Method for preparing maltotetraose

Abstract

Disclosed in the present invention is a method for preparing maltotetraose, comprising the following steps: preparing an aqueous maltodextrin solution; adding maltotetraose-producing enzyme MFAps-ACBM enzyme for primary saccharification; then adding a debranching enzyme for reaction; finally adding MFAps-wild enzyme for secondary saccharification; and further performing yeast fermentation, decolorization with activated carbon, desalting by means of ion exchange, and impurity removal by means of membrane separation, so as to obtain the maltotetraose syrup of the present invention. In the present invention, saccharification is performed in two stages, giving full play to the advantages of two MFAps enzymes, and a debranching enzyme is used to promote substrate conversion, the substrate conversion rate reaching 99% or more, the proportion of maltotetraose in a crude sugar solution being 72% or more, and the purity reaching 98% or more after impurity removal.

Description

A method for preparing maltotetraose TECHNICAL FIELD

[0001] The present application relates to a method for preparing maltotetraose, and belongs to the technical field of functional sugar production. BACKGROUND

[0002] Maltotetraose is a new type of specific functional oligosaccharide, which has excellent food processing performance and good physiological efficacy, and has wide application prospects in food, medicine, daily chemical and other fields, and is known as the most promising maltoligosaccharide.

[0003] In recent years, with the increasing demand for maltotetraose in various industries, the industrial production of maltotetraose has become a trend. The production efficiency of the maltotetraose production process reported at present is low, and there are problems such as low substrate conversion rate, low maltotetraose yield, and high cost. To some extent, this limits the industrial production and further development and application of maltotetraose. Chinese patent with application publication number CN 110144312 A discloses an oligomaltose syrup and a preparation process thereof, which can obtain a maltoligosaccharide syrup with a maltotetraose proportion of 59%-62%, but there are 22%-28% of maltodextrin in the syrup, which increases the cost and brings great pressure to the subsequent separation and purification. Chinese patent with application publication number CN 109549059 A discloses a moisturizing syrup and its preparation method and application, which is prepared by maltotriose enzyme and β-amylase saccharification, and then using pullulanase and maltotetraose enzyme to obtain a maltoligosaccharide syrup with a maltotetraose content of 30%-40%, and the glucose+macromolecular sugar proportion is ≤10%, but the maltotetraose yield is low, and the production process needs frequent temperature adjustment, which consumes a lot of energy and has high cost. Chinese patent with application publication number CN 118006708 A discloses a maltoligosaccharide production method with high maltotetraose content, which is prepared by saccharifying maltotetraose enzyme, alpha amylase and debranching enzyme, and then further improving the maltotetraose content to more than 80% by using a simulated moving bed. But this preparation method needs about 50h for saccharification, which is time-consuming, and the pH of the system needs to be adjusted constantly during the reaction process, which has high cost and low production efficiency.

[0004] Therefore, it is urgent to provide a maltotetraose preparation method with high substrate conversion rate, high maltotetraose yield and short saccharification time. SUMMARY

[0005] In view of the low substrate conversion rate, low maltotetraose yield, great difficulty in subsequent separation and purification, high production cost and low production efficiency of the maltotetraose preparation process in the prior art, the application provides a method for preparing maltotetraose, which adopts the form of two-stage saccharification combined with debranching, and precisely controls the saccharification of maltodextrin, so as to fully utilize the substrate, and improve the substrate conversion rate and the maltotetraose yield. The substrate conversion rate of the maltotetraose prepared by the method is greater than or equal to 99%, the maltotetraose accounts for greater than or equal to 70% in the reaction solution after saccharification, and the purity of the maltotetraose can reach more than 98% after further impurity removal, so that the method has the advantages of low production cost, short time consumption, high product quality and high yield, and has a good industrial application prospect.

[0006] The application is realized by the following technical scheme:

[0007] The application aims to provide a method for preparing maltotetraose, which comprises the following steps:

[0008] (1) taking a maltodextrin aqueous solution as a substrate, adding maltotetraose generating enzyme MFA PS -ΔCBM enzyme to perform primary saccharification, so as to obtain a primary saccharification reaction solution; the primary saccharification reaction solution mainly contains glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), maltoheptaose (G7) and the like straight-chain maltoligosaccharides, and also contains a part of limit dextrin.

[0009] (2) adding a debranching enzyme to the primary saccharification reaction solution obtained in step (1) to perform reaction, so as to obtain a reaction solution after debranching, and the limit dextrin in the system is converted into straight-chain dextrin and small molecule sugar by debranching;

[0010] (3) adding maltotetraose generating enzyme MFA PS -wild enzyme to the reaction solution after debranching obtained in step (2) to perform secondary saccharification, so as to obtain maltotetraose after reaction.

[0011] In an embodiment of the application, in step (1), the maltodextrin aqueous solution is obtained by the following preparation method: dissolving maltodextrin in hot water at 90 DEG C to 95 DEG C, keeping warm for 0.5 h to 1.0 h, and cooling to 45 DEG C to 60 DEG C to obtain the maltodextrin aqueous solution; the concentration of the maltodextrin aqueous solution is 5 wt% to 30 wt%;

[0012] and / or, the pH of the maltodextrin aqueous solution is 6.5 to 7.5;

[0013] and / or, the DE value of the maltodextrin in the maltodextrin aqueous solution is 4 to 15.

[0014] In an embodiment of the application, in step (1), the maltotetraose generating enzyme MFAPS - the amino acid sequence of the ACBM enzyme is shown in SEQ ID NO. 1.

[0015] In one embodiment of the present application, in step (1), the maltotetraohydrolase MFA PS - the addition amount of the ACBM enzyme is 10 U / g to 20 U / g of dry substrate.

[0016] In one embodiment of the present application, in step (1), the time of the primary saccharification is 8 h to 16 h; preferably 10 h to 14 h; more preferably 12 h.

[0017] In one embodiment of the present application, in step (2), the debranching enzyme is selected from one or more of a pullulanase, a dextrin debranching enzyme, an isoamylase, and an oligosaccharide debranching enzyme.

[0018] In one embodiment of the present application, in step (2), the enzyme addition amount of the debranching enzyme is 1 U / g to 4 U / g of dry substrate.

[0019] In one embodiment of the present application, in step (2), the time of the reaction is 6 h to 14 h; preferably 8 h.

[0020] In one embodiment of the present application, in step (3), the MFA PS - the wild enzyme is derived from Pseudomonas saccharophila STB07, and the amino acid sequence is shown in SEQ ID NO. 2.

[0021] In one embodiment of the present application, in step (3), the MFA PS - the enzyme addition amount of the wild enzyme is 10 U / g to 20 U / g of dry substrate.

[0022] In one embodiment of the present application, in step (3), the time of the secondary saccharification is 12 h to 24 h; preferably 16 h.

[0023] In one embodiment of the present application, in step (3), after the secondary saccharification, one or more of yeast fermentation, decolorization, desalination, and impurity removal steps are further included.

[0024] In one embodiment of the present application, the maltoligosaccharide syrup is sequentially subjected to yeast fermentation, activated carbon decolorization, cation and anion exchange resin desalination, membrane separation impurity removal, and concentration to obtain a maltotetraohydrolase syrup with a purity of ≥98%.

[0025] In one embodiment of the present application, the resins used for ion exchange are cation resin D001-FD and anion resin D354-FD, respectively.

[0026] In an embodiment of the present application, the yeast is selected from one or more of baker's yeast, Saccharomyces cerevisiae and brewer's yeast.

[0027] In an embodiment of the present application, the inoculation amount of the yeast is 0.1% to 10% of the mass of the substrate.

[0028] In an embodiment of the present application, the fermentation conditions of the yeast are 30°C to 37°C for 12h to 48h.

[0029] In an embodiment of the present application, the decolorization is activated carbon decolorization; the activated carbon is added in an amount of 1.0% to 2.5%;

[0030] And / or, the pH value of the decolorization is 4 to 6;

[0031] And / or, the temperature of the decolorization is 30°C to 60°C;

[0032] And / or, the time of the decolorization is 30min to 120min.

[0033] In an embodiment of the present application, the impurity removal is membrane separation impurity removal.

[0034] In an embodiment of the present application, the membrane molecular weight used in the membrane separation impurity removal is 250Da and 1000Da, respectively.

[0035] In an embodiment of the present application, the concentration is membrane concentration; the membrane molecular weight used in the membrane concentration is 100Da.

[0036] The maltotetraose prepared by the present application is applied in food and daily chemicals.

[0037] The technical solution of the present application has the following advantages:

[0038] 1. The present application fully utilizes the advantages of two maltotetraose generating enzymes, and combines with a debranching enzyme to fully convert the substrate into small molecule sugars, has the advantages of high substrate conversion rate, high maltotetraose yield, short time consumption and low cost.

[0039] 2. The present application uses maltodextrin as a substrate to prepare maltotetraose, which can use maltodextrin with a substrate concentration of about 30% to produce maltotetraose, and has the advantage of high production efficiency.

[0040] 3. Although the present application uses two-stage saccharification to improve the substrate conversion rate, the enzyme amount is not increased compared with one-step saccharification, and has the characteristics of low cost and high efficiency.

[0041] 4. The maltotetraose content in the malt oligosaccharide syrup produced by the present application is high, and even without subsequent impurity removal process, it still has good application effect.

[0042] 5、The malt oligosaccharide syrup produced by the present application has low content of macromolecular dextrin, greatly reducing the pressure of subsequent separation and purification, and through yeast fermentation and membrane separation, maltotetraose syrup with purity of more than 98% can be obtained.

[0043] 6、The saccharification process and the purification process involved in the present application have been verified through pilot tests, which can greatly improve the production efficiency and capacity of maltotetraose, have good industrial application prospects, and are conducive to the research and development and promotion of maltotetraose related products. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0045] Figure 1 is a small molecule sugar distribution diagram of maltotetraose syrup after purification in Example 6 of the present application;

[0046] Figure 2 is the purity of maltotetraose in the sugar solution at different stages in Example 6 of the present application. DETAILED DESCRIPTION

[0047] The present application will be further described in conjunction with specific examples. These examples are only used to illustrate the present application and not to limit the scope of the present application. In addition, after reading the content taught by the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0048] Raw material source

[0049] The malt dextrin used in the examples was purchased from Shandong Xiangyang Sugar Industry Co., Ltd.;

[0050] The pullulanase used in the examples was purchased from Aladdin Reagent (Shanghai) Co., Ltd.;

[0051] The dextrin debranching enzyme used in the examples was self-made in the laboratory, and the preparation method is shown in CN113430156A;

[0052] The baker's yeast used in the examples was purchased from Angel Yeast Co., Ltd.

[0053] In the present application, MFA PS The amino acid sequence of the CBM enzyme is as follows:

[0054] MFA PSThe amino acid sequence of the wild enzyme is as follows:

[0055] Detection method:

[0056] The determination method of maltotetrahydrolase activity is the DNS method. A 1% (w / v) soluble starch solution prepared with C6H8O7-Na2HPO4 buffer (10 mM, pH 6) is used as a substrate, 100 μL of the enzyme solution diluted 200 times is added to 900 μL of the 1% soluble starch solution, and the reaction is carried out at 50°C for 15 min. Then, 1.0 mL of DNS solution is added to terminate the reaction, and the color is developed in a boiling water bath for 5 min, followed by ice water bath cooling. After being diluted twice with deionized water, the absorbance is measured at 540 nm. The reduced sugar content in the system is calculated according to the glucose standard curve. The amount of enzyme required to produce 1 μmol of reduced sugar (calculated as glucose) per minute is defined as 1 enzyme activity unit (U).

[0057] The determination method of pullulanase activity is the DNS method. A 1% (w / v) pullulan solution prepared with C6H8O7-Na2HPO4 buffer (10 mM, pH 6) is used as a substrate, 20 μL of the enzyme solution diluted is added to 1.98 mL of the substrate, and the reaction is carried out at 60°C for 15 min. Then, 2.0 mL of DNS solution is added to terminate the reaction, and the color is developed in a boiling water bath for 5 min, followed by ice water bath cooling. The absorbance is measured at 540 nm. The reduced sugar content in the system is calculated according to the glucose standard curve. The amount of enzyme required to produce 1 μmol of reduced sugar (calculated as glucose) per minute is defined as 1 enzyme activity unit (U).

[0058] The content of each component in the product is analyzed by high-performance anion exchange chromatography (HPAEC-PAD). The analysis conditions are as follows: a CarboPac PA200 chromatographic column, an electrochemical amperometric detector, a silver ion reference electrode, 0.25 M NaOH, 1 M NaAc and ultrapure water as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 35°C, and a sample injection amount of 25 μL. The calculation methods of the purity of maltotetraose (G4), the proportion of maltotetraose (G4) in the product and the substrate conversion rate are as follows:

[0059] The purity of maltotetraose (G4) = (maltotetraose mass / total solid mass) x 100%;

[0060] The proportion of maltotetraose (G4) in the product = (maltotetraose mass / G1-G7 total mass) x 100%;

[0061] The substrate conversion rate = (G1-G7 total mass / substrate dry basis mass) x 100%.

[0062] The technical solutions of the present application are described in detail below in combination with specific examples. In the following examples, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, or prepared by conventional methods, or commonly used in the industry.

[0063] Example 1

[0064] This example provides a method for preparing maltotetraose, the specific steps are as follows:

[0065] Step (1): Prepare a 30% mass fraction maltodextrin aqueous solution at 95°C, incubate for 1h to completely dissolve the maltodextrin, then cool to 50°C and adjust the pH to neutral;

[0066] Step (2): Add 10 U / g of dry substrate of MFA to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme (preparation method see patent CN116334159A), stirring speed 500 rpm, saccharification at 50°C for 12h, to prepare a maltotetraose primary saccharification liquid;

[0067] Step (3): Add 1 U / g of dry substrate of pullulanase to the maltotetraose primary saccharification liquid prepared in step (2), stirring speed 500 rpm, reaction at 50°C for 2h, to prepare a pullulanase reaction liquid;

[0068] Step (4): Add 10 U / g of dry substrate of MFA to the pullulanase reaction liquid prepared in step (3) PS -wild enzyme, stirring speed 500 rpm, saccharification at 50°C for 12h, to prepare a maltotetraose crude sugar liquid;

[0069] Step (5): Take part of the maltotetraose crude sugar liquid and heat in boiling water for 20min to terminate the saccharification reaction, then centrifuge at 12000 rpm for 10min, pass the supernatant through a 0.22μm water system membrane to remove impurities and dilute, then analyze the distribution of small molecule sugars in the reaction liquid by ion chromatography, and it is measured that the substrate conversion rate of the maltotetraose crude sugar liquid is 94.72%, and the proportion of maltotetraose in the product is 69.12%.

[0070] Example 2

[0071] This example provides a method for preparing maltotetraose, and the time for primary saccharification is optimized, the specific steps are as follows:

[0072] Step (1): Prepare a 30% mass fraction maltodextrin aqueous solution at 95°C, incubate for 1h to completely dissolve the maltodextrin, then cool to 50°C and adjust the pH to neutral;

[0073] Step (2): 10 U / g dry substrate of MFA was added to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50℃ for 6h, 8h, 10h, 12h, 14h, 16h respectively, to prepare maltotetraose primary saccharification liquor;

[0074] Step (3): 1 U / g dry substrate of pullulanase was added to the maltotetraose primary saccharification liquor prepared in step (2), stirring speed 500 rpm, reaction at 50℃ for 2h, to prepare pullulanase reaction liquor;

[0075] Step (4): 10 U / g dry substrate of MFA was added to the pullulanase reaction liquor prepared in step (3) PS -wild enzyme, stirring speed 500 rpm, saccharification at 50℃ for 12h, to prepare maltotetraose primary saccharification liquor;

[0076] Step (5): A portion of the maltotetraose primary saccharification liquor was heated in boiling water for 20 min to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 min. The supernatant was passed through a 0.22 μm water membrane to remove impurities and then diluted. The distribution of small molecule sugars in the reaction liquor was analyzed by ion chromatography. The substrate conversion rate and the proportion of maltotetraose in the product prepared under different primary saccharification times are shown in Table 1. Further extension of the saccharification time after 12h of primary saccharification did not significantly improve the production efficiency of maltotetraose. The highest substrate conversion rate was 94.72%, which did not reach a better level. Therefore, the action time of the debranching enzyme was further extended in Example 3 to enhance the debranching effect and improve the production efficiency.

[0077] Table 1 Production efficiency of maltotetraose under different primary saccharification times

[0078] Example 3:

[0079] The present embodiment provides a method for preparing maltotetraose, and the debranching time is optimized, the specific steps are as follows:

[0080] Step (1): Prepare a 30% mass fraction of maltodextrin aqueous solution at 95℃, and incubate for 1h to completely dissolve the maltodextrin, and then cool to 50℃ and adjust the pH to neutral;

[0081] Step (2): 10 U / g dry substrate of MFA was added to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50℃ for 12h, to prepare maltotetraose primary saccharification liquor;

[0082] Step (3): 1 U / g of dry substrate of pullulanase was added to the maltotetraose primary saccharification solution prepared in step (2), and the reaction was carried out at 50°C for 2h, 4h, 6h, 8h, 10h, 12h, and 14h under the stirring speed of 500 rpm to prepare a pullulanase reaction solution;

[0083] Step (4): 10 U / g of dry substrate of MFA PS -wild enzyme, and the reaction was carried out at 50°C for 12h under the stirring speed of 500 rpm to prepare a maltotetraose primary saccharification solution;

[0084] Step (5): A part of the maltotetraose primary saccharification solution was heated in boiling water for 20 min to terminate the saccharification reaction, and then centrifuged at 12000 rpm for 10 min. The supernatant was filtered through a 0.22 μm water system membrane to remove impurities and diluted, and then the distribution of small molecule sugars in the reaction solution was analyzed by ion chromatography. The substrate conversion rate and the proportion of maltotetraose in the product prepared under different debranching enzyme action times were as shown in Table 2. After further prolonging the debranching enzyme action time, the production efficiency of maltotetraose was obviously improved. When the debranching time was 8h, the production efficiency basically reached the limit, the substrate conversion rate increased by 5%, and the proportion of maltotetraose in the product increased by 1.27%. The production efficiency of maltotetraose did not obviously improve when the debranching time was further prolonged.

[0085] Table 2 Production efficiency of maltotetraose under different debranching times

[0086] Example 4:

[0087] The present embodiment provides a method for preparing maltotetraose, and the time of secondary saccharification is optimized. The specific steps are as follows:

[0088] Step (1): A 30% mass fraction of maltodextrin aqueous solution was prepared at 95°C, and the maltodextrin was completely dissolved after incubation for 1h. Then the temperature was lowered to 50°C and the pH was adjusted to neutral;

[0089] Step (2): 10 U / g of dry substrate of MFA PS -ΔCBM enzyme, and the reaction was carried out at 50°C for 12h under the stirring speed of 500 rpm to prepare a maltotetraose primary saccharification solution;

[0090] Step (3): 1 U / g of dry substrate of pullulanase was added to the maltotetraose primary saccharification solution prepared in step (2), and the reaction was carried out at 50°C for 8h under the stirring speed of 500 rpm to prepare a pullulanase reaction solution;

[0091] Step (4): 10 U / g dry substrate of MFA was added to the pullulanase reaction solution prepared in step (3) PS -wild enzyme, stirring speed 500 rpm, 50℃, saccharification for 12h, 14h, 16h, 18h, 20h respectively, to prepare maltotetraose crude sugar solution;

[0092] Step (5): A part of the maltotetraose crude sugar solution was heated in boiling water for 20 min to terminate the saccharification reaction, and then centrifuged at 12000 rpm for 10 min. The supernatant was passed through a 0.22 μm water membrane to remove impurities and then diluted. The distribution of small molecule sugars in the reaction solution was analyzed by ion chromatography. The substrate conversion rate and the proportion of maltotetraose in the product prepared under different secondary saccharification times are shown in Table 3. Further prolonging the secondary saccharification time, the substrate conversion rate increased limitedly, but the proportion of maltotetraose in the product was further improved from 70.39% to 71.82%, which means that the yield of maltotetraose was improved; and the optimal time was 16h.

[0093] Table 3 Production efficiency of maltotetraose under different secondary saccharification times

[0094] Example 5:

[0095] The present embodiment provides a method for preparing maltotetraose, and the specific steps are as follows:

[0096] Step (1): A 30% mass fraction of maltodextrin aqueous solution was prepared at 95℃, and the maltodextrin was completely dissolved after incubation for 1h. Then the temperature was lowered to 50℃ and the pH was adjusted to neutral;

[0097] Step (2): 10 U / g dry substrate of MFA was added to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme (preparation method see patent CN116334159A), stirring speed 500 rpm, 50℃, saccharification for 12h, to prepare maltotetraose primary saccharification solution;

[0098] Step (3): 750 U / g dry substrate of dextrin debranching enzyme was added to the maltotetraose primary saccharification solution prepared in step (2), stirring speed 500 rpm, 50℃, reaction for 2h, to prepare dextrin debranching enzyme reaction solution;

[0099] Step (4): 10 U / g dry substrate of MFA was added to the dextrin debranching enzyme reaction solution prepared in step (3) PS -wild enzyme, stirring speed 500 rpm, 50℃, saccharification for 12h, to prepare maltotetraose crude sugar solution;

[0100] Step (5): Take part of the maltotetraose crude sugar solution in boiling water and heat for 20 min to terminate the saccharification reaction, then centrifuge at 12000 rpm for 10 min, and then pass the supernatant through a 0.22 μm water system membrane to remove impurities and dilute, and then analyze the distribution of small molecule sugars in the reaction solution by ion chromatography. It is measured that the substrate conversion rate of the maltotetraose crude sugar solution is 99.83%, and the proportion of maltotetraose in the product is 70.82%.

[0101] Example 6

[0102] The present embodiment provides a method for preparing maltotetraose, and the specific steps are as follows:

[0103] Step (1): Prepare a 30% mass fraction maltodextrin aqueous solution at 95°C, and incubate for 1 h to completely dissolve the maltodextrin, and then cool to 50°C and adjust the pH to neutral;

[0104] Step (2): Add 10 U / g of dry substrate of MFA to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50°C for 12 h, to prepare a maltotetraose primary saccharification solution;

[0105] Step (3): Add 1 U / g of dry substrate of pullulanase to the maltotetraose primary saccharification solution prepared in step (2), stirring speed 500 rpm, reaction at 50°C for 8 h, to prepare a pullulanase reaction solution;

[0106] Step (4): Add 10 U / g of dry substrate of MFA to the pullulanase reaction solution prepared in step (3) PS -wild enzyme, stirring speed 500 rpm, secondary saccharification at 50°C for 16 h, to prepare a maltotetraose crude sugar solution, and the distribution of small molecule sugars is shown in FIG. 1 measured by ion chromatography;

[0107] Step (5): Cool the maltotetraose crude sugar solution to 32°C, and add 2% of bread yeast for fermentation for 24 h;

[0108] Step (6): Add 1.5% of activated carbon to the fermentation solution in step (5), and after decolorization for 60 min, centrifugal filtration is performed to remove the yeast cells and activated carbon, to obtain a sugar solution with a transmittance of more than 99.6%;

[0109] Step (7): Pass the decolorized sugar solution in step (6) through anion and cation exchange resin to remove anions and cations therein, and reduce the conductivity to less than 10 μs / cm;

[0110] Step (8): The sugar solution after ion exchange in step (7) is passed through a nanofiltration membrane with a molecular weight of 1000 Da to remove macromolecular impurities therein, and then the collected filtrate is passed through a nanofiltration membrane with a molecular weight of 250 Da to remove small molecular impurities therein, and the sugar solution between 250 Da and 1000 Da is concentrated, thereby obtaining the high-purity maltotetraose syrup in the present application;

[0111] Step (9): The crude sugar solution of step (4), the fermentation liquor of step (5), and the maltotetraose syrup of step (8) are taken to analyze the distribution of small molecular sugars in the sugar solution by ion chromatography, and the purity of maltotetraose in each step is further calculated, as shown in FIG. 2.

[0112] It can be seen that by the method for efficiently preparing maltotetraose provided in the present application, the maltotetraose syrup with a transmittance of 99.6 or above and a purity of 98% or above can be obtained, the substrate conversion rate in the preparation process is 100.75%, and the proportion of maltotetraose in the product is 71.82%, which has the advantages of high substrate conversion rate, high maltotetraose yield, and low production cost.

[0113] Comparative Example 1

[0114] The present comparative example provides a method for preparing maltotetraose, MFA PS -wild enzyme for primary saccharification without debranching enzyme action, and the specific steps are as follows:

[0115] Step (1): A 30% mass fraction maltodextrin aqueous solution is prepared at 95℃, and the maltodextrin is completely dissolved after 1h of incubation, and then the temperature is lowered to 50℃ and the pH is adjusted to neutral;

[0116] Step (2): 20U / g of dry substrate of MFA PS -wild enzyme is added to the maltodextrin aqueous solution prepared in step (1), the stirring speed is 500rpm, and the saccharification is carried out at 50℃ for 24h to obtain a maltotetraose crude sugar solution;

[0117] Step (3): A part of the maltotetraose crude sugar solution is heated in boiling water for 20min to terminate the saccharification reaction, and then centrifuged at 12000rpm for 10min, the supernatant is passed through a 0.22μm water system membrane to remove impurities and diluted, and the distribution of small molecular sugars in the reaction solution is analyzed by ion chromatography, and it is measured that the substrate conversion rate of the maltotetraose crude sugar solution is 84.47%, and the proportion of maltotetraose in the product is 66.82%.

[0118] Comparative Example 2

[0119] The present comparative example provides a method for preparing maltotetraose, MFA PS -ΔCBM enzyme for primary saccharification without debranching enzyme action, and the specific steps are as follows:

[0120] Step (1): Prepare a 30% mass fraction of malt dextrin aqueous solution at 95°C, and keep it at this temperature for 1 h to completely dissolve the malt dextrin, and then cool it to 50°C and adjust the pH to neutral;

[0121] Step (2): Add 20 U / g of dry substrate of MFA to the malt dextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50°C for 24 h, to obtain a maltotetraose crude sugar solution;

[0122] Step (3): Take part of the maltotetraose crude sugar solution and heat it in boiling water for 20 min to terminate the saccharification reaction, then centrifuge it at 12000 rpm for 10 min, pass the supernatant through a 0.22 μm water system membrane to remove impurities, dilute it, and then analyze the distribution of small molecule sugars in the reaction solution by ion chromatography. It is found that the substrate conversion rate of the maltotetraose crude sugar solution is 89.02%, and the proportion of maltotetraose in the product is 66.28%.

[0123] Comparative Example 3:

[0124] This comparative example provides a method for preparing maltotetraose, and the two saccharifications do not perform debranching enzyme action, and the specific steps are as follows:

[0125] Step (1): Prepare a 30% mass fraction of malt dextrin aqueous solution at 95°C, and keep it at this temperature for 1 h to completely dissolve the malt dextrin, and then cool it to 50°C and adjust the pH to neutral;

[0126] Step (2): Add 10 U / g of dry substrate of MFA to the malt dextrin aqueous solution prepared in step (1) PS -wild enzyme, stirring speed 500 rpm, saccharification at 50°C for 12 h, to obtain a maltotetraose primary saccharification solution;

[0127] Step (3): Add 10 U / g of dry substrate of MFA to the maltotetraose primary saccharification solution obtained in step (2) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50°C for 12 h, to obtain a maltotetraose crude sugar solution;

[0128] Step (4): Take part of the maltotetraose crude sugar solution and heat it in boiling water for 20 min to terminate the saccharification reaction, then centrifuge it at 12000 rpm for 10 min, pass the supernatant through a 0.22 μm water system membrane to remove impurities, dilute it, and then analyze the distribution of small molecule sugars in the reaction solution by ion chromatography. It is found that the substrate conversion rate of the maltotetraose crude sugar solution is 87.02%, and the proportion of maltotetraose in the product is 67.65%.

[0129] Comparative Example 4:

[0130] The comparative example provides a method for preparing maltotetraose, and the two saccharifications are not subjected to debranching enzyme action, and the specific steps are as follows:

[0131] Step (1): prepare a 30% mass fraction of malt dextrin aqueous solution at 95°C, heat for 1h to completely dissolve the malt dextrin, and then cool to 50°C and adjust the pH to neutral;

[0132] Step (2): add 10U / g of dry substrate of MFA to the malt dextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500rpm, saccharification at 50°C for 12h, to obtain a maltotetraose primary saccharification liquor;

[0133] Step (3): add 10U / g of dry substrate of MFA to the maltotetraose primary saccharification liquor prepared in step (2) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 12h, to obtain a maltotetraose crude sugar liquor;

[0134] Step (4): take part of the maltotetraose crude sugar liquor to heat in boiling water for 20min to terminate the saccharification reaction, then centrifuge at 12000rpm for 10min, pass the supernatant through a 0.22μm water system membrane to remove impurities, and then dilute, analyze the distribution of small molecule sugars in the reaction liquor by ion chromatography, and measure that the substrate conversion rate of the maltotetraose crude sugar liquor is 91.24%, and the proportion of maltotetraose in the product is 67.40%.

[0135] Comparative Example 5:

[0136] The comparative example provides a method for preparing maltotetraose, and MFA PS -wild enzyme primary saccharification treatment, and the specific steps are as follows:

[0137] Step (1): prepare a 30% mass fraction of malt dextrin aqueous solution at 95°C, heat for 1h to completely dissolve the malt dextrin, and then cool to 50°C and adjust the pH to neutral;

[0138] Step (2): add 20U / g of dry substrate of MFA to the malt dextrin aqueous solution prepared in step (1) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 22h, to obtain a saccharification liquor;

[0139] Step (3): add 1U / g of dry substrate of pullulanase to the saccharification liquor prepared in step (2), saccharification at 50°C for 2h, to obtain a maltotetraose crude sugar liquor;

[0140] Step (4): Take part of the maltotetraose crude sugar solution in boiling water for 20 min to terminate the saccharification reaction, then centrifuge at 12000 rpm for 10 min, pass the supernatant through a 0.22 μm water system membrane to remove impurities and dilute, then analyze the distribution of small molecule sugars in the reaction solution by ion chromatography. The substrate conversion rate of the maltotetraose crude sugar solution is 86.32%, and the proportion of maltotetraose in the product is 66.42%.

[0141] Comparative Example 6:

[0142] This comparative example provides a method for preparing maltotetraose, MFA PS -ΔCBM enzyme primary saccharification treatment, the specific steps are as follows:

[0143] Step (1): Prepare a 30% mass fraction maltodextrin aqueous solution at 95°C, incubate for 1 h to completely dissolve the maltodextrin, and then cool to 50°C and adjust the pH to neutral;

[0144] Step (2): Add 20 U / g of dry substrate of MFA PS -ΔCBM enzyme to the maltodextrin aqueous solution prepared in step (1), stir at a speed of 500 rpm, and saccharify at 50°C for 22 h to obtain a saccharification solution;

[0145] Step (3): Add 1 U / g of dry substrate of pullulanase to the saccharification solution obtained in step (2), saccharify at 50°C for 2 h to obtain a maltotetraose crude sugar solution;

[0146] Step (4): Take part of the maltotetraose crude sugar solution in boiling water for 20 min to terminate the saccharification reaction, then centrifuge at 12000 rpm for 10 min, pass the supernatant through a 0.22 μm water system membrane to remove impurities and dilute, then analyze the distribution of small molecule sugars in the reaction solution by ion chromatography. The substrate conversion rate of the maltotetraose crude sugar solution is 86.32%, and the proportion of maltotetraose in the product is 66.42%.

[0147] As can be seen from the above, compared with single enzyme method and MFA PS -wild enzyme primary saccharification followed by MFA PS -ΔCBM enzyme secondary saccharification, MFA PS -ΔCBM enzyme primary saccharification followed by MFA PS -wild enzyme secondary saccharification has obvious advantages in substrate conversion rate, and the substrate conversion rate is increased by 4.22%. MFA PS -ΔCBM enzyme is added to the system before MFA PS -wild enzyme, avoiding the adverse effects of MFA PS -wild enzyme on MFA PS -ΔCBM enzyme hydrolysis reaction, compared with MFAPS -wild enzyme, MFA PS -ΔCBM enzyme preferentially hydrolyzes medium and short chain dextrins, leaving the remaining substrate mainly composed of long chain dextrins, which can better produce MFA PS -wild enzyme; and MFA PS -After the preliminary treatment of malt dextrin by the ΔCBM enzyme, the viscosity of the sugar solution as a whole decreases, and with the decrease in viscosity, MFA can be promoted to a certain extent PS -wild enzyme and substrate, improving the efficiency of enzymatic reaction.

[0148] Compared with Comparative Example 4, the addition of the debranching enzyme in Example 1 increased the substrate conversion rate by 3.48%, and the proportion of maltotetraose in the product increased by 1.72%, and the production efficiency of maltotetraose was significantly improved. As can be seen from the above, the MFA PS -ΔCBM enzyme is added first, and then MFA PS -wild enzyme, this enzymatic reaction strategy has obvious advantages, but at this time the substrate length is shortened as a whole, the molecular weight is reduced, and many limit dextrins are produced after enzymatic reaction, and there are still a large number of α-1, 6 glycosidic bonds in the substrate. In order to make the substrate be fully converted, it is necessary to add debranching enzyme to debranch the substrate. The timing of adding debranching enzyme is important. MFA PS -ΔCBM enzyme, the substrate composition is relatively complex at this time, the solution viscosity is large, and the substrate is not suitable, which leads to poor debranching effect; if the debranching enzyme is added after the second saccharification, there will be no maltotetraose generating enzyme to continue saccharification, and a large amount of medium and short chain dextrins will remain in the solution, which will cause incomplete reaction. Therefore, when debranching enzyme is used to assist in the preparation of maltotetraose, it should be added after MFA PS -ΔCBM enzyme, at this time the viscosity of the sugar solution as a whole decreases, a large number of α-1, 6 glycosidic bonds are exposed, which is suitable for the debranching reaction of debranching enzyme, and finally MFA PS -wild enzyme, the second saccharification can convert the remaining substrate as much as possible.

[0149] The examples provided above are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the execution order. Those skilled in the art can make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A process for the preparation of maltotetraose, characterized in that, The method comprises the following steps: (1) adding maltotetraose-forming enzyme MFA to a maltodextrin aqueous solution as a substrate PS - performing primary saccharification by the ΔCBM enzyme to obtain a primary saccharification reaction liquid; (2) adding a debranching enzyme to the primary saccharification reaction solution obtained in step (1) to perform a reaction, thereby obtaining a debranched reaction solution; (3) Adding maltotetraose-forming enzyme MFA to the reaction solution obtained in step (2) after the debranching PS - secondary saccharification with wild enzymes, and maltotetraose was obtained after the reaction.

2. The method of claim 1, wherein, In step (1), the concentration of the malt dextrin aqueous solution is 5wt%-30wt%. And / or, the pH of the malt dextrin aqueous solution is 6.5-7.

5. And / or, the DE value of the malt dextrin in the malt dextrin aqueous solution is 4-15.

3. The method of claim 1, wherein, In step (1), the maltotetraose-forming enzyme MFA PS The amino acid sequence of the ΔCBM enzyme is shown in SEQ ID NO.

1.

4. The method of claim 1, wherein, In step (1), the maltotetraose-forming enzyme MFA PS - the CBM enzyme is added in an amount of 10-20 U / g dry substrate.

5. The method of claim 1, wherein, In step (1), the time for the primary saccharification is 8h-16h.

6. The method of claim 1, wherein, In step (2), the debranching enzyme is selected from one or more of pullulanase, dextrin debranching enzyme, isoamylase and oligosaccharide debranching enzyme.

7. The method of claim 1, wherein, In step (2), the enzyme addition amount of the debranching enzyme is 1U / g-4U / g dry substrate.

8. The method of claim 1, wherein, In step (2), the time for the reaction is 6h-14h.

9. The method of claim 1, wherein, In step (3), the MFA PS - The wild enzyme is derived from Pseudomonas saccharophila STB07, and the amino acid sequence is shown as SEQ ID NO.

2.

10. The method of claim 1, wherein, In step (3), the MFA PS - the amount of enzyme added to the wild enzyme is 10 U / g to 20 U / g of dry substrate.

11. The method of claim 1, wherein, In step (3), the time for the secondary saccharification is 12h-24h.

12. The method of claim 1, wherein, In step (3), the secondary saccharification further comprises one or more of the following steps: yeast fermentation, decolorization, desalination and impurity removal.

13. The method of claim 12, wherein, The yeast is selected from one or more of baker's yeast, Saccharomyces cerevisiae and beer yeast.

14. The method of claim 12, wherein, The inoculation amount of the yeast is 0.1%-10% of the substrate mass.

15. The method of claim 12, wherein, The conditions for the yeast fermentation are: 30℃-37℃ fermentation for 12h-48h.

16. The method of claim 12, wherein, The decolorization is activated carbon decolorization; the addition amount of the activated carbon is 1.0%-2.5%; And / or, the pH value for the decolorization is 4-6; And / or, the temperature for the decolorization is 30℃-60℃; And / or, the time for the decolorization is 30min-120min.

17. The method of claim 12, wherein, The impurity removal is membrane separation impurity removal.

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