Method for preparing alpha-cyclodextrin by using amylase

Abstract

The invention discloses a method for preparing alpha-cyclodextrin by using amylase, and belongs to the technical field of cyclodextrin production. The method comprises the following steps: by taking starch or dextrin as a substrate, mixing and gelatinizing, adding CGT enzyme, reacting under proper conditions, deactivating the enzyme after the reaction is finished, adding amylase into the obtained reaction liquid, and purifying alpha-cyclodextrin. According to the method disclosed by the invention, beta-cyclodextrin and gamma-cyclodextrin are hydrolyzed by utilizing amylase with selective hydrolysis capability on cyclodextrin, and the hydrolysis capability on alpha-cyclodextrin is weak, so that the alpha-cyclodextrin is purified. The method provided by the invention breaks through a conventional industrial method which mostly adopts an organic solvent for precipitation, and adopts an enzymatic method for purifying alpha-cyclodextrin, and the method is simple to operate, mild in condition, green and environment-friendly, and has a good application prospect in the field of cyclodextrin production.

Description

TECHNICAL FIELD

[0001] The application relates to a method for preparing alpha-cyclodextrin by using amylase and belongs to the technical field of cyclodextrin production. BACKGROUND

[0002] Cyclodextrin is a cyclic oligosaccharide formed by D-glucopyranose connected by 1,4 glycosidic bonds through the action of cyclodextrin glucosyltransferase (CGTase) on starch or dextrin. Common cyclodextrins are alpha-, beta- and gamma-cyclodextrins formed by 6, 7 and 8 glucose units, and the three cyclodextrins are also the most widely used cyclodextrins in the industry. Due to the slightly conical hollow cylindrical structure, cyclodextrin has the characteristics of external hydrophilicity and internal hydrophobicity, so it can form inclusion compounds with many hydrophobic guest molecules, thereby changing the physicochemical properties of the guest molecules, such as solubility, volatility and stability.

[0003] With the deepening of the research on the structure and properties of cyclodextrin, cyclodextrin has been increasingly widely used in food, medicine, chemical industry and analytical chemistry as a quality improver, stabilizer, molecular carrier and adsorbent. In the food industry, cyclodextrin can stabilize active ingredients in food through inclusion, remove certain odors in food, form stable emulsions in some foods, increase the foaming power of food, and be used as food production aids. Alpha-cyclodextrin has not only high water solubility but also cannot be digested and absorbed in the small intestine, and can significantly promote the growth of lactic acid bacteria and bifidobacteria in the large intestine, and has become one of the preferred dietary fibers in food. In the pharmaceutical industry, cyclodextrin is a new type of drug carrier, and its hollow cylindrical structure can completely or partially include drug molecules to form non-bonding complexes, and its dosage form is similar to microcapsules, which can improve the solubility and stability of drugs, improve the bioavailability of drugs, reduce irritation, toxicity and side effects, mask bitterness, and powderize volatile liquids and solids, oil-like liquids. Although cyclodextrin has been widely used in food, medicine and other industries, the current production cost of alpha-cyclodextrin is high, and the price is expensive.

[0004] Enzymatic production of cyclodextrin:

[0005] A method for producing alpha-cyclodextrin is disclosed in the Chinese patent application for invention with the publication number CN105177082A. The method uses potato starch as raw material to prepare a 10-15% starch milk, gelatinizes the starch milk, and then produces alpha-cyclodextrin through the enzymatic hydrolysis of high-specificity alpha-CGTase under the promotion of n-decanol, the recovery of n-decanol by water vapor distillation, concentration, crystallization and spray drying. The cyclodextrin conversion rate in the enzymatic hydrolysis stage of the method for producing alpha-cyclodextrin is 50-55%, and the alpha-cyclodextrin accounts for 80-87% of the total cyclodextrin.

[0006] A method for producing alpha-cyclodextrin is disclosed in the text of Chinese invention patent application with publication number CN101712972A, which uses alpha-cyclodextrin glucosyltransferase (alpha-CGTase) derived from Bacillus deramificans to produce alpha-CGT; the product produced by this process contains 80% alpha-CGT;

[0007] In the above method for producing alpha-cyclodextrin, alpha-CGTase is used, and the CGTase reaction product is a mixture of various cyclodextrins. At present, the separation and purification of different CDs are mainly solved by "solvent method" in industry, that is, adding an organic solvent as a complexing agent to selectively separate the target cyclodextrin. For example, the use of 1-butanol or n-decanol can precipitate alpha-CD. However, this solvent method is also relatively expensive, flammable and toxic. Moreover, the high boiling point of the solvent is not conducive to recovery. In addition, the proportion of alpha-CGT in the product after enzymatic hydrolysis is about 80% to 90%, and the yield is not high.

[0008] A method for producing alpha-cyclodextrin and its application are disclosed in the text of Chinese invention patent application with publication number CN115873913A; the method includes the following steps: starch is used as raw material to prepare starch milk, CaCl2 and high-temperature-resistant CGTase are added, and stirring is performed at 90-94℃ for 20-25 minutes to complete gelatinization and liquefaction; the feed liquid is cooled to 40-45℃, the pH value is adjusted, CGTase is added and reacted for 2-3 hours, then ethanol is added, and after the reaction is completed, ethanol is removed by nanofiltration membrane filtration; the feed liquid is concentrated and enters the chromatographic separation equipment for separation and purification; after separation, the component feed liquid with high alpha-cyclodextrin concentration is evaporated and concentrated, then alpha-cyclodextrin crystals are obtained by crystallization method; after centrifugation, the white powder of alpha-cyclodextrin is obtained by drying; although ethanol is used as a complexing agent, ethanol is removed by membrane separation, and cyclodextrin products are obtained by adsorption decolorization, ion exchange and chromatographic separation, but the process is complicated and not suitable for industrial production, and the proportion of alpha-CGT in the product after enzymatic reaction is not given, but from the text: after separation, the component feed liquid with high alpha-cyclodextrin concentration is evaporated and concentrated, it can be known that other cyclodextrins exist after enzymatic reaction, therefore, the yield of this method is not high;

[0009] And recorded in the text of the Chinese invention patent application with publication number CN103981238A discloses a preparation method of injection grade alpha-cyclodextrin, using epoxy resin as carrier to prepare alpha-CGTase immobilized enzyme, starch is made into starch milk, alpha-CGTase immobilized enzyme is added to carry out starch liquefaction, and then it is transferred into a packed reactor filled with alpha-CGTase immobilized enzyme for reaction, the conversion liquid is collected by ultrafiltration, the permeate is treated by alpha-amylase and saccharifying enzyme, decolorized by activated carbon, concentrated by reverse osmosis membrane, recrystallized, filtered to remove beta-cyclodextrin, the mother liquor is further concentrated by reverse osmosis membrane, crystallized, filtered and dried and ground to obtain injection grade alpha-cyclodextrin; Although it is a solvent-free production process, alpha-cyclodextrin product is obtained by repeated membrane separation and crystallization, but the process is complicated, which is not suitable for industrial production, and beta-cyclodextrin needs to be removed after reaction, so the yield of alpha-cyclodextrin prepared by this method is not high.

[0010] And recorded in the text of the Chinese invention patent application with publication number CN115873913A discloses a production method of alpha-cyclodextrin and its application; comprising the following steps: starch is used as raw material to prepare starch milk, CaCl2 and high-temperature resistant CGTase are added and stirred at 90-94℃ for 20-25 minutes to complete gelatinization and liquefaction; the feed liquid is cooled to 40-45℃, the pH value is adjusted, CGTase is added and reacted for 2-3 hours, then ethanol is added, and after the reaction is completed, the ethanol is removed by nanofiltration membrane filtration; the conductivity of the feed liquid is reduced by decolorization filtration and ion exchange; the concentrated feed liquid is separated and purified by chromatographic separation equipment; after separation, the alpha-cyclodextrin component feed liquid with high concentration is evaporated and concentrated, and then alpha-cyclodextrin crystals are obtained by crystallization method; after centrifugation, the white powder of alpha-cyclodextrin is obtained. Although there is no organic solvent residue problem in the product, the process is complicated and not suitable for industrial production; and as described in the text, after separation, the alpha-cyclodextrin component feed liquid with high concentration is evaporated and concentrated, so there are other cyclodextrins after enzymatic reaction, therefore, the yield of this method is not high;

[0011] In view of the above problems, it is very important to find a green and efficient method to prepare alpha-CD with only alpha-CD in the product after enzymolysis. SUMMARY

[0012] In view of the problems existing in the prior art, the present application provides a method for purifying alpha-cyclodextrin by using amylase, which removes beta-cyclodextrin and gamma-cyclodextrin by the selective degradation ability of amylase to different cyclodextrins, and the method is simple in operation, mild in conditions, green and environmentally friendly, and can purify alpha-cyclodextrin without adding complexing agents.

[0013] To solve the above problems, the technical scheme adopted by the present application is as follows:

[0014] The present application also provides a method for preparing alpha-cyclodextrin by using amylase, which comprises adding alpha-amylase from Aspergillus niger to a reaction solution containing multiple cyclodextrins as a substrate, and obtaining alpha-cyclodextrin after hydrolysis.

[0015] In an embodiment of the present application, the amino acid sequence of the alpha-amylase is shown in SEQ ID NO. 1.

[0016] In an embodiment of the present application, the reaction solution containing multiple cyclodextrins is a reaction solution containing alpha-cyclodextrin and one or both of beta-cyclodextrin and gamma-cyclodextrin.

[0017] In an embodiment of the present application, the hydrolysis is performed under the conditions of a temperature of 55-65℃ and a pH of 5.0-7.0, and the time is 0.5h-2h.

[0018] In an embodiment of the present application, the temperature is 55-56℃, 56-57℃, 57-58℃, 58-59℃, 59-60℃, 60-61℃, 61-62℃, 62-63℃, 63-64℃, or 64-65℃.

[0019] In an embodiment of the present application, the time is 0.5-0.6h, 0.6-0.7h, 0.7-0.8h, 0.8-0.9h, 0.9-1.0h, 1.0-1.1h, 1.1-1.2h, 1.2-1.3h, 1.3-1.4h, 1.4-1.5h, 1.5h-1.6h, 1.6h-1.7h, 1.7h-1.8h, 1.8h-1.9h, or 1.9h-2.0h.

[0020] In an embodiment of the present application, the reaction solution containing multiple cyclodextrins is a mixture obtained by adding cyclodextrin glucosyltransferase to starch or dextrin as a substrate, performing gelatinization and liquefaction, and then performing cyclization reaction by adding cyclodextrin glucosyltransferase.

[0021] In an embodiment of the present application, the alpha-amylase is added in an amount of 10U / g starch / dextrin-1000U / g starch / dextrin.

[0022] In an embodiment of the present application, the substrate is one or more of corn starch, cassava starch, potato starch, wheat starch, and malt dextrin.

[0023] In an embodiment of the present application, after the slurry is prepared, the mass fraction of the starch or dextrin is 5-25% (w / w).

[0024] In one embodiment of the present application, the conditions for gelatinization and liquefaction are as follows: 2-10 U / g (dry starch basis) of cyclodextrin glucanotransferase is added, the temperature is 80-90°C, and the gelatinization time is 20-30 min.

[0025] In one embodiment of the present application, the conditions for cyclization are as follows: 2-10 U / g (dry starch basis) of cyclodextrin glucanotransferase is added, the temperature is 40-50°C, and the reaction time is 5-48 h.

[0026] The present application also provides a method for purifying α-cyclodextrin using amylase, which comprises adding α-amylase from Aspergillus niger to a reaction solution containing multiple cyclodextrins, and obtaining α-cyclodextrin after reaction.

[0027] Preferably, the amino acid sequence of the α-amylase is shown in SEQ ID NO. 1.

[0028] SEQ ID NO. 1:

[0029] MVAWWSLFLYGLQVAAPALAATPADWRSQSIYFLLTDRFARTDGSTTATCNTADQKYCGGTWQGIIDKLDYIQGMGFTAIWITPVTAQLPQTTAYGDAYHGYWQQDIYSLNENYGTADDLKALSSALHERGMYLMVDVVANHMGYDGAGSSVDYSVFKPFSSQDYFHPFCFIQNYEDQTQVEDCWLGDNTVSLPDLDTTKDVVKNEWYDWVGSLVSNYSIDGLRIDTVKHVQKDFWPGYNKAAGVYCIGEVLDGDPAYTCPYQNVMDGVLNYPIYYPLLNAFKSTSGSMDDLYNMINTVKSDCPDSTLLGTFVENHDNPRFASYTNDIALAKNVAAFIILNDGIPIIYAGQEQHYAGGNDPANREATWLSGYPTDSELYKLIASANAIRNYAISKDTGFVTYKNWPIYKDDTTIAMRKGTDGSQIVTILSNKGASGDSYTLSLSGAGYTAGQQLTEVIGCTTVTVGSDGNVPVPMAGGLPRVLYPTEKLAGSKICSSS

[0030] Preferably, the reaction solution containing multiple cyclodextrins is a reaction solution containing α-cyclodextrin and one or both of β-cyclodextrin and γ-cyclodextrin.

[0031] Preferably, the reaction conditions are as follows: temperature, 55-65°C; pH, 5.0-7.0; and time, 0.5h-2h.

[0032] Preferably, the amount of the added α-amylase is 10U / g starch / dextrin-1000U / g starch / dextrin.

[0033] In one embodiment of the present application, the method comprises:

[0034] (1) adding starch into a stirring tank to prepare starch milk, and adding CGTase to react under suitable conditions to obtain starch liquefaction liquid;

[0035] (2) adding CGTase after the starch liquefaction and cooling, and reacting under suitable conditions to obtain reaction liquid containing different cyclodextrins;

[0036] (3) adding amylase into the cyclodextrin aqueous solution to hydrolyze β-cyclodextrin, γ-cyclodextrin, and unreacted starch and dextrin.

[0037] Preferably, the substrate is one or more of corn starch, cassava starch, potato starch, wheat starch, and malt dextrin.

[0038] Preferably, after the slurry preparation, the mass fraction of the starch or dextrin is 5-25%(w / w).

[0039] Preferably, the gelatinization and liquefaction conditions are as follows: adding 2-10U / g (dry starch basis) of cyclodextrin glucosyltransferase, 80-90°C, and gelatinizing for 20-30min.

[0040] Preferably, the cyclization reaction conditions are as follows: adding 2-10U / g (dry starch basis) of cyclodextrin glucosyltransferase, 40-50°C, and reacting for 5-48h.

[0041] Beneficial effects

[0042] (1) The present application utilizes a starch amylase with selective degradation ability for cyclodextrin to purify α-CD, utilizes its high degradation ability for β-CD and γ-CD, and weak degradation ability for α-CD, selectively hydrolyzes β-CD and γ-CD into glucose and oligosaccharides, and improves the purity of α-CD. The present application breaks through the previous industrial method of using organic solvent precipitation, adopts the method of enzyme purification of α-CD, is simple to operate, green and environmentally friendly, and has good application potential in the field of cyclodextrin processing.

[0043] (2) The cyclodextrin solution obtained by hydrolysis of starch contains not only α-CD, β-CD and γ-CD, but also other macro-molecular substances such as large ring cyclodextrin, unreacted starch and dextrin; although it has been reported that other enzymes (such as CGTase after structural modification) can selectively degrade β-CD and γ-CD while retaining α-CD, but it cannot degrade other macro-molecules; thus, more process steps are required for further purification of α-CD, such as additional addition of amylase to degrade other macro-molecules; that is, the process is more complicated;

[0044] The amylase of the present application can not only be used to prepare α-CD from dextrin mixture, but also can directly degrade other macro-molecules while degrading β-CD and γ-CD in the cyclodextrin solution obtained by hydrolysis of starch as raw material, thus making the further separation and purification of α-CD simpler;

[0045] (3) The present application can completely degrade β-CD and γ-CD to prepare α-CD within 1 hour, thus the method of the present application has great application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Ion chromatogram of the product of Example 3 of the present application after adding amylase for 5 min.

[0047] Figure 2 Ion chromatogram of the product of Example 3 of the present application after adding amylase for 30 min.

[0048] Figure 3 Ion chromatogram of the product of Example 3 of the present application after adding amylase for 60 min.

[0049] Figure 4 Ion chromatogram of the product of Comparative Example 1 of the present application.

[0050] Figure 5 Ion chromatogram of the product of Comparative Example 2 of the present application.

[0051] Figure 6 Ion chromatogram of three cyclodextrin standards. DETAILED DESCRIPTION

[0052] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not limiting to the present application.

[0053] Technical terms:

[0054] α-cyclodextrin

[0055] α-cyclodextrin (α-CD) is a cyclic oligosaccharide formed by 6 D-glucopyranose units linked by α-1, 4-glycosidic bonds, with a truncated cone or barrel shape structure, and a molecular formula of C 36 H 60 O 30 , and a molecular weight of about 972.84.

[0056] α-cyclodextrin has good water solubility. Although the solubility of α-cyclodextrin at 25°C is lower than that of γ-cyclodextrin, it is 8 times that of β-cyclodextrin. Compared with β- and γ-cyclodextrin, α-cyclodextrin is more stable in acidic solution. α-cyclodextrin does not have a non-reducing end and cannot be hydrolyzed by common exo-type amylases such as α-glucosidase and glucoamylase, but can be hydrolyzed by α-amylase derived from fungi or bacteria. Because α-cyclodextrin cannot be hydrolyzed by saliva and pancreatic amylase, it is not easily degraded and absorbed after entering the human intestinal tract, and can be quickly excreted from the body. Since α-cyclodextrin molecules contain some free hydroxyl groups, they can be combined with other functional groups by biological or chemical methods to form various cyclodextrin derivatives.

[0057] α-cyclodextrin (6 glucose units): cavity inner diameter about 0.45-0.6 nm, good water solubility.

[0058] β-cyclodextrin

[0059] β-cyclodextrin (β-CD) is a cyclic oligosaccharide formed by 7 α-D-glucopyranose units connected end to end by α-1, 4-glycosidic bonds, with a molecular formula of C 42 H 70 O 35 , and a molecular weight of about 1134.98. Its overall shape is a truncated cone (torus) with a typical cavity structure of "outer hydrophilic and inner hydrophobic", and is the most widely used, largest produced and lowest priced member of the cyclodextrin family.

[0060] β-cyclodextrin (7 glucose units): cavity inner diameter about 0.7-0.8 nm, most widely used but lower water solubility.

[0061] γ-cyclodextrin

[0062] γ-cyclodextrin (γ-CD) is a cyclic oligosaccharide formed by 8 α-D-glucopyranose units connected end to end by α-1, 4-glycosidic bonds, with a molecular formula of C 48 H 80 O 40 , a molecular weight of about 1297.12, and a CAS number of 17465-86-0.

[0063] γ-cyclodextrin (8 glucose units): cavity inner diameter about 0.85-1.0 nm, highest water solubility.

[0064] Alpha-amylase

[0065] Alpha-amylase (EC 3.2.1.1) is one of the most important hydrolytic enzymes in industry, medicine and scientific research, which is responsible for randomly cutting the internal α-1,4-glucosidic bond of starch, glycogen and other polysaccharides, and belongs to the glycoside hydrolase 13 family (GH13).

[0066] The role of alpha-amylase in cyclodextrin preparation is "liquefaction first and then length control", that is, through random endo-α-1,4-glucosidic bond, the high-viscosity natural starch is quickly degraded into linear oligosaccharide of 6-8 glucose units, which provides ideal substrate for subsequent cyclization reaction.

[0067] In the present application, alpha-amylase is used for product purification.

[0068] Liquefaction stage (alpha-amylase dominant):

[0069] The substrate is natural starch (containing linear / branched) such as corn, cassava, etc.; the purpose is to rapidly reduce the molecular weight and viscosity under high temperature (90-100℃, pH 6-6.5, Ca 2+ ) exists) to generate a dextrin mixture with DE 8-12; as a result, long chains are broken, exposing a large number of non-reducing ends, which facilitates subsequent enzyme action.

[0070] In the present application, cyclodextrin glucanotransferase is used for liquefaction reaction.

[0071] Cyclization stage:

[0072] CGTase (cyclodextrin glucanotransferase) needs relatively short and uniform oligosaccharide chains to efficiently perform transglycosylation-cyclization reaction to generate α-, β-, γ-cyclodextrin with a length of 6, 7, 8 glucose units. If the substrate chain is too long or the viscosity is too high, the diffusion and cyclization efficiency of CGTase will decrease sharply; the pre-stage "trimming" of alpha-amylase is just to eliminate this bottleneck.

[0073] Sequence identity:

[0074] The degree of association between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0075] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) version 5.0.0 or later (preferably 5.0.0 or later), with the parameters set to a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent sequence identity, and is calculated as follows:

[0076] (identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0077] Alternatively, the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent sequence identity, and is calculated as follows:

[0078] (identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment).

[0079] Case 1 provided by the present application:

[0080] A method for preparing α-cyclodextrin by using amylase, which is to take the reaction solution containing multiple cyclodextrins as the substrate, add α-amylase from Aspergillus niger, and obtain α-cyclodextrin after hydrolysis.

[0081] The detection method of the enzyme activity of α-amylase is as follows:

[0082] 1 mL of 1% soluble starch solution and 0.9 mL of 20 mM, pH 6.0 phosphate buffer were thoroughly mixed, preheated at 70°C for 10 min, 0.1 mL of crude enzyme solution was added, mixed well, 3 mL of DNS was added after 5 min of reaction, shaken, boiled for 7 min, quickly cooled, and distilled water was added to 15 mL, and the absorbance was measured at 540 nm (the same operation was carried out with inactivated enzyme solution as catalyst as blank).

[0083] Enzyme activity unit definition: under the above conditions, the amount of enzyme required to catalyze the production of 1 μmol of glucose per minute is defined as one starch hydrolysis activity unit.

[0084] Case 2 provided by the present application:

[0085] According to the method of case 1, the amino acid sequence of the alpha-amylase is shown in SEQ ID NO. 1 or the alpha-amylase having at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO. 1.

[0086] Case 3 provided by the present application:

[0087] According to the method of case 1 or 2, the reaction solution containing a plurality of cyclodextrins is a reaction solution containing alpha-cyclodextrin, and containing one or both of beta-cyclodextrin and gamma-cyclodextrin.

[0088] In one aspect, the content of alpha-cyclodextrin in the reaction solution can be 1-100 parts;

[0089] In one aspect, the content of beta-cyclodextrin in the reaction solution can be 0-100 parts;

[0090] In one aspect, the content of gamma-cyclodextrin in the reaction solution can be 0-100 parts;

[0091] Case 4 provided by the present application:

[0092] According to the method of case 3, the hydrolysis is carried out under the conditions of temperature 55-65℃, pH 5.0-7.0, and time 0.5h-2h.

[0093] In one aspect, the temperature is: 55-56℃, 56-57℃, 57-58℃, 58-59℃, 59-60℃, 60-61℃, 61-62℃, 62-63℃, 63-64℃, 64-65℃.

[0094] In one aspect, the temperature is: 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃.

[0095] In an aspect, the time is: 0.5-0.6 h, 0.6-0.7 h, 0.7-0.8 h, 0.8-0.9 h, 0.9-1.0 h, 1.0-1.1 h, 1.1-1.2 h, 1.2-1.3 h, 1.3-1.4 h, 1.4-1.5 h, 1.5-1.6 h, 1.6-1.7 h, 1.7-1.8 h, 1.8-1.9 h, 1.9-2.0 h.

[0096] In an aspect, the time is: 0.5 h, 0.6 h, 0.7 h, 0.8 h, 0.9 h, 1.0 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2.0 h.

[0097] Case 5 provided by the application:

[0098] According to the method of case 4, the amount of α-amylase added is: 10-1000 U / g starch / dextrin;

[0099] In an aspect, the amount of α-amylase added is: 10-50 U / g starch / dextrin, 50-100 U / g starch / dextrin, 100-150 U / g starch / dextrin, 150-200 U / g starch / dextrin, 200-250 U / g starch / dextrin, 250-300 U / g starch / dextrin, 300-350 U / g starch / dextrin, 350-400 U / g starch / dextrin, 400-450 U / g starch / dextrin, 450-500 U / g starch / dextrin, 500-550 U / g starch / dextrin, 550-600 U / g starch / dextrin, 600-650 U / g starch / dextrin, 650-700 U / g starch / dextrin, 700-750 U / g starch / dextrin, 750-800 U / g starch / dextrin, 800-850 U / g starch / dextrin, 850-900 U / g starch / dextrin, 900-950 U / g starch / dextrin, 950-1000 U / g starch / dextrin.

[0100] Case 6 provided by the application:

[0101] According to the method of case 4, the reaction solution containing a plurality of cyclodextrins is obtained by mixing starch or dextrin as a substrate, adding cyclodextrin glucosyltransferase for gelatinization and liquefaction, and then adding cyclodextrin glucosyltransferase for cyclization reaction.

[0102] In one aspect, all starches as substrates are suitable for the present application. The starch can be derived from any natural source, and the "natural" starch is the unmodified form naturally found. Starch is a carbohydrate widely present in plants, mainly synthesized by amyloplast and stored in the roots, stems, leaves and seeds of plants by glucose molecules. Depending on the plant source, the type and characteristics of starch also vary. The natural source can be cereal starch, legume starch, tuber starch, etc., such as corn, pea, potato, sweet potato, barley, wheat, rice, cassava, sorghum, lotus root, yam.

[0103] In one aspect, the gelatinization refers to the process of starch granules changing from solid state to gelatinous or paste state under the action of heat and water. This process produces significant changes in the physical and chemical properties of starch, and is an important characteristic of starch in food processing and industrial applications; in the embodiments of the present application, the gelatinization is carried out by adding water and reacting at a certain temperature; in the present application, the starch suspension can be gelatinized by direct heating; different types of starch have different gelatinization temperatures, for example, the gelatinization temperature of corn starch is about 60-80℃; the gelatinization temperature of potato starch is about 60-80℃; the gelatinization temperature of wheat starch is about 60-70℃; the gelatinization temperature of rice starch is about 70-85℃; the gelatinization temperature of lotus seed starch is about 70-80℃.

[0104] In a laboratory environment, a water bath is often used, but in actual industrial production and food processing, as long as the starch is added with water and heated for gelatinization reaction, the technical solution of the present application can be realized.

[0105] In one aspect, after the slurry is prepared, the mass fraction of starch or dextrin is 5-25% (w / w).

[0106] In one aspect, after the slurry is prepared, the mass fraction of starch or dextrin is 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 11% (w / w), 12% (w / w), 13% (w / w), 14% (w / w), 15% (w / w), 16% (w / w), 17% (w / w), 18% (w / w), 19% (w / w), 20% (w / w), 21% (w / w), 22% (w / w), 23% (w / w), 24% (w / w), 25% (w / w).

[0107] In one aspect, the mass fraction of starch or dextrin after the size mixing is: 5% to 6% (w / w), 6% to 7% (w / w), 7% to 8% (w / w), 8% to 9% (w / w), 9% to 10% (w / w), 10% to 11% (w / w), 11% to 12% (w / w), 12% to 13% (w / w), 13% to 14% (w / w), 14% to 15% (w / w), 15% to 16% (w / w), 16% to 17% (w / w), 17% to 18% (w / w), 18% to 19% (w / w), 19% to 20% (w / w), 20% to 21% (w / w), 21% to 22% (w / w), 22% to 23% (w / w), 23% to 24% (w / w), 24% to 25% (w / w).

[0108] In one aspect, the conditions of the gelatinization and liquefaction are: 2-10 U / g (dry starch basis) of cyclodextrin glucanotransferase is added, 80-90°C, and gelatinization is performed for 20-30 min.

[0109] In one aspect, the conditions of the cyclization reaction are: 2-10 U / g (dry starch basis) of cyclodextrin glucanotransferase is added, and the reaction is performed at 40-50°C for 5-48 h.

[0110] In one aspect, the temperature of the gelatinization is: 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C.

[0111] In one aspect, the temperature of the gelatinization is: 80°C to 81°C, 81°C to 82°C, 82°C to 83°C, 83°C to 84°C, 84°C to 85°C, 85°C to 86°C, 86°C to 87°C, 87°C to 88°C, 88°C to 89°C, 89°C to 90°C.

[0112] In one aspect, the time of the gelatinization is: 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min.

[0113] In one aspect, the time of the gelatinization is: 20 min to 21 min, 21 min to 22 min, 22 min to 23 min, 23 min to 24 min, 24 min to 25 min, 25 min to 26 min, 26 min to 27 min, 27 min to 28 min, 28 min to 29 min, 29 min to 30 min.

[0114] In an aspect, the cyclodextrin glucosyltransferase is added in an amount of: 2 U / g (dry basis starch), 3 U / g (dry basis starch), 4 U / g (dry basis starch), 5 U / g (dry basis starch), 6 U / g (dry basis starch), 7 U / g (dry basis starch), 8 U / g (dry basis starch), 9 U / g (dry basis starch), 10 U / g (dry basis starch).

[0115] In an aspect, the cyclodextrin glucosyltransferase is added in an amount of: 2 U / g (dry basis starch) to 3 U / g (dry basis starch), 3 U / g (dry basis starch) to 4 U / g (dry basis starch), 4 U / g (dry basis starch) to 5 U / g (dry basis starch), 5 U / g (dry basis starch) to 6 U / g (dry basis starch), 6 U / g (dry basis starch) to 7 U / g (dry basis starch), 7 U / g (dry basis starch) to 8 U / g (dry basis starch), 8 U / g (dry basis starch) to 9 U / g (dry basis starch), 9 U / g (dry basis starch) to 10 U / g (dry basis starch).

[0116] In an aspect, the cyclodextrin glucosyltransferase sequence is as set forth in SEQ ID NO. 2 or a cyclodextrin glucosyltransferase having at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a sequence as set forth in SEQ ID NO. 2.

[0117] Cyclodextrin glucosyltransferase cyclization activity assay:

[0118] Take 0.1 mL of appropriately diluted enzyme solution, add to a 0.9 mL of pre-prepared pH 6.5, 10 mM phosphate buffer (PBS) containing 0.1 mM methyl orange (MO) solution, and mix well.

[0119] In a centrifuge tube prepared with 1% maltodextrin (DE = 4), after preheating for 5 min, react at 50°C for 10 min, add 1.0 mL of 1.0 M hydrochloric acid solution to terminate the reaction, then add 1.0 mL of 0.1 mM methyl orange solution (MO) prepared with pH 6.5, 10 mM PBS, develop color at room temperature for 20 min, and measure the absorbance at a wavelength of 505 nm. Use the inactivated enzyme solution as a blank.

[0120] In an aspect, the cyclization temperature is: 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C.

[0121] In an aspect, the cyclization temperature is: 40°C to 41°C, 41°C to 42°C, 42°C to 43°C, 43°C to 44°C, 44°C to 45°C, 45°C to 46°C, 46°C to 47°C, 47°C to 48°C, 48°C to 49°C, 49°C to 50°C.

[0122] In an aspect, the time for the cyclization is: 5-10h, 10-15h, 15-20h, 20-25h, 25-30h, 30-35h, 35-40h, 40-48h.

[0123] The application provides a case 7:

[0124] A method for purifying α-cyclodextrin by using amylase, the method is, taking a reaction solution containing multiple cyclodextrins as a substrate, adding α-amylase from Aspergillus niger, and obtaining α-cyclodextrin after reaction.

[0125] In an aspect, the amino acid sequence of the α-amylase is shown in SEQ ID NO. 1.

[0126] In an aspect, the reaction solution containing multiple cyclodextrins is a reaction solution containing α-cyclodextrin and one or both of β-cyclodextrin and γ-cyclodextrin.

[0127] In an aspect, the reaction conditions are: the hydrolysis reaction is carried out at a temperature of 55-65℃ and a pH of 5.0-7.0, and the time is 0.5h-2h.

[0128] In an aspect, the temperature is: 55-56℃, 56-57℃, 57-58℃, 58-59℃, 59-60℃, 60-61℃, 61-62℃, 62-63℃, 63-64℃, 64-65℃.

[0129] In an aspect, the temperature is: 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃.

[0130] In an aspect, the time is: 0.5-0.6h, 0.6-0.7h, 0.7-0.8h, 0.8-0.9h, 0.9-1.0h, 1.0-1.1h, 1.1-1.2h, 1.2-1.3h, 1.3-1.4h, 1.4-1.5h, 1.5h-1.6h, 1.6h-1.7h, 1.7h-1.8h, 1.8h-1.9h, 1.9h-2.0h.

[0131] In an aspect, the time is: 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2.0h.

[0132] In an aspect, the amount of the α-amylase added is: 10U / g starch / dextrin-1000U / g starch / dextrin;

[0133] In an aspect, the alpha-amylase is added in an amount of: 10 U / g starch / dextrin to 50 U / g starch / dextrin, 50 U / g starch / dextrin to 100 U / g starch / dextrin, 100 U / g starch / dextrin to 150 U / g starch / dextrin, 150 U / g starch / dextrin to 200 U / g starch / dextrin, 200 U / g starch / dextrin to 250 U / g starch / dextrin, 250 U / g starch / dextrin to 300 U / g starch / dextrin, 300 U / g starch / dextrin to 350 U / g starch / dextrin, 350 U / g starch / dextrin to 400 U / g starch / dextrin, 400 U / g starch / dextrin to 450 U / g starch / dextrin, 450 U / g starch / dextrin to 500 U / g starch / dextrin, 500 U / g starch / dextrin to 550 U / g starch / dextrin, 550 U / g starch / dextrin to 600 U / g starch / dextrin, 600 U / g starch / dextrin to 650 U / g starch / dextrin, 650 U / g starch / dextrin to 700 U / g starch / dextrin, 700 U / g starch / dextrin to 750 U / g starch / dextrin, 750 U / g starch / dextrin to 800 U / g starch / dextrin, 800 U / g starch / dextrin to 850 U / g starch / dextrin, 850 U / g starch / dextrin to 900 U / g starch / dextrin, 900 U / g starch / dextrin to 950 U / g starch / dextrin, 950 U / g starch / dextrin to 1000 U / g starch / dextrin.

[0134] Case 8 provided by the present application:

[0135] The reaction solution containing a plurality of cyclodextrins is a mixture obtained after adding cyclodextrin glucosyltransferase to starch or dextrin as a substrate, adjusting the slurry, gelatinizing and liquefying, and then adding cyclodextrin glucosyltransferase to perform cyclization reaction.

[0136] In an aspect, the substrate is one or more of corn starch, cassava starch, potato starch, wheat starch, and malt dextrin.

[0137] In an aspect, after the slurry is adjusted, the mass fraction of the starch or dextrin is 5-25% (w / w).

[0138] In an aspect, after the slurry is adjusted, the mass fraction of the starch or dextrin is 5% (w / w), 6% (w / w), 7% (w / w), 8% (w / w), 9% (w / w), 10% (w / w), 11% (w / w), 12% (w / w), 13% (w / w), 14% (w / w), 15% (w / w), 16% (w / w), 17% (w / w), 18% (w / w), 19% (w / w), 20% (w / w), 21% (w / w), 22% (w / w), 23% (w / w), 24% (w / w), 25% (w / w).

[0139] In one aspect, the mass fraction of starch or dextrin after the size mixing is: 5%~6% (w / w), 6%~7% (w / w), 7%~8% (w / w), 8%~9% (w / w), 9%~10% (w / w), 10%~11% (w / w), 11%~12% (w / w), 12%~13% (w / w), 13%~14% (w / w), 14%~15% (w / w), 15%~16% (w / w), 16%~17% (w / w), 17%~18% (w / w), 18%~19% (w / w), 19%~20% (w / w), 20%~21% (w / w), 21%~22% (w / w), 22%~23% (w / w), 23%~24% (w / w), 24%~25% (w / w).

[0140] In one aspect, the conditions of the gelatinization and liquefaction are: adding 2-10 U / g (dry starch basis) cyclodextrin glucosyltransferase, 80-90℃, and gelatinizing for 20~30 min.

[0141] In one aspect, the cyclization reaction conditions are: adding 2-10 U / g (dry starch basis) cyclodextrin glucosyltransferase, and reacting for 5-48 h at 40-50℃.

[0142] In one aspect, the temperature of the gelatinization is: 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃.

[0143] In one aspect, the temperature of the gelatinization is: 80℃~81℃, 81℃~82℃, 82℃~83℃, 83℃~84℃, 84℃~85℃, 85℃~86℃, 86℃~87℃, 87℃~88℃, 88℃~89℃, 89℃~90℃.

[0144] In one aspect, the time of the gelatinization is: 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min.

[0145] In one aspect, the time of the gelatinization is: 20 min~21 min, 21 min~22 min, 22 min~23 min, 23 min~24 min, 24 min~25 min, 25 min~26 min, 26 min~27 min, 27 min~28 min, 28 min~29 min, 29 min~30 min.

[0146] In an aspect, the cyclodextrin glucosyltransferase is added in an amount of: 2 U / g (dry basis starch), 3 U / g (dry basis starch), 4 U / g (dry basis starch), 5 U / g (dry basis starch), 6 U / g (dry basis starch), 7 U / g (dry basis starch), 8 U / g (dry basis starch), 9 U / g (dry basis starch), 10 U / g (dry basis starch).

[0147] In an aspect, the cyclodextrin glucosyltransferase is added in an amount of: 2 U / g (dry basis starch) to 3 U / g (dry basis starch), 3 U / g (dry basis starch) to 4 U / g (dry basis starch), 4 U / g (dry basis starch) to 5 U / g (dry basis starch), 5 U / g (dry basis starch) to 6 U / g (dry basis starch), 6 U / g (dry basis starch) to 7 U / g (dry basis starch), 7 U / g (dry basis starch) to 8 U / g (dry basis starch), 8 U / g (dry basis starch) to 9 U / g (dry basis starch), 9 U / g (dry basis starch) to 10 U / g (dry basis starch).

[0148] In an aspect, the cyclization temperature is: 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C.

[0149] In an aspect, the cyclization temperature is: 40°C to 41°C, 41°C to 42°C, 42°C to 43°C, 43°C to 44°C, 44°C to 45°C, 45°C to 46°C, 46°C to 47°C, 47°C to 48°C, 48°C to 49°C, 49°C to 50°C.

[0150] In an aspect, the cyclization time is: 5 to 10 h, 10 to 15 h, 15 to 20 h, 20 to 25 h, 25 to 30 h, 30 to 35 h, 35 to 40 h, 40 to 48 h.

[0151] The corn starch, cassava starch, and soluble starch involved in the following examples were purchased from Shandong Shouguang Ju Neng Corn Development Co., Ltd., Thai Taw Hiap, and Shanghai Reagent Co., Ltd. The alpha-amylase from Bacillus subtilis involved in the following examples was purchased from Sinopharm, with the product number TA044725G; the alpha-amylase from Bacillus licheniformis was purchased from Macklin, with the product number A693385; and the alpha-amylase from Bacillus amyloliquefaciens was purchased from Macklin, with the product number A886083.

[0152] The alpha-CD, beta-CD, and gamma-CD involved in the following examples were purchased from Shanghai Reagent Co., Ltd., with a purity of 98%.

[0153] The CGTase amino acid sequence involved in the following examples is shown in SEQ ID NO. 2, and is as follows:

[0154] SPDTSVDNKVNFSTDVIYQIVTDRFADGDRTNNPAGDAFSGDRSNLKLYFGGDWQGIIDKINDGYLTGMGVTALWISQPVENITSVIKYSGVNNTSYHGYWARDFKQTNDAFGDFADFQNLIDTAHAHNIKVVIDFAPNHTSPADRDNPGFAENGGMYDNGSLLGAYSNDTAGLFHHNGGTDFSTIEDGIYKNLYDLADINHNNNAMDAYFKSAIDLWLGMGVDGIRFDAVKHMPFGWQKSFVSSIYGGDHPVFTFGEWYLGADQTDGDNIKFANESGMNLLDFEYAQEVREVFRDKTETMKDLYEVLASTESQYDYINNMVTFIDNHDMDRFQVAGSGTRATEQALALTLTSRGVPAIYYGTEQYMTGDGDPNNRAMMTSFNTGTTAYKVIQALAPLRKSNPAIAYGTTTERWVNNDVLIIERKFGSSAALVAINRNSSAAYPISGLLSSLPAGTYSDVLNGLLNGNSITVGSGGAVTNFTLAAGGTAVWQYTAPETSPAIGNVGPTMGQPGNIVTIDGRGFGGTAGTVYFGTTAVTGSGIVSWEDTQIKAVIPKVAAGKTGVSVKTSSGTASNTFKSFNVLTGDQVTVRFLVNQANTNYGTNVYLVGNAAELGSWDPNKAIGPMYNQVIAKYPSWYYDVSVPAGTKLDFKFIKKGGGTVTWEGGGNHTYTTPASGVGTVTVDWQN

[0155] In the present invention, the CGTase is used only to gelatinize and liquefy the starch to obtain a dextrin mixture.

[0156] The sequence of the Aspergillus niger-derived alpha-amylase involved in the following examples is shown as SEQ ID NO. 1, and is as follows:

[0157] MVAWWSLFLYGLQVAAPALAATPADWRSQSIYFLLTDRFARTDGSTTATCNTADQKYCGGTWQGIIDKLDYIQGMGFTAIWITPVTAQLPQTTAYGDAYHGYWQQDIYSLNENYGTADDLKALSSALHERGMYLMVDVVANHMGYDGAGSSVDYSVFKPFSSQDYFHPFCFIQNYEDQTQVEDCWLGDNTVSLPDLDTTKDVVKNEWYDWVGSLVSNYSIDGLRIDTVKHVQKDFWPGYNKAAGVYCIGEVLDGDPAYTCPYQNVMDGVLNYPIYYPLLNAFKSTSGSMDDLYNMINTVKSDCPDSTLLGTFVENHDNPRFASYTNDIALAKNVAAFIILNDGIPIIYAGQEQHYAGGNDPANREATWLSGYPTDSELYKLIASANAIRNYAISKDTGFVTYKNWPIYKDDTTIAMRKGTDGSQIVTILSNKGASGDSYTLSLSGAGYTAGQQLTEVIGCTTVTVGSDGNVPVPMAGGLPRVLYPTEKLAGSKICSSS

[0158] The preparation method of the Aspergillus niger-derived alpha-amylase is as follows:

[0159] The alpha-amylase-encoding gene is constructed between the NcoI and XhoI enzyme cutting sites of the plasmid vector pET-20b(+), to obtain a recombinant plasmid, which is named as E. coli BL21(DE3) (amylase / pET-20b(+)). The E. coli BL21(DE3) containing the recombinant plasmid or the mutant plasmid is inoculated into an LB culture medium, and cultured at 37°C until the OD600 is about 0.6. The inoculation is added into a TB culture medium to add IPTG with a final concentration of 0.1 mM, and the protein expression is induced by further culturing for 30 hours.

[0160] The determination method used in the present application is as follows:

[0161] The product in the reaction solution is detected by ion chromatography. The ion chromatography test conditions are as follows: CarPac PA200 ion column, column temperature is 30°C, the detector is an amperometric detector, gold electrode is used as the working electrode, Ag / AgCl is used as the reference electrode, water-NaOH solution (0.25 mol·L -1 )-NaAC solution (0.5 mol·L -1 ) is used as the mobile phase, and the flow rate is 0.4 mL·min -1Gradient elution was performed. The cyclodextrin product was prepared into a 10 mg / L -1 solution, centrifuged and filtered through a 0.22 μm filter membrane.

[0162] The meaning of w / v involved in the following examples is: g / L.

[0163] The hydrolysis rate involved in the following examples is the ratio of the content of cyclodextrin in the solution before reaction to the content of the corresponding cyclodextrin after reaction.

[0164] Example 1: Preparation of α-cyclodextrin using a cyclodextrin mixture as a substrate

[0165] The specific steps are as follows:

[0166] (1) Preparation of a substrate: a mixed solution of 5% (w / v) α-cyclodextrin, 5% (w / v) β-cyclodextrin and 5% (w / v) γ-cyclodextrin was prepared with deionized water as a substrate.

[0167] (2) Preparation of α-cyclodextrin: 300 U / g of cyclodextrin of the α-amylase from Aspergillus niger of the application was added to the cyclodextrin mixed solution obtained in step (1), and the reaction was carried out at 60°C and pH 5.5 for 1 h. After the reaction was completed, the contents of the three cyclodextrins in the reaction solution were determined, and the hydrolysis rate was calculated, and the results are shown in Table 1:

[0168] Table 1: Hydrolysis rate

[0169] Reaction time Beta-cyclodextrin hydrolysis rate (%) Gamma-cyclodextrin hydrolysis rate (%) 5 min 3.4 10.7 20 min 53.3 85.4 30 min 78.7 100 45 min 93.1 100 55 min 100 100 60 min 100 100

[0170] The results show that the hydrolysis rates of β-cyclodextrin and γ-cyclodextrin are both 100%, and all of them are hydrolyzed into glucose and oligosaccharides, while the α-cyclodextrin in the reaction solution has no effect.

[0171] Example 2: Preparation of α-cyclodextrin using corn starch as a substrate

[0172] The specific steps are as follows:

[0173] (1) 100 mL of a starch milk with a concentration of 10% (w / w) was prepared by taking corn starch and adjusting with deionized water, and after stirring, 5 U / g (dry basis of starch) of cyclodextrin glucosyltransferase CGT was added, and the temperature was raised to 90°C, and the liquefaction was carried out for 1 h;

[0174] (2) The liquefied starch was cooled to 45°C, and 3 U / g (dry basis of starch) of cyclodextrin glucosyltransferase CGT was added and reacted for 48 h to obtain a water solution containing different cyclodextrins.

[0175] The detection results show that in the aqueous solution, the proportion of α-cyclodextrin is 60.7%, the proportion of β-cyclodextrin is 35.2%, and the proportion of γ-cyclodextrin is 4.1%. In addition to cyclodextrin, there are also unreacted starch, malt dextrin and other macromolecular substances in the aqueous solution.

[0176] (3) 100 U / g of dry starch of Aspergillus-derived amylase was added to the solution obtained in step (2), the pH was adjusted to 5.5, and the reaction was carried out at 60°C for 1 h. After the reaction was completed, the enzyme was inactivated by boiling for 30 min. During the reaction, samples were taken regularly, the contents of the three cyclodextrins in the reaction solution were determined, and the hydrolysis rates were calculated. The results are shown in Table 2:

[0177] Table 2: Hydrolysis rate

[0178] Reaction time Beta-cyclodextrin hydrolysis rate Gamma-cyclodextrin hydrolysis rate 5 min 3.6 11.2 20 min 54.5 87.6 30 min 77.4 100 45 min 92.9 100 55 min 100 100 60 min 100 100

[0179] The results show that:

[0180] The hydrolysis rates of β-cyclodextrin and γ-cyclodextrin in the reaction solution are 100%, and all of them are hydrolyzed into glucose and oligosaccharides.

[0181] Example 3: Preparation of α-cyclodextrin using cassava starch as substrate

[0182] The specific steps are as follows:

[0183] (1) 100 mL of 10% (w / w) starch milk was prepared by taking cassava starch and adjusting the concentration with deionized water, and then 5 U / g (dry starch basis) of CGTase was added and stirred. The temperature was raised to 90°C, and the starch was liquefied for 1 h;

[0184] (2) The liquefied starch was cooled to 45°C, and then 3 U / g (dry starch basis) of CGTase was added and reacted for 30 h to obtain an aqueous solution containing different cyclodextrins.

[0185] Since the same cyclodextrin glucosyltransferase is used as in Example 2, the detection results show that in the aqueous solution, the proportion of α-cyclodextrin, the proportion of β-cyclodextrin, and the proportion of γ-cyclodextrin are the same as in Example 2. In addition to cyclodextrin, there are also unreacted starch, malt dextrin and other macromolecular substances in the aqueous solution.

[0186] (3) 100 U / g of dry starch of Aspergillus-derived amylase was added to the solution obtained in step (2), the pH was adjusted to 5.5, and the reaction was carried out at 60°C for 1 h. After the reaction was completed, the enzyme was inactivated by boiling for 30 min. During the reaction, samples were taken regularly, the contents of the three cyclodextrins in the reaction solution were determined, and the hydrolysis rates were calculated. The results are shown in Table 3:

[0187] Table 3: Hydrolysis rate

[0188] Reaction time Beta-cyclodextrin hydrolysis rate (%) Gamma-cyclodextrin hydrolysis rate (%) 5 min 3.3 15.7 20 min 64.5 89.4 30 min 77.6 100 45 min 100 100 55 min 100 100 60 min 100 100

[0189] The results show that Figures 1 to 3 ):

[0190] The hydrolysis rates of β-cyclodextrin and γ-cyclodextrin in the reaction solution were 100%, and all were hydrolyzed into glucose and oligosaccharides.

[0191] Example 4: Preparation of α-cyclodextrin using soluble starch as substrate

[0192] The specific steps are as follows: (1) take soluble starch and adjust it to 10% (w / w) starch milk 100 mL with deionized water, stir, add 3 U / g (dry basis of starch) of CGTase, heat to 90°C, and liquefy for 1 h;

[0193] (2) After the liquefaction of the starch, cool it to 45°C, and then add 3 U / g (dry basis of starch) of CGTase and react for 10 h. The solution containing different cyclodextrins is obtained.

[0194] Since the same cyclodextrin glucosyltransferase is used as in Example 2, it is detected that the proportion of α-cyclodextrin in the aqueous solution, the proportion of β-cyclodextrin, and the proportion of γ-cyclodextrin are the same as in Example 2. In addition to cyclodextrin, there are unreacted starch, maltodextrin and other macromolecular substances in the aqueous solution.

[0195] (3) Add 300 U / g of starch dry basis of Aspergillus-derived amylase to the solution obtained in step (2), adjust the pH to 5.5, and react at 60°C for 3 h. During the reaction, periodically take samples to determine the content of the three cyclodextrins in the reaction solution and calculate the hydrolysis rate, as shown in Table 4; take the reaction solutions of 5 min, 1 h, and 3 h respectively, and boil for 30 min. The products under different reaction times are detected by ion chromatography, and the results are shown in Table 5. Figure 1

[0196] Table 4: Hydrolysis rate

[0197] Reaction time Beta-cyclodextrin hydrolysis rate (%) Gamma-cyclodextrin hydrolysis rate (%) 5 min 5.1 14.2 20 min 62.3 86.4 30 min 85.9 100 45 min 97.2 100 55 min 100 100 60 min 100 100

[0198] The results show that

[0199] From Figure 1 It can be seen from the results that as the amylase hydrolysis time is prolonged, β-cyclodextrin and γ-cyclodextrin are gradually hydrolyzed into glucose and maltose and other non-cyclic oligosaccharides, and only α-cyclodextrin is left after 1 h of reaction, which makes α-cyclodextrin purified.

[0200] Comparative Example 1

[0201] (1) Take soluble starch and adjust it to 10% (w / w) starch milk 100 mL with deionized water, stir, add 3 U / g (dry basis of starch) of CGTase, heat to 90°C, and liquefy for 1 h;

[0202] ​(2) The liquefied starch was cooled to 45°C, and 3 U / g (dry starch basis) of CGTase was added and reacted for 10 hours. The resulting solution contained various cyclodextrins.

[0203] (3) To the solution obtained in step (2), 300 U / g (dry starch basis) of the amylase derived from Bacillus subtilis was added, and the pH was adjusted to 5.5, and the reaction was carried out at 60°C for 3 hours. The reaction solution after 3 hours was boiled for 30 minutes, and the product was analyzed by ion chromatography. The results are shown in Table 1. Figure 4

[0204] From the results, it can be seen that the amylase derived from Bacillus subtilis cannot hydrolyze cyclodextrins. Figure 4 Comparative Example 2

[0205] (1) 100 mL of a starch solution of 10% (w / w) concentration was prepared by dissolving soluble starch in deionized water, and 3 U / g (dry starch basis) of CGTase was added and the temperature was raised to 90°C to liquefy the starch for 1 hour.

[0206] (2) The liquefied starch was cooled to 45°C, and 3 U / g (dry starch basis) of CGTase was added and reacted for 10 hours. The resulting solution contained various cyclodextrins.

[0207] (3) To the solution obtained in step (2), 300 U / g (dry starch basis) of the amylase derived from Bacillus subtilis was added, and the pH was adjusted to 5.5, and the reaction was carried out at 60°C for 3 hours. The reaction solution after 3 hours was boiled for 30 minutes, and the product was analyzed by ion chromatography. The results are shown in Table 1.

[0208] Figure 5 From the results, it can be seen that the amylase derived from Bacillus subtilis cannot hydrolyze cyclodextrins.

[0209] Figure 5 Comparative Example 3

[0210] (1) 100 mL of a starch solution of 10% (w / w) concentration was prepared by dissolving soluble starch in deionized water, and 3 U / g (dry starch basis) of CGTase was added and the temperature was raised to 90°C to liquefy the starch for 1 hour.

[0211] (2) The liquefied starch was cooled to 45°C, and 3 U / g (dry starch basis) of CGTase was added and reacted for 10 hours. The resulting solution contained various cyclodextrins.

[0212] (3) To the solution obtained in step (2), 300 U / g (dry starch basis) of the amylase derived from Bacillus subtilis was added, and the pH was adjusted to 5.5, and the reaction was carried out at 60°C for 3 hours. The reaction solution after 3 hours was boiled for 30 minutes, and the product was analyzed by ion chromatography. The results are shown in Table 1.

[0213] ​​​(3) 300 U / g of dry starch of commercially available amylases from Bacillus licheniformis, Aspergillus oryzae, Aspergillus candidus, Trichoderma reesei, Bacillus amyloliquefaciens, Bacillus stearothermophilus were added into the solution obtained in step (2) respectively, the pH was adjusted to 5.5, and the reaction was carried out at 90°C for 3 h. The reaction solution after 3 h was boiled for 30 min, and the product was detected by ion chromatography;

[0214] The results show that other commercially available amylases of different sources cannot hydrolyze cyclodextrin.

[0215] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for producing α-cyclodextrin by using amylase, characterized by, The method is to take a reaction solution containing multiple cyclodextrins as a substrate, add an alpha-amylase from Aspergillus niger, and obtain alpha-cyclodextrin after hydrolysis.

2. The method of claim 1, wherein, The amino acid sequence of the alpha-amylase is shown in SEQ ID NO.

1.

3. The method according to claim 1 or 2, characterized in that, The reaction solution containing multiple cyclodextrins is a reaction solution containing alpha-cyclodextrin and one or both of beta-cyclodextrin and gamma-cyclodextrin.

4. The method of claim 3, wherein, The hydrolysis is performed under the conditions of a temperature of 55-65°C, a pH of 5.0-7.0, and a time of 0.5h-2h.

5. The method of claim 3 or 4, wherein, The temperature is: 55-56°C, 56-57°C, 57-58°C, 58-59°C, 59-60°C, 60-61°C, 61-62°C, 62-63°C, 63-64°C, 64-65°C.

6. The method according to any one of claims 1 to 5, characterized in that, The time is: 0.5-0.6h, 0.6-0.7h, 0.7-0.8h, 0.8-0.9h, 0.9-1.0h, 1.0-1.1h, 1.1-1.2h, 1.2-1.3h, 1.3-1.4h, 1.4-1.5h, 1.5h-1.6h, 1.6h-1.7h, 1.7h-1.8h, 1.8h-1.9h, 1.9h-2.0h.

7. The method according to any one of claims 1 to 6, characterized in that, The addition amount of the alpha-amylase is: 10U / g starch / dextrin-1000U / g starch / dextrin.

8. The method of claim 7, wherein, The reaction solution containing multiple cyclodextrins is a mixture obtained after starch or dextrin is taken as a substrate, slurry is adjusted, a cyclodextrin glucosyltransferase is added for gelatinization and liquefaction, and a cyclodextrin glucosyltransferase is added for cyclization reaction.

9. The method of claim 8, wherein, The substrate is one or more of corn starch, cassava starch, potato starch, wheat starch, and malt dextrin.

10. The method of claim 9, wherein, The amino acid sequence of the cyclodextrin glucosyltransferase is shown in SEQ ID NO.

2.

11. The method according to any one of claims 8 to 10, characterized in that, After the slurry is adjusted, the mass fraction of the starch or dextrin is: 5-25% (w / w).

12. The method according to any one of claims 8 to 10, characterized in that, The gelatinization and liquefaction conditions are: a cyclodextrin glucosyltransferase is added, the addition amount is: 2-10U / g starch, the temperature is 80-90°C, and the gelatinization time is 20-30min.

13. The method of any one of claims 8-10, wherein, The cyclization reaction conditions are: a cyclodextrin glucosyltransferase is added, the addition amount is: 2-10U / g starch, and the reaction is performed at a temperature of 40-50°C for 5-48h.

14. A method for purifying α-cyclodextrin using amylase, characterized by, The method is to take a reaction solution containing multiple cyclodextrins as a substrate, add an alpha-amylase from Aspergillus niger, and obtain alpha-cyclodextrin after purification.

15. The method of claim 14, wherein, The amino acid sequence of the alpha-amylase is shown in SEQ ID NO.

1.

16. The method of claim 15, wherein, The reaction solution containing multiple cyclodextrins is a reaction solution containing alpha-cyclodextrin and one or both of beta-cyclodextrin and gamma-cyclodextrin.

17. The method of claim 16, wherein, The reaction is performed under the conditions of a temperature of 55-65°C, a pH of 5.0-7.0, and a time of 0.5h-2h.

18. The method of claim 17, wherein, The addition amount of the alpha-amylase is: 10U / g starch / dextrin-1000U / g starch / dextrin.

19. The method of claim 18, wherein, The reaction solution containing multiple cyclodextrins is obtained by mixing starch or dextrin as a substrate, adding cyclodextrin glucosyltransferase for gelatinization and liquefaction, and then adding cyclodextrin glucosyltransferase for cyclization reaction.

20. The method of claim 19, wherein, The substrate is one or more of corn starch, cassava starch, potato starch, wheat starch, and malt dextrin. Preferably, the mass fraction of the starch or dextrin after the mixing is 5-25% (w / w). Preferably, the gelatinization and liquefaction conditions are as follows: the addition amount of the cyclodextrin glucosyltransferase is 2-10 U / g of starch, the temperature is 80-90°C, and the gelatinization time is 20-30 min. Preferably, the cyclization reaction conditions are as follows: the addition amount of the cyclodextrin glucosyltransferase is 2-10 U / g of starch, and the reaction is performed at a temperature of 40-50°C for 5-48 h.

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