters on the conversion rate of alkaline protease enzymatic reaction;

[0036] Figure 7. Effect of different concentrations of sucralose-6-ethyl ester on the conversion rate of the enzymatic reaction of immobilized deacetylesterase;

[0037] Figure 8. Effect of different sucralose concentrations on the conversion rate of the immobilized deacetylesterase enzymatic reaction;

[0038] Figure 9. Effect of different ammonium acetate concentrations on the conversion rate of the immobilized deacetylesterase enzymatic reaction;

[0039] Figure 10. Effect of different reaction pH on the conversion rate of the immobilized deacetylesterase enzymatic reaction;

[0040] Figure 11. Effect of different reaction temperatures on the conversion rate of the enzymatic reaction catalyzed by immobilized deacetylesterase;

[0041] Figure 12. Effect of different pH adjusters on the conversion rate of immobilized deacetylesterase enzymatic reaction;

[0042] Figure 13 Effect of different sucralose-6-ethyl ester concentrations on the conversion rate of serine endopeptide enzymatic reaction;

[0043] Figure 14. Effect of different sucralose concentrations on the conversion rate of serine endopeptide enzymatic reaction;

[0044] Figure 15. Effect of different ammonium acetate concentrations on the conversion rate of serine endopeptide enzymatic reaction;

[0045] Figure 16. Effect of different reaction pH on the conversion rate of serine endopeptide enzymatic reaction;

[0046] Figure 17. Effect of different reaction temperatures on the conversion rate of serine endopeptide enzyme-catalyzed reaction;

[0047] Figure 18. Effect of different pH adjusters on the conversion rate of serine endopeptide enzymatic reaction;

[0048] Figure 19. Effect of different concentrations of sucralose-6-ethyl ester on the conversion rate of immobilized lipase enzymatic reaction;

[0049] Figure 20 Effect of different sucralose concentrations on the conversion rate of immobilized lipase enzymatic reaction;

[0050] Figure 21 Effect of different ammonium acetate concentrations on the conversion rate of immobilized lipase enzymatic reaction;

[0051] Figure 22 Effect of different reaction pH on the conversion rate of immobilized lipase enzymatic reaction;

[0052] Figure 23 Effect of different reaction temperatures on the conversion rate of immobilized lipase enzymatic reaction;

[0053] Figure 24. Effect of different pH adjusters on the conversion rate of immobilized lipase enzymatic reaction;

[0054] Figure 25 Effect of different types of biological enzymes on the hydrolysis conversion rate of sucralose-6-ethyl ester;

[0055] Figure 26. Effect of different enzyme-to-base ratios on the conversion rate of immobilized lipase enzymatic reaction;

[0056] Figure 27 Liquid chromatography chromatograms of sucralose-6-ethyl ester and sucralose standard;

[0057] Figure 28. Liquid chromatography spectrum of ammonium acetate standard;

[0058] Figure 29 Liquid phase diagram of the immobilized lipase enzymatic reaction solution;

[0059] Figure 30 Batch stability results of immobilized lipase enzymatic reaction. Detailed Implementation

[0060] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] The method for determining the sucralose content in the following examples is as follows:

[0062] HPLC was used for detection, specifically:

[0063] Column: ShimNex UP C18 (4.6*150mm, 5μm);

[0064] Mobile phase: A: water, B: methanol;

[0065] Evaporative light scattering detector (ELSD);

[0066] Column temperature: 40℃;

[0067] Flow rate: 1.0 ml / min;

[0068] Drift tube temperature: 45℃;

[0069] Injection volume: 10 μL;

[0070] Gradient elution program: T / min (B%): 0 (40), 8 (90), 8.01 (40), 15 (40).

[0071] Molar conversion rate calculation formula:

[0072] Molar conversion rate = (molar amount of sucralose generated / initial molar amount of sucralose-6-ethyl ester added) × 100%.

[0073] Basic Example: A method for preparing sucralose using biological enzymes

[0074] Specifically, the steps include the following:

[0075] Sucralose-6-ethyl ester was dissolved in pure water / 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. A biological enzyme was added to mixture 1 and the pH was adjusted to obtain mixture 2 (the enzyme-to-substrate ratio is an important factor). Sucralose and / or ammonium acetate were added to mixture 2 to obtain mixture 3. Mixture 3 was placed in a shaker at a temperature of 30-60℃, pH 6-8, a rotation speed of 220 rpm, and a reaction time of 2 hours. After the reaction, a sample was taken, diluted 4-fold with pure methanol, and filtered through a 0.22 μm organic filter membrane to obtain the sucralose liquid chromatography sample.

[0076] The alkaline protease is a protein catalysis enzyme purchased from Shanghai Kangdian Biotechnology Co., Ltd., abbreviated as Kangdian alkaline protease, CAS number 9014-01-1; the immobilized deacetylase is cephalosporin C deacetylase produced by Changsha Kaixiao Biotechnology Co., Ltd., specific model: IAE, abbreviated as Kaixiao immobilized deacetylase; the serine endopeptide protease (Novozymes PJN02351-Alcalase) is a serine endopeptide protease produced by Novozymes, specific model: 2.5L, CAS number 9014-01-1; the immobilized lipase is CALB (immobilized lipase), which is lipase B derived from Candida antarctica and expressed in Pichia pastoris through fermentation. This immobilized lipase was purchased from Shanghai Kangdian Biotechnology Co., Ltd., specifically model CalB01.

[0077] In this invention, the following amounts of biological enzymes have the same enzyme activity: Candien alkaline protease: 0.15g; Kaixiao immobilized deacetylase: 0.5g; Novozymes PJN02351-Alcalase: 0.25ml; Candien immobilized lipase: 0.1g.

[0078] Example 1: A method for preparing sucralose using biological enzymes

[0079] Specifically, the steps include the following:

[0080] 1%-40% sucralose-6-ethyl ester substrate was dissolved in 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Connecticut alkaline protease was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0081] The mass fractions of sucralose-6-ethyl ester in the enzymatic reaction system were 5% (5g), 10% (10g), 20% (20g), and 40% (40g).

[0082] The results of the detection of the molar amount of sucralose are shown in Table 1 below.

[0083] Table 1

[0084] Comparative Example 1:

[0085] The difference from Example 1 is that the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1% (1g), while the other steps and operations are the same as in Example 1.

[0086] The results of the detection of the molar amount of sucralose are shown in Table 2 below.

[0087] Table 2

[0088] According to the detection data in Tables 1 and 2 and Figure 1, the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose-6-ethyl ester added affects the molar amount of sucralose formed. When the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1-40%, the addition of 20% sucralose-6-ethyl ester can make the molar amount of sucralose formed reach 6.08 mmol, while the addition of 40% sucralose-6-ethyl ester reduces the molar amount of sucralose formed to 5.45 mmol. The optimal addition amount of sucralose-6-ethyl ester should be controlled within 20%.

[0089] Example 2

[0090] Specifically, the steps include the following:

[0091] 2g of sucralose-6-ethyl ester substrate was dissolved in 50ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Candien alkaline protease was added to the mixture to obtain mixture 2. 1%-40% (1-40g) of sucralose were dissolved in 0.15M PBS buffer (pH 7.0), and after a final concentration of 50ml, the solution was added to mixture 2 to obtain mixture 3. The pH was adjusted to 7.0. Mixture 3 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time at 2h. After the reaction, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0092] The mass fractions of sucralose in the enzymatic reaction system were 1% (1g), 5% (5g), 10% (10g), and 20% (20g).

[0093] The results of the molar conversion rate test are shown in Table 3 below.

[0094] Table 3

[0095] Comparative Example 2:

[0096] The difference from Example 2 is that the mass fraction of sucralose in the enzymatic reaction system is 40% (40g), while the other steps and operations are the same as in Example 2.

[0097] The results of the molar conversion rate test are shown in Table 4 below.

[0098] Table 4

[0099] According to the detection data in Tables 3 and 4 and Figure 2, the mass fraction of sucralose in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose added affects the molar conversion rate of sucralose-6-ethyl ester. When the mass fraction of sucralose in the enzymatic reaction system is 1-40%, the molar conversion rate of sucralose-6-ethyl ester can reach 45.23% to 68.41%, especially when it is 1-20%, the molar conversion rate of sucralose-6-ethyl ester reaches 60.52% to 68.41%. Therefore, the reasonable control range for the sucralose content in the reaction system is within 20%.

[0100] Example 3

[0101] Specifically, the steps include the following:

[0102] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Connecticut alkaline protease was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. 0%-10% (1-10g) ammonium acetate was dissolved in PBS buffer (0.15M), and after a final concentration of 50ml, it was added to mixture 2 to obtain mixture 3. Mixture 3 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0103] The mass fractions of ammonium acetate in the enzyme-catalyzed reaction system were 0%, 1% (1g), 2% (2g), and 3% (3g).

[0104] The results of the molar conversion rate test are shown in Table 5 below.

[0105] Table 5

[0106] Comparative Example 3:

[0107] The difference from Example 3 is that the mass fractions of ammonium acetate in the enzymatic reaction system are 4% (4g), 5% (5g), 8% (8g), and 10% (10g), respectively. The other steps and operations are the same as in Example 3.

[0108] The results of the molar conversion rate test are shown in Table 6 below.

[0109] Table 6

[0110] Based on the detection data in Tables 5 and 6 and Figure 3, it can be seen that the mass fraction of ammonium acetate in the enzymatic reaction system has little effect on the formation of sucralose. The amount of ammonium acetate added will slightly affect the molar conversion rate of sucralose-6-ethyl ester. When the mass fraction of ammonium acetate in the enzymatic reaction system is 0-5%, the molar conversion rate of sucralose-6-ethyl ester can be controlled above 65%. When the mass fraction of ammonium acetate in the enzymatic reaction system is 8-10%, it will affect the molar conversion rate of sucralose-6-ethyl ester, reducing the conversion rate to below 62.42%. Therefore, the reasonable control range for the ammonium acetate content in the reaction system is within 5%.

[0111] Example 4

[0112] Specifically, the steps include the following:

[0113] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Candien alkaline protease was added to the mixture and the pH was adjusted to obtain mixture 2. Mixture 2 was placed in a shaker and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2.5h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0114] The pH values ​​are 6.6, 7.0, 7.4, and 7.8, respectively.

[0115] The results of the molar conversion rate test are shown in Table 7 below.

[0116] Table 7

[0117] Comparative Example 4:

[0118] The difference from Example 4 is that the pH value is 8.2, while the other steps and operations are the same as in Example 4.

[0119] The results of the molar conversion rate test are shown in Table 8 below.

[0120] Table 8

[0121] According to the detection data in Tables 7 and 8 and Figure 4, pH affects the formation of sucralose. The molar conversion rate of sucralose-6-ethyl ester reaches more than 55% when the pH value is 6.6-8.2, and the molar conversion rate of sucralose-6-ethyl ester is 60.44% when the pH value is 7.8.

[0122] Example 5

[0123] Specifically, the steps include the following:

[0124] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Candien alkaline protease was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed and time were set to 220rpm for 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0125] The set temperatures are 30℃, 38℃, 46℃, 54℃, and 58℃.

[0126] The results of the molar conversion rate test are shown in Table 9 below.

[0127] Table 9

[0128] According to the detection data in Table 9 and Figure 5 above, temperature affects the formation of sucralose. When the reaction temperature is 30-58℃, the molar conversion rate of sucralose-6-ethyl ester reaches more than 39%, and when the temperature is 38℃, the molar conversion rate of sucralose-6-ethyl ester is 57.90%.

[0129] Example 6

[0130] Specifically, the steps include the following:

[0131] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and pure water, respectively, and sonicated until clear to obtain mixture 1. 0.1g of Candien alkaline protease was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed, and reaction time were set to 220rpm for 2h (pH was stabilized with ammonia). After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0132] The sample solution was analyzed by HPLC, and the results are shown in Figure 6. The results show that using pure water to dissolve the sucralose-6-ethyl ester substrate (with ammonia water to stabilize the pH) will improve the conversion rate of sucralose-6-ethyl ester compared with using PBS buffer, increasing the conversion rate from 73.47% to 80.37%.

[0133] Example 7: A method for preparing sucralose using biological enzymes

[0134] Specifically, the steps include the following:

[0135] 1%-40% sucralose-6-ethyl ester substrate was dissolved in 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Kaixiao immobilized deacetylesterase was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0136] The mass fractions of sucralose-6-ethyl ester in the enzymatic reaction system were 5% (5g), 10% (10g), 20% (20g), and 40% (40g).

[0137] The results of the detection of the molar amount of sucralose are shown in Table 10 below.

[0138] Table 10

[0139] Comparative Example 7:

[0140] The difference from Example 7 is that the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1% (1g), while the other steps and operations are the same as in Example 7.

[0141] The results of the detection of the molar amount of sucralose are shown in Table 11 below.

[0142] Table 11

[0143] According to the detection data in Tables 10 and 11 and Figure 7, the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose-6-ethyl ester added affects the molar amount of sucralose formed. When the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1-40%, the addition of 20% sucralose-6-ethyl ester can make the molar amount of sucralose formed reach 1.69 mmol, while the addition of 40% sucralose-6-ethyl ester reduces the molar amount of sucralose formed to 1.52 mmol. The optimal addition amount of sucralose-6-ethyl ester should be controlled within 20%.

[0144] Example 8

[0145] Specifically, the steps include the following:

[0146] 2g of sucralose-6-ethyl ester substrate was dissolved in 50ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Kaixiao immobilized deacetylesterase was added to the mixture to obtain mixture 2. 1%-40% (1-40g) of sucralose were dissolved in 0.15M PBS buffer (pH 7.0), and 50ml of the solution was added to mixture 2 to obtain mixture 3. The pH was adjusted to 7.0. Mixture 3 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time at 2h. After the reaction, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0147] The mass fractions of sucralose in the enzymatic reaction system were 1% (1g), 5% (5g), 10% (10g), and 20% (20g).

[0148] The results of the molar conversion rate test are shown in Table 12 below.

[0149] Table 12

[0150] Comparative Example 8:

[0151] The difference from Example 8 is that the mass fraction of sucralose in the enzymatic reaction system is 40% (40g), while the other steps and operations are the same as in Example 8.

[0152] The results of the molar conversion rate test are shown in Table 13 below.

[0153] Table 13

[0154] According to the detection data in Tables 12 and 13 and Figure 8, the mass fraction of sucralose in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose added affects the molar conversion rate of sucralose-6-ethyl ester. When the mass fraction of sucralose in the enzymatic reaction system is 1-40%, the molar conversion rate of sucralose-6-ethyl ester can reach 35-42.63%, especially when it is 1-10%, the molar conversion rate of sucralose-6-ethyl ester reaches 38.90-42.63%. Therefore, the reasonable control range for the sucralose content in the reaction system is within 10%.

[0155] Example 9

[0156] Specifically, the steps include the following:

[0157] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Kashyal immobilized deacetylesterase was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. 0%-10% (1-10g) ammonium acetate was dissolved in PBS buffer (0.15M), and after a final concentration of 50ml, it was added to mixture 2 to obtain mixture 3. Mixture 3 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0158] The mass fractions of ammonium acetate in the enzyme-catalyzed reaction system were 0%, 1% (1g), 2% (2g), and 3% (3g).

[0159] The results of the molar conversion rate test are shown in Table 14 below.

[0160] Table 14

[0161] Comparative Example 9:

[0162] The difference from Example 9 is that the mass fractions of ammonium acetate in the enzymatic reaction system are 4% (4g), 5% (5g), 8% (8g), and 10% (10g), respectively. The other steps and operations are the same as in Example 9.

[0163] The results of the molar conversion rate test are shown in Table 15 below.

[0164] Table 15

[0165] According to the detection data in Tables 14 and 15 and Figure 9, the mass fraction of ammonium acetate in the enzymatic reaction system has little effect on the formation of sucralose. When the mass fraction of ammonium acetate in the enzymatic reaction system is 0-10%, the molar conversion rate of sucralose-6-ethyl ester can be maintained at about 33%. Therefore, the reasonable control range of ammonium acetate content in the reaction system is within 10%.

[0166] Example 10

[0167] Specifically, the steps include the following:

[0168] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Kashyal immobilized deacetylesterase was added to the mixture, and the pH was adjusted to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2.5h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0169] The pH values ​​are 6.6, 7.0, 7.4, and 7.8, respectively.

[0170] The results of the molar conversion rate test are shown in Table 16 below.

[0171] Table 16

[0172] Comparative Example 10:

[0173] The difference from Example 10 is that the pH value is 8.2, while the other steps and operations are the same as in Example 10.

[0174] The results of the molar conversion rate test are shown in Table 17 below.

[0175] Table 17

[0176] According to the detection data in Tables 16 and 17 and Figure 10, pH affects the formation of sucralose. The molar conversion rate of sucralose-6-ethyl ester is 21.05%-30.90% when the pH is 6.6-8.2, and 30.90% when the pH is 7.4.

[0177] Example 11

[0178] Specifically, the steps include the following:

[0179] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Kaixiao immobilized deacetylesterase was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed and time were set to 220rpm for 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0180] The set temperatures are 30℃, 38℃, 46℃, 54℃, and 58℃.

[0181] The results of the molar conversion rate test are shown in Table 18 below.

[0182] Table 18

[0183] According to the detection data in Table 18 and Figure 11 above, temperature affects the formation of sucralose. The molar conversion rate of sucralose-6-ethyl ester is 19.23%-35.2% when the reaction temperature is 30-58℃, and the molar conversion rate of sucralose-6-ethyl ester is 35.20% when the temperature is 58℃.

[0184] Example 12

[0185] Specifically, the steps include the following:

[0186] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and pure water, respectively, and sonicated until clear to obtain mixture 1. 0.1g of Kaixiao immobilized deacetylesterase was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed, and reaction time were set to 220rpm for 2h (pH was stabilized with ammonia). After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid phase detection sample.

[0187] The sample solution was analyzed by HPLC, and Figure 12 was obtained. The results show that using pure water to dissolve the sucralose-6-ethyl ester substrate (with ammonia water to stabilize the pH) will improve the conversion rate of sucralose-6-ethyl ester compared with using PBS buffer, increasing the conversion rate from 34.75% to 42.33%.

[0188] Example 13: A method for preparing sucralose using biological enzymes

[0189] Specifically, the steps include the following:

[0190] 1%-40% sucralose-6-ethyl ester substrate was dissolved in 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1 ml Novozymes PJN02351-Alcalase was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, set at 30℃, 220 rpm, and reacted for 2 hours. After the reaction, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22 μm organic filter membrane to obtain the sucralose liquid chromatography sample.

[0191] The mass fractions of sucralose-6-ethyl ester in the enzymatic reaction system were 5% (5g), 10% (10g), 20% (20g), and 40% (40g).

[0192] The results of the detection of the molar amount of sucralose are shown in Table 19 below.

[0193] Table 19

[0194] Comparative Example 13:

[0195] The difference from Example 13 is that the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1% (1g), while the other steps and operations are the same as in Example 13.

[0196] The results of the detection of the molar amount of sucralose are shown in Table 20 below.

[0197] Table 20

[0198] According to the detection data in Tables 19 and 20 and Figure 13, the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose-6-ethyl ester added affects the molar amount of sucralose formed. When the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1-40%, the addition of 20% sucralose-6-ethyl ester can make the molar amount of sucralose formed reach 5.88 mmol, while the addition of 40% sucralose-6-ethyl ester reduces the molar amount of sucralose formed to 5.36 mmol. The optimal addition amount of sucralose-6-ethyl ester should be controlled within 20%.

[0199] Example 14

[0200] Specifically, the steps include the following:

[0201] 2g of sucralose-6-ethyl ester substrate was dissolved in 50ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1ml of Novozymes PJN02351-Alcalase was added to the mixture to obtain mixture 2. 1%-40% (1-40g) of sucralose were dissolved in 0.15M PBS buffer (pH 7.0), and 50ml of the solution was added to mixture 2 to obtain mixture 3. The pH was adjusted to 7.0. Mixture 3 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time at 2h. After the reaction, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0202] The mass fractions of sucralose in the enzymatic reaction system were 1% (1g), 5% (5g), 10% (10g), and 20% (20g).

[0203] The results of the molar conversion rate test are shown in Table 21 below.

[0204] Table 21

[0205] Comparative Example 14:

[0206] The difference from Example 14 is that the mass fraction of sucralose in the enzymatic reaction system is 40% (40g), while the other steps and operations are the same as in Example 14.

[0207] The results of the molar conversion rate test are shown in Table 22 below.

[0208] Table 22

[0209] According to the detection data in Tables 21 and 22 and Figure 14, the mass fraction of sucralose in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose added affects the molar conversion rate of sucralose-6-ethyl ester. When the mass fraction of sucralose in the enzymatic reaction system is 1-40%, the molar conversion rate of sucralose-6-ethyl ester can reach 46.22%-62.44%, especially when it is 1-10%, the molar conversion rate of sucralose-6-ethyl ester reaches 58.41%-62.44%. Therefore, the reasonable control range for the sucralose content in the reaction system is within 10%.

[0210] Example 15

[0211] Specifically, the steps include the following:

[0212] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1ml of Novozymes PJN02351-Alcalase was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. 0%-10% (1-10g) ammonium acetate was dissolved in PBS buffer (0.15M), and after a final concentration of 50ml, it was added to mixture 2 to obtain mixture 3. Mixture 3 was placed in a shaker, set at 30℃, 220rpm, and reacted for 2h. After the reaction, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0213] The mass fractions of ammonium acetate in the enzyme-catalyzed reaction system were 0%, 1% (1g), 2% (2g), and 3% (3g).

[0214] The results of the molar conversion rate test are shown in Table 23 below.

[0215] Table 23

[0216] Comparative Example 15:

[0217] The difference from Example 15 is that the mass fractions of ammonium acetate in the enzymatic reaction system are 4% (4g), 5% (5g), 8% (8g), and 10% (10g), respectively. Other steps and operations are the same as in Example 15.

[0218] The results of the molar conversion rate test are shown in Table 24 below.

[0219] Table 24

[0220] Based on the detection data in Tables 23 and 24 and Figure 15, it can be seen that the mass fraction of ammonium acetate in the enzymatic reaction system has little effect on the formation of sucralose. The amount of ammonium acetate added will slightly affect the molar conversion rate of sucralose-6-ethyl ester. When the mass fraction of ammonium acetate in the enzymatic reaction system is 0-5%, the molar conversion rate of sucralose-6-ethyl ester can be controlled above 54.01%. When the mass fraction of ammonium acetate in the enzymatic reaction system is 8-10%, it will affect the molar conversion rate of sucralose-6-ethyl ester, reducing the conversion rate to below 51.34%. Therefore, the reasonable control range for the ammonium acetate content in the reaction system is within 5%.

[0221] Example 16

[0222] Specifically, the steps include the following:

[0223] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1ml of Novozymes PJN02351-Alcalase was added to the mixture, and the pH was adjusted to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2.5h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0224] The pH values ​​are 6.6, 7.0, 7.4, and 7.8, respectively.

[0225] The results of the molar conversion rate test are shown in Table 25 below.

[0226] Table 25

[0227] Comparative Example 16:

[0228] The difference from Example 16 is that the pH value is 8.2, while the other steps and operations are the same as in Example 16.

[0229] The results of the molar conversion rate test are shown in Table 26 below.

[0230] Table 26

[0231] According to the detection data in Tables 25 and 26 and Figure 16, pH affects the formation of sucralose. The molar conversion rate of sucralose-6-ethyl ester reaches more than 44% when the pH value is 6.6-8.2, and the molar conversion rate of sucralose-6-ethyl ester is 55.33% when the pH value is 7.0.

[0232] Example 17

[0233] Specifically, the steps include the following:

[0234] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1ml of Novozymes PJN02351-Alcalase was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed, and reaction time were set to 220rpm for 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0235] The set temperatures are 30℃, 38℃, 46℃, 54℃, and 58℃.

[0236] The results of the molar conversion rate test are shown in Table 27 below.

[0237] Table 27

[0238] According to the detection data in Table 27 and Figure 17 above, temperature affects the formation of sucralose. When the reaction temperature is 30-58℃, the molar conversion rate of sucralose-6-ethyl ester reaches more than 41%, and when the temperature is 38℃, the molar conversion rate of sucralose-6-ethyl ester is 52.33%.

[0239] Example 18

[0240] Specifically, the steps include the following:

[0241] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and pure water, respectively, and sonicated until clear to obtain mixture 1. 0.1ml of Novozymes PJN02351-Alcalase was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed, and reaction time were set to 220rpm for 2h (pH was stabilized with ammonia). After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid phase detection sample.

[0242] The sample solution was analyzed by HPLC, and Figure 18 was obtained. The results show that using pure water to dissolve the sucralose-6-ethyl ester substrate (with pH adjusted and stabilized by ammonia) improves the conversion rate of sucralose-6-ethyl ester compared to using PBS buffer, increasing the conversion rate from 72.87% to 70.56%.

[0243] Example 19: A method for preparing sucralose using biological enzymes

[0244] Specifically, the steps include the following:

[0245] 1%-40% sucralose-6-ethyl ester substrate was dissolved in 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Condensate immobilized lipase was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0246] The mass fractions of sucralose-6-ethyl ester in the enzymatic reaction system were 5% (5g), 10% (10g), 20% (20g), and 40% (40g).

[0247] The results of the molar production of sucralose are shown in Table 28 below.

[0248] Table 28

[0249] Comparative Example 19:

[0250] The difference from Example 19 is that the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1% (1g), while the other steps and operations are the same as in Example 19.

[0251] The results of the molar production of sucralose are shown in Table 29 below.

[0252] Table 29

[0253] According to the detection data in Tables 28 and 29 and Figure 19, the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose-6-ethyl ester added affects the molar amount of sucralose formed. When the mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1-40%, the molar amount of sucralose formed can reach more than 0.81g, especially when it is 20%, the molar amount of sucralose formed reaches 2.78g.

[0254] Example 20

[0255] Specifically, the steps include the following:

[0256] 2g of sucralose-6-ethyl ester substrate was dissolved in 50ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Condensate immobilized lipase was added to the mixture to obtain mixture 2. 1%-40% (1-40g) of sucralose were dissolved in 0.15M PBS buffer (pH 7.0), and after a final concentration of 50ml, the solution was added to mixture 2 to obtain mixture 3. The pH was adjusted to 7.0. Mixture 3 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time at 2h. After the reaction, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0257] The mass fractions of sucralose in the enzymatic reaction system were 1% (1g), 5% (5g), 10% (10g), and 20% (20g).

[0258] The results of the molar conversion rate test are shown in Table 30 below.

[0259] Table 30

[0260] Comparative Example 20:

[0261] The difference from Example 20 is that the mass fraction of sucralose in the enzymatic reaction system is 40% (40g), while the other steps and operations are the same as in Example 20.

[0262] The results of the molar conversion rate test are shown in Table 31 below.

[0263] Table 31

[0264] According to the detection data in Tables 30 and 31 and Figure 20, the mass fraction of sucralose in the enzymatic reaction system has a significant impact on the formation of sucralose. The amount of sucralose added affects the molar conversion rate of sucralose-6-ethyl ester. When the mass fraction of sucralose in the enzymatic reaction system is 1-40%, the molar conversion rate of sucralose-6-ethyl ester can reach 57.4%-73.16%, especially when it is 1-20%, the molar conversion rate of sucralose-6-ethyl ester reaches 69.86%-73.16%. Therefore, the reasonable control range for the sucralose content in the reaction system is within 20%.

[0265] Example 21

[0266] Specifically, the steps include the following:

[0267] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Condensate immobilized lipase was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. 0%-10% (1-10g) ammonium acetate was dissolved in PBS buffer (0.15M), and after a final concentration of 50ml, it was added to mixture 2 to obtain mixture 3. Mixture 3 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0268] The mass fractions of ammonium acetate in the enzyme-catalyzed reaction system were 0%, 1% (1g), 2% (2g), and 3% (3g).

[0269] The results of the molar conversion rate test are shown in Table 32 below.

[0270] Table 32

[0271] Comparative Example 21:

[0272] The difference from Example 21 is that the mass fractions of ammonium acetate in the enzymatic reaction system are 4% (4g), 5% (5g), 8% (8g), and 10% (10g), respectively. Other steps and operations are the same as in Example 21.

[0273] The results of the molar conversion rate test are shown in Table 33 below.

[0274] Table 33

[0275] Based on the detection data in Tables 32 and 33 and Figure 21, it can be seen that the mass fraction of ammonium acetate in the enzymatic reaction system has little effect on the formation of sucralose. The amount of ammonium acetate added will slightly affect the molar conversion rate of sucralose-6-ethyl ester. When the mass fraction of ammonium acetate in the enzymatic reaction system is 0-4%, the molar conversion rate of sucralose-6-ethyl ester can be controlled above 67%. When the mass fraction of ammonium acetate in the enzymatic reaction system is 5-10%, it will affect the molar conversion rate of sucralose-6-ethyl ester, reducing the conversion rate to below 63.95%. Therefore, the reasonable control range for the ammonium acetate content in the reaction system is within 4%.

[0276] Example 22

[0277] Specifically, the steps include the following:

[0278] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Condien immobilized lipase was added to the mixture, and the pH was adjusted to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature was set at 30℃, the rotation speed at 220rpm, and the reaction time was 2.5h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0279] The pH values ​​are 6.6, 7.0, 7.4, and 7.8, respectively.

[0280] The results of the molar conversion rate test are shown in Table 34 below.

[0281] Table 34

[0282] Comparative Example 22:

[0283] The difference from Example 22 is that the pH value is 8.2, while the other steps and operations are the same as in Example 22.

[0284] The results of the molar conversion rate test are shown in Table 35 below.

[0285] Table 35

[0286] According to the detection data in Tables 34 and 35 and Figure 22, pH affects the formation of sucralose. The molar conversion rate of sucralose-6-ethyl ester reaches more than 50% when the pH value is 6.6-7.8, and the molar conversion rate of sucralose-6-ethyl ester is 63.47% when the pH value is 7.0.

[0287] Example 23

[0288] Specifically, the steps include the following:

[0289] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. 0.1g of Condensate immobilized lipase was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed, and reaction time were set to 220rpm for 2h. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0290] The set temperatures are 30℃, 38℃, 46℃, 54℃, and 58℃.

[0291] The results of the molar conversion rate test are shown in Table 36 below.

[0292] Table 36

[0293] According to the detection data in Table 36 and Figure 23 above, temperature affects the formation of sucralose. When the reaction temperature is 30-58℃, the molar conversion rate of sucralose-6-ethyl ester reaches more than 54%, and when the temperature is 30℃, the molar conversion rate of sucralose-6-ethyl ester is 67.78%.

[0294] Example 24

[0295] Specifically, the steps include the following:

[0296] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and pure water, respectively, and sonicated until clear to obtain mixture 1. 0.1g of Condensate immobilized lipase was added to the mixture, and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker, and the temperature, rotation speed, and reaction time were set to 220rpm for 2h (pH was stabilized with ammonia). After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22um organic filter membrane to obtain the sucralose liquid chromatography sample.

[0297] The sample solution was analyzed by HPLC, and Figure 24 was obtained. The chromatogram results show that using pure water to dissolve the sucralose-6-ethyl ester substrate (with ammonia water to adjust the pH to stabilize) will improve the conversion rate of sucralose-6-ethyl ester compared with using PBS buffer, increasing the conversion rate from 82.81% to 89.47%.

[0298] Example 25: A method for preparing sucralose using biological enzymes

[0299] Specifically, the steps include the following:

[0300] The optimal reaction system and reaction conditions obtained from Examples 1-24 are as follows:

[0301] Example 25-1

[0302] The reaction was carried out using Kandian alkaline protease at pH 7.8 and temperature 30°C. The initial concentration of sucralose-6-ethyl ester was controlled at 0-20%, the concentration of sucralose in the reaction solution was controlled at 0-20%, the concentration of ammonium acetate was controlled at 0-5%, and the reaction system consisted of 3L of pure water.

[0303] Example 25-2

[0304] Kaixiao immobilized deacetylesterase was selected. The reaction pH was 7.4, the reaction temperature was 58℃, the initial concentration of sucralose-6-ethyl ester was controlled at 0-20%, the concentration of sucralose generated in the reaction solution was controlled at 0-20%, the concentration of ammonium acetate generated was controlled at 0-4%, and the reaction system was 3L of pure water.

[0305] Example 25-3

[0306] Novozymes PJN02351-Alcalase was used. The reaction pH was 7.0, the reaction temperature was 30℃, the initial concentration of sucralose-6-ethyl ester was controlled at 0-20%, the concentration of sucralose generated in the reaction solution was controlled at 0-20%, the concentration of ammonium acetate generated was controlled at 0-4%, and the reaction system was 3L PBS.

[0307] Example 25-4

[0308] Condensed lipase was used. The reaction was carried out at pH 7.0 and temperature 30℃. The initial concentration of sucralose-6-ethyl ester was controlled at 0-20%. The concentration of sucralose and ammonium acetate in the reaction solution was controlled at 0-20% and 0-4% respectively. The reaction system consisted of 3L of pure water.

[0309] For the following steps in Examples 25-1, 25-2, 25-3, and 25-4, 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer or aqueous solution (pH adjusted with ammonia) and sonicated until clear to obtain mixture 1. The same amount of bioenzymes with the same activity (Condian alkaline protease: 0.15g; Kaixiao immobilized deacetylase: 0.5g; Novozymes PJN02351-Alcalase: 0.25ml; Condian immobilized lipase: 0.1g) were added to the mixture to obtain mixture 2, and the pH was adjusted to the optimal pH. Mixture 2 was placed in a shaker, the optimal reaction temperature was set, the rotation speed was 220rpm, and the reaction time was 2h. After the reaction, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22µm organic filter membrane to obtain the sucralose liquid chromatography sample.

[0310] The enzymes used in Examples 25-1, 25-2, 25-3, and 25-4 were respectively Kandian alkaline protease, Kaixiao immobilized deacetylesterase, Novozymes PJN02351-Alcalase, and Kandian immobilized fat. The molar conversion efficiency results are shown in Table 37 below.

[0311] Table 37

[0312] According to the detection data in Table 37 and Figure 25 above, under optimal reaction conditions, the type of enzyme has a significant impact on the formation of sucralose. Using a suitable enzyme can significantly affect the molar conversion rate of sucralose-6-ethyl ester. Using Kandian alkaline protease, Kaixiao immobilized deacetylesterase, Novozymes PJN02351-Alcalase, and Kandian immobilized lipase as enzymes can achieve a molar conversion rate of sucralose-6-ethyl ester of 53.44% to 99.83%. In particular, using Kandian immobilized lipase as an enzyme can achieve a molar conversion rate of 99.83% for sucralose-6-ethyl ester.

[0313] Example 26: A method for preparing sucralose using biological enzymes

[0314] Specifically, the steps include the following:

[0315] 2g of sucralose-6-ethyl ester substrate was dissolved in 100ml of 0.15M PBS buffer (pH 7.0) and sonicated until clear to obtain mixture 1. Condien immobilized lipase was added to the mixture and the pH was adjusted to 7.0 to obtain mixture 2. Mixture 2 was placed in a shaker at 30℃ and 220rpm for 2 hours. After the reaction, a sample was taken, diluted 4-fold with pure methanol, and filtered through a 0.22µm organic filter membrane to obtain the sucralose liquid chromatography sample.

[0316] The amounts of the biological enzyme added were: 0.05g (enzyme-to-enzyme ratio 15:1), 0.1g (enzyme-to-enzyme ratio 30:1), 0.2g (enzyme-to-enzyme ratio 60:1), and 0.4g (enzyme-to-enzyme ratio 120:1), where the enzyme-to-enzyme ratio = enzyme activity (U): sucralose-6-ethyl ester (g). Other types of enzymes can be used by equivalent conversion according to the enzyme activity of this invention.

[0317] The results of the molar conversion rate test are shown in Table 38 below.

[0318] Table 38

[0319] Comparative Example 26:

[0320] The difference from Example 26 is that the amount of biological enzyme added is 0.025g (enzyme-to-base ratio 7.5:1), while the other steps and operations are the same as in Example 26.

[0321] The results of the molar conversion rate test are shown in Table 39 below.

[0322] Table 39

[0323] According to the detection data in Tables 38 and 39 and Figure 26, the amount of added biological enzyme, i.e., the enzyme-to-base ratio, has a significant impact on the formation of sucralose. Using an appropriate enzyme-to-base ratio will affect the molar conversion rate of sucralose-6-ethyl ester. An enzyme-to-base ratio of 15-120:1 can make the molar conversion rate of sucralose-6-ethyl ester reach more than 77%, especially when the enzyme-to-base ratio is 30:1, the molar conversion rate of sucralose-6-ethyl ester reaches 99.18%.

[0324] Example 27: A method for preparing sucralose using biological enzymes

[0325] Specifically, the steps include the following:

[0326] The optimal reaction system and reaction conditions obtained in Examples 1-26 were adopted, specifically: reaction pH 7.0, reaction temperature 30℃, sucralose-6-ethyl ester addition amount 20%, sucralose formation concentration in the reaction solution controlled within the range of 0-20%, ammonium acetate formation concentration controlled within the range of 0-4%, 3L pure water reaction system, enzyme-to-solvent ratio 1:20. According to the batch stability determination method of immobilized enzyme in a 5L fermenter: (1) Weigh the sucralose-6-ethyl ester, pure water / PBS required for the experiment. (2) Dilute ammonia water, dilute 25-28% ammonia water reagent three times for later use. (3) Install the fermenter, adjust and calibrate the pH electrode, and connect the alkali replenishment bottle. (4) Add the prepared materials to the fermenter, set the parameters required for the experiment, temperature, rotation speed, pH. The conversion experiment begins. After the reaction is completed, take a sample, dilute it 4 times with methanol, filter it through a 0.22um organic filter membrane, and prepare a liquid phase detection sample. (5) Enzyme washing after reaction: The conversion liquid in the fermenter is extracted by attaching a filter cloth to the pipette of a peristaltic pump, 1L of pure water is added, the stirring is turned on, and the mixture is stirred and washed for 30 minutes. The mixture is then extracted by the peristaltic pump. (6) The next batch of 5L fermenter conversion experiments is carried out.

[0327] The reaction was carried out for 2.5 hours. After the reaction was completed, a sample was taken, diluted 4 times with pure methanol, and filtered through a 0.22 μm organic filter membrane to obtain a sucralose liquid phase detection sample.

[0328] The sample solution, along with ammonium acetate, sucralose-6-ethyl ester, and sucralose standards, were analyzed by HPLC, yielding spectra 27-29. The spectra showed that only the peaks of ammonium acetate (retention time 1.416 min) and sucralose (3.186 min) were present in the sample, indicating that there were essentially no reactive impurities and the reaction was complete.

[0329] After repeating the method of Example 27 for 120 batches, the conversion rate was tested. The results are shown in Figure 30. As can be seen from Figure 30, after 120 batches of reaction, the enzyme activity did not decrease significantly, and the conversion rate remained at about 99.81%.

[0330] Examples 1, 7, 13, and 19 of this invention provide experiments on the removal of the acetyl group at the 6-position of sucralose-6-ethyl ester using the enzyme alone at different enzyme-to-base ratios; Examples 2, 8, 14, and 20 provide experiments on the removal of the acetyl group at the 6-position of sucralose-6-ethyl ester using the enzyme alone at different enzyme-to-base ratios. When the acetyl group at the 6-position of sucralose was removed from the ethyl ester, sucralose was added to the system. The reaction showed that the conversion rate gradually decreased with increasing sucralose concentration. Examples 3, 9, 15, and 21 showed that when a small amount of ammonium acetate was added to the system for the removal of the acetyl group at the 6-position of sucralose-6-ethyl ester, the conversion rate gradually decreased with increasing ammonium acetate concentration. Examples 4, 10, 16, and 22 showed the effect of pH on the reaction during the removal of the acetyl group at the 6-position of sucralose-6-ethyl ester. Examples 5, 11, 17, and 23 showed the effect of temperature on the reaction during the removal of the acetyl group at the 6-position of sucralose-6-ethyl ester. Examples 6, 12, 18, and 24 showed the effect of using pure water or PBS on the reaction during the removal of the acetyl group at the 6-position of sucralose-6-ethyl ester.

[0331] From the above examples, it can be seen that in Examples 2, 8, 14, 20 and Examples 3, 9, 15, 21, sucralose and ammonium acetate were added respectively, and the conversion rate decreased to varying degrees with increasing concentration. Based on the suggestions from Examples 4, 10, 16, 22 and Examples 5, 11, 17, 23 and Examples 6, 12, 18, 24, suitable reaction process parameters can be found.

[0332] From the basic principle of enzyme-catalyzed reactions, enzymes, as biological catalysts, can accelerate the rate of chemical reactions without changing the total energy change of the reaction. Enzymes bind to substrates to form enzyme-substrate complexes, lowering the activation energy of the reaction and thus promoting its progress. For example, enzymes bind to sucralose-6-ethyl ester to form enzyme-sucralose-6-ethyl ester complexes, which also promote the reaction.

[0333] Theoretically, adding a product (such as sucralose) will bind to the enzyme, forming an enzyme-product complex, which will occupy the enzyme's active site and reduce the enzyme's catalytic ability on the substrate. This usually leads to a decrease in substrate conversion because the enzyme's catalytic effect on the substrate is weakened.

[0334] This invention discovers that the addition of ammonium acetate has an inhibitory effect on enzymatic reactions. As an effector, ammonium acetate affects the conformation or activity of enzymes; or by binding to enzymes, it changes the conformation of enzymes, weakens their ability to bind to substrates, and reduces the catalytic efficiency of enzymes.

[0335] Therefore, when the concentrations of sucralose and ammonium acetate in the reaction system reach a certain value, the conversion rate of sucralose-6-ethyl ester will be significantly reduced. Controlling the concentrations of sucralose and ammonium acetate in each batch of reaction is the key to maintaining the efficiency of the enzyme catalyst.

[0336] As seen in Example 26, the enzyme activity was well maintained after 120 cycles. This is likely a result of reducing the inhibitory effect on the enzyme by controlling the concentrations of sucralose and ammonium acetate in the reaction batches.

[0337] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A process for the production of sucralose using a biological enzyme, characterized in that: Sucralose is obtained by removing the acetyl group at the 6-position of sucralose-6-ethyl ester by adding a biological enzyme to an enzymatic reaction system. The biological enzyme is selected from one or more of alkaline protease, serine endopeptide protease, immobilized lipase, and immobilized deacetylesterase.

2. The method of claim 1, wherein: The enzymatic reaction system includes a biological enzyme and sucralose-6-ethyl ester.

3. The method of claim 2, wherein: The enzyme-catalyzed reaction system also includes sucralose; the mass fraction of sucralose in the enzyme-catalyzed reaction system is 1%-20%, preferably less than 10%.

4. The method of claim 2, wherein: The mass fraction of sucralose-6-ethyl ester in the enzymatic reaction system is 1%-40%; preferably 5-20%; and particularly preferably 10-20%.

5. The method of claim 2, wherein: The enzyme-catalyzed reaction system also includes ammonium acetate, wherein the mass fraction of ammonium acetate in the enzyme-catalyzed reaction system is 0%-10%, preferably less than 4%.

6. The method of claim 1, wherein: The enzyme-to-enzyme ratio of the biological enzyme and sucralose-6-ethyl ester is 7.5-120:1, preferably 15-60:1, and particularly preferably 15-30:

1.

7. The method of claim 1, wherein: The enzymatic reaction conditions include temperature and pH; the temperature is 30-60℃, preferably 46℃; the pH is 6.6-7.7, preferably 7.0-7.8; and particularly preferably 7.0-7.

4.

8. A method for preparing sucralose using a biological enzyme, characterized by, Includes the following steps: Step 1: Dissolve the sucralose-6-ethyl ester substrate in pure water or PBS buffer to obtain mixture 1; Step 2: Add the biological enzyme to the mixture to obtain mixture 2; Step 3: Treat mixture 2 and carry out the reaction.

9. A process for the production of sucralose using a biological enzyme according to claim 8, characterised in that, Includes the following steps: In step 3, the mixture is treated by adding sucralose and ammonium acetate to mixture 2 to obtain mixture 3; the reaction temperature is 30-60 degrees Celsius and the pH value is 6.6-8.

2.

10. The process of preparing trichlorosucrose using a biological enzyme according to claim 9, characterized in that, In step 2, the biological enzyme is selected from one or more of alkaline protease, serine endopeptide protease, immobilized lipase, and immobilized deacetylesterase.

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