Method for preparing maltotetraose

Abstract

The invention discloses a method for preparing maltotetraose, and belongs to the technical field of functional sugar production. Comprising the following steps: preparing a maltodextrin aqueous solution; maltotetraose generating enzyme MFAPS-delta CBM enzyme is added, and primary saccharification is conducted for 8 h to 16 h; then adding debranching enzyme to react for 6-14 hours; finally, adding an MFAPS-wild enzyme, and carrying out secondary saccharification for 12 to 24 hours; and further performing yeast fermentation, activated carbon decoloration, ion exchange desalination and membrane separation impurity removal to obtain the maltotetraose syrup. According to the method, saccharification is divided into two stages, the advantages of two MFAPS enzymes are fully played, substrate conversion is promoted in combination with debranching, the substrate conversion rate reaches 99% or above, maltotetraose accounts for 72% or above in a crude sugar solution, and the purity reaches 98% or above after impurity removal. According to the method, the substrate conversion rate and the maltotetraose yield in the maltotetraose production process are improved, the problems of low conversion rate, high cost, high separation difficulty and the like of a traditional method are solved, the production and purification cost is reduced, and the yield and the industrialization level are improved.

Description

Technical Field

[0001] The invention relates to the technical field of functional sugar production, in particular to a method for preparing maltotetraose. Background Art

[0002] As a new type of specific functional oligomer, maltotetraose has excellent food processing performance and good physiological effects. It has broad application prospects in food, medicine, daily chemicals and other fields, and is known as the most promising maltooligosaccharide.

[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 so far is low, and there are many problems such as low substrate conversion rate, low maltotetraose yield, and high cost. This has limited the industrial production and further development and application of maltotetraose to a certain extent. The Chinese patent with application publication number CN 110144312A discloses a maltooligosaccharide syrup and its preparation process, which can obtain malto-oligosaccharide syrup with 59%-62% maltotetraose, but there is 22%-28% maltodextrin in the syrup, which increases the cost and also brings great pressure to the subsequent separation and purification. The Chinese patent with application publication number CN 109549059A discloses a moisturizing syrup and its preparation method and application. Maltotriose is saccharified by β-amylase in coordination with maltotriose enzyme, and then pullulanase is used to cooperate with maltotetraose enzyme to obtain a malto-oligosaccharide syrup with a maltotetraose content of 30%-40%, and its glucose + macromolecular sugar accounts for ≤10%, but its maltotetraose yield is low, and frequent temperature adjustment is required during the production process, energy consumption is large, and the cost is high. The Chinese patent with application publication number CN 118006708A discloses a method for producing malto-oligosaccharides with a high maltotetraose content, by saccharifying maltotriose enzyme, α-amylase, and debranching enzyme in coordination to obtain a malto-oligosaccharide syrup with a maltotetraose content greater than 75%, and then the maltotetraose content is further increased to more than 80% by a simulated moving bed. However, this preparation method takes about 50 hours to saccharify, which is time-consuming, and the pH of the system needs to be continuously adjusted during the reaction, which is costly and has low production efficiency.

[0004] Therefore, there is an urgent need to provide a method for preparing maltotetraose with high substrate conversion rate, high maltotetraose yield and short saccharification time. Summary of the invention

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

[0006] The present invention is achieved through the following technical solutions:

[0007] The object of the present invention is to provide a method for preparing maltotetraose, comprising the following steps:

[0008] (1) Using maltodextrin aqueous solution as substrate, adding maltotetraose generating enzyme MFA PS -ΔCBM enzyme is used for a saccharification to obtain a saccharification reaction liquid; the saccharification reaction liquid mainly contains linear maltooligosaccharides such as glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), malpentose (G5), maltohexaose (G6), maltoheptaose (G7), etc., and also contains a part of limit dextrin.

[0009] (2) adding a debranching enzyme to the primary saccharification reaction liquid obtained in step (1) to carry out a reaction to obtain a debranched reaction liquid, wherein the limit dextrin in the system is debranched and converted into linear dextrin and small molecular sugars;

[0010] (3) Adding maltotetraose generating enzyme MFA to the debranched reaction solution obtained in step (2) PS -wild enzyme for secondary saccharification, and maltotetraose is obtained after the reaction. The proportion of the obtained maltotetraose in the maltooligosaccharide syrup is more than 75%.

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

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

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

[0014] In one embodiment of the present invention, in step (1), the maltotetraose generating enzyme MFA PS The amino acid sequence of the -ΔCBM enzyme is shown in SEQ ID NO.1.

[0015] In one embodiment of the present invention, in step (1), the maltotetraose generating enzyme MFA PS The addition amount of -ΔCBM enzyme is 10U / g to 20U / g dry substrate.

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

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

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

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

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

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

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

[0023] In one embodiment of the present invention, in step (3), the secondary saccharification further comprises one or more of yeast fermentation, decolorization, desalination and impurity removal on the reaction solution.

[0024] In one embodiment of the present invention, the maltooligosaccharide syrup is sequentially fermented by yeast, decolorized by activated carbon, desalted by anion and cation exchange resins, separated by membranes for impurity removal, and concentrated to obtain maltotetraose syrup with a purity of ≥98%.

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

[0026] In one embodiment of the present invention, the yeast is selected from one or more of baker's yeast, brewer's yeast and beer yeast.

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

[0028] In one embodiment of the present invention, the yeast fermentation conditions are: fermentation at 30° C. to 37° C. for 12 h to 48 h.

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

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

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

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

[0033] In one embodiment of the present invention, the impurity removal is membrane separation impurity removal.

[0034] In one embodiment of the present invention, the membrane molecular weights used in the membrane separation and impurity removal are 250Da and 1000Da respectively.

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

[0036] The maltotetraose prepared by the invention is used in food and daily chemicals.

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

[0038] 1. The present invention gives full play to the advantages of two maltotetraose generating enzymes, and combines with debranching enzymes to fully convert substrates into small molecule sugars, and has the advantages of high substrate conversion rate and high maltotetraose yield.

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

[0040] 3. Although the present invention uses two-stage saccharification to improve substrate conversion rate, it does not increase the amount of enzyme added compared to one-step saccharification, and has the characteristics of low cost and high efficiency.

[0041] 4. The malto-oligosaccharide syrup produced by the present invention has a high maltotetraose content and has a good application effect even without a subsequent impurity removal process.

[0042] 5. The maltooligosaccharide syrup produced by the present invention has a low content of macromolecular dextrin, which greatly reduces the pressure of subsequent separation and purification. Maltotetraose syrup with a purity of more than 98% can be obtained through yeast fermentation and membrane separation.

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

[0044] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

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

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

[0047] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0048] Method for determining the activity of maltotetraose production enzyme: dinitrosalicylic acid method (DNS method). Prepare 1% (w / v) soluble starch solution in C6H8O7-Na2HPO4 buffer (10mM, pH 6) as substrate, add 100μL of enzyme solution diluted 200 times to 900μL of 1% soluble starch, react at 50℃ for 15min, add 1.0mL DNS solution to terminate the reaction, transfer to boiling water bath for color development for 5min, and immediately cool in ice water bath, dilute 2 times with deionized water, measure the absorbance at 540nm, and calculate the reducing sugar content in the system according to the glucose standard curve. The amount of enzyme required to generate 1μmol reducing sugar (in terms of glucose) per minute is defined as 1 enzyme activity unit (U).

[0049] Method for determining pullulanase activity: DNS method is used. Prepare 1% (w / v) pullulan solution in C6H8O7-Na2HPO4 buffer (10mM, pH 6) as substrate, add 20μL diluted enzyme solution to 1.98mL substrate, react at 60℃ for 15min, add 2.0mL DNS solution to terminate the reaction, color in boiling water bath for 5min, immediately cool in ice bath, measure absorbance at 540nm, and calculate reducing sugar content in the system according to glucose standard curve. The amount of enzyme required to generate 1μmol reducing sugar (in terms of glucose) per minute is defined as 1 enzyme activity unit (U).

[0050] The content of each component in the product was analyzed by high performance anion exchange chromatography (HPAEC-PAD). The analysis conditions were: CarboPac PA200 column, electrochemical amperometric detector, silver ion reference electrode, 0.25MNaOH, 1MNaAc and ultrapure water as mobile phase, flow rate set to 0.5mL / min, column temperature 35°C, injection volume 25μL. The purity of maltotetraose (G4), the proportion of maltotetraose (G4) in the product and the substrate conversion rate were calculated as follows:

[0051] Maltotetraose (G4) purity = (maltotetraose mass / total solid mass) × 100%;

[0052] The proportion of maltotetraose (G4) in the product = (mass of maltotetraose / total mass of G1 to G7) × 100%;

[0053] Substrate conversion rate = (total mass of G1 to G7 / substrate dry mass) × 100%.

[0054] Example 1

[0055] This embodiment provides a method for preparing maltotetraose, and the specific steps are as follows:

[0056] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0057] Step (2): Add 10 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme (preparation method see patent CN116334159A), stirring speed 500rpm, saccharification at 50°C for 12h to obtain maltotetraose primary saccharification liquid;

[0058] Step (3): adding 1 U / g of dry substrate pullulanase to the primary saccharification liquid of maltotetraose obtained in step (2), stirring at a speed of 500 rpm, reacting at 50° C. for 2 h, to obtain a pullulanase reaction liquid;

[0059] Step (4): Add 10 U / g dry substrate MFA to the pullulanase reaction solution obtained in step (3) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 12h to obtain maltotetraose crude sugar liquid;

[0060] Step (5): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 94.72%, and the proportion of maltotetraose in the product was 69.12%.

[0061] Example 2

[0062] This embodiment provides a method for preparing maltotetraose and optimizes the time of a saccharification. The specific steps are as follows:

[0063] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0064] Step (2): Add 10 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50°C for 6h, 8h, 10h, 12h, 14h, 16h, respectively, to obtain maltotetraose primary saccharification liquid;

[0065] Step (3): adding 1 U / g of dry substrate pullulanase to the primary saccharification liquid of maltotetraose obtained in step (2), stirring at a speed of 500 rpm, reacting at 50° C. for 2 h, to obtain a pullulanase reaction liquid;

[0066] Step (4): Add 10 U / g dry substrate MFA to the pullulanase reaction solution obtained in step (3) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 12h to obtain maltotetraose crude sugar liquid;

[0067] Step (5): Take part of the maltotetraose crude sugar liquid and heat it in boiling water for 20 minutes to terminate the saccharification reaction, then centrifuge at 12000rpm for 10 minutes, pass the supernatant through a 0.22μm aqueous membrane to remove impurities and dilute it, and then analyze the distribution of small molecular sugars in the reaction solution by ion chromatography. The corresponding substrate conversion rate of the maltotetraose crude sugar liquid obtained under different saccharification times and the proportion of maltotetraose in the product are shown in Table 1. Further extending the saccharification time after a saccharification of 12h did not significantly improve the production efficiency of maltotetraose, and the substrate conversion rate was as high as 94.72%, which did not reach a better level. Therefore, in Example 3, the action time of the debranching enzyme was further extended to enhance the debranching effect and improve production efficiency.

[0068] Table 1 Production efficiency of maltotetraose at different first saccharification times

[0069]

[0070] Example 3

[0071] This example provides a method for preparing maltotetraose and optimizing the debranching time. The specific steps are as follows:

[0072] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0073] Step (2): Add 10 U / g dry substrate 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 obtain maltotetraose primary saccharification liquid;

[0074] Step (3): adding 1 U / g of pullulanase to the primary saccharification liquid of maltotetraose obtained in step (2), stirring at a speed of 500 rpm, and reacting at 50° C. for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 14 h to obtain a pullulanase reaction liquid;

[0075] Step (4): Add 10 U / g dry substrate MFA to the pullulanase reaction solution obtained in step (3) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 12h to obtain maltotetraose crude sugar liquid;

[0076] Step (5): Take part of the maltotetraose crude sugar solution and heat it in boiling water for 20 minutes to terminate the saccharification reaction, then centrifuge at 12000rpm for 10 minutes, pass the supernatant through a 0.22μm aqueous membrane to remove impurities and dilute it, and analyze the distribution of small molecular sugars in the reaction solution by ion chromatography. The corresponding substrate conversion rate and the proportion of maltotetraose in the product of the maltotetraose crude sugar solution obtained under different saccharification times are shown in Table 2. After further extending the action time of the debranching enzyme, the production efficiency of maltotetraose was significantly improved. When the debranching time was 8h, the production efficiency basically reached the limit, the corresponding substrate conversion rate increased by 5%, and the proportion of maltotetraose in the product increased by 1.27%. Continuing to extend the debranching time, the production efficiency of maltotetraose was not significantly improved.

[0077] Table 2 Production efficiency of maltotetraose at different debranching times

[0078]

[0079] Example 4

[0080] This example provides a method for preparing maltotetraose and optimizes the time of secondary saccharification. The specific steps are as follows:

[0081] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0082] Step (2): Add 10 U / g dry substrate 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 obtain maltotetraose primary saccharification liquid;

[0083] Step (3): adding 1 U / g of dry substrate pullulanase to the primary saccharification liquid of maltotetraose obtained in step (2), stirring at a speed of 500 rpm, reacting at 50° C. for 8 h, to obtain a pullulanase reaction liquid;

[0084] Step (4): Add 10 U / g dry substrate MFA to the pullulanase reaction solution obtained in step (3) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 12h, 14h, 16h, 18h, 20h respectively, to obtain maltotetraose crude sugar liquid;

[0085] Step (5): Take part of the maltotetraose crude sugar solution and heat it in boiling water for 20 minutes to terminate the saccharification reaction, then centrifuge at 12000rpm for 10 minutes, pass the supernatant through a 0.22μm water system membrane to remove impurities and dilute it, and analyze the distribution of small molecular sugars in the reaction solution by ion chromatography. The corresponding substrate conversion rate and the proportion of maltotetraose in the product of the maltotetraose crude sugar solution obtained under different primary saccharification times are shown in Table 3. Further extending the secondary saccharification time has limited increase in substrate conversion rate, but the proportion of maltotetraose in the product has been further improved, from 70.39% to 71.82%, which means that the yield of maltotetraose has been improved; among them, the optimal time is 16h.

[0086] Table 3 Production efficiency of maltotetraose under different secondary sugar durations

[0087]

[0088] Example 5

[0089] This embodiment provides a method for preparing maltotetraose, and the specific steps are as follows:

[0090] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0091] Step (2): Add 10 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme (preparation method see patent CN116334159A), stirring speed 500rpm, saccharification at 50°C for 12h to obtain maltotetraose primary saccharification liquid;

[0092] Step (3): adding 750 U / g dry substrate of dextrin debranching enzyme to the primary saccharification liquid of maltotetraose obtained in step (2), stirring at a speed of 500 rpm, reacting at 50° C. for 2 h, to obtain a dextrin debranching enzyme reaction liquid;

[0093] Step (4): Add 10 U / g dry substrate MFA to the dextrin debranching enzyme reaction solution obtained in step (3) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 12h to obtain maltotetraose crude sugar liquid;

[0094] Step (5): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 99.83%, and the proportion of maltotetraose in the product was 70.82%.

[0095] Example 6

[0096] This embodiment provides a method for preparing maltotetraose, and the specific steps are as follows:

[0097] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0098] Step (2): Add 10 U / g dry substrate 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 obtain maltotetraose primary saccharification liquid;

[0099] Step (3): adding 1 U / g of dry substrate pullulanase to the primary saccharification liquid of maltotetraose obtained in step (2), stirring at a speed of 500 rpm, reacting at 50° C. for 8 h, to obtain a pullulanase reaction liquid;

[0100] Step (4): Add 10 U / g dry substrate MFA to the pullulanase reaction solution obtained in step (3) PS -wild enzyme, stirring speed 500rpm, secondary saccharification at 50℃ for 16h, maltotetraose crude sugar solution was obtained, and the distribution of small molecule sugars was measured by ion chromatography. Figure 1 As shown;

[0101] Step (5): cooling the maltotetraose crude sugar solution to 32° C., adding 2% baker's yeast and fermenting for 24 hours;

[0102] Step (6): adding 1.5% activated carbon to the fermentation liquid in step (5), and centrifugally filtering to remove yeast cells and activated carbon after decolorization for 60 minutes to obtain a sugar solution with a transmittance of more than 99.6%;

[0103] Step (7): passing the decolorized sugar solution in step (6) through an anion and cation exchange resin to remove anions and cations therein and reduce the conductivity to below 10 μs / cm;

[0104] Step (8): passing the sugar solution after separation in step (7) through a nanofiltration membrane with a molecular weight of 1000Da to remove macromolecular impurities therein, and then passing the collected filtrate through a nanofiltration membrane with a molecular weight of 250Da to remove small molecular impurities therein, and concentrating the sugar solution between 250Da and 1000Da to obtain the high-purity maltotetraose syrup of the present invention;

[0105] Step (9): Take the raw sugar solution of step (4), the fermentation solution of step (5) and the maltotetraose syrup of step (8), analyze the distribution of small molecular sugars in the sugar solution by ion chromatography, and further calculate the purity of maltotetraose in each step. Figure 2 shown.

[0106] It can be seen that through the method for efficiently preparing maltotetraose of the present invention, maltotetraose syrup with a transmittance of more than 99.6 and a purity of more than 98% 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.

[0107] Comparative Example 1

[0108] This comparative example provides a method for preparing maltotetraose, and the specific steps are as follows:

[0109] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0110] Step (2): Add 20 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 24h to obtain maltotetraose crude sugar liquid;

[0111] Step (3): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 84.47%, and the proportion of maltotetraose in the product was 66.82%.

[0112] Comparative Example 2

[0113] This comparative example provides a method for preparing maltotetraose, and the specific steps are as follows:

[0114] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0115] Step (2): Add 20 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50°C for 24 h to obtain maltotetraose crude sugar liquid;

[0116] Step (3): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 89.02%, and the proportion of maltotetraose in the product was 66.28%.

[0117] Comparative Example 3

[0118] This comparative example provides a method for preparing maltotetraose, and the specific steps are as follows:

[0119] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0120] Step (2): Add 10 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -wild enzyme, stirring speed 500rpm, saccharification at 50°C for 12h to obtain maltotetraose primary saccharification liquid;

[0121] Step (3): Add 10 U / g dry substrate 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 maltotetraose crude sugar liquid;

[0122] Step (4): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 87.02%, and the proportion of maltotetraose in the product was 67.65%.

[0123] Comparative Example 4

[0124] This comparative example provides a method for preparing maltotetraose, and the specific steps are as follows:

[0125] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0126] Step (2): Add 10 U / g dry substrate 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 obtain maltotetraose primary saccharification liquid;

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

[0128] Step (4): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 91.24%, and the proportion of maltotetraose in the product was 67.40%.

[0129] Comparative Example 5

[0130] This comparative example provides a method for preparing maltotetraose, and the specific steps are as follows:

[0131] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0132] Step (2): Add 20 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -wild enzyme, stirring speed 500 rpm, saccharification at 50°C for 22 h to obtain saccharification liquid;

[0133] Step (3): adding 1 U / g of pullulanase to the saccharified liquid obtained in step (2), saccharifying at 50° C. for 2 h to obtain maltotetraose crude sugar liquid;

[0134] Step (4): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 86.32%, and the proportion of maltotetraose in the product was 66.42%.

[0135] Comparative Example 6

[0136] This comparative example provides a method for preparing maltotetraose, and the specific steps are as follows:

[0137] Step (1): preparing a 30% by mass maltodextrin aqueous solution at 95° C., keeping the temperature for 1 hour to completely dissolve the maltodextrin, then cooling to 50° C. and adjusting the pH to neutral;

[0138] Step (2): Add 20 U / g dry substrate MFA to the maltodextrin aqueous solution prepared in step (1) PS -ΔCBM enzyme, stirring speed 500 rpm, saccharification at 50°C for 22 h to obtain saccharification liquid;

[0139] Step (3): adding 1 U / g of pullulanase to the saccharified liquid obtained in step (2), saccharifying at 50° C. for 2 h to obtain maltotetraose crude sugar liquid;

[0140] Step (4): taking a portion of the maltotetraose crude sugar solution and heating it in boiling water for 20 minutes to terminate the saccharification reaction, followed by centrifugation at 12000 rpm for 10 minutes, passing the supernatant through a 0.22 μm aqueous membrane to remove impurities and diluting it, and then analyzing the distribution of small molecular sugars in the reaction solution by ion chromatography. It was measured that the substrate conversion rate of the maltotetraose crude sugar solution was 78.67%, and the proportion of maltotetraose in the product was 71.68%.

[0141] In summary, it can be seen that compared with the single enzyme method and the first MFA PS -Wild enzyme MFA after one saccharification PS -ΔCBM enzymatic secondary saccharification compared to first using MFA PS -ΔCBM enzyme after one saccharification MFA PS -wild enzyme secondary saccharification has obvious advantages in substrate conversion rate, which is increased by 4.22%. MFA PS -ΔCBM enzyme precedes MFA PS -Wild enzyme was added to the system for reaction, avoiding MFA in the system PS -wild enzyme for MFA PS -ΔCBM has adverse effects on enzymatic hydrolysis compared to MFA PS -wild enzyme, MFA PS -ΔCBM enzyme preferentially hydrolyzes medium- and short-chain dextrins, making the remaining substrate mainly composed of long-chain dextrins, which can better play the role of MFA in the subsequent PS -wild enzyme action; and MFA PS After the initial treatment of maltodextrin by -ΔCBM enzyme, the viscosity of the sugar solution decreased overall, and as the viscosity decreased, MFA could be promoted to a certain extent. PS-The combination of wild enzyme and substrate improves the efficiency of enzymatic reaction.

[0142] Compared with Comparative Example 4, the addition of debranching enzyme in Example 1 increased the substrate conversion rate by 3.48%, the proportion of maltotetraose in the product by 1.72%, and the production efficiency of maltotetraose was significantly improved. PS -ΔCBM enzyme followed by MFA supplementation PS The enzymatic reaction strategy of -wild enzyme has obvious advantages, but the substrate length is shortened and the molecular weight is reduced. After the enzymatic reaction, many limit dextrins are produced. There are also a large number of α-1,6 glycosidic bonds in the substrate. In order to fully convert the substrate, it is necessary to add debranching enzyme to debranch the substrate. The timing of adding debranching enzyme is more important. PS -ΔCBM enzyme saccharification before adding pullulanase, at this time the substrate composition is more complex, the solution viscosity is larger, the substrate is not suitable, resulting in poor debranching effect; if the debranching enzyme is added after the secondary saccharification, because there is no subsequent maltotetraose generating enzyme to continue saccharification, a large amount of medium and short chain dextrins will remain in the solution, resulting in incomplete reaction. Therefore, when the debranching enzyme is used to assist in the preparation of maltotetraose, it should be added before MFA PS -ΔCBM enzyme is added to the debranching enzyme after the first saccharification. At this time, the viscosity of the sugar solution decreases as a whole, and a large number of α-1,6 glycosidic bonds are exposed, which is suitable for the debranching reaction of the debranching enzyme. Finally, MFA PS -The secondary saccharification of wild enzyme can convert all the remaining substrates as much as possible.

[0143] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing maltotetraose, characterized in that: The following steps are involved: (1) Using maltodextrin aqueous solution as substrate, adding maltotetraose generating enzyme MFA PS -ΔCBM enzyme performs saccharification once to obtain a saccharification reaction solution; (2) adding a debranching enzyme to the primary saccharification reaction solution obtained in step (1) to carry out a reaction to obtain a debranched reaction solution; (3) Adding maltotetraose generating enzyme MFA to the debranched reaction solution obtained in step (2) PS -wild enzyme for secondary saccharification, and maltotetraose is obtained after the reaction.

2. The method according to claim 1, characterized in that In step (1), the concentration of the maltodextrin aqueous solution is 5wt% to 30wt%; and / or, the pH of the maltodextrin aqueous solution is 6.5 to 7.5; And / or, the DE value of maltodextrin in the maltodextrin aqueous solution is 4-15.

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

1.

4. The method according to claim 1, characterized in that: In step (1), the maltotetraose generating enzyme MFA PS The addition amount of -ΔCBM enzyme is 10U / g to 20U / g dry substrate.

5. The method according to claim 1, characterized in that In step (1), the time of the first saccharification is 8 h to 16 h.

6. The method according to claim 1, characterized in that 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 according to claim 1, characterized in that In step (2), the amount of the debranching enzyme added is 1 U / g to 4 U / g of dry substrate.

8. The method according to claim 1, characterized in that In step (2), the reaction time is 6 h to 14 h.

9. The method according to claim 1, characterized in that: 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.

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

11. The method according to claim 1, characterized in that: In step (3), the secondary saccharification time is 12h to 24h.

12. The method according to claim 1, characterized in that In step (3), after the secondary saccharification, the reaction solution is further subjected to one or more of the steps of yeast fermentation, decolorization, desalination and impurity removal.

13. The method according to claim 12, characterized in that The yeast is selected from one or more of baker's yeast, brewer's yeast and beer yeast.

14. The method according to claim 12, characterized in that The inoculation amount of the yeast is 0.1% to 10% of the substrate mass.

15. The method according to claim 12, characterized in that The yeast fermentation conditions are: fermentation at 30° C. to 37° C. for 12 h to 48 h.

16. The method according to claim 12, characterized in that The decolorization is activated carbon decolorization; the added amount of the activated carbon is 1.0% to 2.5%; And / or, the pH value of the decolorization is 4 to 6; And / or, the decolorization temperature is 30°C to 60°C; And / or, the decolorization time is 30 min to 120 min.

17. The method according to claim 12, characterized in that The impurity removal is membrane separation impurity removal.

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