A process for the preparation of linear maltopentaose

Abstract

The application discloses a method for preparing linear maltopentaose, and belongs to the technical field of functional sugar production. The application adopts the strategy of adjusting the pH of the reaction system after adding linear maltopentaose generating enzyme, so that the proportion of linear maltopentaose is reduced in the enzymolysis reaction step. After further reducing the by-products of glucose, maltose and maltotriose through yeast metabolism, the purity of linear maltopentaose is greatly improved. The substrate conversion rate of the prepared linear maltopentaose is greater than or equal to 99%, the proportion of linear maltopentaose in the reaction solution after yeast treatment is greater than or equal to 80%, and the purity of linear maltopentaose after further impurity removal can be greater than or equal to 83%. The method has the advantages of low production cost, high product quality, high yield and the like, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to a method for preparing linear maltopentose, belonging to the field of functional sugar production technology. Background Technology

[0002] Linear-chain maltopentose (G5), an important member of the maltodextrin family, is a functional oligosaccharide with significant development potential, showing broad application prospects in the food, pharmaceutical, and cosmetic fields. G5 possesses excellent processing characteristics and unique physiological functions, such as low cariogenicity, prebiotic activity, and moisturizing properties, and is considered a representative of the next generation of functional sugar sources. In recent years, with the rapid development of the health food and functional ingredient markets, the demand for high-purity maltopentose has been increasing, making its industrial production technology a research hotspot.

[0003] Currently, the industrial production of linear maltopentose faces three major technical bottlenecks: First, existing production processes generally suffer from low substrate conversion rates, resulting in low raw material utilization. Second, the content of linear maltopentose in the final product is low, often mixed with other maltodextrins of different molecular weights, increasing the difficulty and cost of subsequent purification. Third, the enzyme preparation system lacks selectivity, making it difficult to precisely control the degree of polymerization of sugar chains, resulting in an excessively wide molecular weight distribution of the product, affecting its functional properties and application effects. These factors severely restrict the large-scale production and market promotion of maltopentose.

[0004] In existing technologies, Chinese patent CN108300749A discloses a method for preparing linear maltopentose using a dual-enzyme method. Although the main product, linear maltopentose, accounts for up to 40%, the product still contains significant amounts of glucose (G1), maltose (G2), and maltotriose (G3), increasing separation and purification costs and reducing the product's application value. Another Chinese patent, CN110157688A, discloses a linear maltodextrin-producing enzyme mutant W139Y that can effectively hydrolyze starch to produce maltopentose, with a maltopentose content of 45.86%. However, the product also contains significant amounts of glucose, maltose, maltotriose, maltotetraose (G4), and maltohexaose (G6). These existing technologies collectively reflect the core problem currently facing linear maltopentose production: how to achieve high-efficiency, low-cost industrial production while ensuring high product purity. Breakthrough innovations are urgently needed, particularly in key areas such as process parameter optimization and product separation and purification.

[0005] Therefore, developing a method for preparing maltopentose with high substrate conversion rate and high product yield has become a pressing technical challenge for those skilled in the art. This technological breakthrough will not only promote the industrialization of maltopentose but also open up new possibilities for the market application of functional sugar sources. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing linear maltopentose. The method employs a strategy of adjusting the pH of the reaction system after adding a linear maltopentose generating enzyme. This reduces the proportion of linear maltohexasaccharide during the enzymatic hydrolysis step. Furthermore, by reducing glucose, maltose, and maltotriose byproducts through yeast metabolism, the purity of linear maltopentose is significantly improved.

[0007] This invention is achieved through the following technical solution: The first objective of this invention is to provide a method for preparing linear maltopentose, comprising the following steps: S1. Prepare starch milk or maltodextrin milk as the reaction substrate, add linear maltopentose generating enzyme, then adjust the pH of the reaction system to 5.5~7.0, add debranching enzyme after liquefaction, and carry out double enzymatic hydrolysis to obtain the enzymatic hydrolysis reaction solution. S2. After cooling the enzymatic hydrolysis solution obtained in step S1, yeast is added to carry out the reaction to obtain an oligosaccharide syrup containing linear maltopentose.

[0008] In one embodiment of the present invention, the starch is selected from one or more of cassava starch, ordinary corn starch, waxy corn starch, potato starch, rice starch, and wheat starch.

[0009] In one embodiment of the present invention, the concentration of the starch milk or maltodextrin milk is 10% to 30% w / w, preferably 15% to 25% w / w.

[0010] In one embodiment of the present invention, the amino acid sequence of the linear maltopentose generating enzyme is shown in SEQ ID NO. 1.

[0011] In one embodiment of the present invention, the amount of linear maltopentose generating enzyme added is 25-100 U / g dry substrate. Preferably, it is 50-75 U / g dry substrate.

[0012] In one embodiment of the invention, the liquefaction is performed at 80-100°C for 5-30 minutes. Preferably, it is performed at 85-95°C for 10-20 minutes.

[0013] In one embodiment of the present invention, the debranching enzyme is pullulanase, dextrin debranching enzyme, isoamylase, or oligosaccharide debranching enzyme or one or more.

[0014] In one embodiment of the present invention, the amount of debranching enzyme added is 1-5 U / g dry substrate. Preferably, it is 1-3 U / g dry substrate.

[0015] In one embodiment of the present invention, the reaction temperature of the dual enzymatic hydrolysis reaction is 50-70°C, and the reaction time is 24-96 h. Preferably, the reaction temperature is 55-65°C, and the reaction time is 48-96 h.

[0016] In one embodiment of the present invention, in step S2, the temperature after cooling is 30~40°C, preferably 35°C.

[0017] In one embodiment of the present invention, the amount of yeast added is 1% to 5% of the mass of starch milk or maltodextrin milk. Preferably, it is 1% to 3%. More preferably, it is 2%.

[0018] In one embodiment of the present invention, in step S2, the reaction time for adding yeast is 10-40 h, preferably 20-30 h.

[0019] In one embodiment of the present invention, after adding yeast to carry out the reaction, the process further includes decolorization, filtration, and resin treatment steps.

[0020] In one embodiment of the present invention, the decolorization is performed by adding activated carbon to the reaction solution. Preferably, the amount of activated carbon added is 0.5% to 2% of the solution mass, and the decolorization time is 20 to 60 minutes. More preferably, the amount of activated carbon added is 1% of the solution mass, and the decolorization time is 30 minutes.

[0021] In one embodiment of the present invention, the resin treatment involves first passing the decolorized sugar solution through anion and cation exchange resins, and then through hydrogen ion exchange resins.

[0022] In one embodiment of the present invention, the anion and cation exchange resin treatment involves passing the sugar solution through anion exchange resin and cation exchange resin respectively to remove ions from the sugar solution.

[0023] In one embodiment of the present invention, the elution rate during hydrogen ion resin treatment is 20-40 mL / h.

[0024] A second objective of this invention is to provide the application of the method in the fields of food, medicine, and cosmetics.

[0025] The beneficial effects of this invention are: This invention provides a method for preparing linear maltopentose, which employs a strategy of adjusting the pH of the reaction system after adding a linear maltopentose generating enzyme. This reduces the proportion of linear maltohexasaccharide in the enzymatic hydrolysis step. Furthermore, by reducing glucose, maltose, and maltotriose byproducts through yeast metabolism, the purity of linear maltopentose is significantly improved.

[0026] The linear maltopentose prepared by this invention has a substrate conversion rate of ≥99%, and the linear maltopentose accounts for ≥80% in the reaction solution after yeast treatment. After further impurity removal, the purity of linear maltopentose can reach more than 83%. The method of this invention has the advantages of low production cost, high product quality, and high yield, and has good prospects for industrial application. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a small molecule sugar distribution diagram of the purified linear maltopentose syrup in Example 6 of the present invention; G1 to G6 represent glucose, maltose, linear maltotriose, linear maltotetraose, linear maltopentose, and linear maltohexaose, respectively. Figure 2 The color changes of linear maltopentose syrup after decolorization and desalting in Example 6 of this invention are shown in Figure a, which is the enzymatic hydrolysis solution in step (1), the solution after action of active dry yeast in step (2), and the solution after decolorization and desalting in step (5). Detailed Implementation

[0029] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] Source of raw materials Linear maltopentose synthase originates from Bacillus stearothermophilus The mutant STB04, W139Y (disclosed in CN110157688A), has an amino acid sequence as shown in SEQ ID NO.1. AAPFNGTMMQYFEWYLPDDGTLWTKVANEANNLSSLGITALWLPPAYKGTSRSDVGYGVYDLYDLGEFNQKGTVRTKYGTKAQYLQAIQAAHAAGMQVYADVVFDHKGGADGTEWVDAVEVNPSDRNQ EISGTYQIQAYTKFDFPGRGNTYSSFKWRWYHFDGVDWDESRKLSRIYKFRGIGKAWDWEVDTENGNYDYLMYADLDMDHPEVVTELKNWGKWYVNTTNIDGFRLDAVKHIKFSFFPDWLSYVRSQTGK PLFTVGEYWSYDINKLHNYITKTNGTMSLFDAPLHNKFYTASKSGGAFDMRTLMTNTLMKDQPTLAVTFVDNHDTEPGQALQSWVDPWFKPLAYAFILTRQEGYPGVFYGDYYGIPQYNIPSLKSKIDP LLIARRDYAYGTQHDYLDHSDIIGWTREGVTEKPGSGLAALITDGPGGSKWMYVGKQHAGKVFYDLTGNRSDTVTITSDGWGEFKVNGGSVSVWVPRKTTVSTITRPITTRPWTGEFVRWTEPRLVAWP Pullulanase was purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0031] The corn starch was purchased from Shandong Shengyu Group Co., Ltd.

[0032] The active yeast powder was purchased from Angel Yeast Co., Ltd.

[0033] The starch sugar desalting cation exchange resin D001-FD and anion exchange resin D354-FD were purchased from Zhejiang Zhengguang Industrial Co., Ltd.

[0034] The hydrogen ion exchange resin is Mitsubishi UBK 530 Na type cation exchange resin, which is converted into hydrogen ion exchange resin after being washed with 5% hydrochloric acid.

[0035] Detection method: Product composition analysis: Using G1–G6 standards as quantitative and qualitative standards, the monosaccharide, disaccharide, and oligosaccharide components in the products were detected using HPAEC-PAD. The analytical conditions were as follows: A Thermo Scientific CarboPacPA 200 column was used, with 0.10 M NaOH and 0.50 M NaAc solution as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 35℃, and an injection volume of 25 μL. The percentage of each monosaccharide component's total conversion to the substrate in G1–G6 was calculated as follows:

[0036]

[0037] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0038] Example 1: This embodiment provides a method for preparing linear maltopentose, the specific steps of which are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and incubate at 60°C for 15 min. Add linear maltopentose synthase at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, raise the reaction temperature to 90°C, liquefy for 15 min, and then immediately cool to 60°C for saccharification reaction for 48 h.

[0039] Step (2): After boiling the solution obtained in step (1) in a water bath for 30 min to inactivate the enzyme, cool it to 35°C, add 2% (by weight) of active yeast powder (by weight of corn starch milk), and continue the reaction for 24 h. The distribution of small molecule sugars in the reaction solution was analyzed by ion chromatography. The substrate conversion rate of the linear maltopentose crude sugar solution was found to be 81.36%, and the proportion of linear maltopentose in the product was 76.26%.

[0040] Example 2: This embodiment provides a method for preparing linear maltopentose and optimizes the pH of the reaction system. The specific steps are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and keep it at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 5.0-7.0, adjust the reaction temperature to 90°C, liquefy for 15 min, and then immediately cool down to 60°C for saccharification reaction for 48 h; Step (2): After boiling the solution obtained in step (1) in a water bath for 30 min to inactivate the enzyme, cool it to 35°C, add 2% (by weight) of active yeast powder (by weight of corn starch milk), and continue the reaction for 24 h. Ion chromatography analysis of the distribution of small-molecule sugars in the reaction solution showed that the substrate conversion rate reached a maximum of 81.36% at pH 6.0, with the highest proportion of linear maltopentose at 76.26%.

[0041] Table 1. Effects of different reaction pH on sample components and substrate conversion rates in syrup products.

[0042] Example 3: This embodiment provides a method for preparing linear maltopentose and optimizes the amount of linear maltopentose generating enzyme added. The specific steps are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and keep it at 60°C for 15 min. Add linear maltopentose generating enzyme at enzyme dosages of 25, 50, 75, and 100 U / g of dry substrate, adjust the pH to 6.0, adjust the reaction temperature to 90°C, liquefy for 15 min, and then immediately cool down to 60°C for saccharification reaction for 48 h. Step (2): After boiling the solution obtained in step (1) in a water bath for 30 min to inactivate the enzyme, cool it to 35°C, add 2% (by weight of corn starch milk) of active yeast powder, and continue the reaction for 24 h. Ion chromatography analysis of the distribution of small-molecule sugars in the reaction solution showed that increasing the amount of enzyme added gradually decreased the proportion of linear maltohexasaccharide in the product, but gradually increased the proportion of linear maltotetrasaccharide. Considering both product characteristics and production costs, a 50 U / g linear maltopentose generating enzyme is more suitable.

[0043] Table 2. Effects of enzyme addition amount on sample components and substrate conversion rate in syrup products.

[0044] Example 4: This embodiment provides a method for preparing linear maltopentose and optimizes the enzymatic hydrolysis time of linear maltopentose generating enzyme. The specific steps are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and keep it at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, adjust the reaction temperature to 90°C, liquefy for 15 min, and then immediately cool down to 60°C to carry out the saccharification reaction for 24-96 h; Step (2): After boiling the solution obtained in step (1) in a water bath for 30 min to inactivate the enzyme, cool it to 35°C, add 2% (by weight of corn starch milk) of active yeast powder, and continue the reaction for 24 h. Ion chromatography analysis of the distribution of small-molecule sugars in the reaction solution showed that as the enzymatic hydrolysis time increased, the purity of linear maltopentose gradually increased, while linear maltohexaose had the lowest proportion, making it more suitable for later separation and purification. Therefore, a saccharification time of 96 h should be selected.

[0045] Table 3. Effects of enzyme reaction time on sample components and substrate conversion rate in syrup products.

[0046] Example 5: This embodiment provides a method for preparing linear maltopentose, which involves adding a debranching enzyme (pullulanase) during saccharification and optimizing its addition amount. The specific steps are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and incubate at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, adjust the reaction temperature to 90°C, liquefy for 15 min, and immediately cool down to 60°C. Then add pullulanase at 1, 2, and 3 U / g dry substrate for enzymatic hydrolysis reaction for 96 h. Step (2): After boiling the solution obtained in step (1) in a water bath for 30 min to inactivate the enzyme, cool it to 35°C, add 2% (by weight of corn starch milk) of active yeast powder, and continue the reaction for 24 h. Ion chromatography analysis of the distribution of small-molecule sugars in the reaction solution showed that when pullulanase was added at 2 U / g, the substrate conversion rate was 99.48%, while the proportion of linear maltpentose was 79.55%, which increased the yield by 15.31%.

[0047] Table 4. Effects of different pullulanase dosages on sample components and substrate conversion rates in syrup products.

[0048] Example 6: This embodiment provides a method for preparing linear maltopentose, the specific steps of which are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and incubate at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, adjust the reaction temperature to 90°C, liquefy for 15 min, immediately cool to 60°C, and then add 2 U / g pullulanase to carry out enzymatic hydrolysis for 96 h; Step (2): After boiling the solution obtained in step (1) for 30 min to inactivate the enzyme, cool it to 35°C, add 2% active yeast powder and continue the reaction for 24 h; Step (3): Add 1.0% activated carbon to the fermentation broth in step (2), decolorize at 50℃ for 30 min, then centrifuge and filter to remove yeast cells and activated carbon, and obtain linear maltopentose with a light transmittance of more than 99.5%. Step (4): Pass the decolorized sugar solution from step (3) through anion and cation exchange resin to remove the anions and cations and reduce the conductivity to below 10 μS / cm; Step (5): Pass the sugar solution after ion exchange treatment in step (4) through a hydrogen ion exchange resin and collect the sugar solution at 25°C with an elution rate of 30 mL / h. The distribution of small molecule sugars in the sugar solution was analyzed by ion chromatography. The product recovery rate was 70.22%, and the proportion of linear maltopentose was 84.47%.

[0049] It can be seen that the method for preparing linear maltopentose by means of the present invention can obtain linear maltopentose syrup with a light transmittance of more than 99.5% and a purity of more than 84%. The substrate conversion rate during the preparation process is 99.48% and the yield of linear maltopentose is 49.53%. It has the advantages of high substrate conversion rate, high yield of linear maltopentose and low production cost.

[0050] Comparative Example 1: Prepare 250 g of 20% (w / w) corn starch milk with deionized water and incubate at 60°C for 15 min. Add linear maltopentose synthase at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, raise the reaction temperature to 90°C, liquefy for 15 min, and immediately cool to 60°C for saccharification reaction for 96 h. Inactivate the enzyme by boiling in a water bath for 30 min.

[0051] Comparative Example 2: Prepare 250 g of 20% (w / w) corn starch milk with deionized water, adjust the pH to 6.0, and incubate at 60°C for 15 min. Add linear maltopentose synthase at a dosage of 50 U / g dry substrate, adjust the reaction temperature to 90°C, liquefy for 15 min, and immediately cool to 60°C for saccharification reaction for 96 h. Inactivate the enzyme by boiling in a water bath for 30 min.

[0052] The distribution of small-molecule sugars in the reaction solutions of Comparative Example 1 and Comparative Example 2 was analyzed by ion chromatography. The results showed that in the product of Comparative Example 1, glucose, maltose, and maltotriose accounted for 37.98%, linear maltotetraose accounted for 8.62%, linear maltopentose accounted for 50.45%, and linear maltohexaose accounted for 2.44%; in the product of Comparative Example 2, glucose, maltose, and maltotriose accounted for 37.62%, linear maltotetraose accounted for 8.13%, linear maltopentose accounted for 44.12%, and linear maltohexaose accounted for 10.13%.

[0053] Comparative Example 3: Based on the reaction in Example 4 for 96 h, the pH was adjusted to 6.0 after the starch milk was prepared, but no pH adjustment was performed after the enzyme was added. Other steps and parameters remained the same as in Example 4.

[0054] That is: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water, adjust the pH to 6.0, and keep it at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the reaction temperature to 90°C, liquefy for 15 min, and then immediately cool down to 60°C for saccharification reaction for 96 h.

[0055] Step (2): Inactivate the enzyme in the solution obtained in step (1) by boiling in a water bath for 30 min. Cool to 35°C, add 2% (by weight of corn starch milk) of active yeast powder and continue the reaction for 24 h. The distribution of small molecule sugars in the reaction solution was analyzed by ion chromatography. The substrate conversion rate of the linear maltopentose crude sugar solution was found to be 80.49%, and the proportion of linear maltopentose in the product was 69.27%, while the proportion of linear maltohexaose was 15.90%.

[0056] Comparative Example 3, based on Comparative Example 2, further added yeast for the reaction. After the enzymatic hydrolysis, active dry yeast was added. Glucose, maltose, and maltotriose byproducts were metabolized by the yeast, thus increasing the purity of linear maltopentose. However, because the proportion of linear maltohexasose was high in Comparative Example 2, the proportion of linear maltohexasose in the final product also increased to 15.90% after the glucose, maltose, and maltotriose byproducts were metabolized by yeast in Comparative Example 3. In Comparative Example 1, a strategy of adding linear maltopentose-producing enzyme and adjusting the pH was used to first reduce the proportion of linear maltohexasose. The 96-hour saccharification reaction in Example 4, based on Comparative Example 1, further added yeast for the reaction. In this case, the proportion of linear maltopentose reached 80.67%, while the proportion of linear maltohexasose was only 3.92%. This shows that the strategy of adding linear maltopentose-producing enzyme and adjusting the pH can effectively increase the content of linear maltopentose and reduce the proportion of other maltooligosaccharides.

[0057] Comparative Example 4: Based on Example 5, after preparing the starch milk, the pH was adjusted to 6.0, but no pH adjustment was performed after adding the enzyme. Then, 2 U / g pullulanase was added after liquefaction, while other steps and parameters remained consistent with Example 5.

[0058] That is: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water, adjust the pH to 6.0, and keep it at 60°C for 15 min. Add linear maltopentase at an enzyme dosage of 50 U / g of dry substrate, adjust the reaction temperature to 90°C, liquefy for 15 min, and then immediately cool down to 60°C and add pullulanase at 2 U / g for enzymatic hydrolysis for 96 h.

[0059] Step (2): The solution obtained in step (1) was boiled in a water bath for 30 min to inactivate the enzyme. The temperature was lowered to 35°C, and 2% (by weight of) active yeast powder (by weight of corn starch milk) was added to continue the reaction for 24 h. Ion chromatography analysis of the distribution of small-molecule sugars in the reaction solution showed that the substrate conversion rate of the linear maltopentose crude sugar solution was 99.89%, and the proportion of linear maltopentose in the product was 68.54%. Comparative Example 4 further demonstrated that even after adding pullulanase to improve the substrate conversion rate, it was impossible to further increase the proportion of linear maltopentose.

[0060] Comparative Example 5: This embodiment provides a method for preparing linear maltopentose, the specific steps of which are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and incubate at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, adjust the reaction temperature to 90°C, liquefy for 15 min, immediately cool to 60°C, and then add 2 U / g pullulanase to carry out enzymatic hydrolysis for 96 h; Step (2): Inactivate the enzymes in the solution obtained in step (1) by boiling it in a water bath for 30 min. Cool it down to 35°C, add 2% active yeast powder, and continue the reaction for 24 h. Step (3): Add 1.0% activated carbon to the solution obtained in step (2), decolorize for 30 min, then centrifuge and filter to remove yeast cells and activated carbon; Step (4): The decolorized sugar solution from step (3) was passed through anion and cation exchange resin to remove the anions and cations, reducing the conductivity to below 10 μS / cm. Ion chromatography analysis revealed that the distribution of small-molecule sugars in the solution was highest, with linear maltopentose accounting for 80.04% of the separated product. Comparative Example 5 used only activated carbon and anion and cation exchange resins to treat the product, removing pigments and inorganic salt ions, but having no significant effect on the distribution of small-molecule sugars. In Example 6, further passage through a hydrogen ion exchange resin further improved the purity of linear maltopentose in the product.

[0061] Comparative Example 6: This embodiment provides a method for preparing linear maltopentose, the specific steps of which are as follows: Step (1): Prepare 250 g of 20% (w / w) corn starch milk with deionized water and incubate at 60°C for 15 min. Add linear maltopentose generating enzyme at an enzyme dosage of 50 U / g dry substrate, adjust the pH to 6.0, adjust the reaction temperature to 90°C, liquefy for 15 min, immediately cool to 60°C, and then add 2 U / g pullulanase to carry out enzymatic hydrolysis for 96 h; Step (2): Inactivate the enzymes in the solution obtained in step (1) by boiling it in a water bath for 30 min. Cool it down to 35°C, add 2% active yeast powder, and continue the reaction for 24 h. Step (3): Add 1.0% activated carbon to the solution obtained in step (2), decolorize for 30 min, then centrifuge and filter to remove yeast cells and activated carbon; Step (4): Pass the decolorized sugar solution from step (3) through anion and cation exchange resin to remove the anions and cations and reduce the conductivity to below 10 μS / cm; Step (5): The sugar solution after ion exchange treatment in step (4) was passed through potassium ion exchange resin. The product recovery rate was 45.73%. The distribution of small molecule sugars in the sugar solution was analyzed by ion chromatography. The highest proportion of linear maltopentose in the separated product was found to be 83.59%.

[0062] In Comparative Example 6, potassium ion-type resin, commonly used for maltodextrin purification, was used to purify linear maltopentose, which significantly improved the purity of linear maltopentose, but the product recovery rate was much lower than that of hydrogen ion-type resin.

[0063] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing a linear maltopentose, characterized in that, Includes the following steps: S1. Prepare starch milk or maltodextrin milk as the reaction substrate, add linear maltopentose generating enzyme, then adjust the pH of the reaction system to 5.5~7.0, add debranching enzyme after liquefaction, and carry out double enzymatic hydrolysis to obtain the enzymatic hydrolysis reaction solution. S2. After cooling the enzymatic hydrolysis solution obtained in step S1, yeast is added to carry out the reaction to obtain an oligosaccharide syrup containing linear maltopentose.

2. The preparation method according to claim 1, characterized in that, The starch is selected from one or more of tapioca starch, ordinary corn starch, waxy corn starch, potato starch, rice starch, and wheat starch.

3. The preparation method according to claim 1, characterized in that, The concentration of the starch milk or maltodextrin milk is 10%~30% w / w.

4. The preparation method according to claim 1, characterized in that, The amino acid sequence of the linear maltopentose generating enzyme is shown in SEQ ID NO.

1.

5. The preparation method according to claim 1 or 4, characterized in that, The amount of linear maltopentose generating enzyme added is 25~100 U / g dry substrate.

6. The preparation method according to claim 1, characterized in that, The liquefaction process involves liquefaction treatment at 80-100°C for 5-30 minutes.

7. The preparation method according to claim 1, characterized in that, The debranching enzyme is one or more of pullulanase, dextrin debranching enzyme, isoamylase, or oligosaccharide debranching enzyme.

8. The preparation method according to claim 1, characterized in that, The amount of debranching enzyme added is 1~5 U / g dry substrate.

9. The preparation method according to claim 1, characterized in that, The reaction temperature for the dual enzymatic hydrolysis reaction is 50~70°C, and the reaction time is 24~96 h.

10. The preparation method according to claim 1, characterized in that, In step S2, the temperature after cooling is 30~40°C.

11. The preparation method according to claim 1, characterized in that, The amount of yeast added is 1% to 5% of the mass of starch milk or maltodextrin milk.

12. The preparation method according to claim 1 or 11, characterized in that, In step S2, the yeast is added and the reaction time is 10-40 hours.

13. The preparation method according to claim 1, characterized in that, After adding yeast to initiate the reaction, the process also includes decolorization, filtration, and resin treatment.

14. The preparation method according to claim 13, characterized in that, The decolorization is achieved by adding activated carbon to the reaction solution.

15. The preparation method according to claim 14, characterized in that, The amount of activated carbon added is 0.5% to 2% of the solution mass.

16. The preparation method according to claim 15, characterized in that, The decolorization time is 20~60 min.

17. The preparation method according to claim 13, characterized in that, The resin treatment involves first passing the decolorized sugar solution through anion and cation exchange resins, and then through hydrogen ion exchange resins.

18. The preparation method according to claim 17, characterized in that, Anion and cation exchange resin treatment involves passing the sugar solution through anion exchange resin and cation exchange resin respectively to remove ions from the sugar solution.

19. The preparation method according to claim 17, characterized in that, When treating with hydrogen ion exchange resin, the elution rate is 20~40 mL / h.

20. The application of the method according to any one of claims 1 to 19 in the fields of food, medicine and cosmetics.

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