Method for preparing trehalose from starch
By preparing modified silica particles with uniform morphology and smooth surface as carriers to load enzymes, the problems of large enzyme usage and poor immobilization performance were solved, and efficient preparation and low-cost production of trehalose were achieved.
Patent Information
- Application Number
- PCT/CN2024/117960
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2024-03-13
- Filing Date
- 2024-09-10
- Publication Date
- 2025-09-18
AI Technical Summary
The existing methods for preparing trehalose require a large amount of enzyme, resulting in high production costs and poor enzyme immobilization performance, which leads to a decrease in yield and purity after repeated use.
A specific method is used to prepare the carrier, using mixed acid as a surfactant in combination with specific reaction and post-treatment steps to prepare silica particles with uniform morphology and smooth surface. A modification step is performed to avoid agglomeration and use them as a carrier to load the enzyme, achieving a uniform distribution of the enzyme active centers and improving the immobilization performance.
The yield and purity of trehalose are improved, the production cost is reduced, the enzyme reusability is achieved, and the quality and yield of trehalose are ensured.
Abstract
Description
A method for preparing trehalose from starch Technical Field
[0001] The invention belongs to the technical field of trehalose, and particularly relates to a method for preparing trehalose from starch. Background Art
[0002] Trehalose is a safe and reliable natural sugar. It is a non-reducing sugar composed of two glucose molecules with an α,α-1,1-glycosidic bond and is very stable in nature.
[0003] Trehalose forms a unique protective film on the cell surface under harsh environments such as high temperature, extreme cold, high osmotic pressure, and dehydration, effectively protecting protein molecules from denaturation and inactivation, thereby maintaining the life processes and biological characteristics of living organisms. Other sugars in nature, such as sucrose and glucose, do not have this function. This unique characteristic makes trehalose not only an excellent active protector for protein drugs, enzymes, vaccines and other biological products, but also an important ingredient for maintaining cell activity and moisturizing cosmetics. It can also be used as a unique food ingredient to prevent food deterioration, maintain food freshness and flavor, and improve food quality, expanding the scope of trehalose's application.
[0004] Existing methods for preparing trehalose mainly include microbial extraction, fermentation, genetic recombination, and enzymatic conversion. Microbial extraction, fermentation, and genetic recombination involve high technical requirements and high production costs, making them unsuitable for large-scale industrial production.
[0005] The enzymatic conversion method uses glucose, maltose or starch as substrates and converts them into trehalose through the action of relevant enzymes. Currently, there are two routes for preparing trehalose using starch as raw material. One is to convert starch into trehalose using glucosyltransferase and amylase, and the other is to convert amylose into trehalose using the synergistic action of maltoside trehalose synthase and maltoside trehalose hydrolase.
[0006] However, the existing methods for preparing trehalose all have the problem of large amount of enzyme used and high production cost;
[0007] In this regard, Chinese patent application number 2014102331988 discloses a method for preparing trehalose using cassava starch as a raw material. It specifically discloses the introduction of silica as an enzyme stationary phase to shorten the reaction time and reduce the amount of enzyme used. However, when silica is directly used as the enzyme stationary phase, silica easily agglomerates, affecting the contact between the enzyme and the substrate and the catalytic performance of the enzyme. Moreover, after repeated use, the enzyme easily falls off the silica, thereby greatly reducing the yield of the product after repeated use.
[0008] Therefore, providing a method for preparing trehalose from starch, using a carrier to immobilize the enzyme, reducing the amount of enzyme used, ensuring effective adsorption of the enzyme, reducing enzyme shedding, and enhancing the reusability of the immobilized enzyme are technical difficulties that need to be solved in the existing technology.
[0009] Summary of the Invention
[0010] In order to solve the technical problems existing in the prior art, the present invention provides a method for preparing trehalose from starch, and provides a method for preparing trehalose from starch. The method uses a carrier to immobilize the enzyme, thereby reducing the amount of enzyme used, ensuring the effective adsorption of the enzyme, reducing the shedding of the enzyme, and enhancing the reusability of the immobilized enzyme.
[0011] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0012] A method for preparing trehalose from starch, comprising the steps of preparing starch milk, liquefaction, carrier preparation, enzymatic hydrolysis, filtration, decolorization, ion exchange, crystallization, and centrifugal drying. The specific operations are as follows:
[0013] 1. Preparation of starch milk
[0014] Mix corn starch with deionized water, add polyethylene glycol 400, control the stirring temperature to 60-64°C, stir for 25-30 minutes, and prepare starch milk after stirring.
[0015] The mass ratio of the corn starch, deionized water and polyethylene glycol 400 is 47-50:145-153:1.2-1.4.
[0016] 2. Liquefaction
[0017] Adding a thermostable α-amylase to the starch milk, heating the mixture to 73-78°C at a rate of 0.4-0.6°C / min, stirring for 10-14 minutes at a stirring rate of 76-82 rpm, and continuing to heat the mixture at a heating rate of 0.2-0.4°C / min to 88-92°C after the stirring is completed, stirring the mixture for 8-12 minutes at a stirring rate of 98-110 rpm, and finally completing the stirring to obtain a liquefied liquid;
[0018] The mass ratio of the starch milk to the thermostable α-amylase is 16-19 kg:1.2-1.4 g.
[0019] 3. Preparation of Vector
[0020] (1) Preparation of mixed sulfuric acid solution
[0021] Hexadecyltrimethylammonium bromide, carboxymethyl cellulose and sodium lauryl sulfate were mixed and then ultrasonically treated, with the ultrasonic time controlled at 10-14 minutes and the ultrasonic frequency at 10-12 kHz. After the ultrasonic treatment, the mixture was added to a sulfuric acid solution and stirred to obtain a mixed sulfuric acid solution.
[0022] The mass concentration of the sulfuric acid is 12.5-12.9%;
[0023] The mass ratio of cetyltrimethylammonium bromide, carboxymethyl cellulose, sodium lauryl sulfate and sulfuric acid is 0.8-1.2:0.16-0.18:0.60-0.64:96-104;
[0024] (2) Reaction
[0025] Add 2.8-3.4 times the mass of deionized water to the water glass, then place it in a reaction apparatus, heat it to 82-86°C at a rate of 0.3-0.5°C / min, add a mixed sulfuric acid solution, control the addition rate of the mixed sulfuric acid solution to be 4.8-5.2 g / min, control the pH value at the reaction endpoint to be 5.1-5.3, complete the reaction, and age it for 3.4-3.6 hours to obtain a reaction solution;
[0026] The mass content of Na2O in the water glass is 8.0-8.4%, and the mass content of SiO2 is 16.5-17.0%;
[0027] (3) Post-processing
[0028] The reaction solution is filtered, washed, dried, and calcined in sequence to obtain silica particles;
[0029] The drying is vacuum freeze drying, the freezing temperature is -40 to -36°C, the freezing time is 18 to 22 hours, and the vacuum degree is 23 to 27 Pa;
[0030] The calcination temperature is 305-315° C. and the calcination time is 1.3-1.7 h;
[0031] (4) Modification
[0032] The silica particles were mixed with 6-10 times the mass of an ethanol solution, and the temperature was raised to 48-52°C at a rate of 0.4-0.6°C / min. A modifier was added and stirred for 2.1-2.3 hours at a stirring speed of 174-185 rpm. After the stirring was completed, the mixture was washed and dried to obtain modified silica.
[0033] The mass concentration of the ethanol solution is 30-34%;
[0034] The mass ratio of the ethanol solution to the modifier is 100:1.4-1.6;
[0035] The modifier is a mixture of γ-glycidyloxypropyltriethoxysilane and Tween-20, and the mass ratio of the γ-glycidyloxypropyltriethoxysilane to Tween-20 is 0.8-1.2:0.4-0.6.
[0036] 4. Enzymatic hydrolysis
[0037] (1) Loading of complex enzyme
[0038] The modified silica is mixed with 6-8 times the volume of deionized water, and then maltosyl trehalose synthase and maltosyl trehalose hydrolase are added and stirred at a stirring temperature of 25-27° C. and a pH of 6.7-6.9. After stirring evenly, the mixture is washed, filtered, and vacuum-dried at 30-34° C. to obtain a composite enzyme loaded with modified silica;
[0039] The mass ratio of the maltosyl trehalose synthase, maltosyl trehalose hydrolase and deionized water is 3.2-3.6:1.8-2.0:87-94;
[0040] (2) Catalytic reaction
[0041] Adding the composite enzyme loaded with modified silica to the liquefied liquid, carrying out a catalytic reaction at a temperature of 43-45° C. for 28-32 hours, and controlling the pH at 5.8-6.2 to obtain a catalytic reaction liquid;
[0042] The mass ratio of the liquefied liquid to the complex enzyme loaded with modified silica is 4.0-4.2 kg:3 g.
[0043] 5. Filtration, decolorization, ion exchange
[0044] The catalytic reaction liquid was filtered with a filtration accuracy of 0.25 μm, and the filtration cycle was repeated three times to obtain a filtrate. Activated carbon was added to the filtrate, and the decolorization temperature was 60-64°C, and the decolorization time was 24-27 minutes. The process was repeated twice to obtain a decolorized supernatant. The decolorized supernatant was ion exchanged to obtain a desalted solution with a conductivity of 29-31 μs / cm.
[0045] The mass ratio of the activated carbon to the filtrate is 1.0-1.2:100.
[0046] 6. Crystallization
[0047] The desalted liquid is concentrated until the mass concentration of trehalose is 73-77% to obtain a concentrated solution, which is then cooled to 43-46°C, and trehalose seed crystals are added for cooling crystallization. The cooling rate is controlled at 1.0-1.4°C / min, and the temperature is lowered to 16-20°C to obtain a crystallized solution.
[0048] The particle size of the trehalose seed crystals is 110-130 mesh;
[0049] The mass ratio of the trehalose seed crystals to the concentrated solution is 1.4-1.7:100.
[0050] 7. Centrifugal drying
[0051] The crystal liquid is centrifuged at a centrifugal speed of 2040-2150 rpm for 18-22 minutes, and then placed in an oven for drying for 7-9 hours at a temperature of 76-78° C. to obtain trehalose.
[0052] Compared with the prior art, the present invention achieves the following beneficial effects:
[0053] 1. The present invention adopts a specific method to prepare a carrier, wherein the mixed acid solution mainly serves as a surfactant, which is combined with specific reactions and post-processing steps to make the silica particles uniform in morphology, smooth in surface, and dispersible, thereby ensuring the integrity of the silica particles. Then, through a specific modification step, the agglomeration of silica ions is avoided. Modified silica is used as a carrier, and the enzyme is loaded on the surface and in the pores of the modified silica, thereby achieving the distribution of the enzyme active center on the carrier, giving full play to the catalytic activity of the enzyme active center, improving the immobilization performance of the enzyme, and enhancing the immobilization performance of the enzyme, thereby improving the yield of trehalose, ensuring purity and quality, achieving the reusability of the composite enzyme after loading the modified silica, and reducing production costs.
[0054] 2. The trehalose prepared by the method of the present invention has a purity of 99.24-99.63% and a yield of 60.3-61.2%. According to the method of GB / T 23529-2009, the drying loss is 0.6-0.8% and the chromaticity is 0.05-0.08.
[0055] 3. The method of preparing trehalose from starch of the present invention comprises the following steps: after the reaction is completed, the composite enzyme loaded with modified silica is recovered, washed with purified water, vacuum-dried, and then reused 15 times. The trehalose prepared in the 15th step is measured to have a purity of 99.02-99.50% and a yield of 58.7-60.5%. DETAILED DESCRIPTION
[0056] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.
[0057] Example 1 A method for preparing trehalose from starch
[0058] 1. Preparation of starch milk
[0059] Corn starch was mixed with deionized water, polyethylene glycol 400 was added, the stirring temperature was controlled at 62°C, the stirring time was 27 minutes, and starch milk was prepared after the stirring was completed.
[0060] The mass ratio of the corn starch, deionized water and polyethylene glycol 400 is 49:150:1.3.
[0061] 2. Liquefaction
[0062] Adding a thermostable α-amylase to the starch milk, heating the mixture to 75°C at a rate of 0.5°C / min, stirring for 12 minutes at a stirring rate of 79 rpm, and continuing to heat the mixture at a heating rate of 0.3°C / min to 90°C, stirring for 10 minutes at a stirring rate of 104 rpm, and finally obtaining a liquefied liquid;
[0063] The mass ratio of the starch milk to the thermostable α-amylase is 18kg:1.3g.
[0064] 3. Preparation of Vector
[0065] (1) Preparation of mixed sulfuric acid solution
[0066] Hexadecyltrimethylammonium bromide, carboxymethyl cellulose and sodium lauryl sulfate were mixed and then ultrasonically treated. The ultrasonic time was controlled to be 12 minutes and the ultrasonic frequency was 11 kHz. After the ultrasonic treatment, the mixture was added to the sulfuric acid solution and stirred to obtain a mixed sulfuric acid solution.
[0067] The mass concentration of the sulfuric acid is 12.7%;
[0068] The mass ratio of the hexadecyltrimethylammonium bromide, carboxymethyl cellulose, sodium lauryl sulfate and sulfuric acid is 1.0:0.17:0.62:100;
[0069] (2) Reaction
[0070] Add 3 times the mass of deionized water to the water glass, then place it in a reaction apparatus, heat it to 84°C at a rate of 0.4°C / min, add a mixed sulfuric acid solution, control the addition rate of the mixed sulfuric acid solution to be 5g / min, control the pH value at the reaction end point to be 5.2, terminate the reaction, and age it for 3.5h to obtain a reaction solution;
[0071] The mass content of Na2O in the water glass is 8.2%, and the mass content of SiO2 is 16.8%;
[0072] (3) Post-processing
[0073] The reaction solution is filtered, washed, dried, and calcined in sequence to obtain silica particles;
[0074] The drying is vacuum freeze drying, the freezing temperature is -38°C, the freezing time is 20h, and the vacuum degree is 25Pa;
[0075] The calcination temperature is 310° C. and the calcination time is 1.5 h;
[0076] (4) Modification
[0077] The silica particles were mixed with 8 times the mass of ethanol solution, heated to 50°C at a rate of 0.5°C / min, and a modifier was added and stirred for 2.2 hours at a stirring speed of 180 rpm. After the stirring was completed, the mixture was washed and dried to obtain modified silica.
[0078] The mass concentration of the ethanol solution is 32%;
[0079] The mass ratio of the ethanol solution to the modifier is 100:1.5;
[0080] The modifier is a mixture of γ-glycidyloxypropyltriethoxysilane and Tween-20, and the mass ratio of the γ-glycidyloxypropyltriethoxysilane to Tween-20 is 0.9:0.5.
[0081] 4. Enzymatic hydrolysis
[0082] (1) Loading of complex enzyme
[0083] The modified silica was mixed with 7 volumes of deionized water, and then maltosyl trehalose synthase and maltosyl trehalose hydrolase were added and stirred at 26°C and pH 6.8. After uniform stirring, the mixture was washed, filtered, and vacuum-dried at 32°C to obtain a composite enzyme loaded with modified silica.
[0084] The mass ratio of the maltosyl trehalose synthase, maltosyl trehalose hydrolase and deionized water is 3.4:1.9:90;
[0085] (2) Catalytic reaction
[0086] The composite enzyme loaded with modified silica was added to the liquefied liquid, and the catalytic reaction was carried out at a temperature of 44° C. for 30 hours, and the pH was controlled at 6 to obtain a catalytic reaction liquid.
[0087] The mass ratio of the liquefied liquid to the complex enzyme loaded with modified silica is 4.1 kg:3 g.
[0088] 5. Filtration, decolorization, ion exchange
[0089] The catalytic reaction liquid was filtered with a filtration accuracy of 0.25 μm, and the filtration cycle was repeated three times to obtain a filtrate. Activated carbon was added to the filtrate, and the decolorization temperature was 62°C and the decolorization time was 25 minutes. This was repeated twice to obtain a decolorized supernatant. The decolorized supernatant was subjected to ion exchange to obtain a desalted solution with a conductivity of 30 μs / cm.
[0090] The mass ratio of the activated carbon to the filtrate is 1.1:100.
[0091] 6. Crystallization
[0092] The desalted liquid was concentrated until the trehalose mass concentration was 75% to obtain a concentrated solution, which was then cooled to 45°C, and trehalose seed crystals were added for cooling crystallization. The cooling rate was controlled at 1.2°C / min, and the temperature was lowered to 18°C to obtain a crystallized solution.
[0093] The particle size of the trehalose seed crystals is 120 mesh;
[0094] The mass ratio of the trehalose seed crystals to the concentrated solution is 1.6:100.
[0095] 7. Centrifugal drying
[0096] The crystal liquid was centrifuged at a centrifugal speed of 2100 rpm for 20 minutes, and then dried in an oven at a temperature of 77° C. for 8 hours to obtain trehalose.
[0097] The trehalose prepared by the method of Example 1 had a purity of 99.63% and a yield of 61.2%. According to the method of GB / T 23529-2009, the drying loss was 0.6% and the chromaticity was 0.05.
[0098] After the reaction, the composite enzyme loaded with modified silica was recovered, washed with purified water, and vacuum-dried. The composite enzyme was then reused 15 times according to the method of Example 1. The trehalose obtained in the 15th step had a purity of 99.50% and a yield of 60.5%.
[0099] Example 2 A method for preparing trehalose from starch
[0100] 1. Preparation of starch milk
[0101] Corn starch was mixed with deionized water, polyethylene glycol 400 was added, the stirring temperature was controlled at 60°C, the stirring time was 25 minutes, and starch milk was prepared after the stirring was completed;
[0102] The mass ratio of the corn starch, deionized water and polyethylene glycol 400 is 47:145:1.2.
[0103] 2. Liquefaction
[0104] Adding a thermostable α-amylase to the starch milk, heating the mixture to 73°C at a rate of 0.4°C / min, stirring for 10 minutes at a stirring rate of 76 rpm, and continuing to heat the mixture at a heating rate of 0.2°C / min to 88°C after the stirring, stirring the mixture for 12 minutes at a stirring rate of 98 rpm, and finally obtaining a liquefied liquid;
[0105] The mass ratio of the starch milk to the thermostable α-amylase is 16kg:1.2g.
[0106] 3. Preparation of Vector
[0107] (1) Preparation of mixed sulfuric acid solution
[0108] Hexadecyltrimethylammonium bromide, carboxymethyl cellulose and sodium lauryl sulfate were mixed and then ultrasonically treated. The ultrasonic time was controlled to be 10 minutes and the ultrasonic frequency was 10 kHz. After the ultrasonic treatment, the mixture was added to the sulfuric acid solution and stirred to obtain a mixed sulfuric acid solution.
[0109] The mass concentration of the sulfuric acid is 12.5%;
[0110] The mass ratio of cetyltrimethylammonium bromide, carboxymethyl cellulose, sodium lauryl sulfate and sulfuric acid is 0.8:0.16:0.60:96;
[0111] (2) Reaction
[0112] 2.8 times the mass of deionized water was added to the water glass, and then placed in a reaction apparatus. The temperature was raised to 82°C at a rate of 0.3°C / min, and a mixed sulfuric acid solution was added. The addition rate of the mixed sulfuric acid solution was controlled to be 4.8 g / min. The pH value at the reaction end point was controlled to be 5.1. The reaction was completed and aged for 3.4 hours to obtain a reaction solution.
[0113] The mass content of Na2O in the water glass is 8.0%, and the mass content of SiO2 is 16.5%;
[0114] (3) Post-processing
[0115] The reaction solution is filtered, washed, dried, and calcined in sequence to obtain silica particles;
[0116] The drying is vacuum freeze drying, the freezing temperature is -40°C, the freezing time is 18h, and the vacuum degree is 23Pa;
[0117] The calcination temperature is 305°C and the calcination time is 1.7h;
[0118] (4) Modification
[0119] The silica particles were mixed with 6 times the mass of ethanol solution, heated to 48°C at a rate of 0.4°C / min, and a modifier was added and stirred for 2.3 hours at a stirring speed of 174 rpm. After the stirring was completed, the mixture was washed and dried to obtain modified silica.
[0120] The mass concentration of the ethanol solution is 30%;
[0121] The mass ratio of the ethanol solution to the modifier is 100:1.4;
[0122] The modifier is a mixture of γ-glycidyloxypropyltriethoxysilane and Tween-20, and the mass ratio of the γ-glycidyloxypropyltriethoxysilane to Tween-20 is 0.8:0.4.
[0123] 4. Enzymatic hydrolysis
[0124] (1) Loading of complex enzyme
[0125] The modified silica was mixed with 6 times the volume of deionized water, and then maltosyl trehalose synthase and maltosyl trehalose hydrolase were added and stirred at a stirring temperature of 25°C and a pH of 6.7. After stirring evenly, the mixture was washed, filtered, and vacuum-dried at 30°C to obtain a composite enzyme loaded with modified silica.
[0126] The mass ratio of the maltosyl trehalose synthase, maltosyl trehalose hydrolase and deionized water is 3.2:1.8:87;
[0127] (2) Catalytic reaction
[0128] The composite enzyme loaded with modified silica was added to the liquefied liquid, and the catalytic reaction was carried out at a temperature of 43° C. for 28 hours, and the pH was controlled at 5.8 to obtain a catalytic reaction liquid.
[0129] The mass ratio of the liquefied liquid to the complex enzyme loaded with modified silica is 4.0 kg:3 g.
[0130] 5. Filtration, decolorization, ion exchange
[0131] The catalytic reaction liquid was filtered with a filtration accuracy of 0.25 μm and recycled for three times to obtain a filtrate. Activated carbon was added to the filtrate and the decolorization temperature was 60°C for 27 minutes. This was repeated twice to obtain a decolorized supernatant. The decolorized supernatant was subjected to ion exchange to obtain a desalted solution with a conductivity of 29 μs / cm.
[0132] The mass ratio of the activated carbon to the filtrate is 1.0:100.
[0133] 6. Crystallization
[0134] The desalted liquid was concentrated until the trehalose mass concentration was 73% to obtain a concentrated solution, which was then cooled to 43°C, and trehalose seed crystals were added for cooling crystallization. The cooling rate was controlled at 1.0°C / min, and the temperature was lowered to 16°C to obtain a crystallized solution.
[0135] The particle size of the trehalose seed crystals is 110 mesh;
[0136] The mass ratio of the trehalose seed crystals to the concentrated solution is 1.4:100.
[0137] 7. Centrifugal drying
[0138] The crystal liquid was centrifuged at a centrifugal speed of 2040 rpm for 18 minutes, and then dried in an oven at a temperature of 78° C. for 7 hours to obtain trehalose.
[0139] The trehalose prepared by the method of Example 2 had a purity of 99.24% and a yield of 60.3%. According to the method of GB / T 23529-2009, the drying loss was 0.8% and the chromaticity was 0.08.
[0140] After the reaction, the composite enzyme loaded with modified silica was recovered, washed with purified water, and vacuum-dried. The composite enzyme was then reused 15 times according to the method of Example 2. The purity of the trehalose obtained in the 15th step was 99.02%, and the yield was 58.7%.
[0141] Example 3 A method for preparing trehalose from starch
[0142] 1. Preparation of starch milk
[0143] Corn starch was mixed with deionized water, polyethylene glycol 400 was added, the stirring temperature was controlled at 64°C, the stirring time was 30 minutes, and starch milk was prepared after the stirring was completed.
[0144] The mass ratio of the corn starch, deionized water and polyethylene glycol 400 is 50:153:1.4.
[0145] 2. Liquefaction
[0146] Adding a thermostable α-amylase to the starch milk, heating the mixture to 78°C at a rate of 0.6°C / min, stirring for 14 minutes at a stirring rate of 82 rpm, and continuing to heat the mixture at a heating rate of 0.4°C / min to 92°C, stirring for 8 minutes at a stirring rate of 110 rpm, and finally completing stirring to obtain a liquefied liquid;
[0147] The mass ratio of the starch milk to the thermostable α-amylase is 19kg:1.4g.
[0148] 3. Preparation of Vector
[0149] (1) Preparation of mixed sulfuric acid solution
[0150] Hexadecyltrimethylammonium bromide, carboxymethyl cellulose and sodium lauryl sulfate were mixed and then ultrasonically treated. The ultrasonic time was controlled to be 14 minutes and the ultrasonic frequency was controlled to be 12 kHz. After the ultrasonic treatment, the mixture was added to the sulfuric acid solution and stirred to obtain a mixed sulfuric acid solution.
[0151] The mass concentration of the sulfuric acid is 12.9%;
[0152] The mass ratio of cetyltrimethylammonium bromide, carboxymethyl cellulose, sodium lauryl sulfate and sulfuric acid is 1.2:0.18:0.64:104;
[0153] (2) Reaction
[0154] 3.4 times the mass of deionized water was added to the water glass, and then placed in a reaction apparatus. The temperature was raised to 86°C at a rate of 0.5°C / min, and a mixed sulfuric acid solution was added. The addition rate of the mixed sulfuric acid solution was controlled to be 5.2 g / min, and the pH value at the reaction end point was controlled to be 5.3. The reaction was completed and aged for 3.6 hours to obtain a reaction solution.
[0155] The mass content of Na2O in the water glass is 8.4%, and the mass content of SiO2 is 17.0%;
[0156] (3) Post-processing
[0157] The reaction solution is filtered, washed, dried, and calcined in sequence to obtain silica particles;
[0158] The drying is vacuum freeze drying, the freezing temperature is -36°C, the freezing time is 22h, and the vacuum degree is 27Pa;
[0159] The calcination temperature is 315°C and the calcination time is 1.3h;
[0160] (4) Modification
[0161] The silica particles were mixed with 10 times the mass of an ethanol solution, heated to 52°C at a rate of 0.6°C / min, and a modifier was added and stirred for 2.1 hours at a stirring speed of 185 rpm. After the stirring was completed, the mixture was washed and dried to obtain modified silica.
[0162] The mass concentration of the ethanol solution is 34%;
[0163] The mass ratio of the ethanol solution to the modifier is 100:1.6;
[0164] The modifier is a mixture of γ-glycidyloxypropyltriethoxysilane and Tween-20, and the mass ratio of the γ-glycidyloxypropyltriethoxysilane to Tween-20 is 1.2:0.6.
[0165] 4. Enzymatic hydrolysis
[0166] (1) Loading of complex enzyme
[0167] The modified silica was mixed with 8 times the volume of deionized water, and then maltosyl trehalose synthase and maltosyl trehalose hydrolase were added and stirred at a stirring temperature of 27°C and a pH of 6.9. After stirring evenly, the mixture was washed, filtered, and vacuum-dried at 34°C to obtain a composite enzyme loaded with modified silica.
[0168] The mass ratio of the maltosyl trehalose synthase, maltosyl trehalose hydrolase and deionized water is 3.6:2.0:94;
[0169] (2) Catalytic reaction
[0170] The composite enzyme loaded with modified silica was added to the liquefied liquid, and the catalytic reaction was carried out at a temperature of 45° C. for 32 hours, and the pH was controlled at 6.2 to obtain a catalytic reaction liquid.
[0171] The mass ratio of the liquefied liquid to the complex enzyme loaded with modified silica is 4.2 kg:3 g.
[0172] 5. Filtration, decolorization, ion exchange
[0173] The catalytic reaction liquid was filtered with a filtration accuracy of 0.25 μm and recycled for three times to obtain a filtrate. Activated carbon was added to the filtrate and the decolorization temperature was 64°C for 24 minutes. This was repeated twice to obtain a decolorized supernatant. The decolorized supernatant was subjected to ion exchange to obtain a desalted solution with a conductivity of 31 μs / cm.
[0174] The mass ratio of the activated carbon to the filtrate is 1.2:100.
[0175] 6. Crystallization
[0176] The desalted liquid was concentrated until the trehalose mass concentration was 77% to obtain a concentrated solution, which was then cooled to 46°C, and trehalose seed crystals were added for cooling crystallization. The cooling rate was controlled at 1.4°C / min, and the temperature was lowered to 20°C to obtain a crystallized solution.
[0177] The particle size of the trehalose seed crystals is 130 mesh;
[0178] The mass ratio of the trehalose seed crystals to the concentrated solution is 1.7:100.
[0179] 7. Centrifugal drying
[0180] The crystal liquid was centrifuged at a centrifugal speed of 2150 rpm for 22 minutes, and then dried in an oven at a temperature of 76° C. for 9 hours to obtain trehalose.
[0181] The trehalose prepared by the method of Example 3 had a purity of 99.47% and a yield of 60.9%. According to the method of GB / T 23529-2009, the drying loss was 0.7% and the chromaticity was 0.06.
[0182] After the reaction, the composite enzyme loaded with modified silica was recovered, washed with purified water, and vacuum-dried. The enzyme was then reused 15 times according to the method of Example 3. The purity of the trehalose obtained in the 15th step was 99.28%, and the yield was 59.6%.
[0183] Comparative Example 1
[0184] Based on Example 1, the change is that in the enzymatic hydrolysis reaction, the enzyme loading is omitted and the enzyme is directly added for reaction. The other operations are the same.
[0185] The trehalose prepared by the method of Comparative Example 1 had a purity of 90.26% and a yield of 45.3%. According to the method in GB / T 23529-2009, the drying loss was 1.5% and the chromaticity was 0.17.
[0186] In Comparative Example 1, the enzyme was not loaded, and the enzyme could not be reused, which greatly increased the production cost.
[0187] Comparative Example 2
[0188] On the basis of Example 1, the changes are as follows:
[0189] (1) In the carrier preparation step, the mixed sulfuric acid solution is replaced with sulfuric acid;
[0190] (2) In the post-processing step, vacuum freeze drying was replaced by drying at 80°C for 30 min;
[0191] (3) Omit the modification step;
[0192] The rest of the operations are the same.
[0193] The trehalose prepared by the method of Comparative Example 2 had a purity of 93.14% and a yield of 52.6%. According to the method in GB / T 23529-2009, the loss on drying was 1.1% and the chromaticity was 0.10.
[0194] After the reaction, the composite enzyme loaded with modified silica was recovered, washed with purified water, and vacuum-dried. The enzyme was then reused 15 times in accordance with the method of Comparative Example 2. The purity of the trehalose obtained in the 15th step was 83.68%, and the yield was 45.7%.
[0195] The present invention adopts a specific method to prepare a carrier, wherein the mixed acid solution mainly serves as a surfactant, which is combined with specific reactions and post-processing steps to ensure that the silica particles have uniform morphology, good surface smoothness, and good dispersibility, thereby ensuring the integrity of the silica particles. Then, through a specific modification step, the agglomeration of silica ions is avoided. Modified silica is used as a carrier, and enzymes are loaded on the surface and in the pores of the modified silica, thereby achieving the distribution of enzyme active centers on the carrier, giving full play to the catalytic activity of the enzyme active centers, improving the immobilization performance of the enzyme, and enhancing the immobilized use performance of the enzyme, thereby improving the yield of trehalose, ensuring purity and quality, achieving the reusability of the composite enzyme after loading the modified silica, and reducing production costs.
[0196] As can be seen from Comparative Example 1, the enzyme loading was omitted and the enzyme was directly added for reaction. The enzyme could not be reused, which greatly increased the production cost. In addition, the catalytic effect of the enzyme in the reaction system was poor and the enzyme's reaction activity could not be fully exerted, thereby reducing the yield and quality of trehalose.
[0197] According to Comparative Example 2, the surfactant was omitted, and the drying method was used for drying. In addition, the modification step of the silica particles was omitted. The surface of the silica particles obtained was not smooth and the morphology was not uniform. In addition, many pores collapsed during the drying process. The silica particles had poor dispersibility and were easily agglomerated, which made it impossible to effectively load the enzyme. The enzyme had poor immobilization performance. During repeated use, the enzyme fell off the carrier, resulting in a low yield and purity of trehalose. Moreover, after repeated use, the purity and yield of trehalose decreased significantly.
[0198] Unless otherwise specified, all ratios and percentages described in the present invention are by mass.
[0199] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.
Claims
1. A method for preparing trehalose from starch, characterized in that: The method comprises the steps of preparing starch milk, liquefying, preparing a carrier, enzymatic hydrolysis, filtering, decolorizing, ion exchanging, crystallizing, and centrifugal drying; The carrier preparation step includes preparing a mixed sulfuric acid solution, reaction, post-treatment and modification steps; The enzymatic hydrolysis step includes complex enzyme loading and catalytic reaction steps; The step of preparing the mixed sulfuric acid solution comprises mixing cetyltrimethylammonium bromide, carboxymethyl cellulose and sodium lauryl sulfate, then performing ultrasonic treatment, controlling the ultrasonic time to be 10-14 minutes and the ultrasonic frequency to be 10-12 kHz, adding the mixture to a sulfuric acid solution after the ultrasonic treatment, and stirring the mixture to obtain the mixed sulfuric acid solution. The reaction steps include adding 2.8-3.4 times the mass of deionized water to water glass, then placing the mixture in a reaction device, heating the mixture to 82-86° C. at a rate of 0.3-0.5° C. / min, adding a mixed sulfuric acid solution, controlling the addition rate of the mixed sulfuric acid solution to be 4.8-5.2 g / min, controlling the pH value at the reaction end point to be 5.1-5.3, completing the reaction, and aging the mixture for 3.4-3.6 hours to obtain a reaction solution. The post-processing step is to filter, wash, dry and calcine the reaction solution in sequence to obtain silica particles; the drying is vacuum freeze drying, the freezing temperature is -40 to -36°C, the freezing time is 18 to 22 hours, and the vacuum degree is 23 to 27 Pa; The modification step comprises mixing the silica particles with an ethanol solution of 6-10 times the mass thereof, heating the mixture to 48-52°C at a rate of 0.4-0.6°C / min, adding a modifier and stirring the mixture for 2.1-2.3 hours at a stirring speed of 174-185 rpm, and washing and drying the mixture after the stirring to obtain modified silica. The modifier is a mixture of γ-glycidyloxypropyltriethoxysilane and Tween-20, and the mass ratio of the γ-glycidyloxypropyltriethoxysilane to Tween-20 is 0.8-1.2:0.4-0.
6.
2. The method for preparing trehalose from starch according to claim 1, characterized in that: The starch milk preparation step comprises mixing corn starch with deionized water, adding polyethylene glycol 400, controlling the stirring temperature to be 60-64° C., stirring time to be 25-30 minutes, and completing the stirring to obtain the starch milk; The mass ratio of the corn starch, deionized water and polyethylene glycol 400 is 47-50:145-153:1.2-1.
4.
3. The method for preparing trehalose from starch according to claim 2, characterized in that: The liquefaction step is to add a high-temperature resistant α-amylase to the starch milk, heat it to 73-78°C at a rate of 0.4-0.6°C / min, stir it for 10-14 minutes at a stirring rate of 76-82 rpm, and continue to heat it after the stirring is completed. The temperature was raised at a rate of 0.2-0.4°C / min to 88-92°C, and the mixture was stirred for 8-12 minutes at a rate of 98-110 rpm. The liquefied liquid was obtained after the stirring was completed. The mass ratio of the starch milk to the thermostable α-amylase is 16-19 kg:1.2-1.4 g.
4. The method for preparing trehalose from starch according to claim 3, characterized in that: In the step of preparing the mixed sulfuric acid solution, the mass concentration of the sulfuric acid is 12.5-12.9%; The mass ratio of the hexadecyltrimethylammonium bromide, carboxymethyl cellulose, sodium lauryl sulfate and sulfuric acid is 0.8-1.2:0.16-0.18:0.60-0.64:96-104.
5. The method for preparing trehalose from starch according to claim 4, characterized in that: In the reaction step, the mass content of Na2O in the water glass is 8.0-8.4%, and the mass content of SiO2 is 16.5-17.0%; In the post-treatment step, the calcination temperature is 305-315°C and the calcination time is 1.3-1.7h; In the modification step, the mass concentration of the ethanol solution is 30-34%; The mass ratio of the ethanol solution to the modifier is 100:1.4-1.
6.
6. The method for preparing trehalose from starch according to claim 5, characterized in that: The loading step of the complex enzyme comprises mixing the modified silica with 6-8 times the volume of deionized water, then adding maltosyl trehalose synthase and maltosyl trehalose hydrolase and stirring at a stirring temperature of 25-27° C. and controlling the pH to be 6.7-6.9, stirring evenly, washing, filtering, and vacuum drying at 30-34° C. to obtain the complex enzyme loaded with the modified silica; The mass ratio of the maltosyl trehalose synthase, maltosyl trehalose hydrolase and deionized water is 3.2-3.6:1.8-2.0:87-94.
7. The method for preparing trehalose from starch according to claim 6, characterized in that: The catalytic reaction step comprises adding the complex enzyme loaded with modified silica to the liquefied liquid, carrying out a catalytic reaction at a temperature of 43-45° C. for 28-32 hours, and controlling the pH to be 5.8-6.2 to obtain a catalytic reaction liquid; The mass ratio of the liquefied liquid to the complex enzyme loaded with modified silica is 4.0-4.2 kg:3 g.
8. The method for preparing trehalose from starch according to claim 7, characterized in that: The filtration, decolorization and ion exchange steps are as follows: filtering the catalytic reaction liquid with a filtration accuracy of 0.25 μm, circulating the filtration three times to obtain a filtrate, adding activated carbon to the filtrate, decolorizing at a temperature of 60-64° C. for 24-27 minutes, repeating twice to obtain a decolorized clear solution, and performing ion exchange on the decolorized clear solution. A desalted solution with a conductivity of 29-31 μs / cm was obtained; The mass ratio of the activated carbon to the filtrate is 1.0-1.2:
100.
9. The method for preparing trehalose from starch according to claim 8, characterized in that: The crystallization step comprises concentrating the desalted liquid until the trehalose mass concentration reaches 73-77% to obtain a concentrated solution, cooling the solution to 43-46°C, adding trehalose seed crystals for cooling crystallization, controlling the cooling rate to 1.0-1.4°C / min, and lowering the temperature to 16-20°C to obtain a crystallized solution; The particle size of the trehalose seed crystals is 110-130 mesh; The mass ratio of the trehalose seed crystals to the concentrated solution is 1.4-1.7:
100.
10. The method for preparing trehalose from starch according to claim 9, characterized in that: The centrifugal drying step comprises the following steps: subjecting the crystallized liquid to centrifugal separation at a centrifugal speed of 2040-2150 rpm and a centrifugal time of 18-22 min, and then placing the crystallized liquid in an oven for drying for 7-9 h at a temperature of 76-78° C. to obtain trehalose.