Process for the preparation of oligoisomaltose

Abstract

The application provides a preparation method of oligoisomaltose, and belongs to the technical field of oligoisomaltose; the preparation method comprises the steps of preparing starch milk, liquefaction, primary saccharification, alpha-glucosidase conversion, filtration and decolorization, ion exchange and concentration; the liquefaction step is that ultrasonic alpha-amylase spraying liquefaction is added into the starch milk to obtain a liquefied solution with a DE value of 13-25%; the preparation method of the ultrasonic alpha-amylase is that alpha-amylase is subjected to ultrasonic irradiation treatment, the ultrasonic time is controlled to be 115-125s each time, the interval between each ultrasonic irradiation is controlled to be 55-65s, and the operation is repeated for 10 times to obtain the ultrasonic alpha-amylase; the oligoisomaltose prepared by the method has high content and low energy consumption.

Description

Technical Field

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

[0002] Isomaltooligosaccharides refer to a type of oligosaccharide with 2-6 monosaccharides formed by glucose groups linked by α-1,6 glycosidic bonds. Its main components are isomaltose, panose, isomaltotriose and isomaltotetraose. Among them, isomaltose, panose and isomaltotriose are the main functional components of isomaltooligosaccharides. Their content reflects the quality of the product and also affects the product application and price prospects.

[0003] There are two ways to prepare oligosaccharides of isomaltooligosaccharide: one is to use the reverse reaction of saccharifying enzyme to generate oligosaccharides such as isomaltose and maltose in a high-concentration glucose solution; however, due to the disadvantages of low yield, complex products and long production cycle, it is difficult to promote industrialization on a large scale;

[0004] The second method is to use high-concentration glucose syrup made from starch as a substrate, and obtain it through the α-glucosyl transfer reaction catalyzed by α-glucosidase; the industrial production of oligosaccharides generally uses starch as raw material and adopts a full enzyme process. The production process is roughly as follows: starch → spray liquefaction → β-amylase saccharification → α-glucosidase conversion → decolorization → ion exchange → vacuum concentration or spray drying → finished product.

[0005] When using starch as raw material and adopting the whole enzyme process to prepare oligosaccharides of isomaltooligosaccharide in the existing technology, the following problems usually exist:

[0006] First, the key to controlling this process technology lies in mastering the jet liquefaction technology, which is the foundation of production. To control the degree of starch hydrolysis, the DE value of the liquefied liquid is particularly important, as it directly affects the quality of the product. A high DE value means that some sugars will be further broken down into other small molecular compounds, and the saccharification enzyme cannot fully function, reducing the quality of the product. A low DE value means that the degree of starch hydrolysis is insufficient, and some starch still retains a larger molecular structure, resulting in high viscosity of the liquefied liquid, which is not conducive to subsequent saccharification. In addition, the number of substrates exposed at the non-reducing end is limited, and the saccharification enzyme does not have enough substrate to act on, which affects the saccharification efficiency and ultimately the product yield.

[0007] Second, α-glucosidase has poor stability and low activity, resulting in low conversion rate of glucose to isomaltooligosaccharides and low production efficiency;

[0008] Third, in the separation of low oligosaccharide isomaltulose from the saccharification liquid, usually first using plate and frame filter to remove suspended solids and residual starch, etc., and then using a large number of anion and cation exchange resin to remove salt in the saccharification liquid, and finally vacuum concentration or spray drying to obtain the product; but this process produces more waste water, and the resin is easily damaged, consumes a lot of energy, and has low working efficiency. SUMMARY

[0009] In order to solve the technical problems existing in the prior art, the present application provides a preparation method of oligosaccharide isomaltulose, which has high purity and low energy consumption.

[0010] In view of the above technical problems, the present application adopts the following technical solutions:

[0011] A preparation method of oligosaccharide isomaltulose, comprising the steps of preparing starch milk, liquefaction, initial saccharification, alpha-glucosidase conversion, filtration and decolorization, ion exchange and concentration, and the specific operation is as follows:

[0012] 1. Preparing starch milk

[0013] The corn starch is added to the deionized water to prepare a slurry, and the mass concentration of the slurry is controlled to be 15-30%, then dodecyl glucoside is added, and uniform stirring is continued, the pH value is adjusted to be 4-5, the temperature is raised to 40-55℃, and the temperature is kept and stirred for 0.5-1.5h to obtain the starch milk.

[0014] The addition amount of the dodecyl glucoside is 0.1-0.5wt% of the slurry.

[0015] 2. Liquefaction

[0016] The ultrasonic alpha-amylase is added to the starch milk, the pH value is adjusted to be 4.0-6.5, and the starch milk is sprayed and liquefied by a liquefaction sprayer after stirring for 0.2-0.5h, the liquefaction temperature is controlled to be 102-115℃, and the liquefaction time is 1-3h to obtain a liquefied liquid with a DE value of 13-25%;

[0017] The addition amount of the ultrasonic alpha-amylase is 0.1-0.7wt% of the mass of the corn starch;

[0018] The preparation method of the ultrasonic alpha-amylase is that the alpha-amylase is subjected to ultrasonic irradiation treatment, the ultrasonic time is controlled to be 115-125s each time, the interval between each ultrasonic is 55-65s, the operation is repeated for 10 times, the ultrasonic frequency is controlled to be 10-15kHz, and the ultrasonic power is 82-90W, and after the ultrasonic treatment, the ultrasonic alpha-amylase is obtained.

[0019] 3. Initial saccharification

[0020] controlling the temperature of the liquefied liquid to be 45-60 DEG C, adjusting the pH value to be 6.5-8.0, adding primary saccharifying enzyme to carry out primary saccharification reaction, and incubating for 15-35 h to obtain a crude saccharified liquid, and controlling the DE value of the crude saccharified liquid to be 40-47%;

[0021] 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;

[0022] the adding amount of the primary saccharifying enzyme is 0.1-0.7 wt% of the mass of the corn starch;

[0023] the preparation method of the functional amylase comprises a modified amylase step, a preparation of amylase carrier step and a compounding step;

[0024] the modified amylase step is to add alpha-fungal amylase into deionized water to stir uniformly, to raise the temperature to 40-50 DEG C, then to add glutaraldehyde solution, to stir for 2-3 h to obtain modified amylase;

[0025] the mass concentration of the glutaraldehyde solution is 8-12%;

[0026] the mass ratio of the alpha-fungal amylase, deionized water and glutaraldehyde solution is 8-12:100:12-18;

[0027] the preparation of amylase carrier step is to put chitin into dimethyl sulfoxide, to stir uniformly, then to add citric anhydride and N-hydroxysuccinimide, to continue stirring uniformly, then to carry out microwave reaction, to control the microwave temperature to be 62-66 DEG C, the microwave time to be 85-95 min, and the microwave power to be 175-185 W, after the microwave reaction, to wash, then to vacuum dry at 82-87 DEG C for 15.0-18.0 h to obtain primary carrier;

[0028] 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;

[0029] the primary carrier is added into ethanol solution, after stirring uniformly, 1, 4-butanediol diglycidyl ether is added, the pH value is adjusted to 8.2-8.7, the temperature is raised to 38-42 DEG C to carry out ultrasonic reaction, the ultrasonic frequency is controlled to be 42-50 kHz, the ultrasonic power is controlled to be 100-110 W, the ultrasonic time is controlled to be 4.8-5.3 h, after the ultrasonic reaction, the temperature is raised to 47-54 DEG C, gelatin and L-lysine are added, and incubation reaction is carried out for 3.2-3.7 h, after filtration, washing and drying, the amylase carrier is obtained;

[0030] The mass ratio of the primary carrier, the 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;

[0031] The mass concentration of the ethanol solution is 22-27%;

[0032] The compounding step is that the amylase carrier is added to deionized water, and after stirring uniformly, the modified amylase is added, and stirring reaction is carried out at 5.8-6.2℃ for 9.0-11.0h, after the reaction is completed, functional amylase is obtained through filtration and drying;

[0033] The mass ratio of the deionized water, the amylase carrier and the modified amylase is 100:6.5-7.5:2.2-2.6.

[0034] 4. α-glucosidase transferase conversion

[0035] The α-glucosidase transferase is added to the crude saccharification liquid, the pH value is adjusted to 7-9, the temperature is controlled to 45-60℃, and incubation is carried out for 50-100h, so that the crude oligoisomaltose sugar liquid is obtained;

[0036] The addition amount of the α-glucosidase transferase is 0.3-0.5wt% of the mass of the corn starch.

[0037] 5. Filtration and decolorization

[0038] The crude oligoisomaltose sugar liquid is filtered through a plate and frame filter, the filtration precision is controlled to be 200-400um, the pressure is controlled to be 10-35kPa, the primary filtrate of the saccharification liquid is collected, then the modified activated carbon is added, stirring is carried out at 240-260rpm for 0.2-0.5h, after the stirring is completed, standing is carried out for 1.5-2.5h, after the standing is completed, filtration is carried out, the secondary filtrate of the saccharification liquid is collected, the secondary filtrate of the saccharification liquid is maintained at 42-48℃, nanofiltration treatment is carried out through a nanofiltration membrane, the feeding pressure is controlled to be 1.8-2.2MPa, and the cut-off amount is controlled to be 275-285MW, so that the saccharification filtrate is obtained;

[0039] The mass-volume ratio of the modified activated carbon and the primary filtrate of the saccharification liquid is 2.0-6.0g:100mL;

[0040] The preparation method of the modified activated carbon is that the activated carbon is incubated at 220-240℃ for 2.3-2.8h, after natural cooling, deionized water is added, after stirring uniformly, the temperature is controlled to be 25-30℃, hydrogen peroxide is added, and stirring is continued for 1-2h, the modified activated carbon is obtained through filtration and drying;

[0041] The mass ratio of the activated carbon, the deionized water and the hydrogen peroxide is 5.0-8.0:100:20-25.

[0042] 6. Ion exchange

[0043] The ion exchange is carried out by using a continuous simulation moving bed, and the saccharification filtrate is treated by using a strong acid cation exchange resin, the saccharification filtrate flow rate is 4-6 bed volumes / h, and the temperature is 45-50 DEG C, so that a refined oligoisomaltose solution is obtained;

[0044] The strong acid cation exchange resin is of LX-160 type, and is purchased from Xi'an Lanxiao Science and Technology New Material Co., Ltd.

[0045] The mass concentration of the refined oligoisomaltose solution is 15-25%.

[0046] 7. Concentration

[0047] The refined oligoisomaltose solution is evaporated and concentrated to 48-52% of the original volume, the evaporation temperature is 70-90 DEG C, so that oligoisomaltose is obtained.

[0048] Compared with the prior art, the present application has the following beneficial effects:

[0049] 1.The present application adopts a specific method to prepare oligoisomaltose, controls the appropriate DE value in the liquefaction step, ensures that the hydrolysis of starch is within a certain range, so that the saccharification reaction can be carried out fully, and in the liquefaction step, the amylase is subjected to ultrasonic irradiation treatment, the ultrasonic wave can change the enzyme molecular structure, enhance the catalytic efficiency and stability of amylase; In the primary saccharification step, a functional enzyme carrier is prepared by a specific method, specifically, glutaraldehyde solution is first used to treat amylase, glutaraldehyde can cross-link with the amino group of amylase, so that the amylase molecules are cross-linked to form a network structure, which can improve the stability of amylase while maintaining the activity of amylase, then chitin is used as the carrier matrix, first treated with acid anhydride, and then the carboxyl group is introduced onto the surface of chitin by reacting the acid anhydride group with the amino group of chitin, then 1, 4-butanediol diglycidyl ether is added, which can cross-link with the carboxyl group on the surface of the primary carrier, and under the action of gelatin and L-lysine, the epoxy group of 1, 4-butanediol diglycidyl ether is cross-linked again, thereby obtaining an amylase carrier with a three-dimensional stable network structure, in the complexing step of modified amylase, the amylase carrier and the modified amylase are cross-linked again to realize stable cross-linking, and the functional amylase prepared finally can not only maintain high activity of amylase, but also ensure its stability, effectively ensuring the progress of saccharification reaction and the recovery performance and reuse performance of functional amylase; synergistic pullulanase, the liquefied liquid is converted into a crude saccharification liquid with a higher DE value, the content of glucose and oligosaccharides in the sugar liquid is increased, and sufficient substrate is provided for the conversion reaction of alpha-glucosidase, further converting the saccharides in the crude saccharification liquid into the target product oligoisomaltose, and increasing the content of oligoisomaltose; in the filtration and decolorization step, the pore size and initial chemical structure of activated carbon are changed by treating the activated carbon, and then the polarity is modified, so that the activated carbon has strong adsorption capacity for polar substances in the sugar liquid, the absorption performance of the sugar liquid is improved, the pigments and proteins in the sugar liquid are effectively removed, and after the subsequent treatment steps, a product with high oligoisomaltose content is obtained, and the content of impurities is reduced;

[0050] 2.Detecting each component in the product, the mass content of oligoisomaltose is 98.17-98.45%, the total mass content of isomaltose and isomaltotriose is 74.68-74.83%, and the mass content of impurities is 0.22-0.29%;

[0051] 3. The functional amylase is recovered at the end of each initial saccharification step to obtain recovered functional amylase, and the recovered functional amylase is reused for 25 times, and the oligoisomaltose prepared in the 25th reuse is measured, and the mass content of the oligoisomaltose is 92.37-93.82%, the mass content of isomaltose and isomaltotriose is 71.09-72.05%, and the mass content of the miscellaneous proteins is 0.40-0.45%;

[0052] The recovery method is that the crude saccharification liquid is centrifuged at 4000 rpm for 8 min to obtain solid matter; the solid matter is washed with 4 times the mass of deionized water for 3 times, and then stirred and washed with 4 times the mass of phosphate buffer for 60 min at a stirring speed of 100 rpm, the molar concentration of the phosphate buffer is 0.1 mol / L, and the pH value is 7.0; then the solid matter is stirred and washed with 0.5 mol / L sodium chloride solution for 20 min at a stirring speed of 100 rpm, and the recovered functional amylase is obtained after drying after washing. DETAILED DESCRIPTION

[0053] In order to more clearly understand the technical features, objectives and effects of the present application, the specific embodiments of the present application will be described.

[0054] Example 1: A preparation method of oligoisomaltose

[0055] 1. Preparation of starch milk

[0056] The corn starch is added to deionized water for slurry preparation to obtain a slurry, the mass concentration of the slurry is controlled to be 30%, then dodecyl glucoside is added, and uniform stirring is continued, the pH value is adjusted to be 5, the temperature is increased to 55℃, and the slurry is stirred and kept at 55℃ for 1.5 h to obtain starch milk.

[0057] The addition amount of the dodecyl glucoside is 0.5 wt% of the slurry.

[0058] 2. Liquefaction

[0059] The ultrasonic α-amylase is added to the starch milk, the pH value is adjusted to be 6.5, and the starch milk is stirred for 0.5 h, then the starch milk is sprayed and liquefied by a liquefaction sprayer, the liquefaction temperature is controlled to be 115℃, and the liquefaction time is 3 h to obtain a liquefied liquid with a DE value of 25%;

[0060] The addition amount of the ultrasonic α-amylase is 0.7 wt% of the mass of the corn starch;

[0061] The preparation method of the ultrasonic alpha-amylase is that the alpha-amylase is subjected to ultrasonic irradiation treatment, the ultrasonic time is controlled to be 125 s each time, the interval between each ultrasonic is 65 s, the operation is repeated for 10 times, the ultrasonic frequency is controlled to be 15 kHz, and the ultrasonic power is 90 W, after the ultrasonic treatment, the ultrasonic alpha-amylase is obtained.

[0062] 3. Primary saccharification

[0063] The temperature of the liquefied liquid is controlled to be 60 DEG C, the pH value is adjusted to be 8.0, the primary saccharification enzyme is added to carry out the primary saccharification reaction, and the incubation is carried out for 35 h, so that the crude saccharification liquid is obtained, and the DE value of the crude saccharification liquid is controlled to be 47%;

[0064] The primary saccharification enzyme is a mixed enzyme of functional amylase and pullulanase, and the mass ratio of the functional amylase and the pullulanase is 5:1;

[0065] The adding amount of the primary saccharification enzyme is 0.7 wt% of the mass of the corn starch;

[0066] The preparation method of the functional amylase comprises a modified amylase step, a preparation of amylase carrier step and a compounding step.

[0067] The modified amylase step is that 12 g of alpha-fungal amylase is added into 100 g of deionized water to be stirred uniformly, the temperature is increased to 50 DEG C, then 18 g of glutaraldehyde solution is added, and the stirring reaction is carried out for 3 h, so that the modified amylase is obtained.

[0068] The mass concentration of the glutaraldehyde solution is 12%;

[0069] The preparation of amylase carrier step is that 11.0 g of chitin is placed in 500 g of dimethyl sulfoxide, after being stirred uniformly, 6.0 g of citric anhydride and 5.0 g of N-hydroxysuccinimide are added, after being continuously stirred uniformly, the microwave reaction is carried out, the microwave temperature is controlled to be 66 DEG C, the microwave time is 95 min, the microwave power is 185 W, after the microwave reaction, after being washed, the primary carrier is obtained by being dried under vacuum at 87 DEG C for 15.0 h.

[0070] 8.5 g of the primary carrier is added into 200 g of 27 wt% ethanol solution, after being stirred uniformly, 1.8 g of 1, 4-butanediol diglycidyl ether is added, the pH is adjusted to 8.7, the temperature is increased to 42 DEG C to carry out the ultrasonic reaction, the ultrasonic frequency is controlled to be 50 kHz, the ultrasonic power is 110 W, and the ultrasonic time is 5.3 h, after the ultrasonic reaction, the temperature is increased to 54 DEG C, 2.2 g of gelatin and 0.8 g of L-lysine are added, the incubation reaction is carried out for 3.7 h, after being filtered, washed and dried, the amylase carrier is obtained.

[0071] The compounding step is that 7.5 g of amylase carrier is added to 100 g of deionized water, stirred uniformly, and then 2.6 g of modified amylase is added, stirred and reacted at 6.2 ℃ for 9.0 h, after the reaction is completed, functional amylase is obtained through filtration and drying.

[0072] 4. Alpha-glucosidase transferase conversion

[0073] Alpha-glucosidase transferase is added to the crude saccharification liquid, the pH value is adjusted to 9, the temperature is controlled at 60 ℃, and the temperature is kept for 100 h to obtain a crude oligoisomaltose sugar solution;

[0074] The addition amount of the alpha-glucosidase transferase is 0.5 wt% of the mass of corn starch.

[0075] 5. Filtration and decolorization

[0076] The crude oligoisomaltose sugar solution is filtered through a plate and frame filter, the filtration precision is controlled at 400 um, the pressure is controlled at 35 kPa, the primary filtrate of the saccharification liquid is collected, then modified activated carbon is added, stirring is carried out at 260 rpm for 0.5 h, after the stirring is completed, the solution is allowed to stand for 2.5 h, after the standing is completed, the secondary filtrate of the saccharification liquid is collected by filtration, the secondary filtrate of the saccharification liquid is kept at 48 ℃, and nanofiltration treatment is carried out through a nanofiltration membrane, the feeding pressure is controlled at 2.2 MPa, and the cut-off amount is controlled at 285 MW to obtain a saccharification filtrate.

[0077] The mass-volume ratio of the modified activated carbon to the primary filtrate of the saccharification liquid is 6.0 g:100 mL.

[0078] The preparation method of the modified activated carbon is that 8.0 g of activated carbon is kept at 240 ℃ for 2.8 h, after natural cooling, 100 g of deionized water is added, stirring is carried out uniformly, the temperature is controlled at 30 ℃, 25 g of hydrogen peroxide is added, and stirring is continued for 2 h, and the modified activated carbon is obtained through filtration and drying.

[0079] 6. Ion exchange

[0080] Ion exchange is carried out by using a continuous simulated moving bed, a strong acid cation exchange resin is used to treat the saccharification filtrate, the flow rate of the saccharification filtrate is 6 bed volumes / h, and the temperature is 50 ℃ to obtain a refined oligoisomaltose solution.

[0081] The model of the strong acid cation exchange resin is LX-160, which is purchased from Xi'an Lanxiao Science and Technology New Material Co., Ltd.

[0082] The mass concentration of the refined oligoisomaltose solution is 25%.

[0083] 7. Concentration

[0084] The refined oligoisomaltose solution is concentrated by evaporation to 52% of the original volume at an evaporation temperature of 90℃ to obtain oligoisomaltose.

[0085] The components in the product of Example 1 are detected, and the mass content of oligoisomaltose is 98.32%, the total mass content of isomaltose and isomaltotriose is 74.77%, and the mass content of miscellaneous proteins is 0.26%;

[0086] The functional amylase is recovered at the end of each initial saccharification step to obtain recovered functional amylase, which is repeatedly used for 25 times. The oligoisomaltose prepared in the 25th repeated use is detected, and the mass content of oligoisomaltose is 93.01%, the total mass content of isomaltose and isomaltotriose is 71.55%, and the mass content of miscellaneous proteins is 0.43%.

[0087] Example 2: A preparation method of oligoisomaltose

[0088] 1. Preparation of starch milk

[0089] The corn starch is added to the deionized water to prepare a slurry, and the mass concentration of the slurry is controlled to be 25%. Then, dodecyl glucoside is added, and the stirring is continued until uniform. The pH value is adjusted to be 4.5, the temperature is increased to 50℃, and the slurry is kept at 50℃ for 1.0h to obtain the starch milk.

[0090] The addition amount of the dodecyl glucoside is 0.3wt% of the slurry.

[0091] 2. Liquefaction

[0092] The ultrasonic α-amylase is added to the starch milk, and the pH value is adjusted to be 5.2. After stirring for 0.4h, the starch milk is sprayed and liquefied by a liquefaction sprayer. The liquefaction temperature is controlled to be 110℃, and the liquefaction time is 2h to obtain a liquefied liquid with a DE value of 18%.

[0093] The addition amount of the ultrasonic α-amylase is 0.5wt% of the mass of the corn starch.

[0094] The preparation method of the ultrasonic α-amylase is as follows: the α-amylase is subjected to ultrasonic irradiation treatment, the ultrasonic time is controlled to be 120s each time, the interval between each ultrasonic irradiation is 60s, the operation is repeated for 10 times, the ultrasonic frequency is controlled to be 12kHz, and the ultrasonic power is 85W. After the ultrasonic irradiation, the ultrasonic α-amylase is obtained.

[0095] 3. Initial saccharification

[0096] The temperature of the liquefied liquid is controlled to be 52℃, the pH value is adjusted to be 7.0, and the initial saccharification enzyme is added for initial saccharification reaction. The initial saccharification reaction is kept for 25h to obtain a crude saccharification liquid, and the DE value of the crude saccharification liquid is controlled to be 43%.

[0097] The primary saccharifying enzyme is a mixture of functional amylase and pullulanase, and the mass ratio of the functional amylase and the pullulanase is 4:1;

[0098] The adding amount of the primary saccharifying enzyme is 0.4wt% of the mass of the corn starch;

[0099] The preparation method of the functional amylase comprises a modified amylase step, a preparation of amylase carrier step and a complexing step.

[0100] The modified amylase step is to add 10g of α-fungal amylase into 100g of deionized water, stir uniformly, raise the temperature to 45℃, then add 15g of glutaraldehyde solution, stir for 2.5h to obtain the modified amylase.

[0101] The mass concentration of the glutaraldehyde solution is 10%.

[0102] The preparation of amylase carrier step is to put 10.0g of chitin into 500g of dimethyl sulfoxide, stir uniformly, then add 5.7g of citric anhydride and 4.8g of N-hydroxysuccinimide, continue to stir uniformly, then perform microwave reaction, control the microwave temperature to be 64℃, the microwave time to be 90min, and the microwave power to be 180W, after the microwave reaction, wash, and then vacuum dry at 85℃ for 16.0h to obtain the primary carrier.

[0103] Add 8.0g of the primary carrier into 200g of 25wt% ethanol solution, stir uniformly, then add 1.6g of 1,4-butanediol diglycidyl ether, adjust the pH to be 8.5, raise the temperature to 40℃ to perform ultrasonic reaction, control the ultrasonic frequency to be 45kHz, the ultrasonic power to be 105W, and the ultrasonic time to be 5.0h, after the ultrasonic reaction, raise the temperature to 50℃, add 2.0g of gelatin and 0.7g of L-lysine, and keep the temperature for 3.5h, after filtration, washing and drying, the amylase carrier is obtained.

[0104] The complexing step is to add 7.0g of the amylase carrier into 100g of deionized water, stir uniformly, then add 2.5g of the modified amylase, stir for 10.0h at 6.0℃, after the reaction, filter and dry to obtain the functional amylase.

[0105] 4. α-glucosidase transferase conversion

[0106] Add α-glucosidase transferase into the primary saccharifying liquid, adjust the pH to be 8, control the temperature to be 50℃, and keep the temperature for 75h to obtain the primary low oligomaltose liquid;

[0107] The adding amount of the α-glucosidase transferase is 0.4wt% of the mass of the corn starch.

[0108] 5. Filtration decolorization

[0109] The crude oligoisomaltose solution was filtered through a plate and frame filter, the filtration precision was controlled at 300 um, the pressure was 25 kPa, the primary filtrate of the saccharification solution was collected, then modified activated carbon was added, stirring at 250 rpm for 0.4 h, after stirring, it was placed for 2.0 h, after standing, it was filtered, the secondary filtrate of the saccharification solution was collected, the secondary filtrate of the saccharification solution was maintained at 45℃, nanofiltration treatment was performed through a nanofiltration membrane, the feed pressure was controlled at 2.0 MPa, the cut-off was 280 MW, and the saccharification filtrate was obtained;

[0110] The mass-volume ratio of the modified activated carbon to the primary filtrate of the saccharification solution was 4.0 g: 100 mL;

[0111] The preparation method of the modified activated carbon was as follows: 7.0 g of activated carbon was placed at 230℃ for 2.5 h, after natural cooling, 100 g of deionized water was added, after uniform stirring, the temperature was controlled at 27℃, 23 g of hydrogen peroxide was added, and the stirring was continued for 1.5 h, and the modified activated carbon was obtained by filtration and drying.

[0112] 6. Ion exchange

[0113] The ion exchange was performed by a continuous simulated moving bed, the saccharification filtrate was treated by a strong acid cation exchange resin, the flow rate of the saccharification filtrate was 5 bed volumes / h, and the temperature was 48℃, and the refined oligoisomaltose solution was obtained;

[0114] The type of the strong acid cation exchange resin was LX-160, which was purchased from Xi'an Lanxiao Science and Technology New Material Co., Ltd.;

[0115] The mass concentration of the refined oligoisomaltose solution was 20%.

[0116] 7. Concentration

[0117] The refined oligoisomaltose solution was evaporated and concentrated to 50% of the original volume, the evaporation temperature was 80℃, and the oligoisomaltose was obtained.

[0118] The components in the product of Example 2 were detected, the mass content of oligoisomaltose was 98.45%, the total mass content of isomaltose and isomaltotriose was 74.83%, and the mass content of impurities was 0.22%;

[0119] The functional amylase is recovered at the end of each initial saccharification step to obtain recovered functional amylase, and the recovered functional amylase is repeatedly used, and the oligoisomaltose prepared in the 25th repeated use is measured, and the mass content of the oligoisomaltose is 93.82%, the total mass content of isomaltose and isomaltotriose is 72.05%, and the mass content of the impurity protein is 0.40%.

[0120] Example 3: A preparation method of oligoisomaltose

[0121] 1. Preparing starch milk

[0122] The corn starch is added to the deionized water to prepare a slurry, the mass concentration of the slurry is controlled to be 15%, then the dodecyl glucoside is added, and uniform stirring is continued, the pH value is adjusted to be 4, the temperature is increased to 40℃, and the temperature is kept for 0.5h under stirring to obtain the starch milk;

[0123] The addition amount of the dodecyl glucoside is 0.1wt% of the slurry.

[0124] 2. Liquefaction

[0125] The ultrasonic α-amylase is added to the starch milk, the pH value is adjusted to be 4.0, and after stirring for 0.2h, the starch milk is sprayed and liquefied through a liquefaction sprayer, the liquefaction temperature is controlled to be 102℃, and the liquefaction time is 1h to obtain a liquefied liquid with a DE value of 13%;

[0126] The addition amount of the ultrasonic α-amylase is 0.1wt% of the mass of the corn starch;

[0127] The preparation method of the ultrasonic α-amylase is that the α-amylase is subjected to ultrasonic irradiation treatment, the ultrasonic time is controlled to be 115s each time, the interval between each ultrasonic irradiation is 55s, the operation is repeated for 10 times, the ultrasonic frequency is controlled to be 10kHz, and the ultrasonic power is 82W, and after the ultrasonic irradiation, the ultrasonic α-amylase is obtained.

[0128] 3. Initial saccharification

[0129] The temperature of the liquefied liquid is controlled to be 45℃, the pH value is adjusted to be 6.5, the initial saccharification enzyme is added for initial saccharification reaction, and the temperature is kept for 15h to obtain a crude saccharification liquid, and the DE value of the crude saccharification liquid is controlled to be 40%;

[0130] The initial saccharification enzyme is a mixture of functional amylase and pullulanase, and the mass ratio of the functional amylase and the pullulanase is 10:3;

[0131] The addition amount of the initial saccharification enzyme is 0.1wt% of the mass of the corn starch;

[0132] The preparation method of the functional amylase includes the steps of modifying the amylase, preparing an amylase carrier and compounding;

[0133] The modified amylase step comprises adding 8 g of α-fungal amylase to 100 g of deionized water and stirring evenly, raising the temperature to 40° C., then adding 12 g of glutaraldehyde solution, stirring and reacting for 2 hours to obtain the modified amylase;

[0134] The mass concentration of the glutaraldehyde solution is 8%;

[0135] The step of preparing the amylase carrier comprises: placing 9.0 g of chitosan in 500 g of dimethyl sulfoxide, stirring evenly, adding 5.5 g of citric anhydride and 4.5 g of N-hydroxysuccinimide, continuing to stir evenly, and performing a microwave reaction, wherein the microwave temperature is controlled to be 62° C., the microwave time is controlled to be 85 min, and the microwave power is controlled to be 175 W. After the microwave reaction is completed, the mixture is washed and vacuum dried at 82° C. for 18.0 h to obtain a primary carrier;

[0136] 7.5 g of the primary carrier was added to 200 g of a 22 wt% ethanol solution, stirred evenly, and then 1.4 g of 1,4-butanediol diglycidyl ether was added. The pH was adjusted to 8.2, and the temperature was raised to 38° C. for ultrasonic reaction. The ultrasonic frequency was controlled at 42 kHz, the ultrasonic power was 100 W, and the ultrasonic time was 4.8 h. After the ultrasonic reaction, the temperature was raised to 47° C., 1.8 g of gelatin and 0.5 g of L-lysine were added, and the reaction was kept warm for 3.2 h. After filtration, washing, and drying, an amylase carrier was obtained.

[0137] The compounding step comprises adding 6.5 g of amylase carrier to 100 g of deionized water, stirring evenly, adding 2.2 g of modified amylase, stirring and reacting at 5.8° C. for 11.0 h, and filtering and drying after the reaction to obtain functional amylase.

[0138] 4.α-glucosidase conversion

[0139] α-glucosidase was added to the crude saccharification solution, the pH value was adjusted to 7, the temperature was controlled to 45°C, and the temperature was kept at 45°C for 50 hours to obtain crude isomaltooligosaccharide solution;

[0140] The added amount of the α-glucosidase is 0.3 wt% of the mass of the corn starch.

[0141] 5. Filtration and decolorization

[0142] The crude oligoisomaltose sugar solution was filtered through a plate and frame filter, the filtration accuracy was controlled to be 200 um, the pressure was 10 kPa, the primary filtrate of the saccharification liquid was collected, then modified activated carbon was added, stirring was carried out at 240 rpm for 0.2 h, after the stirring was completed, 1.5 h of standing was carried out, after the standing was completed, filtration was carried out, the secondary filtrate of the saccharification liquid was collected, the secondary filtrate of the saccharification liquid was maintained at 42℃, nanofiltration treatment was carried out through a nanofiltration membrane, the feeding pressure was controlled to be 1.8 MPa, the cut-off amount was 275 MW, and the saccharification filtrate was obtained;

[0143] The mass-volume ratio of the modified activated carbon to the primary filtrate of the saccharification liquid was 2.0 g: 100 mL;

[0144] The preparation method of the modified activated carbon was that 5.0 g of activated carbon was treated at 220℃ for 2.3 h, after natural cooling, 100 g of deionized water was added, after uniform stirring, the temperature was controlled to be 25℃, 20 g of hydrogen peroxide was added, and stirring was continued for 1 h, and the modified activated carbon was obtained after filtration and drying.

[0145] 6. Ion exchange

[0146] The ion exchange was carried out using a continuous simulated moving bed, the saccharification filtrate was treated using a strong acid cation exchange resin, the flow rate of the saccharification filtrate was 4 bed volumes / h, the temperature was 45℃, and the refined oligoisomaltose solution was obtained;

[0147] The model of the strong acid cation exchange resin was LX-160, which was purchased from Xi'an Lanxiao Science and Technology New Material Co., Ltd.;

[0148] The mass concentration of the refined oligoisomaltose solution was 15%.

[0149] 7. Concentration

[0150] The refined oligoisomaltose solution was evaporated and concentrated to 48% of the original volume, the evaporation temperature was 70℃, and the oligoisomaltose was obtained.

[0151] Each component in the product of Example 3 was detected, the mass content of oligoisomaltose was 98.17%, the total mass content of isomaltose and isomaltotriose was 74.68%, and the mass content of miscellaneous proteins was 0.29%;

[0152] The functional starch enzyme was recovered at the end of each initial saccharification step to obtain recovered functional starch enzyme, the recovered functional starch enzyme was repeatedly used, the oligoisomaltose prepared by the 25th repeated use was measured, the mass content of oligoisomaltose was 92.37%, the total mass content of isomaltose and isomaltotriose was 71.09%, and the mass content of miscellaneous proteins was 0.45%.

[0153] Example 4

[0154] The recovery method of the functional amylase is that the crude saccharification liquid is centrifuged at 4000 rpm for 8 min to obtain solid matter; the solid matter is washed with 4 times the mass of deionized water for 3 times, and then stirred and washed with 4 times the mass of phosphate buffer for 60 min, the stirring speed is 100 rpm, the molar concentration of the phosphate buffer is 0.1 mol / L, and the pH value is 7.0, and then stirred and washed with 0.5 mol / L sodium chloride solution for 20 min, the stirring speed is 100 rpm, and after washing, drying is performed to obtain the recovered functional amylase.

[0155] Comparative Example 2.1

[0156] On the basis of Example 2, the following changes are made:

[0157] 1. In the liquefaction step, the preparation of the ultrasonic α-amylase is omitted, and the ultrasonic α-amylase is replaced with an equal amount of α-amylase without any treatment;

[0158] 2. In the primary saccharification step, in the preparation method of the functional amylase, the preparation step of the modified amylase is omitted, and the modified amylase is replaced with an equal amount of α-fungal amylase without any treatment;

[0159] The remaining operations are the same.

[0160] The components in the product of Comparative Example 2.1 are detected, and the mass content of oligoisomaltose is 87.31%, the total mass content of isomaltose and isomaltotriose is 67.32%, and the mass content of miscellaneous proteins is 1.17%;

[0161] The functional amylase is recovered at the end of each primary saccharification step to obtain recovered functional amylase, which is repeatedly used for 25 times, and the oligoisomaltose prepared in the 25th repeated use is measured, and the mass content of oligoisomaltose is 80.33%, the total mass content of isomaltose and isomaltotriose is 62.41%, and the mass content of miscellaneous proteins is 1.68%.

[0162] Comparative Example 2.1 omits the ultrasonic treatment of amylase in the liquefaction step, and omits the modification treatment of amylase in the preparation of functional amylase, which on the one hand affects the activity of the enzyme, making the liquefaction reaction incomplete, thereby greatly reducing the content of oligoisomaltose, and on the other hand, the binding between the amylase and the amylase carrier is poor, to some extent, affecting the recovery performance of the enzyme, and in the process of repeated use, part of the enzyme will fall off, thereby slightly reducing the retention rate of oligoisomaltose content after repeated use for multiple times.

[0163] Comparative Example 2.2

[0164] On the basis of Example 2, the following changes are made:

[0165] In the initial saccharification step, in the preparation method of the functional amylase, the amylase carrier is replaced by an equivalent amount of chitin component, and the rest of the operations are the same.

[0166] The components in the product of Comparative Example 2.2 are detected, and the mass content of oligoisomaltose is 92.68%, the mass content of isomaltulose and isomaltotriose is 71.22%, and the mass content of impurities is 0.31%;

[0167] The functional amylase is recovered at the end of each initial saccharification step to obtain recovered functional amylase, and the recovered functional amylase is repeatedly used for 25 times. The mass content of oligoisomaltose in the oligoisomaltose prepared in the 25th repeated use is 72.85%, the total mass content of isomaltulose and isomaltotriose is 57.05%, and the mass content of impurities is 1.95%.

[0168] Comparative Example 2.2 uses chitin as the amylase carrier, which can be cross-linked with the modified amylase to achieve the solidification of the amylase, but during the recovery and repeated use, due to incomplete binding, most of the enzyme falls off, resulting in poor repeated use performance.

[0169] Unless otherwise specified, the ratios described in the present application are mass ratios, and the percentages described are mass percentages.

[0170] Finally, it should be noted that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Although the present application 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 make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing isomaltooligosaccharide, characterized in that: The method comprises the steps of preparing starch milk, liquefaction, initial saccharification, α-glucosidase conversion, filtration and decolorization, ion exchange and concentration; The starch emulsion is prepared by adding corn starch to deionized water for slurry adjustment to obtain a slurry, controlling the mass concentration of the slurry to be 15-30%, then adding dodecyl glucoside, continuing to stir evenly, adjusting the pH value to 4-5, raising the temperature to 40-55° C., and stirring at this temperature for 0.5-1.5 hours to obtain the starch emulsion; The liquefaction step comprises adding ultrasonic α-amylase to the starch milk for spray liquefaction at a liquefaction temperature of 102-115° C. for a liquefaction time of 1-3 hours to obtain a liquefied liquid with a DE value of 13-25%; The ultrasonic α-amylase preparation method comprises the following steps: subjecting the α-amylase to ultrasonic irradiation treatment, controlling the ultrasonic frequency to 10-15 kHz, the ultrasonic power to 82-90 W, the ultrasonic time for each treatment to 115-125 seconds, the interval between each treatment to 55-65 seconds, and repeating the operation 10 times to obtain the ultrasonic α-amylase; The initial saccharification step comprises controlling the temperature of the liquefied liquid to 45-60° C., adjusting the pH value to 6.5-8.0, adding an initial saccharifying enzyme to carry out an initial saccharification reaction, and keeping the temperature for 15-35 hours to obtain a crude saccharified liquid, wherein the DE value of the crude saccharified liquid is controlled to be 40-47%; The primary saccharifying enzyme is a mixed enzyme of functional amylase and pullulanase, and the preparation method of the functional amylase includes the steps of modifying amylase, preparing an amylase carrier and a compounding step; The modified amylase step comprises adding α-fungal amylase to deionized water, raising the temperature to 40-50° C., and adding glutaraldehyde solution to obtain the modified amylase; The steps of preparing the amylase carrier are as follows: placing chitin in dimethyl sulfoxide, adding citric anhydride and N-hydroxysuccinimide, carrying out microwave reaction, the microwave temperature is 62-66° C., the microwave time is 85-95 minutes, and the microwave power is 175-185W, to obtain a primary carrier; adding the primary carrier to an ethanol solution, adding 1,4-butanediol diglycidyl ether, carrying out ultrasonic reaction at 38-42° C. for 4.8-5.3 hours, the ultrasonic frequency is 42-50kHz, the ultrasonic power is 100-110W, raising the temperature to 47-54° C., adding gelatin and L-lysine, and keeping the temperature for reaction for 3.2-3.7 hours to obtain the amylase 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; 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 step comprises adding an amylase carrier to deionized water, stirring evenly, adding a modified amylase, stirring and reacting at 5.8-6.2° C. for 9.0-11.0 hours, and filtering and drying after the reaction to obtain a functional amylase; The mass ratio of the deionized water, the amylase carrier and the modified amylase is 100:6.5-7.5:2.2-2.6; The α-glucosidase conversion step comprises adding α-glucosidase to the crude saccharification solution, adjusting the pH to 7-9, controlling the temperature to 45-60° C., and keeping the temperature for 50-100 hours to obtain a crude isomaltooligosaccharide solution; The added amount of the α-glucosidase is 0.3-0.5 wt% of the mass of the corn starch.

2. The method for preparing isomaltooligosaccharide according to claim 1, wherein In the step of preparing starch milk, the amount of lauryl glucoside added is 0.1-0.5 wt % of the slurry.

3. The method for preparing isomaltooligosaccharide according to claim 1, wherein In the initial saccharification step, the mass ratio of the functional amylase to the pullulanase in the initial saccharification enzyme is 4-10:1-3; The amount of the primary saccharifying enzyme added is 0.1-0.7 wt% of the mass of the corn starch.

4. The method for preparing isomaltooligosaccharide according to claim 1, wherein In the modified amylase step, 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 method for preparing isomaltooligosaccharide according to claim 1, wherein The amount of ultrasonic α-amylase added is 0.1-0.7 wt% of the mass of corn starch.

6. The method for preparing isomaltooligosaccharide according to claim 1, wherein The filtration and decolorization step comprises filtering the crude isomaltooligosaccharide solution through a plate and frame filter, controlling the filtration precision to 200-400 μm and the pressure to 10-35 kPa, collecting the primary filtrate of the saccharification solution, then adding modified activated carbon, stirring at 240-260 rpm for 0.2-0.5 h, standing for 1.5-2.5 h after the stirring is completed, filtering and collecting the secondary filtrate of the saccharification solution, maintaining the secondary filtrate of the saccharification solution at 42-48° C., performing nanofiltration treatment through a nanofiltration membrane, controlling the feed pressure to 1.8-2.2 MPa, and the retention rate to 275-285 MW, to obtain the saccharification filtrate; The mass volume ratio of the modified activated carbon to the primary filtrate of the saccharification solution is 2.0-6.0 g:100 mL; The modified activated carbon preparation method comprises the following steps: placing the activated carbon at 220-240° C. for insulation treatment for 2.3-2.8 hours, cooling it naturally, adding deionized water, stirring it evenly, controlling the temperature to 25-30° C., adding hydrogen peroxide, continuing stirring for 1-2 hours, and filtering and drying it to 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.

7. The method for preparing isomaltooligosaccharide according to claim 1, wherein: The ion exchange step comprises the following steps: performing ion exchange using a continuous simulated moving bed, treating the saccharification filtrate with a strong acidic cation exchange resin, the saccharification filtrate flow rate being 4-6 bed volumes / h, and the temperature being 45-50° C., to obtain a refined isomaltooligosaccharide solution; The model of the strong acid cation exchange resin is LX-160, which was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd. The mass concentration of the refined isomaltooligosaccharide solution is 15-25%; The concentration step comprises evaporating and concentrating the refined isomaltooligosaccharide solution to 48-52% of the original volume at an evaporation temperature of 70-90° C. to obtain isomaltooligosaccharide.