WO2021169096A1 - 一种多酶耦合生产单一聚合度麦芽糊精的方法 - Google Patents

一种多酶耦合生产单一聚合度麦芽糊精的方法 Download PDF

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WO2021169096A1
WO2021169096A1 PCT/CN2020/097622 CN2020097622W WO2021169096A1 WO 2021169096 A1 WO2021169096 A1 WO 2021169096A1 CN 2020097622 W CN2020097622 W CN 2020097622W WO 2021169096 A1 WO2021169096 A1 WO 2021169096A1
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maltodextrin
cyclodextrin
starch
solution
reaction solution
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江波
张涛
郑露华
姚小琳
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江南大学
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    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A23L29/35—Degradation products of starch, e.g. hydrolysates, dextrins; Enzymatically modified starches
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
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    • A61K8/732—Starch; Amylose; Amylopectin; Derivatives thereof
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    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01054—Cyclomaltodextrinase (3.2.1.54), i.e. cyclodextrinase
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    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2800/10—General cosmetic use

Definitions

  • the invention relates to a method for coupling multiple enzymes to produce maltodextrin with a single degree of polymerization, and belongs to the field of biotechnology.
  • Maltodextrin refers to a product that uses starch as a raw material to hydrolyze the starch with a low degree of acid or enzymatic method to obtain a DE value of less than 20%. Its main components are dextrin with a degree of polymerization (ie DP value) above 10 and oligosaccharides with a degree of polymerization below 10.
  • maltodextrin can be added to foods as thickeners, emulsifiers or stabilizers, it can be added to medicines as excipients, fillers and other ingredients, and it can be added to daily chemical products as an excellent carrier. , Maltodextrin has important applications in the fields of food, medicine and daily chemicals, which also means that maltodextrin has a large market.
  • maltodextrins with a high degree of polymerization (generally maltodextrins with a DE value of 4% to 12%) contain more direct Stranddextrin and the DP value distribution is not uniform, and the solution added with this type of maltodextrin is prone to precipitation after being placed for a period of time (for details, see References: Li Caiming, Li Yang, Gu Zhengbiao, Hong Yan, Cheng Li, Li Zhaofeng. Maltodextrin.
  • maltodextrin with DE value of 13%-20%
  • It has high reducing power, easily reacts with other substances, and easily produces Maillard reaction when it coexists with amino acids or proteins, which reduces the quality of products containing this type of maltodextrin.
  • the maltodextrins currently produced are all mixtures of various substances (including glucose, maltose, oligosaccharides and polysaccharides, etc.), which greatly improves the uniformity of the degree of polymerization. It is difficult to separate and purify maltodextrin products, which in turn increases the cost of separation and purification of maltodextrin products with a uniform degree of polymerization.
  • the technical problem to be solved by the present invention is to provide a low-cost method that can be used to produce non-reducing maltodextrin with a uniform degree of polymerization.
  • the present invention provides a method for producing maltodextrin, the method is to first combine cyclodextrin glucosyltransferase (CGTase), cyclodextrin degrading enzyme (CDase) and/or malto-oligosaccharide Trehalose synthase (MTSase) is added to starch and/or cyclodextrin to react to obtain a reaction solution containing maltodextrin, and then maltodextrin is obtained from the reaction solution containing maltodextrin.
  • CCTase cyclodextrin glucosyltransferase
  • CDase cyclodextrin degrading enzyme
  • MTSase malto-oligosaccharide Trehalose synthase
  • the method is to first add starch to water or buffer to obtain a starch solution, then gelatinize the starch solution to obtain a gelatinized starch solution, and then transfer the cyclodextrin glucose base Enzymes, cyclodextrin degrading enzymes and malto-oligosaccharyl trehalose synthase are added to the gelatinized starch solution to react to obtain a reaction solution containing maltodextrin, and finally maltodextrin is obtained from the reaction solution containing maltodextrin;
  • the method is to first add cyclodextrin to water or buffer to obtain a cyclodextrin solution, and then add cyclodextrin degrading enzyme and malto-oligosaccharyl trehalose synthase to the cyclodextrin to react to obtain Maltodextrin reaction solution, and finally maltodextrin is obtained from the reaction solution containing maltodextrin;
  • the method is to first add starch to water or buffer to obtain a starch solution, then gelatinize the starch solution to obtain a gelatinized starch solution, and then add cyclodextrin glucosyltransferase and cyclodextrin degrading enzyme The reaction is carried out in the gelatinized starch solution to obtain a reaction solution containing maltodextrin, and finally maltodextrin is obtained from the reaction solution containing maltodextrin;
  • the method is to first add cyclodextrin to water or buffer to obtain a cyclodextrin solution, then add the cyclodextrin degrading enzyme to the cyclodextrin for reaction to obtain a reaction solution containing maltodextrin, and finally Maltodextrin is obtained from the reaction solution containing maltodextrin.
  • amino acid sequence of the cyclodextrin glucosyltransferase is shown in SEQ ID NO:1.
  • amino acid sequence of the cyclodextrin degrading enzyme is shown in SEQ ID NO: 2.
  • the amino acid sequence of the malto-oligosaccharyl trehalose synthase is shown in SEQ ID NO: 3.
  • nucleotide sequence of the gene encoding the cyclodextrin glucosyltransferase is shown in SEQ ID NO: 4.
  • nucleotide sequence of the gene encoding the cyclodextrin degrading enzyme is shown in SEQ ID NO: 5.
  • nucleotide sequence of the gene encoding the malto-oligosaccharyl trehalose synthase is shown in SEQ ID NO: 6.
  • the addition amount of the cyclodextrin glucosyltransferase in the gelatinized starch solution is 6-10 U/g starch ; the cyclodextrin degrading enzyme is more effective in the gelatinized starch solution addition amount of 3 ⁇ 7U / g starch; maltooligosyl the trehalose synthase addition amount 10 ⁇ 100U / g starch in gelatinized starch solution.
  • the cyclodextrin in cyclodextrin degradative enzyme solution is added in an amount 0.5 ⁇ 5U / g-cyclodextrin; maltooligosyl the trehalose synthase cyclodextrin
  • the addition amount in the solution is 10-100 U/g cyclodextrin .
  • the temperature of the reaction is 25-65°C and the pH is 5.0-8.5.
  • the gelatinization is to stir the starch solution in a boiling water bath at a speed of 100 to 200 r/min for 20 to 40 minutes.
  • the starch is corn starch, sweet potato starch, potato starch and/or wheat starch.
  • the cyclodextrin is ⁇ -cyclodextrin, ⁇ -cyclodextrin and/or ⁇ -cyclodextrin.
  • the maltodextrin is reducing maltodextrin and/or non-reducing maltodextrin.
  • the method is to first add starch to water or buffer to obtain a starch solution, then gelatinize the starch solution to obtain a gelatinized starch solution, and then transfer the cyclodextrin glucose base Enzymes, cyclodextrin degrading enzymes and malto-oligosaccharyl trehalose synthetase are added to the gelatinized starch solution to react to obtain a reaction solution containing maltodextrin, and finally non-reducing maltodextrin is obtained from the reaction solution containing maltodextrin Refined;
  • the method is to first add cyclodextrin to water or buffer to obtain a cyclodextrin solution, and then add cyclodextrin degrading enzyme and malto-oligosaccharyl trehalose synthase to the cyclodextrin to react to obtain The reaction solution of maltodextrin, and finally the non-reducing maltodextrin is obtained from the reaction solution containing maltodextrin;
  • the method is to first add starch to water or buffer to obtain a starch solution, then gelatinize the starch solution to obtain a gelatinized starch solution, and then add cyclodextrin glucosyltransferase and cyclodextrin degrading enzyme The reaction is carried out in the gelatinized starch solution to obtain a reaction solution containing maltodextrin, and finally reducing maltodextrin is obtained from the reaction solution containing maltodextrin;
  • the method is to first add cyclodextrin to water or buffer to obtain a cyclodextrin solution, then add the cyclodextrin degrading enzyme to the cyclodextrin for reaction to obtain a reaction solution containing maltodextrin, and finally Reducing maltodextrin is obtained from the reaction solution containing maltodextrin.
  • the non-reducing maltodextrin is maltotetraosyl trehalose (4-O- ⁇ -maltopentaosyl ⁇ -D-glucoside), maltopentaosyl trehalose (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside) and/or maltohexaosyl trehalose (4-O- ⁇ -maltoheptosyl ⁇ -D-glucoside).
  • the non-reducing maltodextrin is maltopentaosyl trehalose (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside).
  • the invention also provides the maltodextrin prepared by the method.
  • the maltodextrin is reducing maltodextrin and/or non-reducing maltodextrin.
  • the non-reducing maltodextrin is maltotetraosyl trehalose (4-O- ⁇ -maltopentaosyl ⁇ -D-glucoside), maltopentaosyl trehalose (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside) and/or maltohexaosyl trehalose (4-O- ⁇ -maltoheptosyl ⁇ -D-glucoside).
  • the non-reducing maltodextrin is maltopentaosyl trehalose (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside).
  • the invention also provides the application of the above method in preparing maltodextrin, food containing maltodextrin, medicine containing maltodextrin or daily chemical product containing maltodextrin.
  • the present invention also provides the application of cyclodextrin glucosyltransferase, cyclodextrin degrading enzyme and/or malto-oligosaccharyl trehalose synthase in the production of non-reducing maltodextrin.
  • amino acid sequence of the cyclodextrin glucosyltransferase is shown in SEQ ID NO:1.
  • amino acid sequence of the cyclodextrin degrading enzyme is shown in SEQ ID NO: 2.
  • the amino acid sequence of the malto-oligosaccharyl trehalose synthase is shown in SEQ ID NO: 3.
  • nucleotide sequence of the gene encoding the cyclodextrin glucosyltransferase is shown in SEQ ID NO: 4.
  • nucleotide sequence of the gene encoding the cyclodextrin degrading enzyme is shown in SEQ ID NO: 5.
  • nucleotide sequence of the gene encoding the malto-oligosaccharyl trehalose synthase is shown in SEQ ID NO: 6.
  • the non-reducing maltodextrin is maltotetraosyl trehalose (4-O- ⁇ -maltopentaosyl ⁇ -D-glucoside), maltopentaosyl trehalose (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside) and/or maltohexaosyl trehalose (4-O- ⁇ -maltoheptosyl ⁇ -D-glucoside).
  • the non-reducing maltodextrin is maltopentaosyl trehalose (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside).
  • the present invention provides a method that can be used to produce non-reducing low-polymerization maltodextrin with a uniform degree of polymerization.
  • the method of the present invention can be used to react for 2-6 hours to make the maltopentaosyl trehalose in the reaction liquid.
  • the content of (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside) is as high as 57.2-77.3%, accounting for 50-90% of the total amount of maltodextrin in the reaction solution.
  • the non-reducing low-polymerization degree maltodextrins are only maltopentaosyl trehalose (4-O- ⁇ -maltohexaosyl ⁇ -D-glucoside), and
  • the content of non-reducible low-polymerization maltodextrin can reach 50-90% of the total amount of maltodextrin. Therefore, the method of the present invention is used to prepare non-reducible maltodextrin.
  • the degree of polymerization maltodextrin only needs to be filtered and does not require redundant separation and purification steps to obtain high-purity non-reducing maltodextrin with a low degree of polymerization, and the production cost is low.
  • the present invention provides a method that can be used to produce reducing maltodextrin with a low degree of polymerization.
  • the content of reducing maltodextrin in the reaction solution can be as high as 19.1-44.9 %.
  • Figure 1 The liquid phase distribution diagram of the standard (70% acetonitrile concentration).
  • Figure 2 Liquid phase distribution diagram of products in reaction solution 4 (70% acetonitrile concentration).
  • Figure 3 Liquid phase distribution diagram of products in reaction solution 11 (70% acetonitrile concentration).
  • Figure 4 Liquid phase distribution diagram of products in reaction solution 13 (65% acetonitrile concentration).
  • Escherichia coli DH5 ⁇ and E.coli BL21(DE3) involved in the following examples were purchased from General Biotechnology Co., Ltd.; the pET-28a(+) vector involved in the following examples was purchased from Invitrogen; The corn starch involved in the above examples was purchased from Shanghai Aladdin Co., Ltd.; the ⁇ -cyclodextrin involved in the following examples was purchased from Shanghai Shenggong Co., Ltd.
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, add 100 ⁇ g/mL kanamycin before use.
  • LB solid medium tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar 15g/L, add 100 ⁇ g/mL kanamycin before use.
  • CCTase cyclodextrin glucosyltransferase
  • CDase cyclodextrin degrading enzyme
  • the crude enzyme solution of cyclodextrin degrading enzyme shown is named CDase.
  • MTSase malto-oligosaccharide-based trehalose synthase
  • the preparation method of gelatinized starch solution is as follows:
  • Corn starch was added to a sodium phosphate buffer with a concentration of 20 mM to obtain a corn starch solution with a corn starch concentration of 30 g/L; the corn starch solution was stirred in a boiling water bath at a speed of 150 r/min for 30 minutes to obtain a gelatinized starch solution.
  • ⁇ -cyclodextrin was added to a sodium phosphate buffer with a concentration of 20 mM to obtain a cyclodextrin solution with a ⁇ -cyclodextrin concentration of 20 g/L.
  • HPLC high performance liquid chromatography
  • Chromatographic column amino column (ShodexNH2P-504E);
  • Sample preparation Centrifuge the reaction solution at 12000r/min for 10 minutes, filter it with a 0.22 ⁇ m microporous membrane, and collect the filtrate for determination;
  • Sample measurement First flush the pipeline with mobile phase at a flow rate of 1 mL/min for 30 minutes, install the chromatographic column, before the formal sample injection analysis, enter the mobile phase used into the reference cell for 60 minutes, take the baseline, and wait until the baseline becomes stable.
  • the standard solution and the prepared sample were injected into 10 ⁇ L respectively; the sugar component in the sample was qualitatively determined according to the retention time of the standard, and the ratio of the sugar component was calculated by the internal standard method according to the peak area of the sample.
  • cyclodextrin glucosyltransferase enzyme activity at a temperature of 55°C and a pH of 7.0, the amount of enzyme required to produce 1 ⁇ mol cyclodextrin by acting on gelatinized starch within 1 min is an enzyme activity unit (1U) .
  • the definition of the enzyme activity of cyclodextrin degrading enzyme under the conditions of temperature of 35°C and pH of 7.5, the amount of enzyme required to produce 1 ⁇ mol of maltoheptaose by acting on ⁇ -cyclodextrin within 1 minute is an enzyme activity unit (1U) .
  • malto-oligosaccharyl trehalose synthase enzyme activity at a temperature of 45°C and a pH of 7.0, the amount of enzyme required to act on maltodextrin to convert 1 ⁇ mol ⁇ -1,4 glycosidic bond within 1 min is an enzyme activity Unit (1U).
  • DE value (reduction value) definition the ratio of the amount of reducing sugar in the system to the total amount of solids.
  • the non-reducing maltoheptaose ie maltohexaosyl trehalose, 4-O- ⁇ -maltoheptosyl ⁇ -D-glucoside
  • NDP7 reaction solution 1 to The polymerization degree distribution of maltodextrin in 5 is shown in Table 1, and the liquid phase distribution of the product in reaction solution 4 is shown in Fig. 2).
  • the polymerization degree distribution of maltodextrin in reaction solution 6-8 was detected (the polymerization degree distribution of maltodextrin in reaction solution 6-8 is shown in Table 2).
  • Solution 1 First add CGTase to the gelatinized starch solution at an amount of 7U/g starch , and react with enzymes at a temperature of 55°C and a pH of 7.0 for 3 hours, and then add CDase to the paste at an amount of 5U/g starch.
  • the enzymatic reaction was carried out at a temperature of 35° C. and a pH of 7.5 for 4 hours to obtain a reaction solution 9.
  • Program III CDase first to add an amount 1.5U / g ⁇ - cyclodextrin cyclodextrin solution was added, at a temperature of 35 °C, pH 7.5 under the conditions of the enzymatic reaction 4h, then MTSase at 15U / g ring The added amount of dextrin was added to the ⁇ -cyclodextrin solution, and the enzyme reaction was carried out at a temperature of 45° C. and a pH of 7.0 for 4 hours to obtain a reaction solution 11.
  • the polymerization degree distribution of maltodextrin in the reaction solution 9-11 was detected (the polymerization degree distribution of the maltodextrin in the reaction solution 9-11 is shown in Table 3, and the liquid phase distribution of the product in the reaction solution 11 is shown in Fig. 3).
  • MTSase is the key to the production of non-reducing low-polymerization maltodextrin. Without MTSase, non-reducing low-polymerization maltodextrin cannot be generated; and, from Table 3, it can be seen that the non-reducing low-polymerization maltodextrin is prepared by a step-by-step method. The yield and uniformity of reducing maltodextrin with low polymerization degree are far inferior to the second scheme (one-step method) in Example 2.
  • maltodextrin with a single degree of polymerization can be produced only when the double or triple enzymes are reacted in series.
  • Reaction Solution 9 9.8 38.1 14.2 / 12.8 9.5 / / / 15.6 / Reaction solution 10 14.2 2.9 10.5 / 20.2 35.4 / / / 3.8 13 Reaction solution 11 / 42.5 / / 3.9 3.0 3.9 / / 7.5 39.2
  • reaction solution 12-16 On the basis of the second solution of Example 2, the addition amount of CDase was replaced with 0.5U/g cyclodextrin , 1.5U/g cyclodextrin , 2.5U/g cyclodextrin , 3.5U/g cyclodextrin, respectively , 4.5U/g cyclodextrin to obtain reaction solution 12-16.
  • reaction time was replaced with 30 min, 1 h, 2 h, 4 h, 6 h to obtain the reaction solution 17-21.

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Abstract

一种多酶耦合生产单一聚合度麦芽糊精的方法，属于生物技术领域。一种可用于生产聚合度均一的非还原性低聚合度麦芽糊精的方法，利用上述方法反应2～6h，即可使反应液中麦芽五糖基海藻糖的含量高达57.2～77.3％，占反应液中麦芽糊精总量的50～90％。利用上述方法制备得到的麦芽糊精中，非还原性低聚合度麦芽糊精仅有麦芽五糖基海藻糖一种，且非还原性低聚合度麦芽糊精的含量可达麦芽糊精总量的50～90％，因此，利用上述方法制备非还原性低聚合度麦芽糊精仅需过滤，无需多余的分离纯化步骤，即可获得纯度很高的非还原性低聚合度麦芽糊精，生产成本较低。

Description

一种多酶耦合生产单一聚合度麦芽糊精的方法 技术领域
本发明涉及一种多酶耦合生产单一聚合度麦芽糊精的方法，属于生物技术领域。
背景技术
麦芽糊精是指以淀粉为原料，将淀粉经酸法或酶法低程度水解得到的DE值在20％以下的产品。其主要成分为聚合度(即DP值)在10以上的糊精和聚合度在10以下的低聚糖。
由于麦芽糊精可作为增稠剂、乳化剂或稳定剂等配料添加到食品中，可作为赋形剂、填充剂等配料添加到药品中，且可作为优良载体添加到日化产品中，因此，麦芽糊精在食品、医药和日化等领域均具有重要的应用，这也意味着麦芽糊精具有很大的市场。
但是，受到现有麦芽糊精制备方法的局限，目前生产得到的麦芽糊精中，高聚合度的麦芽糊精(一般为DE值在4％～12％的麦芽糊精)含有较多的直链糊精且DP值分布不均匀，添加了此类麦芽糊精的溶液放置一段时间后容易出现沉淀(具体可见参考文献：李才明,李阳,顾正彪,洪雁,程力,李兆丰.麦芽糊精的支化修饰及其特性研究进展[J].中国食品学报.2018(10):1-8)，低聚合度的麦芽糊精(一般为DE值在13％～20％的麦芽糊精)则具有很高的还原能力，易与其他物质发生反应，并且，当其与氨基酸或蛋白质共存时易产生美拉德反应，降低了含有此类麦芽糊精的产品的质量。
另外，受到现有麦芽糊精制备方法的局限，目前生产得到的麦芽糊精均是多种物质(包括葡萄糖、麦芽糖、低聚糖和多聚糖等)的混合物，这大大提高了聚合度均一的麦芽糊精产品的分离纯化难度，进而增加了聚合度均一的麦芽糊精产品的分离纯化成本。
上述缺陷均大大限制了麦芽糊精市场的进一步发展。因此，急需找到一种成本低的可用于生产聚合度均一的非还原性低聚合度麦芽糊精的方法。
发明内容
[技术问题]
本发明要解决的技术问题是提供一种成本低的可用于生产聚合度均一的非还原性低聚合度麦芽糊精的方法。
[技术方案]
为解决上述技术问题，本发明提供了一种生产麦芽糊精的方法，所述方法为先将环糊精葡萄糖基转移酶(CGTase)、环糊精降解酶(CDase)和/或麦芽寡糖基海藻糖合成酶(MTSase)加入淀粉和/或环糊精中进行反应，获得含有麦芽糊精的反应液，然后从含有麦芽糊精的反应 液中获得麦芽糊精。
在本发明的一种实施方式中，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶、环糊精降解酶和麦芽寡糖基海藻糖合成酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精；
或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶和麦芽寡糖基海藻糖合成酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精；
或者，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶和环糊精降解酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精；
或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶的氨基酸序列如SEQ ID NO:1所示。
在本发明的一种实施方式中，所述环糊精降解酶的氨基酸序列如SEQ ID NO:2所示。
在本发明的一种实施方式中，所述麦芽寡糖基海藻糖合成酶的氨基酸序列如SEQ ID NO:3所示。
在本发明的一种实施方式中，编码所述环糊精葡萄糖基转移酶的基因的核苷酸序列如SEQ ID NO:4所示。
在本发明的一种实施方式中，编码所述环糊精降解酶的基因的核苷酸序列如SEQ ID NO:5所示。
在本发明的一种实施方式中，编码所述麦芽寡糖基海藻糖合成酶的基因的核苷酸序列如SEQ ID NO:6所示。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶在糊化淀粉溶液中的添加量为6～10U/g 淀粉；所述环糊精降解酶在糊化淀粉溶液中的添加量为3～7U/g 淀粉；所述麦芽寡糖基海藻糖合成酶在糊化淀粉溶液中的添加量为10～100U/g 淀粉。
在本发明的一种实施方式中，所述环糊精降解酶在环糊精溶液中的添加量为0.5～5U/g 环 糊精；所述麦芽寡糖基海藻糖合成酶在环糊精溶液中的添加量为10～100U/g 环糊精。
在本发明的一种实施方式中，所述反应的温度为25～65℃、pH为5.0～8.5。
在本发明的一种实施方式中，所述糊化为将淀粉溶液在沸水浴中以100～200r/min的速度搅拌20～40min。
在本发明的一种实施方式中，所述淀粉为玉米淀粉、红薯淀粉、马铃薯淀粉和/或小麦淀粉。
在本发明的一种实施方式中，所述环糊精为α-环糊精、β-环糊精和/或γ-环糊精。
在本发明的一种实施方式中，所述麦芽糊精为还原性麦芽糊精和/或非还原性麦芽糊精。
在本发明的一种实施方式中，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶、环糊精降解酶和麦芽寡糖基海藻糖合成酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得非还原性麦芽糊精；
或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶和麦芽寡糖基海藻糖合成酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得非还原性麦芽糊精；
或者，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶和环糊精降解酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得还原性麦芽糊精；
或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得还原性麦芽糊精。
在本发明的一种实施方式中，所述非还原性麦芽糊精为麦芽四糖基海藻糖(4-O-α-maltopentaosylα-D-glucoside)、麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)和/或麦芽六糖基海藻糖(4-O-α-maltoheptosylα-D-glucoside)。
在本发明的一种实施方式中，所述非还原性麦芽糊精为麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)。
本发明还提供了利用方法制备得到的麦芽糊精。
在本发明的一种实施方式中，所述麦芽糊精为还原性麦芽糊精和/或非还原性麦芽糊精。
在本发明的一种实施方式中，所述非还原性麦芽糊精为麦芽四糖基海藻糖(4-O-α-maltopentaosylα-D-glucoside)、麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)和/或麦芽六糖基海藻糖(4-O-α-maltoheptosylα-D-glucoside)。
在本发明的一种实施方式中，所述非还原性麦芽糊精为麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)。
本发明还提供了上述方法在制备麦芽糊精、含有麦芽糊精的食品、含有麦芽糊精的药品或含有麦芽糊精的日化产品中的应用。
本发明还提供了环糊精葡萄糖基转移酶、环糊精降解酶和/或麦芽寡糖基海藻糖合成酶在生产非还原性麦芽糊精中的应用。
在本发明的一种实施方式中，所述环糊精葡萄糖基转移酶的氨基酸序列如SEQ ID NO:1所示。
在本发明的一种实施方式中，所述环糊精降解酶的氨基酸序列如SEQ ID NO:2所示。
在本发明的一种实施方式中，所述麦芽寡糖基海藻糖合成酶的氨基酸序列如SEQ ID NO:3所示。
在本发明的一种实施方式中，编码所述环糊精葡萄糖基转移酶的基因的核苷酸序列如SEQ ID NO:4所示。
在本发明的一种实施方式中，编码所述环糊精降解酶的基因的核苷酸序列如SEQ ID NO:5所示。
在本发明的一种实施方式中，编码所述麦芽寡糖基海藻糖合成酶的基因的核苷酸序列如SEQ ID NO:6所示。
在本发明的一种实施方式中，所述非还原性麦芽糊精为麦芽四糖基海藻糖(4-O-α-maltopentaosylα-D-glucoside)、麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)和/或麦芽六糖基海藻糖(4-O-α-maltoheptosylα-D-glucoside)。
在本发明的一种实施方式中，所述非还原性麦芽糊精为麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)。
[有益效果]
(1)本发明提供了一种可用于生产聚合度均一的非还原性低聚合度麦芽糊精的方法，利用本发明的方法反应2～6h，即可使反应液中麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)的含量高达57.2～77.3％，占反应液中麦芽糊精总量的50～90％。
(2)利用本发明的方法制备得到的麦芽糊精中，非还原性低聚合度麦芽糊精仅有麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)一种，并且，利用本发明的方法制备得到的麦芽糊精中，非还原性低聚合度麦芽糊精的含量可达麦芽糊精总量的50～90％，因此，利用本发明的方法制备非还原性低聚合度麦芽糊精仅需过滤，无需多余的分离纯化步骤，即可获 得纯度很高的非还原性低聚合度麦芽糊精，生产成本较低。
(3)本发明提供了一种可用于生产还原性低聚合度麦芽糊精的方法，利用本发明的方法反应2～6h，即可使反应液中还原性麦芽糊精的含量高达19.1～44.9％。
附图说明
图1：标准品的液相分布图(70％乙腈浓度)。
图2：反应液4中产物的液相分布图(70％乙腈浓度)。
图3：反应液11中产物的液相分布图(70％乙腈浓度)。
图4：反应液13中产物的液相分布图(65％乙腈浓度)。
具体实施方式
下述实施例中涉及的大肠杆菌(Escherichia coli)DH5α、E.coli BL21(DE3)购自通用生物技术有限公司；下述实施例中涉及的pET-28a(+)载体购自Invitrogen公司；下述实施例中涉及的玉米淀粉购自上海阿拉丁有限公司；下述实施例中涉及的β-环糊精购自上海生工有限公司。
下述实施例中涉及的培养基如下：
LB液体培养基：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L，使用前添加100μg/mL的卡那霉素。
LB固体培养基：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L、琼脂15g/L，使用前添加100μg/mL的卡那霉素。
下述实施例中涉及的制备方法如下：
环糊精葡萄糖基转移酶(CGTase)的制备方法如下：
合成编码氨基酸序列如SEQ ID NO:1所示的环糊精葡萄糖基转移酶的基因(核苷酸序列如SEQ ID NO:4所示)；使用限制性内切酶HindⅢ和Nde I对合成得到的基因与pET-28a(+)载体进行酶切后，使用T 4连接酶将得到的两个酶切产物连接，得到连接产物；将获得的连接产物于16℃下过夜连接15h后，转化至大肠杆菌DH5α感受态细胞中；将转化后的大肠杆菌DH5α感受态细胞涂布LB固体培养基(含有10μg/mL的卡那霉素)，37℃倒置培养24h；挑取阳性转化子，提取质粒，测序验证结果表明连接成功，获得重组质粒pET-28a(+)-CGTase；将获得的重组质粒pET-28a(+)-CGTase导入大肠杆菌E.coli BL21(DE3)中，获得重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-CGTase；将获得的重组大肠杆菌E.coli BL21(DE3)/pET-28a(+)-CGTase划线于LB固体培养基上，于37℃恒温培养箱中培养18h，获得单菌落；挑取单菌落分别接种于LB液体培养基中，于37℃、200rpm的摇床中培养14h，连续活化3 代，获得活化好的菌液；将活化好的菌液按1％(v/v)的接种量分别接种至LB液体培养基中，于温度为37℃、转速为200rpm的条件下培养12h，获得发酵液；将发酵液离心，获得发酵上清液，此发酵上清液即为氨基酸序列如SEQ ID NO:1所示的环糊精葡萄糖基转移酶的粗酶液，命名为CGTase。
环糊精降解酶(CDase)的制备方法如下：
合成编码氨基酸序列如SEQ ID NO:2所示的环糊精降解酶的基因(核苷酸序列如SEQ ID NO:5所示)；参照制备CGTase的方法获得氨基酸序列如SEQ ID NO:2所示的环糊精降解酶的粗酶液，命名为CDase。
麦芽寡糖基海藻糖合成酶(MTSase)的制备方法如下：
合成编码氨基酸序列如SEQ ID NO:3所示的麦芽寡糖基海藻糖合成酶的基因(核苷酸序列如SEQ ID NO:6所示)；参照制备CGTase的方法获得氨基酸序列如SEQ ID NO:3所示的麦芽寡糖基海藻糖合成酶的粗酶液，命名为MTSase。
糊化淀粉溶液的制备方法如下：
将玉米淀粉加入浓度为20mM的磷酸钠缓冲液中，得到玉米淀粉浓度为30g/L的玉米淀粉溶液；将玉米淀粉溶液在沸水浴中以150r/min的速度搅拌30min，得到糊化淀粉溶液。
环糊精溶液的制备方法如下：
将β-环糊精加入浓度为20mM的磷酸钠缓冲液中，得到β-环糊精浓度为20g/L的环糊精溶液。
下述实施例中涉及的检测方法如下：
反应液中葡萄糖、麦芽糖、麦芽三糖、麦芽四糖、麦芽五糖、麦芽六糖、麦芽七糖、α-环糊精、β-环糊精或γ-环糊精含量的检测方法：
采用高效液相色谱(HPLC)法；
色谱柱：氨基柱(ShodexNH2P-504E)；
流动相：乙腈：水＝60～70％(v/v)；
标准品：称取葡萄糖(DP1)、麦芽糖(DP2)、麦芽三糖(DP3)、麦芽四糖(DP4)、麦芽五糖(DP5)、麦芽六糖(DP6)、麦芽七糖(DP7)、α-环糊精(α-CD)、β-环糊精(β-CD)或γ-环糊精(γ-CD)(纯度＝99.5％)的标准品0.5g，精确至0.0001g，用超纯水溶解并定容至50mL，摇匀，用0.22μm微孔滤膜过滤，收集滤液供测定用(标准品的液相分布见图1)；
样品制备：将反应结束的反应液12000r/min离心10min，用0.22μm微孔滤膜过滤，收集滤液供测定用；
试样的测定：先用流动相以1mL/min的流速冲洗管路30min，装上色谱柱，正式进样分析前，将所用流动相输入参比池60min，走基线，待基线走稳后，将标准溶液和制备好的试样分别进样10μL；根据标准品的保留时间定性样品中的糖组分，根据样品的峰面积，以内标法计算糖组分的比例。
环糊精葡萄糖基转移酶酶活的检测方法：
在250μL浓度为30g/L的糊化淀粉溶液中加入10μL CGTase，然后用20mM磷酸缓冲液补足至500μL，得到反应体系；将反应体系在温度为55℃、pH为7.0的条件下反应10min后煮沸灭酶后，通过液相检测环糊精的生成量，得到CGTase的环糊精葡萄糖基转移酶酶活。
环糊精葡萄糖基转移酶酶活的定义：在温度为55℃、pH为7.0的条件下，1min内作用于糊化淀粉生成1μmol环糊精所需的酶量为一个酶活力单位(1U)。
环糊精降解酶酶活的检测方法：
在250μL浓度为20g/L的β-环糊精溶液中加入10μL CDase，然后用20mM磷酸缓冲液补足至500μL，得到反应体系；将反应体系在温度为35℃、pH为7.5的条件下反应30min后煮沸灭酶后，通过液相检测麦芽七糖的生成量，得到CDase的环糊精葡萄糖基转移酶酶活。
环糊精降解酶酶活的定义：在温度为35℃、pH为7.5的条件下，1min内作用于β-环糊精生成1μmol麦芽七糖所需的酶量为一个酶活力单位(1U)。
麦芽寡糖基海藻糖合成酶酶活的检测方法：
在250μL浓度为20g/L的麦芽糊精溶液中加入5μL MTSase，然后用20mM磷酸缓冲液补足至500μL，得到反应体系；将反应体系在温度为45℃、pH为7.0的条件下反应10min后煮沸灭酶后，通过DNS法检测反应液中还原力的下降量，得到MTSase的麦芽寡糖基海藻糖合成酶酶活。
麦芽寡糖基海藻糖合成酶酶活的定义：在温度为45℃、pH为7.0的条件下，1min内作用于麦芽糊精转化1μmolα-1,4糖苷键所需的酶量为一个酶活力单位(1U)。
反应液DE值(还原值)的检测方法(DNS比色法)：
具体可见参考文献：彦繁鹤,周金梅,吴如春.DNS法测定甘蔗渣中还原糖含量[J].食品研究与开发.36(02):126-128。
DE值(还原值)的定义：体系中还原糖的量占固形物总量的比值。
实施例1：麦芽糊精的制备(单酶)
具体步骤如下：
方案一：将CGTase以7U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为55℃、pH为7.0 的条件下酶反应3h，获得反应液1。
方案二：将CDase以5U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为35℃、pH为7.5的条件下酶反应4h，获得反应液2。
方案三：将MTSase以45U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为45℃、pH为7.0的条件下酶反应4h，获得酶反应3。
方案四：将CDase以1.5U/g 环糊精的添加量加入β-环糊精溶液中，于温度为35℃、pH为7.5的条件下酶反应4h，获得反应液4。
方案五：将MTSase以15U/g 环糊精的添加量加入β-环糊精溶液中，于温度为45℃、pH为7.0的条件下酶反应4h，获得酶反应5。
检测反应液1～5中麦芽糊精的聚合度分布，非还原性麦芽七糖(即麦芽六糖基海藻糖，4-O-α-maltoheptosylα-D-glucoside)用NDP7表示(反应液1～5中麦芽糊精的聚合度分布见表1，反应液4中产物的液相分布见图2)。
由表1可知，CGTase、CDase和MTSase无法单独作用于淀粉生成聚合度均一的非还原性低聚合度麦芽糊精，CDase和MTSase也无法单独作用于环糊精生成聚合度均一的非还原性低聚合度麦芽糊精，其中，由CDase反应获得的反应液中还原性低聚合度麦芽糊精的含量最高且聚合度最均一。
表1反应液1～5中麦芽糊精的聚合度分布
Figure PCTCN2020097622-appb-000001
实施例2：麦芽糊精的制备(酶耦合+一步法)
具体步骤如下：
方案一：将CGTase、CDase分别以7U/g 淀粉和5U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为40℃、pH为7.0的条件下酶反应4h，获得反应液6。
方案二：将CDase、MTSase分别以1.5U/g 环糊精和15U/g 环糊精的添加量加入β-环糊精溶液中，于温度为40℃、pH为7.0的条件下酶反应4h，获得反应液7。
方案三：将CGTase、CDase、MTSase分别以7U/g 淀粉、5U/g 淀粉和45U/g 淀粉的添加量加 入糊化淀粉溶液中，于温度为40℃、pH为7.0的条件下酶反应4h，获得反应液8。
检测反应液6～8中麦芽糊精的聚合度分布(反应液6～8中麦芽糊精的聚合度分布见表2)。
由表2可知，MTSase是生成非还原性低聚合度麦芽糊精的关键，若无MTSase，则无法生成非还原性低聚合度麦芽糊精；并且，由表2可知，以环糊精为底物，利用CDase和MTSase一步法生成非还原性低聚合度麦芽糊精的产量和均一性都很高。
表2反应液6～8中麦芽糊精的聚合度分布
Figure PCTCN2020097622-appb-000002
实施例3：麦芽糊精的制备(酶耦合+分步法)
具体步骤如下：
方案一：先将CGTase以7U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为55℃、pH为7.0的条件下酶反应3h，然后将CDase以5U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为35℃、pH为7.5的条件下酶反应4h，获得反应液9。
方案二：先将CGTase以7U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为55℃、pH为7.0的条件下酶反应3h，然后将CDase以5U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为35℃、pH为7.5的条件下酶反应4h，最后将MTSase以45U/g 淀粉的添加量加入糊化淀粉溶液中，于温度为45℃、pH为7.0的条件下酶反应4h，获得反应液10。
方案三：先将CDase以1.5U/g 环糊精的添加量加入β-环糊精溶液中，于温度为35℃、pH为7.5的条件下酶反应4h，然后将MTSase以15U/g 环糊精的添加量加入β-环糊精溶液中，于温度为45℃、pH为7.0的条件下酶反应4h，获得反应液11。
检测反应液9～11中麦芽糊精的聚合度分布(反应液9～11中麦芽糊精的聚合度分布见表3，反应液11中产物的液相分布见图3)。
由表3可知，MTSase是生成非还原性低聚合度麦芽糊精的关键，若无MTSase，则无法生成非还原性低聚合度麦芽糊精；并且，由表3可知，利用分步法制备非还原性低聚合度麦芽糊精的产量和均一性远不如实施例2中的方案二(一步法)。
以淀粉为底物时，只有当双酶或三酶串联反应时才能生成单一聚合度的麦芽糊精。
表3检测反应液9～11中麦芽糊精的聚合度分布
  α-环糊精 β-环糊精 γ-环糊精 DP1 DP2 DP3 DP4 DP5 DP6 DP7 NDP7
   (％) (％) (％) (％) (％) (％) (％) (％) (％) (％) (％)
反应液9 9.8 38.1 14.2 / 12.8 9.5 / / / 15.6 /
反应液10 14.2 2.9 10.5 / 20.2 35.4 / / / 3.8 13
反应液11 / 42.5 / / 3.9 3.0 3.9 / / 7.5 39.2
实施例4：酶添加量对麦芽糊精产率的影响
在实施例2的方案二的基础上，将CDase的添加量分别替换为0.5U/g 环糊精、1.5U/g 环糊精、2.5U/g 环糊精、3.5U/g 环糊精、4.5U/g 环糊精，获得反应液12～16。
检测反应液12～16中麦芽糊精的聚合度分布(检测结果见表4，反应液13中产物的液相分布见图4)。
由表4可知，当CDase的添加量为1.5U/g 环糊精的添加量时，反应液中低聚合度非还原性麦芽糊精的含量最高且聚合度最均一。
表4检测反应液12～16中麦芽糊精的聚合度分布
Figure PCTCN2020097622-appb-000003
实施例5：酶反应时间对麦芽糊精产率的影响
在实施例2的方案二的基础上，将反应时间分别替换为30min，1h，2h，4h，6h获得反应液17～21。
检测反应液17～21中麦芽糊精的聚合度分布(检测结果见表5)。
由表5可知，当反应进行到4h时，反应液中低聚合度非还原性麦芽糊精的含量最高且聚合度最均一。
表5检测反应液17～21中麦芽糊精的聚合度分布
Figure PCTCN2020097622-appb-000004
Figure PCTCN2020097622-appb-000005
实施例6：非还原性麦芽七糖DE值的测定
取实施例2中的反应液7，将反应液7通过半制备液相得到纯度92％(v/v)的NDP7，通过DNS比色法测定NDP7的DE值，测定结果见表6。
由表6可知，NDP7的DE值很低，仅有3％，说明NDP7确为非还原性的糖类。
表6 NDP7DE值的测定
糖制剂 纯度(％) DE值(％)
NDP7 92 3
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (20)

  1. 一种生产麦芽糊精的方法，其特征在于，所述方法为先将环糊精葡萄糖基转移酶、环糊精降解酶和/或麦芽寡糖基海藻糖合成酶加入淀粉和/或环糊精中进行反应，获得含有麦芽糊精的反应液，然后从含有麦芽糊精的反应液中获得麦芽糊精。
  2. 如权利要求1所述的一种生产麦芽糊精的方法，其特征在于，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶、环糊精降解酶和麦芽寡糖基海藻糖合成酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精；
    或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶和麦芽寡糖基海藻糖合成酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精；
    或者，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶和环糊精降解酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精；
    或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得麦芽糊精。
  3. 如权利要求1或2所述的一种生产麦芽糊精的方法，其特征在于，所述环糊精葡萄糖基转移酶的氨基酸序列如SEQ ID NO:1所示。
  4. 如权利要求1-3任一所述的一种生产麦芽糊精的方法，其特征在于，所述环糊精降解酶的氨基酸序列如SEQ ID NO:2所示。
  5. 如权利要求1-4任一所述的一种生产麦芽糊精的方法，其特征在于，所述麦芽寡糖基海藻糖合成酶的氨基酸序列如SEQ ID NO:3所示。
  6. 如权利要求2-5任一所述的一种生产麦芽糊精的方法，其特征在于，所述环糊精葡萄糖基转移酶在糊化淀粉溶液中的添加量为6～10U/g 淀粉；所述环糊精降解酶在糊化淀粉溶液中的添加量为3～7U/g 淀粉；所述麦芽寡糖基海藻糖合成酶在糊化淀粉溶液中的添加量为10～100U/g 淀粉。
  7. 如权利要求2-6任一所述的一种生产麦芽糊精的方法，其特征在于，所述环糊精降解酶在环糊精溶液中的添加量为0.5～5U/g 环糊精；所述麦芽寡糖基海藻糖合成酶在环糊精溶液中的添加量为10～100U/g 环糊精。
  8. 如权利要求1-7任一所述的一种生产麦芽糊精的方法，其特征在于，所述反应的温度为25～65℃、pH为5.0～8.5。
  9. 如权利要求2-8任一所述的一种生产麦芽糊精的方法，其特征在于，所述麦芽糊精含有还原性和/或非还原性麦芽糊精。
  10. 如权利要求2-9任一所述的一种生产麦芽糊精的方法，其特征在于，所述非还原性麦芽糊精为麦芽四糖基海藻糖(4-O-α-maltopentaosylα-D-glucoside)、麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)和/或麦芽六糖基海藻糖(4-O-α-maltoheptosylα-D-glucoside)。
  11. 如权利要求2-10任一所述的一种生产麦芽糊精的方法，其特征在于，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶、环糊精降解酶和麦芽寡糖基海藻糖合成酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得非还原性麦芽糊精；
    或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶和麦芽寡糖基海藻糖合成酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得非还原性麦芽糊精；
    或者，所述方法为先将淀粉加入水或缓冲液中，得到淀粉溶液，然后将淀粉溶液进行糊化，得到糊化淀粉溶液，再将环糊精葡萄糖基转移酶和环糊精降解酶加入糊化淀粉溶液中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得还原性麦芽糊精；
    或者，所述方法为先将环糊精加入水或缓冲液中，得到环糊精溶液，然后将环糊精降解酶加入环糊精中进行反应，获得含有麦芽糊精的反应液，最后从含有麦芽糊精的反应液中获得还原性麦芽糊精。
  12. 利用权利要求1-11任一所述的方法制备得到的麦芽糊精。
  13. 如权利要求12所述的麦芽糊精，其特征在于，所述麦芽糊精为还原性麦芽糊精和/或非还原性麦芽糊精。
  14. 如权利要求12或13所述的麦芽糊精，其特征在于，所述非还原性麦芽糊精为麦芽四糖基海藻糖(4-O-α-maltopentaosylα-D-glucoside)、麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)和/或麦芽六糖基海藻糖(4-O-α-maltoheptosylα-D-glucoside)。
  15. 权利要求1-11任一所述的方法在制备麦芽糊精、含有麦芽糊精的食品、含有麦芽糊精的药品或含有麦芽糊精的日化产品中的应用。
  16. 环糊精葡萄糖基转移酶、环糊精降解酶和/或麦芽寡糖基海藻糖合成酶在生产非还原性麦芽糊精中的应用。
  17. 如权利要求16所述的应用，其特征在于，所述环糊精葡萄糖基转移酶的氨基酸序列 如SEQ ID NO:1所示。
  18. 如权利要求16或17所述的应用，其特征在于，所述环糊精降解酶的氨基酸序列如SEQ ID NO:2所示。
  19. 如权利要求16-18任一所述的应用，其特征在于，所述麦芽寡糖基海藻糖合成酶的氨基酸序列如SEQ ID NO:3所示。
  20. 如权利要求16-19任一所述的应用，其特征在于，所述非还原性麦芽糊精为麦芽四糖基海藻糖(4-O-α-maltopentaosylα-D-glucoside)、麦芽五糖基海藻糖(4-O-α-maltohexaosylα-D-glucoside)和/或麦芽六糖基海藻糖(4-O-α-maltoheptosylα-D-glucoside)。
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CN111304270B (zh) * 2020-02-24 2022-03-18 江南大学 一种多酶耦合生产单一聚合度麦芽糊精的方法
CN112708649B (zh) * 2021-02-06 2022-09-06 江南大学 一种多酶耦合生产低聚异麦芽糖的方法
CN113061632B (zh) * 2021-03-05 2023-04-28 江南大学 一种生产混合型非还原性麦芽糊精的方法
CN113475577B (zh) * 2021-07-08 2022-07-05 江南大学 一种以麦芽五糖基海藻糖代替麦芽糊精的奶粉及制备方法
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