Preparation method of isomaltooligosacharide

Abstract

The invention provides a preparation method of isomaltooligosacharide, and belongs to the technical field of isomaltooligosacharide. The preparation method comprises the steps of starch milk preparation, liquefaction, primary saccharification, alpha-glucoside transferase conversion, filtration and decoloration, ion exchange and concentration. In the liquefaction step, ultrasonic alpha-amylase is added into the starch milk for jet liquefaction, and liquefied liquid with the DE value being 13-25% is obtained; the preparation method of the ultrasonic alpha-amylase comprises the following steps: performing ultrasonic irradiation treatment on alpha-amylase, controlling the ultrasonic time of each time to be 115-125s and the ultrasonic interval to be 55-65s, and repeating the operation for 10 times to obtain the ultrasonic alpha-amylase. The isomaltooligosacharide prepared by the method disclosed by the invention is high in content and low in energy consumption.

Description

Technical Field

[0001] The present invention belongs to the technical field of isomaltooligosaccharides, and particularly relates to a preparation method of isomaltooligosaccharides. Background Art

[0002] Isomaltooligosaccharides refer to a class of oligosaccharides with 2 - 6 monosaccharides bonded by α-1,6 glycosidic bonds. Its main components include isomaltose, panose, isomaltotriose, and isomaltotetraose, etc. Among them, isomaltose, panose, and isomaltotriose are the main components reflecting the functionality of isomaltooligosaccharides. The content of these components reflects the quality of the product, and also affects the product application and price prospects.

[0003] There are generally the following two ways to prepare isomaltooligosaccharides: One is to use the reverse synthesis of glucoamylase to reversely synthesize oligosaccharides such as isomaltose and maltose in a high-concentration glucose solution. However, due to disadvantages such as low yield, complex products, and long production cycle, it is difficult to be widely promoted industrially. The other is to use high-concentration glucose syrup prepared from starch as a substrate and carry out an α-glucosyltransferase-catalyzed α-glucosyl transfer reaction. Generally, industrial production of isomaltooligosaccharides uses a starch raw material and a whole-enzymatic process. The production process flow is roughly: starch → jet liquefaction → β-amylase saccharification → α-glucosyltransferase conversion → decolorization → ion exchange → vacuum concentration or spray drying → finished product.

[0004] When the prior art uses a whole-enzymatic process with starch as a raw material to prepare isomaltooligosaccharides, the following problems usually exist: First, the key to controlling this process technology lies in mastering the jet liquefaction technology, which is the basis of production. To control the hydrolysis degree of starch, the DE value of the liquefied liquid is relatively important, which directly affects the quality of the product. When the DE value is relatively high, some sugars will be further decomposed into other small-molecule compounds, and glucoamylase cannot fully act, reducing the quality of the product. A lower DE value means insufficient hydrolysis of starch, and some starch still maintains a large molecular structure, resulting in a high viscosity of the liquefied liquid, which is not conducive to subsequent saccharification. Moreover, the number of non-reducing end substrates exposed is limited, and there is not enough substrate for glucoamylase to act, affecting the saccharification efficiency and ultimately the product yield. Second, α-glucosyltransferase has poor stability and low activity, resulting in a low conversion rate from glucose to isomaltooligosaccharides and low production efficiency. Third, when separating and extracting isomaltooligosaccharides from the saccharified liquid, usually, first, plate-and-frame filtration is used to remove suspended solids and residual starch, etc., then a large amount of anion and cation exchange resins are used to remove salts in the saccharified liquid, and finally, vacuum concentration or spray drying is carried out to obtain the product. However, this process generates more wastewater, and the resin is easily damaged, with high energy consumption and low working efficiency. Summary of the Invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides a preparation method of isomaltooligosaccharide, and the product has a relatively high purity and low energy consumption.

[0006] In view of the above technical problems, the present invention adopts the following technical solutions: A preparation method of isomaltooligosaccharide includes steps of preparing starch milk, liquefaction, primary saccharification, α-glucosidase conversion, filtration and decolorization, ion exchange and concentration. The specific operations are as follows: 1. Preparation of starch milk Add corn starch into deionized water to make slurry, control the mass concentration of the slurry to be 15 - 30%, then add dodecyl glucoside, continue to stir evenly, adjust the pH value to 4 - 5, raise the temperature to 40 - 55 °C, and keep stirring for 0.5 - 1.5 h to obtain starch milk; The addition amount of the dodecyl glucoside is 0.1 - 0.5 wt% of the slurry.

[0007] 2. Liquefaction Add ultrasonic α-amylase into the starch milk, adjust the pH value to 4.0 - 6.5, stir for 0.2 - 0.5 h, then inject the starch milk through a liquefaction injector for jet liquefaction, control the liquefaction temperature to be 102 - 115 °C, and the liquefaction time to be 1 - 3 h to obtain a liquefied liquid with a DE value of 13 - 25%; The addition amount of the ultrasonic α-amylase is 0.1 - 0.7 wt% of the mass of the corn starch; The preparation method of the ultrasonic α-amylase is to perform ultrasonic irradiation treatment on α-amylase, control the ultrasonic time each time to be 115 - 125 s, the interval between each ultrasonic time to be 55 - 65 s, repeat the operation 10 times, control the ultrasonic frequency to be 10 - 15 kHz, and the ultrasonic power to be 82 - 90 W. After the ultrasonic treatment, the ultrasonic α-amylase is obtained.

[0008] 3. Primary saccharification Control the temperature of the liquefied liquid to be 45 - 60 °C, adjust the pH value to 6.5 - 8.0, add primary saccharifying enzyme for primary saccharification reaction, keep warm for 15 - 35 h to obtain a crude saccharified liquid, and control the DE value of the crude saccharified liquid to be 40 - 47%; The primary saccharifying enzyme is a mixed enzyme of functional amylase and pullulanase, and the mass ratio of the functional amylase to the pullulanase is 4 - 10:1 - 3; The addition amount of the primary saccharifying enzyme is 0.1 - 0.7 wt% of the mass of the corn starch; The preparation method of the functional amylase includes steps of modified amylase, preparation of amylase carrier and compounding step; The steps for the modified amylase are as follows: Add α-fungal amylase to deionized water and stir evenly. Raise the temperature to 40 - 50 °C, then add glutaraldehyde solution and stir for reaction for 2 - 3 h to obtain the modified amylase; The mass concentration of the glutaraldehyde solution is 8 - 12%; The mass ratio of the α-fungal amylase, deionized water, and glutaraldehyde solution is 8 - 12:100:12 - 18; The steps for preparing the amylase carrier are as follows: Place chitin in dimethyl sulfoxide and stir evenly. Then add citric anhydride and N-hydroxysuccinimide, continue to stir evenly, and carry out microwave reaction. Control the microwave temperature at 62 - 66 °C, the microwave time at 85 - 95 min, and the microwave power at 175 - 185 W. After the microwave reaction ends, wash and then vacuum dry at 82 - 87 °C for 15.0 - 18.0 h to obtain the primary carrier; The mass ratio of the chitin, dimethyl sulfoxide, citric anhydride, and N-hydroxysuccinimide is 9.0 - 11.0:500:5.5 - 6.0:4.5 - 5.0; Add the primary carrier to an ethanol solution, stir evenly, then add 1,4-butanediol diglycidyl ether, adjust the pH to 8.2 - 8.7, raise the temperature to 38 - 42 °C for ultrasonic reaction. Control the ultrasonic frequency at 42 - 50 kHz, the ultrasonic power at 100 - 110 W, and the ultrasonic time at 4.8 - 5.3 h. After the ultrasonic reaction ends, raise the temperature to 47 - 54 °C, add gelatin and L-lysine, and keep warm for reaction for 3.2 - 3.7 h. After filtration, washing, and drying, obtain the amylase carrier; The mass ratio of the primary carrier, ethanol solution, 1,4-butanediol diglycidyl ether, gelatin, and L-lysine is 7.5 - 8.5:200:1.4 - 1.8:1.8 - 2.2:0.5 - 0.8; The mass concentration of the ethanol solution is 22 - 27%; The compounding steps are as follows: Add the amylase carrier to deionized water, stir evenly, then add the modified amylase, and stir for reaction at 5.8 - 6.2 °C for 9.0 - 11.0 h. After the reaction ends, filter and dry to obtain the functional amylase; The mass ratio of the deionized water, amylase carrier, and modified amylase is 100:6.5 - 7.5:2.2 - 2.6.

[0009] 4. α-Glucosyltransferase conversion Add α-glucosyltransferase to the crude saccharified liquid, adjust the pH value to 7 - 9, control the temperature at 45 - 60 °C, and keep warm for 50 - 100 h to obtain the crude oligo-isomaltose sugar liquid; The addition amount of the α-glucosidase is 0.3-0.5 wt% of the mass of corn starch.

[0010] 5. Filtration and decolorization The crude isomaltooligosaccharide solution is filtered through a plate and frame filter, with the filtration accuracy controlled at 200-400 μm and the pressure at 10-35 kPa. The primary filtrate of the saccharification liquid is collected, and then modified activated carbon is added. Stir at 240-260 rpm for 0.2-0.5 h. After stirring, let it stand for 1.5-2.5 h. After standing, filter and collect the secondary filtrate of the saccharification liquid. Keep the secondary filtrate of the saccharification liquid at 42-48 °C and perform nanofiltration treatment through a nanofiltration membrane, with the feed pressure controlled at 1.8-2.2 MPa and the retention volume at 275-285 MW to obtain the saccharification filtrate; The mass-volume ratio of the modified activated carbon to the primary filtrate of the saccharification liquid is 2.0-6.0 g:100 mL; The preparation method of the modified activated carbon is as follows: Place the activated carbon at 220-240 °C for heat preservation treatment for 2.3-2.8 h. After natural cooling, add deionized water, stir evenly, control the temperature at 25-30 °C, add hydrogen peroxide, and continue to stir for 1-2 h. After filtration and drying, obtain the modified activated carbon; The mass ratio of the activated carbon, deionized water, and hydrogen peroxide is 5.0-8.0:100:20-25.

[0011] 6. Ion exchange Ion exchange is carried out with a continuous simulated moving bed. The saccharification filtrate is treated with a strongly acidic cation exchange resin. The flow rate of the saccharification filtrate is 4-6 bed volumes / h, and the temperature is 45-50 °C to obtain a refined isomaltooligosaccharide solution; The model of the strongly acidic cation exchange resin is LX-160, purchased from Xi'an BlueSail New Materials Co., Ltd.; The mass concentration of the refined isomaltooligosaccharide solution is 15-25%.

[0012] 7. Concentration The refined isomaltooligosaccharide solution is evaporated and concentrated to 48-52% of the original volume, and the evaporation temperature is 70-90 °C to obtain isomaltooligosaccharide.

[0013] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention prepares isomaltooligosaccharide by a specific method. In the liquefaction step, an appropriate DE value is controlled to ensure that the hydrolysis of starch is within a certain range, so as to enable the full progress of the saccharification reaction. And in the liquefaction step, the amylase is treated by ultrasonic irradiation. Ultrasonic waves can change the enzyme molecular structure, enhancing the catalytic efficiency and stability of the amylase; in the primary saccharification step, a functional enzyme carrier is prepared by a specific method. Specifically, the amylase is first treated with a glutaraldehyde solution. Glutaraldehyde can crosslink with the amino groups of the amylase, causing the amylase molecules to crosslink with each other to form a network structure, which can improve the stability of the amylase while maintaining its activity. Then, chitin is used as the carrier matrix. First, chitin is treated with an acid anhydride. Through the reaction of the acid anhydride group with the amino group of chitin, carboxyl groups are introduced onto the surface of chitin. Then, 1,4-butanediol diglycidyl ether is added, which can crosslink with the carboxyl groups on the surface of the primary carrier. Under the action of gelatin and L-lysine, it crosslinks again with the epoxy groups of 1,4-butanediol diglycidyl ether, thus obtaining an amylase carrier with a three-dimensional stable network structure. In the step of compounding with the modified amylase, stable crosslinking is achieved again between the amylase carrier and the modified amylase. The finally prepared functional amylase can not only maintain a relatively high activity of the amylase, but also ensure its stability, effectively ensuring the progress of the saccharification reaction and the recovery performance and reusability of the functional amylase; cooperating with pullulanase, the liquefied liquid is converted into a crude saccharified liquid with a higher DE value, increasing the content of glucose and oligosaccharides in the sugar solution, providing sufficient substrates for the α-glucosyltransferase conversion reaction, and further converting the saccharides in the crude saccharified liquid into the target product isomaltooligosaccharide, increasing the content of isomaltooligosaccharide; in the filtration and decolorization step, by treating the activated carbon, the pore size and initial chemical structure of the activated carbon are changed, and then through polar modification, it has a strong adsorption capacity for polar substances in the sugar solution, achieving the effect of improving the absorption performance of the sugar solution, effectively removing components such as pigments and proteins in the sugar solution. After subsequent treatment steps, a product with a relatively high content of isomaltooligosaccharide is obtained, reducing the content of heteroproteins; 2. Each component in the product is detected. The mass content of isomaltooligosaccharide is 98.17 - 98.45%, the total mass content of isomaltose and isomaltotriose is 74.68 - 74.83%, and the mass content of heteroproteins is 0.22 - 0.29%; 3. Each time at the end of the primary saccharification step, the functional amylase is recovered to obtain the recovered functional amylase, and the recovered functional amylase is reused 25 times. When measuring the isomaltooligosaccharide prepared in the 25th reuse, the mass content of isomaltooligosaccharide is 92.37 - 93.82%, the total mass content of isomaltose and isomaltotriose is 71.09 - 72.05%, and the mass content of heteroproteins is 0.40 - 0.45%; The recovery method is as follows: centrifuging the crude saccharified liquid at 4000 rpm for 8 min to obtain solid matter; washing the solid matter 3 times with deionized water with a mass 4 times that of the solid matter, then stirring and washing with a phosphate buffer solution with a mass 4 times that of the solid matter for 60 min at a stirring speed of 100 rpm. The molar concentration of the phosphate buffer solution is 0.1 mol / L and the pH value is 7.0. Then, stirring and washing with a 0.5 mol / L sodium chloride solution for 20 min at a stirring speed of 100 rpm. After washing, drying is carried out to obtain the recovered functional amylase. Detailed implementation mode

[0014] For a clearer understanding of the technical features, objectives and effects of the present invention, the detailed implementation mode of the present invention is now described.

[0015] Example 1 A method for preparing isomaltooligosaccharide 1. Preparation of starch milk Corn starch is added to deionized water to prepare a slurry, and the mass concentration of the slurry is controlled to be 30%. Then, dodecyl glucoside is added, and stirring is continued until evenly mixed. The pH value is adjusted to 5, the temperature is raised to 55 °C, and stirring is carried out for 1.5 h to obtain starch milk; The addition amount of the dodecyl glucoside is 0.5 wt% of the slurry.

[0016] 2. Liquefaction Ultrasonic α-amylase is added to the starch milk, the pH value is adjusted to 6.5, and after stirring for 0.5 h, the starch milk is sprayed and liquefied through a liquefaction ejector. The liquefaction temperature is controlled to be 115 °C and the liquefaction time is 3 h to obtain a liquefied liquid with a DE value of 25%; The addition amount of the ultrasonic α-amylase is 0.7 wt% of the mass of the corn starch; The preparation method of the ultrasonic α-amylase is as follows: α-amylase is subjected to ultrasonic irradiation treatment, the ultrasonic time per time is controlled to be 125 s, the interval between each ultrasonic treatment is 65 s, and the operation is repeated 10 times. The ultrasonic frequency is controlled to be 15 kHz and the ultrasonic power is 90 W. After the ultrasonic treatment, the ultrasonic α-amylase is obtained.

[0017] 3. Primary saccharification The temperature of the liquefied liquid is controlled to be 60 °C, the pH value is adjusted to 8.0, and primary saccharification enzyme is added for primary saccharification reaction. After holding for 35 h, a crude saccharified liquid is obtained, and the DE value of the crude saccharified liquid is controlled to be 47%; The primary saccharification enzyme is a mixed enzyme of functional amylase and pullulanase, and the mass ratio of the functional amylase to the pullulanase is 5:1; The addition amount of the primary saccharification enzyme is 0.7 wt% of the mass of the corn starch; The preparation method of the functional amylase includes the steps of modifying amylase, preparing an amylase carrier, and compounding; In the step of modifying amylase, 12 g of α-fungal amylase is added to 100 g of deionized water and stirred evenly. The temperature is raised to 50 °C, and then 18 g of glutaraldehyde solution is added. After stirring and reacting for 3 h, modified amylase is obtained; The mass concentration of the glutaraldehyde solution is 12%; In the step of preparing the amylase carrier, 11.0 g of chitin is placed in 500 g of dimethyl sulfoxide and stirred evenly. Then, 6.0 g of citric anhydride and 5.0 g of N-hydroxysuccinimide are added. After continuing to stir evenly, microwave reaction is carried out. The microwave temperature is controlled at 66 °C, the microwave time is 95 min, and the microwave power is 185 W. After the microwave reaction is completed, it is washed and then vacuum dried at 87 °C for 15.0 h to obtain a primary carrier; 8.5 g of the primary carrier is added to 200 g of 27 wt% ethanol solution and stirred evenly. Then, 1.8 g of 1,4-butanediol diglycidyl ether is added, the pH is adjusted to 8.7, and the temperature is raised to 42 °C for ultrasonic reaction. The ultrasonic frequency is controlled at 50 kHz, the ultrasonic power is 110 W, and the ultrasonic time is 5.3 h. After the ultrasonic reaction is completed, the temperature is raised to 54 °C, 2.2 g of gelatin and 0.8 g of L-lysine are added, and the reaction is carried out under insulation for 3.7 h. After filtration, washing, and drying, an amylase carrier is obtained; In the compounding step, 7.5 g of the amylase carrier is added to 100 g of deionized water and stirred evenly. Then, 2.6 g of modified amylase is added, and the reaction is carried out by stirring at 6.2 °C for 9.0 h. After the reaction is completed, functional amylase is obtained by filtration and drying.

[0018] 4. Conversion by α-glucosidase α-Glucosidase is added to the crude saccharified liquid, the pH value is adjusted to 9, the temperature is controlled at 60 °C, and the mixture is kept warm for 100 h to obtain a crude isomaltooligosaccharide sugar solution; The addition amount of the α-glucosidase is 0.5 wt% of the mass of corn starch.

[0019] 5. Filtration and decolorization The crude isomaltooligosaccharide sugar solution is filtered through a plate and frame filter, the filtration accuracy is controlled at 400 μm, the pressure is 35 kPa, and the primary filtrate of the saccharified liquid is collected. Then, modified activated carbon is added, and the mixture is stirred at 260 rpm for 0.5 h. After stirring is completed, it is allowed to stand for 2.5 h. After standing is completed, it is filtered, and the secondary filtrate of the saccharified liquid is collected. The secondary filtrate of the saccharified liquid is kept at 48 °C and subjected to nanofiltration treatment through a nanofiltration membrane. The feed pressure is controlled at 2.2 MPa, and the cut-off molecular weight is 285 MW to obtain a saccharified filtrate; The mass-volume ratio of the modified activated carbon to the primary filtrate of the saccharified solution is 6.0 g:100 mL; The preparation method of the modified activated carbon is as follows: Place 8.0 g of activated carbon in an oven at 240 °C for heat preservation for 2.8 h. After natural cooling, add 100 g of deionized water, stir evenly, control the temperature at 30 °C, add 25 g of hydrogen peroxide, continue stirring for 2 h, and then obtain the modified activated carbon through filtration and drying.

[0020] 6. Ion exchange Ion exchange is carried out using a continuous simulated moving bed. The saccharified filtrate is treated with a strongly acidic cation exchange resin. The flow rate of the saccharified filtrate is 6 bed volumes / h, and the temperature is 50 °C to obtain a refined isomaltooligosaccharide solution; The model of the strongly acidic cation exchange resin is LX-160, purchased from Xi'an BlueSail New Materials Co., Ltd.; The mass concentration of the refined isomaltooligosaccharide solution is 25%.

[0021] 7. Concentration The refined isomaltooligosaccharide solution is evaporated and concentrated to 52% of its original volume at an evaporation temperature of 90 °C to obtain isomaltooligosaccharide.

[0022] Each component in the product of Example 1 is detected. The mass content of isomaltooligosaccharide is 98.32%, the total mass content of isomaltose and isomaltotriose is 74.77%, and the mass content of miscellaneous proteins is 0.26%; At the end of each primary saccharification step, the functional amylase is recovered to obtain the recovered functional amylase, which is reused 25 times. When measuring the isomaltooligosaccharide prepared in the 25th reuse, the mass content of isomaltooligosaccharide is 93.01%, the total mass content of isomaltose and isomaltotriose is 71.55%, and the mass content of miscellaneous proteins is 0.43%.

[0023] Example 2 A method for preparing isomaltooligosaccharide 1. Preparation of starch milk Corn starch is added to deionized water to prepare a slurry, and the mass concentration of the slurry is controlled at 25%. Then, dodecyl glucoside is added, and stirring is continued until evenly mixed. The pH value is adjusted to 4.5, the temperature is raised to 50 °C, and stirring is carried out for heat preservation for 1.0 h to obtain starch milk; The addition amount of the dodecyl glucoside is 0.3 wt% of the slurry.

[0024] 2. Liquefaction Add ultrasonic α - amylase to the starch milk, adjust the pH value to 5.2, stir for 0.4 h, then spray - liquefy the starch milk through a liquefaction injector, control the liquefaction temperature at 110 °C and the liquefaction time at 2 h to obtain a liquefied liquid with a DE value of 18%; The addition amount of the ultrasonic α - amylase is 0.5 wt% of the mass of corn starch; The preparation method of the ultrasonic α - amylase is as follows: subject α - amylase to ultrasonic irradiation treatment, control the ultrasonic time for each time at 120 s, with a 60 - s interval between each ultrasonic treatment, repeat the operation 10 times, control the ultrasonic frequency at 12 kHz and the ultrasonic power at 85 W. After the ultrasonic treatment, the ultrasonic α - amylase is obtained.

[0025] 3. Primary saccharification Control the temperature of the liquefied liquid at 52 °C, adjust the pH value to 7.0, add primary saccharification enzyme for primary saccharification reaction, keep warm for 25 h to obtain a crude saccharified liquid, and control the DE value of the crude saccharified liquid at 43%; The primary saccharification enzyme is a mixed enzyme of functional amylase and pullulanase, and the mass ratio of the functional amylase to the pullulanase is 4:1; The addition amount of the primary saccharification enzyme is 0.4 wt% of the mass of corn starch; The preparation method of the functional amylase includes steps of modifying amylase, preparing an amylase carrier and a compounding step; The step of modifying amylase is as follows: add 10 g of α - fungal amylase to 100 g of deionized water and stir evenly, raise the temperature to 45 °C, then add 15 g of glutaraldehyde solution, stir and react for 2.5 h to obtain modified amylase; The mass concentration of the glutaraldehyde solution is 10%; The step of preparing the amylase carrier is as follows: put 10.0 g of chitin in 500 g of dimethyl sulfoxide, stir evenly, add 5.7 g of citric anhydride and 4.8 g of N - hydroxysuccinimide, continue to stir evenly, then carry out a microwave reaction, control the microwave temperature at 64 °C, the microwave time at 90 min, and the microwave power at 180 W. After the microwave reaction, after washing, vacuum - dry at 85 °C for 16.0 h to obtain a primary carrier; Add 8.0 g of the primary carrier to 200 g of 25 wt% ethanol solution, stir evenly, add 1.6 g of 1,4 - butanediol diglycidyl ether, adjust the pH to 8.5, raise the temperature to 40 °C for ultrasonic reaction, control the ultrasonic frequency at 45 kHz, the ultrasonic power at 105 W, and the ultrasonic time at 5.0 h. After the ultrasonic reaction, raise the temperature to 50 °C, add 2.0 g of gelatin and 0.7 g of L - lysine, keep warm and react for 3.5 h, and after filtration, washing and drying, obtain the amylase carrier; The composite step is as follows: add 7.0 g of amylase carrier to 100 g of deionized water, stir evenly, then add 2.5 g of modified amylase, stir and react at 6.0 °C for 10.0 h. After the reaction, filter and dry to obtain functional amylase.

[0026] 4. Conversion by α-glucosidase Add α-glucosidase to the crude saccharified liquid, adjust the pH value to 8, control the temperature at 50 °C, and keep warm for 75 h to obtain a crude isomaltooligosaccharide solution. The addition amount of the α-glucosidase is 0.4 wt% of the mass of corn starch.

[0027] 5. Filtration and decolorization Filter the crude isomaltooligosaccharide solution through a plate and frame filter, control the filtration accuracy at 300 μm and the pressure at 25 kPa, collect the primary filtrate of the saccharified liquid, then add modified activated carbon, stir at 250 rpm for 0.4 h. After stirring, let it stand for 2.0 h. After standing, filter and collect the secondary filtrate of the saccharified liquid. Keep the secondary filtrate of the saccharified liquid at 45 °C and perform nanofiltration treatment through a nanofiltration membrane, control the feed pressure at 2.0 MPa and the cut-off molecular weight at 280 MW to obtain a saccharified filtrate. The mass-to-volume ratio of the modified activated carbon to the primary filtrate of the saccharified liquid is 4.0 g:100 mL. The preparation method of the modified activated carbon is as follows: place 7.0 g of activated carbon at 230 °C for heat preservation treatment for 2.5 h. After natural cooling, add 100 g of deionized water, stir evenly, control the temperature at 27 °C, add 23 g of hydrogen peroxide, continue to stir for 1.5 h, filter and dry to obtain the modified activated carbon.

[0028] 6. Ion exchange Perform ion exchange with a continuous simulated moving bed, use a strongly acidic cation exchange resin to treat the saccharified filtrate, the flow rate of the saccharified filtrate is 5 bed volumes / h, and the temperature is 48 °C to obtain a refined isomaltooligosaccharide solution. The model of the strongly acidic cation exchange resin is LX-160, purchased from Xi'an BlueSail New Materials Co., Ltd. The mass concentration of the refined isomaltooligosaccharide solution is 20%.

[0029] 7. Concentration Evaporate and concentrate the refined isomaltooligosaccharide solution to 50% of the original volume, and the evaporation temperature is 80 °C to obtain isomaltooligosaccharide.

[0030] Detect each component in the product of Example 2. The mass content of isomaltooligosaccharide is 98.45%, the total mass content of isomaltose and isomaltotriose is 74.83%, and the mass content of miscellaneous proteins is 0.22%. At the end of each initial saccharification step, the functional amylase was recovered to obtain the recovered functional amylase, which was reused 25 times. When measured for the 25th reuse, the obtained isomaltooligosaccharide had a mass content of 93.82%, the total mass content of isomaltose and isomaltotriose was 72.05%, and the mass content of miscellaneous proteins was 0.40%.

[0031] Example 3 A method for preparing isomaltooligosaccharide 1. Preparation of starch milk Corn starch was added to deionized water to prepare a slurry, and the mass concentration of the slurry was controlled at 15%. Then, dodecyl glucoside was added, and stirring was continued until uniform. The pH value was adjusted to 4, the temperature was raised to 40 °C, and stirring was carried out for 0.5 h to obtain starch milk; The addition amount of the dodecyl glucoside was 0.1 wt% of the slurry.

[0032] 2. Liquefaction Ultrasonic α-amylase was added to the starch milk, the pH value was adjusted to 4.0, and after stirring for 0.2 h, the starch milk was sprayed and liquefied through a liquefaction injector. The liquefaction temperature was controlled at 102 °C, and the liquefaction time was 1 h to obtain a liquefied liquid with a DE value of 13%; The addition amount of the ultrasonic α-amylase was 0.1 wt% of the mass of corn starch; The preparation method of the ultrasonic α-amylase was as follows: α-amylase was subjected to ultrasonic irradiation treatment, the ultrasonic time per time was controlled at 115 s, the interval between each ultrasonic treatment was 55 s, and the operation was repeated 10 times. The ultrasonic frequency was controlled at 10 kHz, and the ultrasonic power was 82 W. After the ultrasonic treatment, ultrasonic α-amylase was obtained.

[0033] 3. Initial saccharification The temperature of the liquefied liquid was controlled at 45 °C, the pH value was adjusted to 6.5, and initial saccharification enzyme was added for an initial saccharification reaction. After holding for 15 h, a crude saccharified liquid was obtained, and the DE value of the crude saccharified liquid was controlled at 40%; The initial saccharification enzyme was a mixed enzyme of functional amylase and pullulanase, and the mass ratio of the functional amylase to the pullulanase was 10:3; The addition amount of the initial saccharification enzyme was 0.1 wt% of the mass of corn starch; The preparation method of the functional amylase included a modified amylase step, a step of preparing an amylase carrier, and a compounding step; The modified amylase step was as follows: 8 g of α-fungal amylase was added to 100 g of deionized water and stirred evenly. The temperature was raised to 40 °C, and then 12 g of glutaraldehyde solution was added, and stirring reaction was carried out for 2 h to obtain modified amylase; The mass concentration of the glutaraldehyde solution is 8%; The steps for preparing the amylase carrier are as follows: Place 9.0 g of chitin in 500 g of dimethyl sulfoxide, stir evenly, add 5.5 g of citric anhydride and 4.5 g of N-hydroxysuccinimide, continue to stir evenly, and then carry out a microwave reaction. Control the microwave temperature at 62 °C, the microwave time at 85 min, and the microwave power at 175 W. After the microwave reaction is completed, wash it, and then vacuum dry it at 82 °C for 18.0 h to obtain a primary carrier; Add 7.5 g of the primary carrier to 200 g of a 22 wt% ethanol solution, stir evenly, add 1.4 g of 1,4-butanediol diglycidyl ether, adjust the pH to 8.2, raise the temperature to 38 °C for an ultrasonic reaction, control the ultrasonic frequency at 42 kHz, the ultrasonic power at 100 W, and the ultrasonic time at 4.8 h. After the ultrasonic reaction is completed, raise the temperature to 47 °C, add 1.8 g of gelatin and 0.5 g of L-lysine, keep the temperature for reaction for 3.2 h, and after filtration, washing, and drying, obtain the amylase carrier; The compounding steps are as follows: Add 6.5 g of the amylase carrier to 100 g of deionized water, stir evenly, add 2.2 g of modified amylase, and stir and react at 5.8 °C for 11.0 h. After the reaction is completed, filter and dry to obtain the functional amylase.

[0034] 4. α-Glucosyltransferase conversion Add α-glucosyltransferase to the crude saccharified liquid, adjust the pH value to 7, control the temperature at 45 °C, and keep the temperature for 50 h to obtain a crude isomaltooligosaccharide sugar solution; The addition amount of the α-glucosyltransferase is 0.3 wt% of the mass of corn starch.

[0035] 5. Filtration and decolorization Filter the crude isomaltooligosaccharide sugar solution through a plate and frame filter, control the filtration accuracy at 200 μm and the pressure at 10 kPa, collect the primary filtrate of the saccharified liquid, then add modified activated carbon, stir at 240 rpm for 0.2 h, after stirring, let it stand for 1.5 h, after standing, filter, collect the secondary filtrate of the saccharified liquid, keep the secondary filtrate of the saccharified liquid at 42 °C, and carry out nanofiltration treatment through a nanofiltration membrane, control the feed pressure at 1.8 MPa and the retention volume at 275 MW to obtain the saccharified filtrate; The mass-volume ratio of the modified activated carbon to the primary filtrate of the saccharified liquid is 2.0 g:100 mL; The preparation method of the modified activated carbon is as follows: Place 5.0 g of activated carbon at 220 °C for heat preservation treatment for 2.3 h, after natural cooling, add 100 g of deionized water, stir evenly, control the temperature at 25 °C, add 20 g of hydrogen peroxide, continue to stir for 1 h, and after filtration and drying, obtain the modified activated carbon.

[0036] 6. Ion exchange Ion exchange was carried out using a continuous simulated moving bed. The saccharified filtrate was treated with a strongly acidic cation exchange resin. The flow rate of the saccharified filtrate was 4 bed volumes / h, and the temperature was 45 °C to obtain a refined isomaltooligosaccharide solution; The model of the strongly acidic cation exchange resin was LX-160, purchased from Xi'an LX Technology New Materials Co., Ltd.; The mass concentration of the refined isomaltooligosaccharide solution was 15%.

[0037] 7. Concentration The refined isomaltooligosaccharide solution was evaporated and concentrated to 48% of the original volume at an evaporation temperature of 70 °C to obtain isomaltooligosaccharide.

[0038] Each component in the product of Example 3 was detected. The mass content of isomaltooligosaccharide was 98.17%, the total mass content of isomaltose and isomaltotriose was 74.68%, and the mass content of miscellaneous proteins was 0.29%; Each time at the end of the primary saccharification step, the functional amylase was recovered to obtain the recovered functional amylase, and the recovered functional amylase was reused. After 25 times of reuse, the isomaltooligosaccharide prepared in the 25th reuse was measured. The mass content of isomaltooligosaccharide was 92.37%, the total mass content of isomaltose and isomaltotriose was 71.09%, and the mass content of miscellaneous proteins was 0.45%.

[0039] Example 4 The recovery method of the functional amylase was as follows: The crude saccharified liquid was centrifuged at 4000 rpm for 8 min to obtain a solid; the solid was washed 3 times with deionized water with a mass 4 times that of the solid, and then stirred and washed with a phosphate buffer solution with a mass 4 times that of the solid for 60 min. The stirring speed was 100 rpm. The molar concentration of the phosphate buffer solution was 0.1 mol / L, and the pH value was 7.0. Then it was stirred and washed with a 0.5 mol / L sodium chloride solution for 20 min. The stirring speed was 100 rpm. After washing, it was dried to obtain the recovered functional amylase.

[0040] Comparative Example 2.1 On the basis of Example 2, the following changes were made: 1. In the liquefaction step, the preparation of ultrasonic α-amylase was omitted, and the ultrasonic α-amylase was replaced with α-amylase without any treatment in equal amount; 2. In the primary saccharification step, in the preparation method of the functional amylase, the preparation step of the modified amylase was omitted, and the modified amylase was replaced with α-fungal amylase without any treatment in equal amount; The remaining operations were the same.

[0041] The components in the product of Comparative Example 2.1 were detected. The mass content of isomaltooligosaccharide was 87.31%, the total mass content of isomaltose and isomaltotriose was 67.32%, and the mass content of heteroprotein was 1.17%. At the end of each primary saccharification step, the functional amylase was recovered to obtain the recovered functional amylase, and the recovered functional amylase was reused 25 times. The isomaltooligosaccharide prepared in the 25th reuse was measured. The mass content of isomaltooligosaccharide was 80.33%, the total mass content of isomaltose and isomaltotriose was 62.41%, and the mass content of heteroprotein was 1.68%.

[0042] In Comparative Example 2.1, the ultrasonic treatment of amylase was omitted in the liquefaction step, and in the process of preparing the functional amylase, the modification treatment of amylase was omitted. On the one hand, it would affect the enzyme activity, making the liquefaction reaction incomplete, thus greatly reducing the content of isomaltooligosaccharide. On the other hand, the binding between the amylase and the amylase carrier was poor, which affected the enzyme recovery performance to a certain extent. During multiple reuse processes, part of the enzyme would fall off, resulting in a slightly lower retention rate of the isomaltooligosaccharide content after multiple reuse.

[0043] Comparative Example 2.2 Based on Example 2, the following changes were made: In the primary saccharification step, in the preparation method of the functional amylase, the amylase carrier was replaced with chitin component in equal amount, and the rest of the operations were the same.

[0044] The components in the product of Comparative Example 2.2 were detected. The mass content of isomaltooligosaccharide was 92.68%, the total mass content of isomaltose and isomaltotriose was 71.22%, and the mass content of heteroprotein was 0.31%. At the end of each primary saccharification step, the functional amylase was recovered to obtain the recovered functional amylase, and the recovered functional amylase was reused 25 times. The isomaltooligosaccharide prepared in the 25th reuse was measured. The mass content of isomaltooligosaccharide was 72.85%, the total mass content of isomaltose and isomaltotriose was 57.05%, and the mass content of heteroprotein was 1.95%.

[0045] Comparative Example 2.2 used chitin as the amylase carrier, which could crosslink with the modified amylase to achieve the immobilization of the amylase. However, during the recovery and reuse processes, most of the enzyme fell off due to incomplete binding, resulting in extremely poor reuse performance.

[0046] Unless otherwise specified, the ratios described in the present invention are all mass ratios, and the percentages are all mass percentages.

[0047] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of isomaltooligosaccharide, characterized in that, It includes steps of preparing starch milk, liquefaction, primary saccharification, α-glucosidase conversion, filtration and decolorization, ion exchange, and concentration; The liquefaction step is to add ultrasonic α-amylase to the starch milk for jet liquefaction to obtain a liquefied liquid with a DE value of 13 - 25%; The preparation method of the ultrasonic α-amylase is to perform ultrasonic irradiation treatment on α-amylase, control the ultrasonic time for each time to be 115 - 125 s, with an interval of 55 - 65 s between each ultrasonic treatment, and repeat the operation 10 times to obtain the ultrasonic α-amylase; The primary saccharification step is to add primary saccharifying enzyme to the liquefied liquid to obtain a crude saccharified liquid; The primary saccharifying enzyme is a mixed enzyme of functional amylase and pullulanase, and the preparation method of the functional amylase includes steps of modifying amylase, preparing an amylase carrier, and a compounding step; The step of modifying amylase is to add α-fungal amylase to deionized water, raise the temperature to 40 - 50 °C, and add glutaraldehyde solution to obtain modified amylase; The step of preparing the amylase carrier is to place chitin in dimethyl sulfoxide, add citric anhydride and N-hydroxysuccinimide for microwave reaction to obtain a primary carrier; add the primary carrier to an ethanol solution, add 1,4-butanediol diglycidyl ether, perform ultrasonic reaction at 38 - 42 °C for 4.8 - 5.3 h, raise the temperature to 47 - 54 °C, add gelatin and L-lysine, and keep the temperature for reaction for 3.2 - 3.7 h to obtain the amylase carrier.

2. The preparation method of isomaltooligosaccharide according to claim 1, characterized in that The step of preparing starch milk is to add corn starch to deionized water to make a slurry, control the mass concentration of the slurry to be 15 - 30%, then add dodecyl glucoside, continue to stir evenly, adjust the pH value to 4 - 5, raise the temperature to 40 - 55 °C, and keep stirring for 0.5 - 1.5 h to obtain starch milk; The addition amount of the dodecyl glucoside is 0.1 - 0.5 wt% of the slurry.

3. The preparation method of isomaltooligosaccharide according to claim 2, characterized in that The primary saccharification step is to control the temperature of the liquefied liquid to be 45 - 60 °C, adjust the pH value to 6.5 - 8.0, add the primary saccharifying enzyme for primary saccharification reaction, keep the temperature for 15 - 35 h to obtain a crude saccharified liquid, and control the DE value of the crude saccharified liquid to be 40 - 47%; In the primary saccharifying enzyme, the mass ratio of the functional amylase to the pullulanase is 4 - 10:1 - 3; The addition amount of the primary saccharifying enzyme is 0.1 - 0.7 wt% of the mass of the corn starch.

4. The preparation method of isomaltooligosaccharide according to claim 1, characterized in that In the step of modifying amylase, the mass concentration of the glutaraldehyde solution is 8 - 12%; The mass ratio of the α-fungal amylase, deionized water, and glutaraldehyde solution is 8 - 12:100:12 - 18.

5. The preparation method of isomaltooligosaccharide according to claim 1, characterized in that In the step of preparing the amylase carrier, in the microwave reaction, the microwave temperature is 62 - 66°C, the microwave time is 85 - 95 min, and the microwave power is 175 - 185 W; In the ultrasonic reaction, the ultrasonic frequency is 42 - 50 kHz, and the ultrasonic power is 100 - 110 W; The mass ratio of chitin, dimethyl sulfoxide, citric anhydride, and N-hydroxysuccinimide is 9.0 - 11.0:500:5.5 - 6.0:4.5 - 5.0; The mass ratio of the primary carrier, ethanol solution, 1,4-butanediol diglycidyl ether, gelatin, and L-lysine is 7.5 - 8.5:200:1.4 - 1.8:1.8 - 2.2:0.5 - 0.8; The mass concentration of the ethanol solution is 22 - 27%.

6. According to the method for preparing isomaltooligosaccharide as claimed in claim 1, characterized in that In the compounding step, amylase carrier is added to deionized water, and after stirring evenly, modified amylase is added, and the mixture is stirred and reacted at 5.8 - 6.2°C for 9.0 - 11.0 h. After the reaction is completed, functional amylase is obtained through filtration and drying; The mass ratio of deionized water, amylase carrier, and modified amylase is 100:6.5 - 7.5:2.2 - 2.

6.

7. According to the method for preparing isomaltooligosaccharide as claimed in claim 2, characterized in that In the liquefaction step, in the jet liquefaction, the liquefaction temperature is 102 - 115°C, and the liquefaction time is 1 - 3 h; In the method for preparing ultrasonic α-amylase, in the ultrasonic irradiation treatment, the ultrasonic frequency is 10 - 15 kHz, and the ultrasonic power is 82 - 90 W; The addition amount of the ultrasonic α-amylase is 0.1 - 0.7 wt% of the mass of corn starch.

8. According to the method for preparing isomaltooligosaccharide as claimed in claim 2, characterized in that In the α-glucosyltransferase conversion step, α-glucosyltransferase is added to the crude saccharified liquid, the pH value is adjusted to 7 - 9, the temperature is controlled at 45 - 60°C, and it is kept warm for 50 - 100 h to obtain a crude isomaltooligosaccharide sugar solution; The addition amount of the α-glucosyltransferase is 0.3 - 0.5 wt% of the mass of corn starch.

9. According to the method for preparing isomaltooligosaccharide as claimed in claim 1, characterized in that In the filtration and decolorization step, the crude isomaltooligosaccharide sugar solution is filtered through a plate and frame filter, the filtration accuracy is controlled at 200 - 400 μm, the pressure is 10 - 35 kPa, the primary filtrate of the saccharified liquid is collected, then modified activated carbon is added, and the mixture is stirred at 240 - 260 rpm for 0.2 - 0.5 h. After stirring is completed, it is allowed to stand for 1.5 - 2.5 h. After standing is completed, it is filtered, and the secondary filtrate of the saccharified liquid is collected. The secondary filtrate of the saccharified liquid is kept at 42 - 48°C and subjected to nanofiltration treatment through a nanofiltration membrane. The feed pressure is controlled at 1.8 - 2.2 MPa, and the retention volume is 275 - 285 MW to obtain a saccharified filtrate; The mass-volume ratio of the modified activated carbon to the primary filtrate of the saccharified liquid is 2.0 - 6.0 g:100 mL; The preparation method of the modified activated carbon is as follows: place the activated carbon at 220 - 240 °C for heat preservation treatment for 2.3 - 2.8 h. After natural cooling, add deionized water, stir evenly, control the temperature at 25 - 30 °C, add hydrogen peroxide, continue to stir for 1 - 2 h, and obtain the modified activated carbon after filtration and drying; The mass ratio of the activated carbon, deionized water and hydrogen peroxide is 5.0 - 8.0:100:20 - 25.

10. According to the preparation method of a kind of isomaltooligosaccharide described in claim 1, characterized in that, The ion exchange step is as follows: perform ion exchange with a continuous simulated moving bed, use a strongly acidic cation exchange resin to treat the saccharified filtrate, the flow rate of the saccharified filtrate is 4 - 6 bed volumes / h, and the temperature is 45 - 50 °C to obtain a refined isomaltooligosaccharide solution; The model of the strongly acidic cation exchange resin is LX - 160, which is purchased from Xi'an Lantian New Material Technology Co., Ltd.; The mass concentration of the refined isomaltooligosaccharide solution is 15 - 25%; The concentration step is as follows: evaporate and concentrate the refined isomaltooligosaccharide solution to 48 - 52% of the original volume, and the evaporation temperature is 70 - 90 °C to obtain isomaltooligosaccharide.